Jane Gibbs BA Gdiped Gdippsych

Home , Backhousia, Corymbia dallachiana, Corymbia ficifolia, Eucalyptus bancroftii, Eucalyptus brassiana, Eucalyptus bridgesiana

Asthma and Plants: Chemotaxonomic Relationships and Patterns of Asthma Incidence and Respiratory Symptoms, in Urban Coastal Versus Rural Highland Areas in South-East Queensland, Australia, with Special Reference to the Family Myrtaceae

Author Gibbs, Jane

Published 2007

Thesis Type Thesis (PhD Doctorate)

School School of Public Health

DOI https://doi.org/10.25904/1912/3128

Downloaded from http://hdl.handle.net/10072/366726

Griffith Research Online https://research-repository.griffith.edu.au

ASTHMA AND PLANTS: CHEMOTAXONOMIC RELATIONSHIPS AND PATTERNS OF ASTHMA INCIDENCE AND RESPIRATORY SYMPTOMS, IN URBAN COASTAL VERSUS RURAL HIGHLAND AREAS IN SOUTH-EAST QUEENSLAND, AUSTRALIA, WITH SPECIAL REFERENCE TO THE FAMILY MYRTACEAE.

Jane Gibbs BA GDipEd GDipPsych

School of Public Health Faculty of Health Science Griffith University

Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy on 22 December 2006 ii

STATEMENT OF ORIGINALITY

This work has not been previously submitted for a degree or diploma in a university. To the best of my knowledge and belief, this thesis contains no material previously published or written by another person except where due reference is made in the thesis itself.

Jane Gibbs

iii

Dedication

To my daughter Anna, and all the children who live with asthma, for providing the reason to begin this task, and to her grandmother, and my mother, Marie Gibbs (1926-1995), for providing the persistence required to finish it. iv

Abstract This thesis represents an exploratory and iterative study into the relationships of Australian native plants from the family Myrtaceae, with respiratory symptoms, specifically asthma. This relationship is explored from a chemo-taxonomic stance and the connections with other plants with related chemotaxonomy are underlined. The research was performed against a background of geographical comparison between an urban coastal area and a rural mountainous area 90 kilometres just north of Brisbane, Queensland, Australia. The focus was the possible contribution of Melaleuca quinquenervia to the occurrence of autumn respiratory symptoms, especially asthma.

The research challenges current beliefs that wind-pollinated plants are the only sources of allergens and the major botanical health threat for those who suffer with asthma. There is some critical analysis of the current understanding of world patterns of asthma, with particular reference to the Global Initiative for Asthma (GINA) geographic compilations.

In this study 380 children were skin-tested with a battery of commercial allergens including Eucalyptus. Comparisons among children from three coastal schools and students from one mountain school were made with a view to testing the hypothesis that proximate vegetation would affect responses to allergens. As hypothesised, significant differences were detected between the coastal and range students in responses to Eucalyptus, which flowers prolifically on the coast. Coastal children, compared to the rural children from the mountain area exhibited a significantly greater percentage of skin-test responses (p≤.05) to Eucalyptus, when all responses greater than zero were measured.

In the next phase more comparisons were undertaken with a smaller group of adults and adolescents in the coast and range communities. A range of terpenes and oxidized terpenes was applied during skin tests along with commercial allergens. Coastal and range reaction profiles revealed that coastal

Abstract v

participants exhibit greater skin reactivity. When a 3mm wheal cut-off is employed alpha-pinene response in the range group is notable.

During this second phase ambient air sampling was carried out at the same time as participants measured peak expiratory flow (PEF) and recorded respiratory symptoms. Chemicals trapped on Tenax over spring and autumn were analysed via GCMS and the results combined with symptom variables to ascertain significant predictors of symptom change.

Using stepwise and general linear regression significant relationships were demonstrated between PEF and beta pinene and limonene in ambient air. In both types of modelling several terpenes were shown to be significant predictors of respiratory symptoms. In the coastal group the addition of alpha or beta pinene to a General Linear Regression model predicting standardized peak flow, accounted to an additional 20% of the variance in autumn. The model consisted of ozone, nitrogen dioxide and particulates less than 10 micron, all lagged three days. For spring, the additional variance explained was only 6 % and for spring an autumn combined it was 5%. Significant relationships were also demonstrated between linalool levels and preventer and reliever usage. The results support the hypotheses regarding the role of terpenes as having a possible role in the acquisition and exacerbation of asthma.

Additionally a number of floral studies resulted in chemical profiles being elucidated for flowers from popular plants in Australia. Tenax collections of vapours from some household and lifestyle products resulted in chemical profiles for popular items.

In conclusion, some observations about vegetation and patterns of asthma have been made and a new model of asthma acquisition proposed.

Abstract vi

TABLE OF CONTENTS

LIST OF TABLES ...... ix

LIST OF FIGURES...... xii

LIST OF APPENDICES...... xvi

ABBREVIATIONS ...... xviii

GLOSSARY...... xxi

ACKNOWLEDGEMENTS...... xxiv

1 . Introduction ...... 1

2. Current Knowledge ...... 7

2.1 Asthma and allergy ...... 7

2.1.1 Asthma definition and world and Australian incidence...... 8 2.1.2 Some host risk factors for development of asthma...... 20

2.1.3 Important processes in asthma...... 23

2.1.4 Anthropogenic and ‘natural’ asthma contributors...... 31

2.2 Possible role of terpenes ...... 51

2.2.1 What are terpenes? ...... 51 2.2.2 Important structural groups in plant chemistry ...... 60

2.2.3 Essential Oils...... 64

2.2.4 Terpene distributions ...... 73

2.2.5 Plant related irritants and allergens ...... 105

2.2.6 Chemical structures implicated in asthma ...... 122

Table of Contents vii

3. Study objectives...... 134

3.1 Summary of current knowledge ...... 134

3.1.1 Terpenes as asthma triggers ...... 134 3.1.2 Pollination problems and paradoxes...... 135

3.2 Rationale for the investigation...... 140 3.3 Objectives of the current investigation ...... 154 3.4 Strategy for the investigation...... 155

4. Methodologies...... 166

4.1 Geography and vegetation of coastal and range areas...... 166

4.1.1 Description of coastal area under investigation ...... 166 4.1.2 Description of mountain range area under investigation...... 169

4.2 Procedures for allergen testing in coastal and range children...... 176 4.3 Procedures for allergen and terpene testing in coastal and Range groups of adults and adolescents in phase 2...... 180 4.4 Procedures for air sampling and respiratory diary analysis for groups of adults and adolescents ...... 182

4.4.1 Participant numbers and location ...... 182 4.4.2 Airspora ...... 185

4.4.3 Air pollution...... 185

4.4.4 Meteorological variables...... 186

4.4.5 Air sampling and respiratory diaries...... 186

4.5 Procedures for plant and fungi analyses...... 188

4.5.1 Observational studies of fungi and flowers ...... 188 4.5.2 Procedures for fungi and flower analyses...... 190

4.5.3 GCMS Analysis Of Tenax Tube Air Samples ...... 193

4.6 Procedures for analysis of household and lifestyle products ...... 198 4.7 Statistical procedures...... 199

Table of Contents viii

5 . Outcomes of investigations of asthma in coastal and range communities, and their flora...... 201

5.1 Occurrence and discussion of reactions to allergens in coastal and range schoolchildren...... 201 5.2 Occurrence and discussion of reactions to allergens and terpenes in coastal and range adults and adolescents...... 209 5.3 Outcomes and discussion of air sampling and respiratory diary analysis in coastal and highland adults and adolescents...... 213 5.4 Outcomes and discussion of plant and fungi analyses ...... 248

5.4.1 Headspace analyses ...... 248 5.4.2 Flower and leaf extractions...... 249

5.4.3 Flower and fungi studies...... 251

5.4.4 Tenax samples of flowers ...... 259

5.5 Outcomes and discussion of analysis of household and personal care products ...... 263

6. New models relating environmental variables and asthma ...... 265

6.1 A chemotaxonomic model for understanding world asthma patterns265 6.2 Revision of the hygiene theory...... 273

7. Conclusions and recommendations...... 276

References...... 279

Table of Contents ix

LIST OF TABLES Table 2.01 Different representations of asthma in GINA 2004...... 10 Table 2.02 All age asthma and allergy prevalence (%) rates for the South East Queensland area...... 19 Table 2.03 Fungal spores and some health issues...... 43 Table 2.04 Meteorological variables and respiratory symptoms ...... 46 Table 2.05 Allergenic pollens in different part of the world...... 49 Table 2.06 Chemical variation in Eucalyptus and Melaleuca oils...... 69 Table 2.07 Volatile emissions from forests...... 75 Table 2.08 Percentage composition of flowers and leaves ...... 78 Table 2.09 Chemical variation in a European Myrtaceae species...... 79 Table 2.10 Sesquiterpenes from parts of Eucalyptus viminalis ...... 90 Table 2.11 Terpenes in abraded leaf particles ...... 92 Table 2.12 Oxidation products from terpenes ...... 102 Table 2.13 Terpene/ozone mixes and SOA formation: Fan et al.564...... 103 Table 2.14 Adverse effects of additives and terpenes...... 116 Table 2.15 Components (%) of leaf and berry oils from Juniperus communis298 ...... 131 Table 4.01 Numbers and sex of children recruited in phase 1 SPT ...... 177 Table 4.02 Age range and sex of phase 2 participants ...... 180 Table 4.03 Participant numbers in range and coastal groups ...... 183 Table 5.01 Symptom group numbers for Phase 1 children ...... 202 Table 5.02 Mean wheal and confidence limits of allergen tests ...... 203 Table 5.03 Wilcoxon non-parametric analyses of group differences...... 204 Table 5.04a Statistically significant predictive variables for symptoms in coastal group of participants...... 218 Table 5.04b Statistically significant predictive variables for symptoms in coastal group of participants...... 219 Table 5.04c Statistically significant predictive variables for symptoms in coastal group of participants...... 220 Table 5.04d Statistically significant predictive variables for symptoms in coastal group of participants...... 221

List of Tables x

Table 5.04e Statistically significant predictive variables for symptoms in coastal group of participants...... 222 Table 5.04f Statistically significant predictive variables for symptoms in coastal group of participants...... 223 Table 5.04g Statistically significant predictive variables for symptoms in coastal group of participants...... 224 Table 5.04h Statistically significant predictive variables for symptoms in coastal group of participants...... 225 Table 5.04i Statistically significant predictive variables for symptoms in coastal group of participants...... 226 Table 5.05a Statistically significant predictive variables for symptoms in Range group of participants...... 227 Table 5.05b Statistically significant predictive variables for symptoms in Range group of participants...... 228 Table 5.05c Statistically significant predictive variables for symptoms in Range group of participants...... 229 Table 5.05d Statistically significant predictive variables for symptoms in Range group of participants...... 230 Table 5.05e Statistically significant predictive variables for symptoms in Range group of participants...... 231 Table 5.05f Statistically significant predictive variables for symptoms in Range group of participants...... 232 Table 5.05g Statistically significant predictive variables for symptoms in Range group of participants ...... 233 Table 5.05h Statistically significant predictive variables for symptoms in Range group of participants ...... 234 Table 5.06 Partial R sq for stepwise regression on coast SPEF variable... 236

Table 5.07 NOX and ozone as predictors of Coast SPEF in combined seasons and separately in spring and autumn ...... 240 Table 5.08 Significant predictors of SPEF for Coast air samples...... 241 Table 5.09 Asthma score seasonal predictors for coast and range...... 242 Table 5.10 Partial R-square for Range group contributing to SPEF score .. 243

List of Tables xi

Table 5.10 Partial R-square for Range group contributing to SPEF score 243

Table 5.11 Linalool seasonal effects upon reliever usage for coast and Range groups 246 Table 5.12 Linalool seasonal effects upon preventer usage for Coast and Range groups 246 Table 5.13 Flower leaf extract comparisons of compounds 251 Table 5.14 Flower substrates for fungal growth: results of experiments 254 Table 5.15 Quantification of ion count for Cladosporium fungal infection of Melaleuca leucadendra stamens 255 Table 5.16 Tenax Flower samples A 260 Table 5.17 Tenax flower samples B 261 Table 5.18 A sample of floral emissions before and after a storm……..….264 Table 5.19 Tenax desorbs from lifestyle products………………………… 263

List of Tables xii

LIST OF FIGURES Figure 2.01 Comparison of asthma measures ...... 11 Figure 2.02 Prevalence of current wheeze in children, asthma fatality rate and health expenditure in international currency...... 12 Figure 2.03 Melaleuca viridiflora...... 48 Figure 2.04 Isoprene ...... 52 Figure 2.05 Citral...... 54 Figure 2.06 Limonene ...... 54 Figure 2.07 Delta-3-Carene...... 55 Figure 2.08 Alpha Pinene and Beta Pinene ...... 55 Figure 2.09 Camphor ...... 56 Figure 2.10 Sabinene ...... 57 Figure 2.11 Alpha-Farnesene...... 58 Figure 2.12 Selinene ...... 59 Figure 2.13 Alpha and Beta Cubebene ...... 60 Figure 2.14 Buds and flowers from Corymbia ficifolia/calophylla hybrid (cultivated specimen- formerly named Eucalyptus)...... 63 Figure 2.15 Relative percentages of terpenes in oil of Melaleuca species. .... 67 Figure 2.16 Callistemon viminalis and rainbow lorikeet pollinator ...... 70 Figure 2.17 Biomes of the World...... 74 Figure 2.18 Pine tree floral structures (Flaxton, S.E. Queensland) ...... 82 Figure 2.19 ‘Bluetop’, Ageratum houstonianum, showing trichomes...... 85 Figure 2.20 Stomata485 ( natural size 0.055 mm ) ...... 85 Figure 2.21 Structure of the flower of Melaleuca leucadendra495 (With permission from the illustrator, Murray Fagg)...... 88 Figure 2.22 Thryptomene calycina 494 (Source: CSIRO Journals, with permission)...... 88 Figure 2.23 Flower buds from Eucalyptus tereticornis (Forest & Kim Starr (USGS)with permission) and flowers from Eucalyptus viminalis (Morwell National Park , Victoria with permission from Ken Harris500)...... 90

List of Figures xiii

Figure 2.24 Leaf litter from mixed woodland: Eucalyptus and rainforest species in highland S.E. Queensland...... 91 Figure 2.25 Delayed sensitivity response to washing-up liquid ...... 109 Figure 2.26 Backhousia citriodora...... 129 Figure 3.01 The Myrtaceae family showing some popular garden genera ... 143 Figure 3.02 Distribution of Melaleuca quinquenervia798...... 144 Figure 3.03 Row of Melaleuca leucadendra and Melaleuca quinquenervia planted as a road divider in Redcliffe City. (M. quinquenervia is shown in bloom.) ...... 146 Figure 3.04 Distribution of Callistemon viminalis798 ...... 147 Figure 3.05 Schematic view of asthma in Brisbane against flowering of bottlebrushes...... 148 Figure 3.06 Melaleuca leucadendra fully grown in street landscaping by local government authority...... 149 Figure 3.07 Distribution of and Melaleuca leucadendra798 ...... 150 Figure 3.08 Acacia concurrens (wattle) and Melaleuca quinquenervia ...... 157 Figure 3.09 Urban footprint and rural land in south-east Queensland804 ...... 158 Figure 3.10 Flowers of Melaleuca quinquenervia...... 163 Figure 3.11 Flowers of small leaf privet, Ligustrum sinense...... 163 Figure 4.01 Melaleuca forest divided by road cutting...... 168 Figure 4.02 An urban street in south-east Queensland coastal suburbs ...... 169 Figure 4.03 Coastal view from range ridge at Maleny ...... 170 Figure 4.04 Glossy or Large-Leaved Privet (Ligustrum lucidum) ...... 171 Figure 4.05 Maleny dairy cow with Small-Leaf Privet ...... 172 Figure 4.06 Red Natal Grass (Melinis repens) ...... 172 Figure 4.07 Japanese or Mexican sunflower (Tithonia diversifolia) on the Blackall Range ...... 173 Figure 4.08 Wet sclerophyll woodland at Flaxton...... 175 Figure 4.09 Cypress ‘fence’ of acreage home at Flaxton ...... 176 Figure 4.10 Skin-prick test in progress ...... 179 Figure 4.11 Participants’ ages of coast and range groups of phase 2 SPT.. 181

List of FIgures xiv

Figure 4.12 Location of participants (coast and range), air sampling sites (Redcliffe & Flaxton), meteorological sites (Brisbane Airport & Maleny) and pollutant sampling site at Deception Bay...... 184 Figure 4.13 Sampling of Grevillea during storm build-up...... 189 Figure 4.14 Melaleuca stamens and Cladosporium plated sample and Cladosporium base culture...... 192 Figure 4.15 Melaleuca flowers before and after stripping of stamens ...... 192 Figure 4.16 Use of foil wrapping during sampling of live flowers...... 195 Figure 5.01 Positive response to allergens Phase 1 for all schools ...... 201 Figure 5.02 Percentage of wheals greater than zero all schools Phase 1.... 206 Figure 5.03 Children with asthma: allergen responses (wheals >0, ≥3mm) . 208 Figure 5.04 ‘Healthy’ children: allergen responses (wheals>0, ≥3mm) ...... 208 Figure 5.05 Comparison of percentage of common allergens response in Phase 1 and Phase2, wheal >0...... 209 Figure 5.06 Allergen percentage responses wheal ≥ 3mm for Phase 2 participants Coast and Range groups’ skin tests...... 210 Figure 5.07 Terpene and methanol positive responses ≥3mm Phase 2 Coast and Range groups skin tests...... 210 Figure 5.08 All responses greater than 1mm to terpenes and methanol...... 212

Figure 5.09 NOX in Brisbane 1995-2004 showing seasonal patterns of nitrogen oxide and nitrogen dioxide (Source: Graph constructed from figures from Environmental Protection Authority, Queensland Government)...... 215 Figure 5.10 Pinus (top graph) and Myrtaceae (bottom graph) pollens and Coast SPEF ...... 235 Figure 5.11 Coast group mean SPEF error...... 237 Figure 5.12 Coast SPEF, precipitation and some terpene volatiles...... 238 Figure 5.13 Rainy day volatile emissions ...... 239 Figure 5.14 Coast group benzoic acid and benzaldehyde and SPEF ...... 239 Figure 5.15 Range group error for SPEF mean...... 242 Figure 5.16 Range terpenes, SPEF, thunder score & precipitation...... 244

List of FIgures xv

Figure 5.17 Benzoic acid and benzaldehyde for range area ...... 244 Figure 5.18 Total volatiles in coast and range areas...... 247 Figure 5.19 Ascomycetes emerging from Melaleuca quinquenervia stamen 257 Figure 5.20 Strings of spores from Melaleuca quinquenervia ...... 258 Figure 5.21 Unidentified structure attached to a fresh Callistemon viminalis tamen...... 258 Figure 6.01 Areas of high asthma incidence in New Zealand ...... 268 Figure 6.02 High sesquiterpene chemotypes of Leptospermum scoparium268 Figure 6.03 Heathland in New Zealand (after Specht819) ...... 269 Figure 6.04 Worst asthma states in the United States ...... 270 Figure 6.05 World distribution of conifers ...... 271 Figure 6.06 World Myrtaceae distribution...... 272 Figure 6.07 Limonene emissions from flowers and lifestyle products...... 273 Figure 6.08 A new model of asthma acquisition...... 275

List of FIgures xvi

LIST OF APPENDICES

Appendix A Seasonal variation on rainfall in Australia1. Appendix B Some better known Myrtaceae subfamily Leptospermoideae genera 2

Appendix C Eucalypts in Moreton region of south east Queensland 3

Appendix D Casuarinaceae species in South-East Queenland3

Appendix E Pinaceae species in South-East Queensland3 Appendix F Native and introduced Olea and Ligustrum naturally occurring in South-east Queensland3

Appendix G Caloundra Shire flora of local significance 4 Appendix H Skin – prick testing report form Phase 1 children Appendix I ISAAC QUESTIONNAIRE Part a Appendix J Skin prick test results form for phase 2 participants Appendix K Chemicals used in SPTs in phase 2 Appendix L Respiratory diary sheet (part of monthly sheet) Appendix M Air sampling equipment Appendix N Rankings assigned to categories of volatiles in samples. Appendix O Significance of variable correlations for Coast group- all symptoms Appendix P Significance of variable correlations for Range group- all symptoms Appendix Q Program and results for stepwise regression lag 3 Coast group SPEF Appendix R Principal components analysis of volatiles in coastal analysis Appendix S General Linear Model Regression Coast group SPEF Appendix T Programs used in SAS for determining predictive capabilities of variables. Appendix U Range stepwise regression for Lag 4 using significant predictors of SPEF

Appendix V General Linear Model results for Asthma Score for coastal group

List of Appendices xvii

APPENDICES CONTINUED Appendix W Wind Rose information for January in Brisbane Source: Bureau of Meteorology, Australian Government Appendix X Wind rose for Brisbane May Appendix Y Flower and leaf extract example: Eucalyptus Appendix Z GCMS of SPME sampled Melaleuca quinquenervia infected and control stamens AppendixAA Methanol extracts of Melaleuca quinquenervia showing sitostenone Appendix AB Regression models predicting SPEF in COAST group for spring and autumn Appendix AC Regression models predicting SPEF in COAST group for spring Appendix AD Regression models predicting SPEF in COAST group for autumn

List of Appendices xviii

ABBREVIATIONS & And ACAM Australian Centre for Asthma Monitoring ACD Allergic contact dermatitis AHR Airway hyperresponsiveness AIDS Acquired Immune Deficiency Syndrome AIHW Australian Institute of Health and Welfare Altern fungi Alternaria fungal spores Altern. Alternaria Aster pollen Asteraceae pollen Atm pres Atmospheric pressure Atmos Atmosphere Aust Australia Aut Autumn Av Average Benzald Benzaldehyde BHR Bronchial hyperresponsiveness Ca Circa i.e. About Caspase Cysteine aspartate-specific protease Casuar pollen Casuar pollen Chem Chemotype Clad fungi Cladosporium fungal spores Cladosp. Cladosporium COPD Chronic obstructive pulmonary disease Cult Cultivated Ddowns Darling Downs DIR Direction of effect Dist District Dist. Distribution EAR Early allergic response ETS Environmental tobacco smoke

FEV1 Forced expiratory volume in one minute

Abbreviations xix

Flw Flowers GC/MS Gas chromatograph/mass spectrometry GINA Global Initiative for Asthma Gr Div Range Great Dividing Range

HO2 Hydroperoxy radical

HOx Collective term for OH and HO2 ICD Irritant contact dermatitis JNK C-Jun N-terminal kinase LPR Late phase reaction Min Minutes Myrtac pollen Myrtaceae pollen MYRTAC. Myrtaceae pollen N.A. Not applicable n.d Not dated

N2O Nitrous oxide NE North-East NF- ĸB Nuclear factor kappa B Nitro Dioxide Nitrogen dioxide Nitro Oxide Nitrogen oxide NO Nitrogen oxide

NO2 Nitrogen dioxide

NO3 Nitrogen trioxide

NOX Collective term for NO + NO2 NZ New Zealand

O3 Ozone Occ Occasionally OH Hydroxyl radical PAF Platelet-activating factor PAR Photosynthetically active radiation PEFR Peak expiratory flow rate

PM10 Particles under 10 microns in size

PM2.5 Particles under 2.5 microns in size PNG Papua New Guinea

Abbreviations xx

PPPV Part per billion by volume PRECIP Precipitation R Humid Relative humidity R2 Correlation coefficient squared Ref Reference SE South-East SE Qld South-East Queensland SL Sesquiterpene lactone SL’s Sesquiterpene lactones

SO2 Sulphur dioxide SOA Secondary organic aerosol Spr Spring SPT Skin prick test Ssp Sub species Subsp Subspecies Sum Summer SW South-West Syn Synonym Syn Synonym Temp Temperature Th'out Throughout Thund Score Thunder score TNFR Tumor necrosis factor receptor TNFα Tumor necrosis factor alpha TTO Tea Tree oil VOC Volatile organic compound Win Winter Wind Spd Wind speed W'spread Widespread α-pinene Alpha pinene β-pinene Beta pinene

Abbreviations xxi

GLOSSARY A compound formed by an addition Adduct reaction Anacardiaceae Cashew and mango family of trees Anemophilous Wind pollinated Anthesis When a flower opens Antigen A substance that the body recognises as foreign and can evoke an immune response (usually a protein). BSP Particles which backscatter light

C 15 H 24 O Oxygenated sesquiterpene

C 15 H 26 O Oxygenated sesquiterpene Carbonyl group A carbonyl group is a carbon-oxygen double bond Caspase Caspases are a group of cysteine proteases, enzymes with a crucial cysteine residue that can cleave other proteins, Caspase Aspartate-specific cysteine protease Commensal Fungi that live in association with other micro-organisms to the benefit of one and not to the detriment of the other Cytokine Low molecular-weight protein involved in cell-to-cell communication Dyspnoea Shortness of breath Electrophile Electron loving- an affinity for negatively charged regions of a molecule. An electrophile must be electron deficient. Entomophilous Insect pollinated Epicuticular Outer surface of a plant Erythema Redness

Headspace Gas space in a chromatography vial above the sample.

Glossary xxii

Hemiacetal Functional groups derived from carbonyl groups by addition of one molecule of an alcohol. Humidity % Relative Humidity at 9am % Hydrolysis of an ester When water breaks the ester into a alcohol and a carboxylic acid group. Hydrophilic A substance that can dissolve in water more readily than oil - 'water loving' Lipophilic A substance than can dissolve in an oil easier than it can water -' lipid loving' Nitrate radical Formed from the reaction of ozone with nitrogen dioxide

NOx Nitrogen oxides Nucleophile Nucleus loving - affinity for positively charged centres. A nucleophile must be electron rich. Oedema Swelling Ontogeny Stages of growth Ozonolysis Reaction whereby ozone oxidizes an alkene forming carbonyl compounds Phloem Living tissue that carries organic nutrients - phloem is underneath and difficult to distinguish from bark.

PM10 Particulate material less than 10 micrometres in diameter. The standard

for PM10 is 50 micrograms per cubic metre averaged over 24 hours.

Glossary xxiii

Particulate material less than 2.5

PM2.5 micrometres in diameter. The advisory

reporting standard for PM2.5 is 25 micrograms per cubic metre averaged over 24 hours. Pressure Mean Daily Station Level Pressure (from synoptic observations) (hPa) Pruritus Severe itching Radical A charged group of atoms

SO2 Sulphur dioxide Spasmogenic Causing spasms Spasmolytic Relieves or prevents spasms Stoma A small opening, many of which are found on the epidermal layers of plants, allowing access for carbon dioxide and egress for water (ox bot) Stomata Plural of 'stoma' Storax A vanilla-scented resin from various trees of the genus Styrax Temperature Mean Daily Air Temperature {from synoptic observations} (°C) Transcription factor A protein that regulates gene transcription in the eukaryotic nucleus Wallum Swamp Windspeed Mean Daily Wind Speed {from synoptic observations} (km/h) Zoophilous Animal pollinated

Glossary xxiv

ACKNOWLEDGEMENTS

This lengthy project began in 1998 with a grant of $ 26,500 from SEQROC (South East Queensland Regional Organization of Councils) facilitated by the then Lord Mayor of Brisbane, Jim Soorley. A two-year scholarship from NRCET (National Research Centre for Environmental Toxicology, now ENTOX) enabled this project to begin and I thank Prof Michael Moore for assistance and support. Several scholarships and project funds for $55,000 were afforded by the Asthma Foundation of Queensland. I thank past CEO Peter Lonsdale and current CEO Paul McGregor for consistent support.

Thanks Prof Rod Simpson for support in the earlier stages of the project and to Pam Harvey and Shane Casson, for frequent assistance with logistics at Griffith University. Elaine Bella provided guidance regarding statistical procedures and Margaret Dauth helped with skin-testing procedures at schools. I am grateful to both who assisted in their respective capacities as employees of Queensland Health at Princess Alexandra Hospital.

Thankyou Emeritus Professor Ray Specht for your learned assistance and Dr Roger Spencer, Horticultural Taxonomist from Melbourne Botanic Gardens. Special thanks to Peter Bostock, Chief Botanist from the Queensland Herbarium for generosity of time and intellect and the use of the HERBRECS software to produce the floral distribution maps. Thankyou to Don Neal and Derek Johnston for help with pollution data from Queensland Environmental Protection Agency. Thankyou Michael Priest from Orange Agricultural Institute and Heidi Martin at Department of Primary Industries, Brisbane for fungi identification. Thanks to Medical Developments Australia for assistance with peak flow meters, and to Qantas for an airfare to Tasmania in the early stages of the project.

Acknowledgements xxv

Thanks to Gail Hill for her wonderful photographs of trichomes and pine flowers. To Michelle Montgomery and Anna Wells thankyou for so much help with administrative details. Profound appreciation to Matt Gregory at the University of Tasmania for extended patience with my questions about the air sampling. Your cheerful responses were always appreciated.

Thanks to Emeritus Prof Des Connell for taking me on very late in the PhD journey as my Griffith University supervisor and for your guidance towards greater clarity in planning the thesis structure. I am grateful too for help with problems related to chemistry. Thankyou to Assoc Prof Charles Mitchell from the University of Queensland for taking me on at the beginning when good sense must have decreed otherwise. Your patience with me has always been a source of wonder and your continued support made it possible to keep going all this time.

To Michelle, Dan and Anna, my three children with asthma, for being so stoic during all the pilot testing and skin-pricking. Thankyou Dan and Anna for living most of your childhood in the shadow of this project and only ever complaining about the field trips!

To Michelle, Judy, Colleen, Bob and the late Retta Cashen, for your interest and for cheering me on. Unending gratitude to Dr Shannon Rutherford first a PhD colleague and now a friend. You took this novice under your wing and generously shared your knowledge and showed me what to do, so often. Thanks especially for your assistance during the skin-testing at schools. It is obvious to me that I would not have been able to complete this project without your guidance and help.

Special acknowledgement for my faithful canine friends who kept me company all those long nights – Charlie my Golden Retriever whose great golden head rested on my feet under the desk for years and for most of this project, and now Jessie, my Border collie who has kept the tradition going.

Acknowledgements xxvi

Most of all thankyous to the participants – all 480 in totals who were part of this journey. Special thanks to the respiratory diary keepers who kept many sets of diaries day after day, month after month. Your good natured selflessness was an amazing contribution. My deepest appreciation for your efforts and continued faith in me.

Jane Gibbs Griffith University 22 December 2006

Scholarships received: • Two-year scholarship from the National Research Centre for Environmental Toxicology (1998-1999). • Two year scholarship from the Asthma Foundation of Queensland (2000- 2001). • Half-year scholarship 2002 from Asthma Foundation Qld. • Half-year scholarship 2003 from the Asthma Foundation Qld. • Half-year scholarship from Griffith University 2006 Total $110,000 in scholarships

Additional funds obtained for the project costs: $26,500 from SEQROC 1998 $15,000 from Asthma Foundation (Queensland) 2000 $5,000 NRCET/GU equipment grant (National Research Centre for Environmental Toxicology/ Griffith University) 2001

Total financial assistance for the project = $ 156,500.

Acknowledgements 1

1 . Introduction This thesis is about the possible contribution of some gaseous emissions from plants to patterns of allergic respiratory disease. The aim here is to highlight terpenes and aromatic compounds for the worldwide health research community to examine the relevance of these atmospheric compounds to their own location, be it urban or rural. The application of the theoretical connections proposed will be demonstrated by investigations carried out just north of Brisbane in South-East Queensland, Australia.

The contribution of Australian plants to seasonal asthma, particularly in South- East Queensland, Australia is the focus of enquiry. Of special interest is the relationship between the seasonal peak of asthma in autumn and the blooming of the creamy-yellow coloured bottlebrush, Melaleuca quinquenervia. Melaleuca quinquenervia is a member of the Myrtaceae family of plants. Other better known genera are the Eucalypts, Leptospermums and Callistemons. They are also commonly known as gum trees, tea trees and bottle brushes.

Additionally, this thesis is about the contribution of the chemicals found in the members of the Myrtaceae plant family, and other essential-oil plants, to asthma and allergic disease, world-wide. Some of these are already well established as associated with respiratory symptoms, for example, the hyper- oxides of delta-3-carene in turpentine5. Such allergic reactions are appearing more frequently 6 and warrant further investigation.

The seasonal nature of asthma exacerbation in South-East Queensland has not been adequately explained to date, nor have the asthma patterns worldwide been adequately explained by the current knowledge about asthma causation7.

Australian seasonal asthma patterns have been examined largely in terms of pollination patterns of plants occurring in Europe and the United States of America. Northern hemisphere grasses, trees and weeds predominate in

Chapter 1: Introduction 2

allergy screening tests in Australia. Australian native plants are regarded as benign in relation to asthma exacerbation, but this position is not addressed by published research, and seems to be based only upon assumption. The assumption is canvassed widely without benefit of testing or of any evidence of their benign nature. Recent pollen counts in Europe, U.S.A. and Asia are showing enough Eucalyptus pollen to engender interest in any possible associated effects8-11.

Most plants that originate in Australia are predominantly insect and/or animal- pollinated, with notable exceptions being the Acacia (wattle), Casuarina (she- oak) and Callitris (Australian cypress) genera. Most Myrtaceae pollens are much smaller than pollens investigated in the northern hemisphere. They are sticky and clump together with sweet nectar as an enticement to creatures to transport the pollens while they move from flower to flower consuming nectar. They are dispersed differently from wind-born pollens which are light and easily blow away to their future growth location. Myrtaceae pollens are coated with oily mixtures consisting predominantly of terpenes.

Very little has been written about Australian pollens and respiratory disease. This thesis represents a research journey which was begun to investigate the contribution of smaller pollens, from Australian Melaleucas in particular and members of the Myrtaceae family in general, to patterns of asthma incidence in Australia and overseas. The journey ended in a different, and, hopefully, useful place with the role of pollens occupying a less prominent place than first thought in new models for understanding plant-related causes of asthma.

Respiratory data from groups of adults and children in coastal and highland regions were compared and contrasted to determine relationships to environmental variables. Pollen, fungi, anthropogenic pollutants and meteorological variables were an integral part in analyses which compared all those measures against ambient air samples examined for terpene content over several seasons. Results of analyses invited the construction of new models for

Chapter 1: Introduction 3

understanding some aspects of the process of asthma acquisition and exacerbation. The research process is reflected in the following chapters:

1.2 Current knowledge Chapter 2 provides an outline of the prevailing view of asthma, its prevalence in Australia and the world, and some of the important processes involved. Predisposing factors are mentioned as are the triggers for asthma exacerbation, both exogenous and endogenous. Asthma as an allergic phenomenon is the focus of this chapter. The literature regarding substances that trigger an allergic response is presented. In particular, literature referring to plant-associated somatic responses is covered. Natural and anthropogenic allergens are presented. Fungal spores as allergens are included in the discussion.

This chapter also investigates the possible role of terpenes in allergic diseases, especially asthma. Australian plants (the Myrtaceae family in particular) are rich in essential oils which are predominantly blends of terpenes. The contribution of Australian plants to allergy has not yet been adequately assessed in terms of symptom variation. Fungi associated with oil-bearing plants may be inhaled and may cause different responses in humans compared to fungi found on other plants, because of the different chemical composition of the fungal growth substrate. Very little investigation of these phenomena is apparent in the literature. Given the commercial and popular interest in essential oils, a better knowledge of terpenes, the plants that contain them and the micro-organisms that grow on them, is timely.

The function of terpenes in the natural world is discussed, as is the application of these substances by humans. The health implications of human exposure to various oil-bearing plants, woods, and substances from a range of plants from all over the world are presented. Functional groups in essential oil substances that may play a role in allergy are presented as are incidences of natural occurrences and anthropogenic concentrations. These may act as triggers for allergic diseases ranging from dermatitis to asthma.

Chapter 1: Introduction 4

1.3 Study objectives and investigation strategy This third chapter begins with a summary of the current knowledge that will be especially relevant to the investigations reported in this thesis. Using pollens as the sole explanation for seasonal allergic symptoms reveals a number of inconsistencies which will be spelled out here. Such inconsistencies invite new explanations of seasonally related allergic symptoms, many of which affect either the upper or lower respiratory system.

The rationale for the investigations reported here follows directly from the inconsistencies just mentioned. Other related observations set the scene for the objectives to be outlined. The strategy for the investigation is then presented.

1.4 Methodologies Chapter 4 describes methodologies employed for the investigations reported in this thesis. The initial research project in this series of investigations involved skin-testing of children, with and without respiratory symptoms, in three coastal schools and one in a highland location. Contrasting topography and vegetation in those two areas is described. Methods involved in allergen testing for groups of school children in coastal and highland regions are described.

Similar methods were employed for a later study involving adolescents and adults. These are also described. Air sampling procedures employed in coastal and highland regions are described as are methods of spore and pollen collection. These spores and pollens were gathered concurrently with air samples and respiratory diary data from the adolescent and adult participants. The participants had previously undergone skin-tests with allergens and terpenes from essential oils.

Plants and fungi were analysed to determine the quality and quantity of terpene composition, with a view to providing a connection between plant substances occurring naturally and those detected in ambient air and/or household

Chapter 1: Introduction 5

products. Methods of plant and fungi analysis are described as are procedures for the analysis of household and commercial products that contain essential oils and/or terpenes. Chapter 7 recounts a set of investigations regarding the chemical content of fungi grown on various substrates.

1.5 Outcomes of investigations into asthma in coastal and highland communities, and the flora located there Chapter 5 covers the outcomes of the previously described procedures. The outcomes and discussion of the skin-testing with allergens for the children and for the adolescents are presented. Detailed reporting of the panel study is presented in this chapter. Statistical analyses of predictors and associations reveal the relationships among health, weather and air quality variables. Outcomes and discussion of the plants and fungi analysis together with those for the household products and personal care items are presented.

1.6 New models for understanding the effects of environmental factors on asthma This chapter provides new models for understanding the effects of environmental factors on asthma by including plant volatile substances as important sources of irritancy or allergenic reactions in their own right. The combination of pollutants and plant volatiles is also discussed as an additional source of asthma triggers. The methods of dispersal are summarized and new explanations of world asthma patterns are offered. The explanations are in terms of chemotaxonomy with a focus on the common chemical threads woven through different regions of the world, differentiated in terms of vegetation and climate combinations.

Asthma patterns from all over the world are presented, highlighting terpene associations. Of special interest are patterns of Myrtaceae plants in Australia and other parts of the world. Also of special interest are plants containing terpenes which may be important in the asthmatic response. A list of plants that are implicated in asthma exacerbation is offered.

Chapter 1: Introduction 6

Following the same chemical trail, a new version of ‘The Hygiene Theory’ of asthma acquisition is offered. Here the relationships between the hygiene and lifestyle practices of the developed world and increased asthma are explored. Some lifestyle practices in developing countries are relevant to this issue also: changing practices in hygiene and diet pave the way for a greater exposure to a range of terpenes. The chapter deals with the possible contribution of particular terpenes that are found in Australian plants and other oil-bearing plants to respiratory disease, especially asthma. Those chemicals are related to the contents of common household cleansers and lifestyle products.

A different slant on “The Thunderstorm Effect” of pollen burst in situations of plummeting air pressure, and a new model for environmental asthma triggers, is presented.

1.7 Conclusions and recommendations The last chapter, Chapter 7 relates the issues from earlier chapters to modern lifestyle and offers some alternatives and recommendations. A new model for environmental asthma triggers is presented.

This thesis touches on several academic disciplines: the interplay of medical, botanical, entomological, environmental and chemical is complex and potentially confusing. Because this thesis cuts across disciplines, minimal reader familiarity with the subject has been assumed. The unfolding of the web of relationships between respiratory symptoms and terpenes begins with some epidemiological observations.

Chapter 1: Introduction 7

2. Current Knowledge

2.1 Asthma and allergy Asthma is the end product of a complex set of triggers, and subsequent cascade of biological processes that result in symptoms that include wheeze, cough, breathlessness and chest tightness. Many of the relationships between these processes are not well understood. Further, the relationships between the processes, their triggers, allergic and irritant responses, and the epidemiology of asthma are often complex and puzzling.

Meteorological, pollutant and botanically-related asthma triggers, and biological responses to them, will be explored in this chapter. When they become a trigger for allergic responses, they are commonly referred to as allergens. An allergen is a substance that can cause an allergic reaction in genetically predisposed individuals who are described as atopic. Allergens can be contracted through the skin, inhaled into the lungs, swallowed or injected. Allergens can take the form of chemicals, micro-organisms, foods, pollutants, pollens and animal dander. The nature of allergic reactions is discussed in more detail in section 2.2.5 after asthma’s physical processes have been briefly explained. Triggers for non-allergic asthma, as well as industrial pollutants, will also be explored.

The term “botanically-related” casts a wide net ranging from pollens to perfumes. It includes discussion of fungi, insects, animals and humans in their relationship with, and responses to, plants and their products. The biological differences between allergic and irritant processes will be addressed in section 2.2.5 in the context of exploring reactions to terpenes. For now, a formal description of asthma and its prevalence is appropriate. Patterns of asthma prevalence are fundamentally important to the arguments framed in this thesis.

Chapter 2: Current knowledge: Asthma and allergy 8

2.1.1 Asthma definition and world and Australian incidence

2.1.1.1 Asthma defined: the GINA perspective “Asthma is a chronic inflammatory disorder of the airways in which many cells and cellular elements play a role. The chronic inflammation causes an associated increase in airway hyperresponsiveness that leads to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing, particularly at night or in the early morning. The episodes are usually associated with widespread but variable airflow obstruction that is often reversible either spontaneously or with treatment.” 12

Further, the authors of the report for the Global Initiative for Asthma (GINA), the leading world source regarding asthma, state that

“A greater understanding of asthma management has been achieved by accepting the persistence of the chronic inflammatory response, with variations in the magnitude of the inflammation reflecting the clinical activity of asthma”12.

This thesis is about environmental triggers, natural and cultural, which may contribute to those variations in the magnitude of inflammation and/or contribute to the maintenance or exacerbation of the asthmatic response. Of special interest are those components that are plant-associated. Associations between some plants and their products and inflammation have been established for some time. In this thesis, the less obvious connection between plants and lifestyle products, manufactured from their constituents, facilitates a greater understanding of the connection between plants and respiratory disease.

Additionally, later in this thesis it will become apparent that some of the plants under scrutiny here may have a role in altering biological processes other than inflammation. The role of plants, and their constituent chemicals, in the maintenance of inflammation will be examined. Asthma is perpetuated by

Chapter 2: Current knowledge: Asthma and allergy 9

reduced apoptosis, programmed cell death. The role of plant components in apoptotic processes is a complex and potentially important one that will be explored later in this chapter.

Before dealing with some of those processes, exploration of recent estimates of the prevalence of asthma is appropriate.

2.1.1.2. Prevalence of asthma world-wide: GINA Many of the matters discussed in this thesis will have bearing upon epidemiological issues so it is of value to assess the contribution of the most comprehensive collation of asthma information published so far. GINA, in their 2004 publication “The Global Burden of Asthma”7, have allocated global estimates of asthma prevalence to many countries and regions. The probable inaccuracy of allocating a global prevalence figure, in any country, is acknowledged by the authors. Some interpretative issues are worthy of consideration.

2.1.1.3. Inter-country asthma prevalence comparisons There are a number of tables and charts in the GINA 2004 report. The difficulty in comparing the data sets is highlighted in Table 2.01 which shows the differing numbers of countries in each data set. It is easy to see that none of these sets of data are comparable with each other. It is tempting to draw conclusions about available information as if it is representative of all participant countries. Many of the world’s 192 countries were not represented at all in the data sets.

What comparisons can be made from those countries with comparable sets of data? Figure 2.01 shows the differences that become evident when some of these measures are compared within countries and across countries. In order to present this chart many countries have been omitted. Only the countries shown in the Figure 2.01 have all three measures available for comparison. The coloured bars represent prevalence of symptoms of asthma as measured by various instruments : response to a video showing an asthma scene, a self report scale for adolescents and a one for adults. In some cultures limited

Chapter 2: Current knowledge: Asthma and allergy 10

literacy of the population means that more information can be gleaned using additional media to illustrate the symptoms i.e. video. Some countries exhibit a large disparity between measures (Estonia and Poland) while others are consistent in the prevalence of symptoms in groups of adults and children (Australia and Canada).

Table 2.01 Different representations of asthma in GINA 2004

Ranking of the symptoms in childhood by country (I). (Written questionnaire: self-reported wheezing in the previous 12- month period, in 13- 14 year old children) 84 countries listed

Ranking of the prevalence of current asthma symptoms in childhood by country (II). (Video questionnaire: Positive response to clinical asthma scene, in 13- 14 year-old children.) 44 countries listed

Ranking of the prevalence of current asthma symptoms in adults by country. Written questionnaire: Self reported wheezing in the previous 12 month period, in 20 to 44 year old adults.) 41 countries listed

Prevalence of clinical asthma 81 countries listed

Ranking of asthma mortality by country. (Asthma deaths per 100,000 in 5 to 34-year-olds.) 68 countries listed

World map of asthma case fatality rates. (Asthma deaths per 100,000 people with asthma.) 49 countries listed

Chapter 2: Current knowledge: Asthma and allergy 11

Figure 2.01 Comparison of asthma measures

Asthma symptom measure comparisons by country

10 Prevalence of symptom ( %)

N A U C A S T P F G A I E P B I T

t ndi

hai i ai

us r pa or us s ol an

e . a

S ge

w n

oni and

and a

. m

t t

i gl

ad ug

A l

r n r

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a i al

i de

a s

nd Country

Video-positive response to clinical asthma scene in 13-14 year olds Self reported wheeze in previous 12 months in 13-14 yr olds Self- reported wheeze in previous 12 months in 20 to 44 year- olds

Source: Interpreted from GINA 2004

Figure 2.02 has been constructed in order to visually explore some variables involved in the ‘Hygiene Hypothesis’. A common observation of those who seek to explain world asthma patterns is that the greatest amount of asthma is found in ‘wealthy’ countries where childhood infection is reduced. The theory behind the ‘hygiene hypothesis’ is that exposure to some infectious diseases in childhood confers a protective effect against later development of allergic diseases13. The extension to the argument is that asthma is related to lifestyle – this is the genesis of the ‘hygiene hypothesis’ 14-16 . Specifically the hypothesis relies heavily upon investigations into rural farming communities versus urbanised Western communities. Simply, this hypothesis connects a lack of hygiene, reduced wealth and protection against allergic disease. Further, it connects hygiene-conscious, wealthy, Western urban cultures with increased allergic disease. This theory will be addressed further in Chapter 7.

Chapter 2: Current knowledge: Asthma and allergy 12

Figure 2.02 represents only those countries that can exhibit comparable information. The purpose of this chart is to graphically represent the evidence regarding the association of wealth and asthma. Here, as a measure of ‘wealth’, the expenditure on health in international currency has been chosen. The data sets constructed for this chart have been obtained by selecting those countries for which data could be obtained from the same source. For this chart, the World Health Organization (2005) 17was the source of the country expenditure information, and GINA 20047 was the source of the asthma information. Thus, some countries do not appear on the chart because some data was absent from either GINA or WHO data groups. The countries represented are included ONLY because matching data sets for the categories selected were available.

Figure 2.02 Prevalence of current wheeze in children, asthma fatality rate and health expenditure in international currency

Prevalence of wheeze in children, health expenditure and asthma fatalities

40 6000

35 5000

4000 25

20 3000 asthmatics 15 2000

10 Health expenditure / international $ 1000 5 Prevalence ofPrevalence asthma %/ Asthma deaths per 100,000

0 0 Greece Romania Russia China Denmark Mexico Poland Latvia Italy Portugal Singapore Uzbekistan Estonia Chile Spain Argentina Austria Belgium Germany Thailand France Norway Japan Columbia Finland Ecuador Czech Republic Malta South Africa Ukraine Israel Uruguay U.S.A. Brazil Costa Rica Canada Ireland Australia New Zealand Scotland (U.K.)

Country

Ranking of prevalence of current asthma symptoms in childhood by country (I) questionnaire - self reported wheeze in previous 12 months in 13-14 yr olds Asthma fatality rates ( Asthma deaths per 100,000 asthmatics) Total health expenditure per capita in international currency ( $,2002)

Source: WHO 2005 17 & GINA 2004 7

Chapter 2: Current knowledge: Asthma and allergy 13

Figure 2.02 illustrates the inadequacy of the hygiene hypothesis in explaining world asthma patterns when it is applied to countries represented in the chart. China, Uzbekistan, Ecuador and the Ukraine expend a similar amount on health per capita and these countries have markedly different asthma fatality rates. Conversely, why are Germany and Austria so far below New Zealand, Australia and Canada in the incidence of childhood wheeze? The issues to be presented in this thesis will address the influence of environmental variables that may have bearing on these patterns. As yet this information is undetected in available literature.

The question of whether the rates of asthma measured in recent years actually reflect more asthma, or whether heightened awareness accounts for some of an apparent increase in its prevalence, is one that could occupy epidemiologists for a long time. That problem will not be addressed here except to mention a related issue of self-reported symptoms.

A concern when examining the rates of asthma prevalence is the difference between self-reported symptoms and measured symptoms. Most of the studies that report asthma prevalence rely upon questionnaires and self-report only, and may be measuring a phenomenon different from that experienced by those who have been diagnosed as hyperresponsive via a histamine or metacholine challenge, as well as experiencing wheeze. Airway hyperresponsiveness and symptoms of asthma (wheeze, chest tightness and cough) may measure different abnormalities in the airways. If both are present there is greater risk of persistent disease12.

The criteria of bronchial hyperresponsiveness, BHR, plus current wheeze are the “gold standard” for identifying clinical asthma in population-based studies. Using this as a measure avoids the problem of over-reporting of mild symptoms. The diagnosis of clinical asthma appears to be between 40% and 60% of those reporting current wheeze7. Such validity issues regarding the research that has been collated to give a global picture of asthma cannot be resolved here. There

Chapter 2: Current knowledge: Asthma and allergy 14

are some other limitations regarding global statistics to be considered.

2.1.1.4 Problems with ‘global’ statistics Both the reports of world asthma prevalence and the Australian report are limited to describing information that has been collected. This becomes something more than stating the obvious when seasonal changes are addressed. Both the GINA (Global Initiative for Asthma) 7and ACAM (Australian

Centre for Asthma Monitoring) 18 reports feature compilations of data, about which general statements are made. For example, in the ACAM report NSW and Victorian asthma statistics are grouped together to demonstrate hospital emergency department visits attributed to asthma. This is in the context of a discussion of seasonal trends.

The nominal reference to seasons is clear and the discussion of asthma fluctuations is also set against the calendar of school vacations: peak asthma periods are after school starts each term, especially in February, and during winter. This thesis will demonstrate the relevance of closer examination of climatic seasonal variants and the effect on asthma symptoms. In this ACAM example, most of the centres in NSW record their greatest rainfall in summer. In Victoria, receiving more rain in winter is the experience for most centres (see appendix ‘A’ for map1). The flow-on effects in the natural environment are considerable. The reality of the seasonal trends is likely to be much more complex than is represented in the ACAM report. Such matters are usually masked by ‘global ‘statistics whether at the local or world level.

Similar issues are involved with the GINA report which uses studies that have taken place in a particular country and have been grouped together in order to determine a ‘global’ figure for that country. For example the ‘Southern Asia’ summary page provides a summary of the research in that region and a figure of 3.5% as the mean prevalence of clinical asthma. Two studies that form part of that ‘mean’ are from Bombay and Delhi: Bombay receives on average 1400mm more rain than Delhi each year19. Again the difference in the natural

Chapter 2: Current knowledge: Asthma and allergy 15

environments, which may influence asthma symptoms, must be considerable.

This thesis will address the complexities of seasonal and climatic variation and the related matters of topography and soils later in this chapter. Further examination of Australian and world asthma incidence patterns as they relate to natural environmental issues, especially climate, will feature in Chapter 6.

Some of the important conclusions reached by the authors of the GINA report should be mentioned: • An estimated 300 million people in the world currently have asthma; • Asthma has become more common in recent decades in children and adults; • The rate of asthma increases as communities become increasingly urbanized; • It is estimated that asthma accounts for one in every 250 deaths world-wide; • The United Kingdom, Australia, New Zealand, parts of South America and the U.S.A lead the world in the prevalence of clinical asthma. None of these countries ranks among those with the highest mortality; • Central Asia is the region with the highest asthma mortality. The fact that no standardized data was available for more than one hundred countries should be remembered when discussing global patterns. Notably, the islands of Micronesia and Tristan da Cunha do not appear as separate entities. Until recently, those places appeared in published research articles 20, 21 about world asthma patterns, in the context of having the highest world asthma rates. Their absence should not be construed as a sudden solution to the problem of asthma in those places but can serve as a reminder that much variation is subsumed under larger regional groupings.

Chapter 2: Current knowledge: Asthma and allergy 16

Another important issue regarding such compilations is that “Wide variations in the prevalence of current asthma symptoms are often observed between centres within the same country. This indicates that the asthma symptom prevalence reported for each country is dependent to some extent on the number of centres studied7.” There are many reasons why symptoms might vary from one place to another. Proximity to coastal breezes, exposure to dusty landscapes, exposure to high levels of industrial pollutions and variations in local pollens are just few commonly reported variants.

Thus the geographical location will influence symptom profiles for an area, as will the population density. While in some areas asthma symptoms are higher in urban areas, this is not always the case22. To date the issue of urban versus rural location has been addressed in terms of proximity to industrial pollution. Later in this thesis the matter will be addressed in terms of vegetative differences as well. The matter of geographical location is of paramount importance to the theoretical position taken during the research that will be reported in this thesis: components of geography (landform, soil, and climate) influence settlement patterns and vegetative patterns. Settlement patterns and land usage are related to anthropogenic pollutants and the change in the natural vegetation. Both of these have an effect on air quality and its components. These matters must be taken into account in the interpretation of asthma rates in the GINA report.

Additionally the GINA report provides a map showing the proportion of the population worldwide with access to essential drugs7. Many of the countries with the highest level of clinical asthma are also those with the most access to essential drugs. There are several exceptions though. Greece, for example, has high drug accessibility and low asthma prevalence. Australia’s position in the various rankings in the GINA report varies according to the measure.

Chapter 2: Current knowledge: Asthma and allergy

2.1.1.5 Prevalence of asthma in Australia Here are the relative rankings for Australia taken from data conveyed in the GINA report7: • Australia ranks 8th out of 84 countries in prevalence of current asthma in childhood as determined by self-reported wheezing in the previous 12 month period, in a written questionnaire (13- 14 year olds); • Australia ranks 3rd out of 44 countries in the prevalence of current asthma in childhood as determined by a positive response to a video questionnaire showing a clinical asthma scene (13-14 year olds); • Australia ranks 2nd out of 41 countries in the prevalence of current asthma symptoms in adults as measured by a self-reported wheezing in the previous 12 months, in a written questionnaire; • Australia ranks 8th out of 81 countries in the prevalence of clinical asthma as represented by the proportion of the population; • Australia ranks 22nd out of 68 for asthma mortality measured by asthma deaths per 100,000 populations in 5 to 34 year olds; • Australia ranks 33rd out of 49 for asthma fatality measured by fatality rate per 100,000 people with asthma; • Australia has a 95% access to essential drugs as measured by the proportion of the population deemed to be able to access them; Australia manages asthma better than many other countries but the prevalence is comparatively high.

Features of asthma patterns in Australia are listed in the GINA report7: • Asthma has become more common in Australia in recent decades; • Children born in Australia have twice the rate of asthma than those living in Australia but born elsewhere; • Asthma is the most common cause of hospital admission in children; • In Australia more than 60,000 persons with asthma are admitted to hospitals annually;

Chapter 2: Current knowledge: Asthma and allergy

A report (2004) form the Australian Bureau of Statistics describes facts about asthma in Australia in 200123. Much of this information is also found in the later report by ACAM. The National Health Scheme in 2001 indicated that 12% of Australians (2.2 million people) reported current asthma. In 1989 it was 8%. This increase may reflect heightened awareness of asthma. Some reported statistics were:

• In children (0-14 yrs) more boys (15%) have asthma than girls (12%); • In adults (> 20 yrs) more women (12%) report asthma than men (9%); • Asthma prevalence was lower in people born overseas (8 %) than those born in Australia (13%);

• The pattern of asthma prevalence was similar for non-indigenous and indigenous Australian men up until age 55 . Children of both groups were not different in asthma prevalence either. It was significantly higher for indigenous women when compared to other Australian women especially in the 45+ age group.

• In 2001, 21% of Indigenous people aged 55 years and over reported asthma compared with 9% of non-Indigenous people in the same age group.

Local variation in asthma prevalence within Australia has been noted for years24-27. For several decades 26, 28 South-East Queensland has been identified as an area with high rates of asthma, even for Australia29. Within

South-East Queensland there is further variation29. Table 2.02 shows rates of asthma and related respiratory disease in the various local government authorities surrounding the capital, Brisbane, at the time that the current project was conceptually conceived.

Chapter 2: Current knowledge: Asthma and allergy

Table 2.02 All age asthma and allergy prevalence (%) rates for the South East Queensland area

Either asthma Statistical Subdivision Asthma Hay fever or hay fever Brisbane City 13.0 11.4 20.8 Caboolture Shire Part A 11.9 11.5 21.4

Ipswich City # 14.3 13.0 22.7 Logan City 15.5 11.7 23.0 Pine Rivers Shire 17.7 - 21.4 Redcliffe City 18.9 - 29.1 Redland Shire 13.6 13.4 23.3 Gold Coast & - - 12.0 Beaudesert * Total 13.5 11.4 21.3

# Some of Ipswich City is in Moreton statistical division * includes the hinterland of the Gold Coast (Source: Australian Bureau of Statistics, unpublished data)

Matters pursued in this thesis are about possible environmental causes of asthma and related allergy based conditions, but the focus will be asthma. Key concepts regarding asthma involve the likelihood of its acquisition, the inflammatory process that is triggered by particular factors, and the maintenance, exacerbation or resolution of the inflammation. Before examining some environmental triggers for the asthma and the processes that result from those, the contribution of risk factors to the asthma process need to be considered. As the premier compilation of international views regarding asthma, the 2004 update to the “Global Strategy for Asthma Management and Prevention” 12will be the source of information for this overview of asthma.

Chapter 2: Current knowledge: Asthma and allergy

2.1.2 Some host risk factors for development of asthma

2.1.2.1 Atopy and airway hyper response Atopy is an important host factor that predisposes a person to develop allergic asthma. Atopy is the production of abnormal amounts of IgE antibodies in response to contact with environmental allergens12. The response is evident in increased serum IgE and positive skin-prick tests. Atopic diseases like asthma, hay fever and eczema occur in families. Atopy in an individual’s family increases the likelihood that that individual will develop asthma and/or another allergic disease12. A formal definition of atopy is fundamental to understanding the difference between the two major groups of asthma sufferers: allergic and non-allergic.

Allergic and non-allergic asthma In the past these groups have been referred to as ‘extrinsic’ and ‘intrinsic’ asthma sufferers. Romanet-Manent 30 in 2002 suggested that those terms be replaced by ‘allergic’ and ‘non-allergic’. People with non-allergic asthma exhibit negative skin-tests and their serum immunoglobulin E (IgE) falls within normal range.12 Arguments persist as to the relative contribution of allergic elements but a simple distinction will suffice here. In their study in which different types of asthma were examined, the researchers confirmed the greater severity of non – allergic asthma.

Higher age and female sex are associated with non-allergic asthma as compared to allergic asthma. History of hay fever was more frequent in allergic patients; however more hay fever was reported in non-allergic asthma patients than non-allergic controls. Patterns of asthma attacks by month and season were different for each type of asthma group. The prevalence of rhinitis was not statistically different for the groups but there was a significantly greater incidence of sinusitis in the non-allergic group who also exhibited nasal polyps. People with allergic asthma are likely to experience hay fever and seasonal exacerbations. Patients who experienced aspirin induced asthma were found in

Chapter 2: Current knowledge: Asthma and allergy

each group equally. The argument regarding the different causes of these two manifestations of asthma will not be advanced further here. Eosinophilic airway inflammation is common to atopic, non-atopic, and occupational asthma31.Suffice to say that no matter what the cause of non-allergic asthma, it is not pathologically distinct from allergic asthma, as determined by airway biopsy12.

Airway hyperresponsiveness When airways respond to stimuli too much and too easily, compared to most people, then airways are said to be hyperresponsive. Airway inflammation, increased total serum IgE and airway hyperresponse are all closely related12. Airway hyperresponsiveness without symptoms is also associated with airway inflammation and remodelling of airways. This suggests that inflammation may precede the onset of asthma12. Atopic individuals are most likely to exhibit airway hyperresponsiveness.

2.1.2.2 Gender and ethnicity According to GINA, world-wide, race and ethnic differences that have been evident in comparative studies around the world are attributable to socioeconomic and exposure differences rather than a fundamental race or ethnicity difference when compared to others. Gender differences between the sexes disappear after age 10 when boys no longer demonstrate higher incidence of asthma. After puberty more females than males develop asthma. Australian observations regarding race and gender have been noted already. Some age and ethnicity groups are more likely to develop asthma than others. How much of that imbalance is due to factors that relate to socioeconomic factors and related dietary and allergen exposure issues, is not clear. The relationship of these factors to the maintenance of optimal immune status and, therefore, lesser vulnerability to allergic disease will not be addressed in this thesis.

Chapter 2: Current knowledge: Asthma and allergy

2.1.2.3 Diet The interest in diet as a variable in the manifestation of allergic diseases has increased over recent decades. Until recently, rates of asthma were escalating in the Western world, Australia in particular32, and have now reduced18 although levels remain high. Increased use and effectiveness of corticosteroids33 is part of the reason for the slight reduction in the rates of asthma in Australia and other parts of the developed world.

Arguably, there are more additive and preservative-free choices at the supermarket so that the ‘aware’ sector of the population can make better dietary choices for health. At the low end of the socio-economic scale in developed countries, the least expensive and most filling food is also the food with the most additives and omega-6 fats. For those countries with little access to medication, and a lot of access to additive-filled carbonated drinks, the respiratory risk may increase.

Responses to ingested substances are thought to affect respiratory response by: directly causing allergy; facilitating allergy; modifying the immune response; or triggering an irritant response34. Dietary zinc has been identified as important in protecting against asthma35, 36 while Picado 37 found no protective effect of micronutrients against asthma. There is evidence that additives and preservatives in food may trigger asthma 38-40 though others dispute that evidence 41citing a strong psychological component in somatic reactions. Substances identified as problematic additives will be mentioned in section 2.2.5.6.

Another area of dietary research interest has focussed on ingestion of lipids. Fish oils (omega-3 fats) reduce the production of harmful arachidonic acid, a building block of prostaglandins involved in inflammation. There has been a reduction of Omega-3 fats in the diet since the 1950s due to the increase in Omega-6 fats which are readily obtained from convenience food. Reduction of

Chapter 2: Current knowledge: Asthma and allergy

ingestion of fish in favour of meat and chicken, especially in ‘fast’ food has been blamed. A 2005 study showed a dose dependent relationship between ingestion of hamburgers and asthma symptoms42. Similarly, the same researchers demonstrated a positive relationship between frequent ‘take-away’ consumption and bronchial hyperresponsiveness, BHR.

Benefits of Omega-3 oils have been demonstrated for respiratory symptoms and arthritis43-45. Wijga et al. in 2006 showed that in susceptible children, their mother’s intake of omega-3 fats reduced their risk of allergic symptoms, but not sensitization 46 through the child’s intake of breast milk. The benefit may only be for those deficient in Omega-3 since fish consumption was not a significant predictor of respiratory symptoms for young Norwegians who traditionally have a high level of fish intake47. As the numbers of obese individuals increases, a greater focus on dietary research will likely benefit those with respiratory diseases in the long term. Meanwhile a continued focus by researchers remains on the processes involved in asthma.

2.1.3 Important processes in asthma

This thesis will chiefly be concerned with the environmental stimuli that may be responsible for the range of respiratory symptoms and physiological changes that are called allergic asthma. Non-allergic asthma is not generally regarded as being a response to environmental stimuli although some researchers consider that an undetected allergen may be to blame12. Subsequent discussion of environmental triggers to asthma will frequently be framed in terms of the physiology of the asthmatic response and/or some part of the process of that response. Understanding the processes at the cellular level will provide some background against which some of the allergen information can be framed. Cellular processes underpin the more obvious physiological changes that will be described afterwards.

Chapter 2: Current knowledge: Asthma and allergy

2.1.3.1 Cellular changes related to lung function

Acute inflammation – its course Inhaled allergens trigger an early phase response in allergic individuals susceptible to asthma. Within minutes of substantial exposure to an antigen, cough, wheeze, dyspnoea will begin31. This immediate response that resolves in 30-60 minutes is mediated by histamine and the leukotrienes, among others. Mast cells feature in the early response when an antigen binds to surface- bound IgE, and mast cells release histamine, leukotrienes, prostaglandins, thromboxane, platelet-activating factor (PAF) and possibly many more cytokines and chemokinesis.

In about half of the individuals who undergo the initial response, the early phase response (EAR) is followed by a late-phase reaction (LPR) which begins 3-4 hours after exposure to an antigen reaching a peak in 4-8 hours and resolving in 12-24 hours. The symptoms of LPR are similar to EAR and include cough, chest tightness, wheezing and dyspnoea. The late response is marked by obstructions to airflow with decreased peak expiratory flow that responds less to inhaled bronchodilators than the early phase response31. Non-allergic occupational sensitizers can cause a late-phase reaction.

Structures involved in airway symptoms

Eosinophils Bronchial biopsies of people with asthma feature increased numbers of activated eosinophils. There is a significant association between the activation of the eosinophils and the severity of asthma and airway responsiveness12. Eosinophils can be activated for the secretion of a number of mediators which will be of relevance later.

Mast Cells These cells are found in the bronchi of people without asthma and are often found in a de-granulated state in the airways of people with asthma12. Mast

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cells are a source of proteases which can alter protein substrates.

Neutrophils These are found in the airways of people with chronic and severe asthma. Their ability to release a number of enzymes and cytokines makes them important contributors to the asthma process.

Macrophages Products secreted from macrophages play an important role in the process of injury and repair of bronchial tissue. They may also be involved in airway remodelling. Mediators released from these structures play important roles in the maintenance of inflammation and the regulation of apoptosis, programmed cell death.

Neural controls Reflex bronchoconstriction can be affected by irritant stimuli like cold, sulphur dioxide, dust and fog. Increased activity in the parasympathetic nervous system is thought to play a role in this reaction in people with asthma.

Nitric oxide (NO) is a potent vasodilator and bronchodilator which may serve as a neuro-regulator of nerves that regulate airway smooth muscle tone, pulmonary blood flow and local immune responses12. It is thought that abnormality of NO production and/or breakdown may be relevant to asthma pathophysiology12.

Physical and chemical change at the cellular level combines to produce a number of functional changes that are characteristic of asthma.

2.1.3.2 Functional changes related to asthma symptoms

Airway obstruction Airway obstruction refers to the limitation of airflow in and out of the lungs. This

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may be due to narrowing of the airway, remodelling of the airways or may be because of mucus plugs. Airway obstruction varies spontaneously or as a result of treatment. It is associated with cough, wheeze, chest tightness and airway hyperresponsiveness to bronchoconstrictor stimuli12. Severe airway obstruction can cause blood gas abnormalities that can require aggressive management in extreme cases12.

Airway hyperresponsiveness Airway hyperresponsiveness is the tendency for an individual’s airways to react more strongly to stimuli relative to the population as a whole. This is assessed clinically by delivering a progressively increasing dose of a pharmacologic stimulant such as histamine, until a measure of lung function changes by a predetermined amount12. The most commonly used endpoint is the dose required to elicit a 20% fall in FEV1 (forced expiratory volume in 1 second). Bronchial hyperresponsiveness was the only significant predictor of asthma onset in patients who presented with cough-variant asthma in a recent study investigating risk factors for asthma onset48.

Contraction of airway smooth muscle

The state of smooth muscle tone is of vital importance in asthma12. The exact way in which smooth muscle contraction functioned in asthma has been the subject of investigation for some time. Very recent research by Brown et al. in 2006 has demonstrated that the magnitude of the hyperresponsiveness in asthma is a function of the structure of the airways. Those researchers concluded that hyperresponsiveness from narrowing of the large airways was related to the “degree of hyperinflation that was present before the increase in smooth muscle tone”. The exact mode of function and influence of airway smooth muscle in the asthmatic response has yet to be clarified.

Mucus hypersecretion The production of mucus is part of the defence process for the respiratory tract. When mucus is overproduced it can obstruct airways. Excessive mucus

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secretion is common to asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis although there are differences in mucus pathophysiology. Air pollution increases mucus production 49 and other ‘lifestyle’ substances have been identified as affecting its production50, 51. More effects of man-made substances are mentioned in Section 2.2.4.2. For the purposes here, it is sufficient to note that many of the processes involved in mucous secretion are related to those involved in apoptosis. Calcium channels and anti-apoptotic factor Bcl-2 appear to have a role in regulating secretion51, 52.

Irreversible airway limitation Airway remodelling is now thought to be a consequence of persistent asthma. Until recently the structural changes that are evident in the lungs of people with asthma were thought to be reversible53. The processes involved in airway remodelling are not well understood but chronic inflammation is presumed to be a major contributor12.

Exacerbations The worsening of asthma symptoms episodically is an important feature of asthma. The GINA authors refer to ‘inciter’ triggers that produce bronchoconstriction only: cold air, fog, exercise; and ‘inducers’ which promote airway inflammation: allergens, pollutants, respiratory virus infections. More detail about some of these inducers will follow later in section 2.1.4.

Nocturnal asthma

As many as 40% of asthma sufferers experience nocturnal symptoms54. Circadian variations in lung volumes and peak expiratory flow have been demonstrated but the processes involved remain incompletely explained. Research has focussed on internal physiological states and indoor allergens (dust mites and cockroaches). Geographical differences55 were demonstrated in Turkey where nocturnal asthma was reduced in the East when compared to the West. More asthma attacks were recorded in the East than any other region. This study lends support to the possibility that geographical

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environmental factors may be influential in nocturnal asthma despite the lack of recognition evident in reported studies to date.

The functional changes observed in the person with asthma are maintained, facilitated or resolved chiefly by the processes of inflammation or apoptosis. A brief explanation of these processes is appropriate.

2.1.3.3 Inflammation and the resolution role of apoptosis

Eosinophil proliferation Control of inflammatory cell numbers is regulated by the balance of cell proliferation and cell death. In asthma, eosinophil proliferation is a feature56. It also plays a central role in cough-variant asthma 57 and the development of nasal polyps58. Eosinophils increase during the inflammatory process following contact with allergens and industrial pollutants59 as well as cockroach allergens60. People presenting with asthma occurring after a thunderstorm also showed elevated eosinophils61. Exposure to formaldehyde following exposure to dust mite allergen has been shown to further increase eosinophil inflammation than that provoked by the mite allergen alone.62. This additive inflammation caused by an allergen and exacerbated by an aldehyde, is of particular importance to the arguments that will be made later in this thesis.

Resolution of asthma requires that the eosinophilic inflammation be reduced. Pro-inflammatory cytokines, or cell messengers, provide a point of intervention pharmaceutical intervention. Drug research is directed at inhibiting important ones like the MAPK family63 that can be activated by house-dust mite allergens64. MAPK signalling factors may belong to at least three groups: extracellular signal-regulated kinase (ERK), MAPK, and c-Jun NH2-terminal kinase (JNK). Inhibition of those factors has reduced inflammatory cells in animal models63.

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Otherwise, reduction of unwanted cells can occur in two main ways: necrosis and apoptosis.

Necrosis and apoptosis Necrosis is a form of cell death resulting from cell injury and it typically involves inflammation, cell swelling and rupture of the cell membrane. Necrosis is not the focus here. Apoptosis is “programmed cell death” and is sometimes referred to as ‘cell suicide’65. This form of cell death is of special interest to the issues related in this thesis, especially in the context of the action of particular terpenes that are discussed later in this chapter. Apoptosis eliminates cells that have been produced in excess, have developed improperly or have genetic damage. Such cells are eliminated without any accompanying inflammation65, 66 that features in necrosis.

Apoptosis is part of normal physiologic processes like embryonic development, death of immune cells and cell death in resolving inflammation. It is also associated with pathological events like the growth of cancers when its suppression may enable tumour growth. An enormous number of research projects have centred on the way in which apoptosis controls tumor growth utilizing chemicals from plants like wormwood67, cotton 68and ginger69. Loss of cells in neurodegenerative disorders like Alzheimer’s disease70 is thought to be due to apoptosis.

Apoptosis is a complex process involving different kinds of structures, including signalling factors. Some of these have a pro-apoptotic effect and others inhibit apoptosis. Transcription factors have a role in signalling apoptotic processes, and include nuclear factor NF-kappa B and activating protein 1 (AP-1)71. These may signal aspartate-specific cysteine proteases (caspases). Caspases are enzymes that play a vital role in apoptosis – some caspases are regarded as ‘initiators’ and others as ‘executioners’. Caspase 3 , together with caspase 6 and caspase 7, are regarded as ‘executioners’, and caspase 8 and caspase 9 as ‘initiators’ of the apoptosis cascade72.

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There are two main pathways of caspase-mediated cell death: one involving death receptors such as Tumor Necrosis Factor-1 (TNF-1) and the second depends upon mitochondria and responds to the Bcl-2 family member of proteins. The Bcl-2 family consist of pro-apoptotic members like Bid73, Bax73,

Bak and Bok and anti-apoptotic members like Bcl-2, Bcl-XL and more72.

The complexity of determining pro or anti apoptotic roles for the various components of the apoptosis process has been demonstrated in a 2006 review of the pro-apoptotic role of NF- ĸB. This transcription factor has been generally regarded as having an anti-apoptotic role. It is often activated in response to chemotherapy, and promotes chemo-resistance of tumours74. The authors reported that in come situations NF- ĸB displays pro-apoptotic properties. Importantly, they note that this duality of function is common to several other proteins. This duality is relevant to the concerns of this thesis as it strikes at the heart of the popular view that plant-derived ‘natural’ substances are beneficial: the action of plant chemicals is a complex one consisting of a set of processes of infinite variety. Cytokines also influence the apoptotic process and can be very sensitive to modification by chemicals from inside or outside the cell. Notably, calcium influx and efflux affects enzymes 75 that affect the cellular calcium balance, which in turn affects the mechanisms of apoptosis. Members of the Bcl-2 family facilitate the replenishment of intracellular calcium, thus protecting cells from death76. Many researchers have demonstrated the effects of natural and synthetic substances on apoptosis in a variety of cell types77-85. Some substances inhibit apoptosis while others facilitate the process, and others have no effect. Substances that can affect apoptosis by various means will be explored in section 2.2.6 of this chapter.

Corticosteroids that are used in the treatment of asthma intervene in the

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apoptotic process65, 86. They inhibit the prolonged survival of eosinophils and lymphocytes by inducing apoptosis. At the same time they suppress the release of cytokines that support their survival. Corticosteroids provide some protection from the inflammation resulting from contact with a number of anthropogenic and naturally occurring allergens and irritants. They may act as triggers for an inflammatory response and/or may perpetuate already established inflammation.

2.1.4 Anthropogenic and ‘natural’ asthma contributors

2.1.4.1 Anthropogenic contributors to the asthma There are a number of pollutants that exist as the by-products of the industrial revolution. The ones of most interest to researchers because of their ability to elicit or exacerbate asthma are: sulphur dioxide, nitrogen oxides, ozone and particulates. All have become problematic in varying degrees in their effect on asthma in industrialized areas of the world. It is useful to know a little about each since the latter three have special relevance to the themes of this thesis.

Sulphur dioxide This pollutant arises predominantly from sulphur-containing fuels. Its atmospheric distribution is dependent upon the nature of the fuel and the location of the combustion. Sulphur dioxide, SO2, was a major problem in Europe and North America until about 1960, when controls on combustion sources led to cleaner fuels87. In developing countries where coal is still used extensively, the levels of SO2 are still high. Patterns are still changing in some 88 parts of Asia. In Japan, SO2 levels are decreasing slightly . This finding in 2005 is consistent with a Beijing study in the same year, where particulates 89 were nominated as the major pollutant instead of SO2 .

Sulphur dioxide is a strong inducer of bronchospasm in people with asthma. For the asthma sufferer, symptoms of bronchoconstriction begin rapidly after exposure and become maximal in 5-10 minutes. Exercise enhances

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90 bronchoconstriction caused by SO2 in those with asthma and they can expect to experience wheeze, chest discomfort and breathlessness. SO2 has been widely associated with asthma in various parts of the world including the United Kingdom91, Sweden92, China89, Eastern Europe93, Hawaii94 and Chile 95 and the U.S.A96.

Distribution of SO2 varies according to industrial activity, and anthropogenic activities are more likely to produce oxidized compounds of sulphur than reduced compounds which are more likely to emanate from biogenic sources97.

Seasonal variations are not consistent. Higher levels of SO2 are more likely to be a summer phenomenon in the U.S.A as identified in a study by Mueller et al. 96 in 2004, in which movement of SO2 was tracked in different regions of that country. In contrast, in a study in the following year in Canada, 98 Rainham et al. identified heightened sulphur dioxide levels in winter.

In 1996, a large study comparing people with asthma in West and East Germany enabled researchers to conclude that exposure to indoor allergens and irritants was more important than exposure to SO2 and particulates for the 99 development of asthma and atopy . Nevertheless, SO2 is problematic for those with respiratory problems. Higgins et al. examined the effect of SO2 on participants with asthma and chronic obstructive pulmonary disease (COPD). Although reduction in peak flow measures were modest, they were significant even though WHO recommended safety levels were not breached100. In a study in 2004 examining emergency room attendance for asthma and 101 pollutants, no association was found for SO2 .

Sulphur dioxide, in combination with nitrogen dioxide, augmented responses to 102 inhaled dust mite allergen , and when SO2 is used in combination with other problematic substances in foods as a preservative 103 a similar reduced FEV1 resulted. Freedman showed decades ago 104 that sulphur dioxide elicited a fall

Chapter 2: Current knowledge: Asthma and allergy

in FEV1 in participants with asthma but the compound is still widely used in foods and drinks. The use of sulphites in preservatives is an increasing health problem in developing countries as accessibility to ‘junk’ food increases105.

Concentrations of SO2 inside houses increases when coal is burnt although

Krieger notes that burning coal in a house is unlikely to produce levels of SO2 that would be problematic106. Thus regions of the world where there is a lot of coal burning register higher levels of SO2. In Australia, this pollutant occurs at low levels 107, 108 compared to other countries. A 2005 comparison of air pollution effects in four Australian cities by Simpson et al.109, did not compare

SO2 although NOx and ozone were compared. They noted that effects of pollutants were unlikely to be confounded by SO2.

Nitrogen Dioxide

Nitrogen dioxide, NO2, is a precursor to photochemical smog and is found in ambient outdoor air in industrial and urban areas. In conjunction with sunlight and hydrocarbons, it results in the production of ozone110. Motor vehicles are the most significant source of outdoor nitrogen oxides, NOx. Gas cooking is responsible for some of the NO2 that is found in homes, a forms a positive association with respiratory symptoms that has been investigated by many researchers. Erdei et al. 111found a strong association between high indoor

NO2 levels and immune biomarkers in Hungarian children. Phoa, in Australia in 2004112, found that exposure to fume heaters early in life was associated with airway hyperresponsiveness (AHR) although there was no association between current use and AHR.

113 Experiments on isolated human bronchi established that NO2 alters human airway smooth muscle and that the mechanism of its contraction is the same following exposure to ozone and acrolein. Nicolai et al. 114 looked at the association of urban measures of soot, benzene, NO2 and found that cough was associated with NO2 but that there was no association with allergic

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sensitization.

The influence of NOx as an additive on factors that contribute to respiratory symptoms has been investigated by several researchers. Wang 115 et al. examined the contribution of NO2 to measures of a nasal allergic response.

These researchers showed that “acute exposure to NO2 at concentrations found at the curb side in heavy traffic during episodes of pollution, may ‘prime’ eosinophils for subsequent activation by allergen in individuals with a history of seasonal allergic rhinitis."

An examination of pollutants associated with the popular practice of burning candles has revealed that nitrogen oxides levels increased, with gel candles producing almost twice the NO2 of beeswax candles. The levels of NO2 for both 116 were below international air quality targets for NO2. Such ‘lifestyle’ emissions become important when added to a background of NO2 from ambient air. More lifestyle issues will be addressed later.

In a Brisbane study investigating the associations between outdoor pollution and hospital admissions117 over the period 1987-1994, no overall association was found for NO2. There were, however, significant positive associations for asthma and respiratory disease in autumn, winter and spring. In an earlier study in the same city, NOX was not found to be significantly associated with respiratory mortality, and there were no interaction effects with ozone108.

Simpson et al.118, in a 2005 study, have continued their investigation of the relationship between air pollutants and mortality, both cardiovascular and respiratory. A comparison of air pollution and mortality in four Australian cities,

Brisbane, Perth, Melbourne and Sydney, has shown that NO2 had the largest impact on total daily mortality (cardiovascular and respiratory) during 1996 to 1999. Comparing measures of particles and ozone, NO2 was associated with a greater daily increase in mortality across all the cities.

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Ozone Ozone is not emitted directly in substantial amounts but is formed in the atmosphere from the reactions in sunlight between NOX and volatile organic compounds (VOCs). It is understandable then that more ozone is formed in summer119. The relationship of ozone to respiratory symptoms has been investigated for several decades and many researchers have demonstrated significant associations with respiratory symptoms.

A multi-centre study involving 23 cities showed the almost linear relationship between ozone and total asthma mortality during summer for European 119 cities. The ozone effects were larger for Southern European cities, where concentrations are higher. This relationship was independent of the effect of particulates less than 10 microns in size. Only 19 out of 23 cities demonstrated the ozone association with increased mortality in the warmer season; four did not and demonstrated a non-significant association with decreased mortality. The authors of that study suggest that there may be different seasonal inter correlations of ozone with other pollutants119. This possibility has meaning for the results and observations recorded later in this thesis.

A smaller comparison of cities in Australia found no significant differences for ozone measures. Simpson et al. 118 in their recent comparison of Melbourne, Sydney, Perth and Brisbane health measures examined effects of particle pollution, nitrogen dioxide and ozone for same day and one-day lag upon respiratory and cardiovascular function. They found no significant differences for effect estimates for comparisons among the cities. Significant ozone effects for all cities for respiratory mortality were noted.

Ozone has been shown to combine with other pollutants to form increasingly problematic pollutant blends. In 2005 Jang et al. demonstrated that ozone combines with diesel particulates to result in an additive effect on bronchial hyperresponsiveness in mice120. Summer fires have been shown to result in

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increased ozone readings in a recent Lithuanian study121. It is ozone’s capacity to influence the oxidation of other compounds which is of most interest in this thesis and this issue will be addressed in section 2.1.4.

Volatile organic compounds (VOCs) These compounds include a variety of chemical compounds that comprise hydrocarbons (alkanes, alkenes and aromatic compounds) and oxygenated compounds (alcohols, aldehydes, ketones and ethers122. Much of the contribution of VOCs is via natural emissions from plants and, to a lesser extent, animals123.

Man-made VOCs predominantly come from solvent use, road transport and industrial processes. These sources frequently come from large numbers of small sources and their contribution to ozone formation can vary according to the chemical components. Two of the most important anthropogenic VOC sources are benzene and 1, 3-butadiene, of which benzene is the most relevant to the issues in this thesis.

Benzene The main sources of benzene in the atmosphere are the production, distribution and use of automotive fuels. The related aldehyde and acid, benzaldehyde and benzoic acid124-126 also occur naturally 127, 128 and are produced by plants and by wood-burning129.

Petrol vehicles emit more benzene than diesel fuels. Benzene is present in petrol and can evaporate into the air from fuel storage containers during refuelling as indicated by exposure levels of fuel station attendants130. Many studies relate increased ambient benzene to traffic and it is now recognized that the mix of pollutants may vary from one city to the next131. Characterization of the exact nature of vehicle emissions in various cities is a growing area of interest. Changes to fuel mixes have also brought changes in the levels of ambient benzene in urban traffic129, 131.

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Cough, current asthma and current wheeze were associated with benzene levels in a large study with children in Munich114. Several researchers also report associations with respiratory symptoms but not with atopy or sensitisation132. Levels of benzene indoors were positively correlated moderately with immune biomarkers for respiratory disease in a study with Hungarian children111. Indoors, environmental benzene increased the risk of respiratory infection in six-week-old babies as shown by Diez et al.133 in a study regarding the effects of painting and smoking on respiratory health of young children. Rumchev134 showed that higher levels of benzene is a risk factor for asthma, while Cakmak et al. 135in 2004 showed that exposure to solvents is a risk factor for asthma. Breath concentrations of benzene were associated with asthma symptoms in a small study by Delfino et al.136 involving children in Los Angeles.

Other VOCs like formaldehyde and acetone137are increasing in profile in respiratory research projects. In recent years more attention has been directed to indoor air quality in pursuit of better understanding of these relationships133,

134, 138, 139. The issue of VOC air toxics will be further addressed later in this thesis.

Particulates Particles in ambient air have been researched for several decades and there is a large body of evidence to attest to the relationship of many kinds of particles with respiratory symptoms. Most commonly, smoke, vehicle exhaust and other industrial effluxes are researched in relation to respiratory health. Distinctions between the sizes of the particles in the air are important because smaller sized particles have deeper access to the inner branches of the lung. Traditionally, 140, 141 particles less than 10 microns (PM10) have been of interest and more recently examination of smaller particles less than 2.5 microns (PM2.5) has been included107, 142.

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For occupational particulates there are four important aspects143 to consider:

• the type of particulate and its biological effect; • the concentration of airborne particulates in the breathing zone of the worker; • the size of particles present in the breathing zone; • the duration of the exposure. Particulates and their ability to combine with other substances will be discussed in section 2.2.4.

Indoor Air In developed countries, many people spend most of their time breathing indoor air. Heating, cooling and filtering systems of varying complexity and efficiency alter qualities of the indoor air that is breathed. Much of the world’s population in colder climates re-breathes air that is poorly circulated and often contaminated with tobacco smoke and pathogens and pollutants. Domestic air quality is modified by the traffic and industrial activity nearby. Bascom and Ouellette144 list the determinants of indoor environment as: climate, geology, region, siting, structure, surfaces, heating and ventilation, occupants and activities. All these determinants represent an infinite capacity for variation between different air environments. The complexities involved in measuring all the variables that influence air quality, contribute to the frustrations of researchers trying to separate the causal asthma variables from the co variables and the confounding ones.

Exposure to environmental tobacco smoke (ETS) is a recognised risk factor for development of asthma and for worsening of pre-existing asthma145, 146. In Australia in 2001, a higher proportion of children under 5 with asthma lived with a smoker than those of similar age and sex without asthma18.

Gas stoves have been associated with increased respiratory symptoms 147 in Australian children. Similarly, exposure to fume emitting heaters in the first year of life increased the risk of airway hyperresponsiveness later in childhood112 .

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Paint fumes133 and fumes from furniture polishes148 are common problematic contributors to the indoor air environment. Along with formaldehyde149 emitted from fitted carpet adhesives150 these substances are associated with an increase in a range of respiratory symptoms.

Many of the lifestyle products used in homes today are potential allergens or irritants. Electronic perfume emitters, aromatherapy oil burners, and perfumed cleansers all are sources that have increased in usage in recent years. These items are usually perfumed with substances obtained from plants, or synthetic analogues of them. Before examining their contents in more detail, it is fitting to describe common natural contributors to the asthmatic response.

2.1.4.2 ‘Natural’ contributors to the asthmatic response Research into the relationship of ‘natural’ elicitors of asthma has been concentrated on insects and their faeces, pollens and fungi. Investigation into relationships between asthma and other animals, and other aspects of plants has taken second place in comparison. Meteorological variables and their relationship to asthma have also been of interest to researchers for some years.

Non-pollen plant components have traditionally been of little interest as possible sources of allergens. These will be explored further in the next section, Terpenes. Recent lifestyle trends that have resulted in more use of fragrances have resulted in increasing interest in the allergic properties of cosmetic products in particular. These products emanate from plants. Similarly the popularity of some essential oil products like tea tree oil has been reflected recently in increasing research interest. For now, it is appropriate to summarize the relationships between the well known allergens and respiratory disease.

Insects: house dust mites and cockroaches For several decades, reduction of exposure to house dust mite (HDM) has been advocated as a means of reducing bronchial hyperactivity151. The most prominent of these are Dermatophagoides pteronyssinus and D.farinae.

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Research interest has focussed on HDMs and cockroaches, and specific allergens from both of these insect groups have been identified152-154.

In recent decades allergen avoidance has been the advice for those seeking to reduce respiratory symptoms155-157. While this continues to be the case158, there has been more interest in dietary change and immunotherapy in recent years 157, 159 , as ways to augment allergen avoidance in pursuit of a reduced allergen response.42, 157, 160.

HDM numbers are regulated by humidity161 and although HDM is recognised as an important associate of respiratory symptoms162, the seasonal fluctuations in their numbers do not adequately explain patterns of asthma exacerbation in South-East Queensland since the peak asthma seasons are in the drier months. Mite numbers should be less in drier months.

Humidity is a regulator of cockroach aggregation 163 and development 164 and their numbers are greatest in South-East Queensland in summer, not the peak asthma season of autumn. Cockroaches found in Australia are usually Blatella germanica or B.americana.

While recognising the importance of these allergens to the indoor environment as shown by decades of research, these allergen sources will not be of prime interest here although they do relate to issues in section 2.2 regarding terpenes. The terpene content of insect faeces and their probable addition of terpenes to the atmosphere will be included in the discussion of bio-aerosols. Larger creatures in the form of pets provide a much more obvious source of allergens.

Fungal spores The numbers of fungal spores in ambient air on days of high fungal load can be very great, as can the variety. Fungal spores of most interest to respiratory

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researchers are those belonging to the Alternaria, Cladosporium, Aspergillus , Penicillium165 ; these are studied in environments in Turkey 166 , Finland167,

France167, the U.S.A.142, Taiwan168 and Australia169, 170, to name just a few. The ubiquitous nature of fungal spores in both inside and outside environments makes the task of assessment of their role in an individual’s symptoms very difficult171.

Fungal spores generally increase in warmer weather168 and yet in the Brisbane area fungal spores are greatest at the end of April through May172, a period which coincides with the asthma peak26. Fungal spores predominate over pollens during this time.172.

Rees in 1963, in a comprehensive catalogue or airspora in Brisbane over one year, recorded the maximum Cladosporium as occurring in late April and May169. The basidiospores or sooty moulds and smuts were also prominent, featuring in the wetter summer months169. Rees surmised that the Cladosporium source was the grass clippings from Brisbane lawns. This theory was supported by previous research173 showing that mowed parklands produced Cladosporium and it has found its way into journals decades afterwards. Agar plates exposed by Rees developed mostly Cladosporium herbarium on them. Epicoccum nigrum was apparent during spring on the plates and Penicillium was most obvious in late March and April.

Subsequently in 2000 Rutherford et al. showed that ambient fungal spores 174 were strongly associated with decreased lung function in allergic adults, but only in those months where the spores peaked. Other studies have shown increased fungal spores in ambient air at the time of thunderstorms165, 175. This phenomenon is perhaps a co-variable to the bursting of pollen grains that has been recorded during storms176 and the asthma exacerbations that have been recorded during these episodes177, 178.

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Some fungi are associated with damp conditions, like Penicillium171 for example, while others favour dry conditions, such as Alternaria which has been associated with increased asthma162. Serology testing of patients in New York revealed allergic sensitivity to Alternaria tenuis and Cladosporium herbarum spores to be as associated with asthma, after adjusting for mite and cat allergy179. Some fungi are particularly associated with occupations ranging from timber industries180, 181 and farming182 183to those associated with biotechnology184 and as such may pose a variety of health threats.

Worthy of note is a methodological problem in fungal spore associated illness. Although there is a lot of circumstantial evidence relating fungi to various illnesses, hard evidence is scarce. Fungi are ubiquitous and establishing causal relationships is difficult. Frequently, evidence of Alternaria spores is recovered from the nasal cavities during sinus surgery. As Bush points out in a 2006 article185, these spores are often found in the nasal passages of healthy individuals. Table 2.03 provides a summary of some associations between fungal spores and health measures.

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Table 2.03 Fungal spores and some health issues

Symptom and/or Fungal spores effects Location Reference

Alternaria, Cladosporium, Helminthosporium, Southern Aspergillus, California, Penicillium, rusts, Increased asthma USA Hyphal fragments severity 186 A range of Wisconsin, Aspergillus increased USA Penicillium respiratory & CNS Cladosporium symptoms 187 Increased BHR Melbourne, but lower Australia sensitization to Total airborne fungi fungi 188 Lung symptoms- Germany Pleurotus - Florida- fever & cough oyster mushroom “farmer’s lung” 189 Alternaria Asthma Italy 190 Fungal debris & Sinusitis and Arkansas hyphae in sinuses asthma in children USA 191 Mould allergy Severe asthma 192 Mould related to asthma and sinusitis –evidence largely Kansas – circumstantial for Irritant effects; Position Alternaria. Several Toxicity; Mucus paper only fungi implicated membrane irritants 185 Allergic Saudi Arabia bronchopulmonary Aspergillus mycosis 193 Decrease in lung Brisbane Fungal spores function Australia 174 Third important sensitizer after dust mites and Isle of Wight Cladosporium & grass in 4 year Alternaria olds 194 Sensitivity to Manhattan, moulds was New York Cladosporium & associated with Alternaria asthma 179

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While insect and fungal spores are an important source of allergen, the family pet often provides a persistent and proximate challenge.

Animal allergens

Animal dander and fur are a source of allergens for many people195 although some animals provoke much more reaction than others. Cat allergy is one of the most frequently reported 156, 188, 196-200 and has even been detected in the absence of cats on the island of Tristan da Cunha where cats were eliminated201 more than 20 years before the investigation. Cat allergen is notorious for its endurance in the atmosphere, remaining six months after removal of the cat202.

Horse allergen provokes a strong reaction and is less frequently investigated probably because fewer people keep horses compared to cats. Horse allergen may cross-react with other furred animals203, all of which are recommended to be removed from the immediate environment for reduction of asthma symptoms158. In contradiction, some studies reveal associations that suggest that pet ownership is associated with protection against asthma204.

Acknowledging the inconsistent views regarding allergy and pets, Simpson and Custovic205, suggest that a dog might be the better choice. A study by

Sandin206 lends support to the notion that dog ownership may be protective against wheeze. Critics of these studies might suggest that the participants are less likely to be atopic as highly allergic people are more likely to choose to be without a pet and therefore would never be involved in one of these studies.

Dogs traditionally produced less allergic responses than cats and breeding for fur/wool type to reduce allergens is common. Controlling environmental variables in the home in order to assess the contribution of pets to allergic status is difficult but investigations into retention of the allergen in the household have been successful. Household surfaces retain animal allergens

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differently207, with carpet being the most problematic208.

Guinea pigs, rats, mice, birds and many other animals can cause allergic responses for some people. The pet allergy scene is set to become more varied as exotic pets become popular : iguana allergy and has now been reported209. Pet allergens tend to be fairly consistent in abundance unless a concerted effort is made to reduce the access to the pet and/or its access to the home. The weather is much more variable as a predictor of allergens compared to pet proximity and consistent patterns among those variables and respiratory symptoms are elusive.

Meteorological variables

Although associations among meteorological variables and respiratory symptoms are inconsistent, some recurring themes are apparent. Thunderstorms and cool dry weather feature more frequently than other variables in study linking respiratory symptoms and weather. In Brisbane there is usually a seasonal peak in autumn and a slightly smaller in spring. A four year study of climate and asthma attendances at hospital, in the 1960s is the most comprehensive record of climate and asthma patterns to date. Derrick26 showed that that although those peaks are discernible each year, they do vary in form and timing. The peak can begin in March some years and in late April in others. The spring peak varies from the beginning of October until early November. Subsequently winter has been identified as a time of increased asthma117. Summer is has always been a time of reduced asthma symptoms in Brisbane, compared to other seasons. Some climatic relationships to respiratory symptoms are shown in Table 2.04.

Chapter 2: Current knowledge: Asthma and allergy

Table 2.04 Meteorological variables and respiratory symptoms Respiratory Meteorological symptom variable associations Location References Reduced asthma Southern 186 Higher temperatures symptoms California Temperatures, fall in Significant but small barometric pressure, associations with New Zealand 210 relative humidity PEFR Associated with New England, 211 Rainfall morning dyspnoea U.S.A. No relationships after 212 controlling for season None Spain Association with Japan respiratory symptoms 3 and 7 213 Temperature fall days after Increases in asthma Cuba 214 Cold days occurrence Strong winds, fog & temperature Associated with 215 inversions asthma France Brisbane, 216 Temperature fall Asthma increase Australia Strong association Washington 217 Air stagnation with asthma State, U.S.A. Strong association with asthma and soy dust presence in New Orleans, 218 Low wind speed harbour U.S.A. Barometric pressure, Predictors of temperature paediatric asthma difference, minimum visits to hospital Trinidad, temperature, wind emergency West Indies 219 speed department Emergency room visits higher than no Ottawa, 177 Thunderstorms activity periods Canada Emergency room London, visits much higher United than no activity Kingdom 220 Thunderstorms periods Accompany increase in asthma; Asthma decreases during rainy periods – measured by Cold dry changes: hospital emergency Brisbane, 26 Rain attendances. Australia

Chapter 2: Current knowledge: Asthma and allergy

In the 1990s the relationship between meteorological variables and asthma was delineated more clearly when episodes of thunderstorm related asthma were reported. This phenomenon has already been mentioned, and represents only one of the ways that pollens have been implicated in respiratory symptom exacerbation.

Pollen Pollens and their fragments are deemed to be the cause of seasonal hay-fever and most allergic respiratory symptoms, including asthma, by means of inducing hypersensitivity reactions 171 which occur when pollens are absorbed via mucosal surfaces on the respiratory tract. Movement into the lung is restricted by the size of the pollen, pine pollen and grass pollen being larger than some weed pollens. Most pollens fall into the 20-50 micron range. Hibiscus pollen is 210 µ and a Parietaria officinalis is only 12µ. Physical characteristics of pollens are discussed further in Chapter 3.

Problematic pollens are deemed to come from wind-pollinated plants while insect –pollinated plants are not regarded as clinically relevant 221 because their pollens are not airborne171. Gardeners have been encouraged to plant insect- pollinated plants in an effort to reduce allergens222-224. Under the heading “Golden Rules”, a pamphlet aimed at reducing allergens in gardens reads: “choose bird or insect pollinated plants rather than wind-pollinated plants”222. One such recommended plant from the Myrtaceae family is Melaleuca viridiflora the flower of this popular garden cultivar for warmer regions is seen in Figure 2.03.

Problematic pollens are thought to cause seasonal nose-itching, sneezing, rhinitis, sinusitis, blocked nose and allergic upper respiratory conditions. Many countries have ‘pollen calendars’ advising when pollen seasons may be expected according to plant type9, 224-228.

Chapter 2: Current knowledge: Asthma and allergy

Figure 2.03 Melaleuca viridiflora

Advice on what plants to avoid varies according to locality. Troublesome plants may be grasses, trees, shrubs or weeds and there are hundreds that could be listed. Some of the common culprits to appear on ‘do not grow ‘ lists for low- allergen gardens in many countries are : several grasses, wattles , ash, she- oak, cypress , pines, birch, juniper, olive, privet, poplar, willow, walnut, plane tree, liquid amber, elm , mesquite, cedar, cypress222. Species from many families are represented. More detail on the taxonomy of allergenic plants follow in Chapter 3 and Chapter 6.

Weed species are problematic and many belong to the family Asteraceae. Ragweed and mugwort allergy 229 are commonly encountered and ragweed has been blamed for Brisbane’s autumn hay fever230. Parthenium and Senecio

(fireweed) are well established in Queensland 231 232and many parts of the world233, 234. Table 2.05 shows some of the other allergenic pollens of the world.

Chapter 2: Current knowledge: Asthma and allergy

Table 2.05 Allergenic pollens in different part of the world Important Pollens country Associated with Reference Prosopis juliflora, Ricinus communis, Morus, Mallotus, Alnus, Quercus, Cedrus, Argemone, Amaranthus, Chenopodium, Holoptelea, Brassica, Cocos, Cannabis, Parthenium, Cassia and grasses; Allergically 11, 235 Eucalyptus India important Cupressus Allergically 236 sempervirens France important

Saudi Arabia Asthma , rhinitis 237 Phoenix dactylifera L urticaria Asthma ; 64% allergic to grass pollen had 238 Grass pollen mix United Kingdom asthma Parietaria, cypress, grasses, Platanus, Allergically 8, 9, 239 olive, birch, Eucalyptus Spain important Grasses, Cupressus, Acacia, Ligustrum; Grevillea, Casuarina, Jacaranda, Pinus, Plantago, Ricinus, Suspected hay- Rumex, Tithonia, Brisbane , fever causing 240 Coreopsis Australia plants 241 Chenopodiaceae Kuwait Increased rhinitis Juniperus, Ligustrum, Crytomeria, Alnus, 226, 242 Acer, Juglans U.S.A. Allergenic pollens Artemesia, Rumex, Urtica, Plantago, Parietaria, Hebe, 224 Juglans United Kingdom Allergenic pollens Parietaria, Cupressus, 243 Olea, Betula Italy Allergenic pollens Betula, Juniperus, Alnus , Fraxinus, 225 Corylus , Poaceae, Croatia Allergenic pollens

Not all allergies that are associated with plants are related to pollen that is inhaled: plant allergens can be contracted directly.

Chapter 2: Current knowledge: Asthma and allergy

Contact allergens from plants Contact allergy is associated with particular groups of plants particularly Rhus, and plants that produce a milky sap which is a skin irritant244. Such plants belong to the Euphorbiaceae family and include plants like poinsettia (Euphorbia pulcherrima) and Jatropha. Less well known is the capacity of the Australian native Grevillea245-247 to be a skin irritant . Allergens can also be absorbed by inhaling gases emitted from plants. This is an important process and the central position of this thesis. The processes involved will be revealed in the next section of this chapter, terpenes.

Plants may cause allergic reactions resulting from contact from plant hairs (trichomes) or spines246, 248 which may pierce the skin effecting contact from allergens contained therein. Herbs like oregano, mint and sage 249 have trichomes 250 251which often contain phytotoxic oils252. Trichomes also feature in many troublesome weeds like Parthenium, and they provide another vector for plant allergens to enter the respiratory tract. These will be further in the context of vectors for plant chemicals in Section 2.2.4.1.

Gaseous emissions from plants Detailed work regarding allergic gaseous emissions from plants is scarce and perhaps the most influential work to date is that connected with canola, or oilseed rape. This allergy is a substantial problem in the United Kingdom and Canada, both of which grow large amounts of canola. Butcher et al. in 1995 examined whether the gases emitted from oilseed rape during the growing period might have the potential to modify human proteins. They found three such compounds: dimethyl disulphide, thiocyanic acid methyl ester and 2- methyl-propanenitrile253. The terpenes also characterized did not have the property of modifying proteins but their potential to form oxidised products that may act as irritants was noted. Butcher et al. suggested that the monoterpenes emitted from the canola during growth would oxidize to aldehydes and ketones under certain conditions. The examination of plant emissions as a possible

Chapter 2: Current knowledge: Asthma and allergy

source of irritants or allergens is the subject of the next section: the role of terpenes in respiratory symptoms.

2.2 Possible role of terpenes The aim here is show how terpenes might relate to many aspects of respiratory inflammatory disease and to ask some relevant questions that may sharpen the focus in the areas of allergy and respiratory research. This is not an attempt to address the complexities of terpene chemistry in depth, nor all the biologic ramifications of contact with terpenes. With that in mind, some understanding of terpenes and their function is useful.

2.2.1 What are terpenes?

The study of terpene chemistry and their role in plants is decades old, and until a recent resurgence which began in the 1990s, terpenes had a negligible profile in matters of allergen research. The exception was interest in the sesquiterpene lactones which was sparked in the 1970s. This allergy-related group was identified as being causal in symptoms of dermatitis in florists254.

Perhaps the most easily understood explanation of terpenes and plants has come from Josep Penuelas255 and Joan Llusia, researchers from Spain: “… we might regard terpenes as a chemical ‘language’ of plants. Like in any language, terpenoids are induced and emitted in response to internal (genetic and biochemical) and external (ecological) factors, both abiotic and biotic. Their information or effect is received and responded to by other parts of the plant, by other plants, animals and micro-organisms. “ Broadly, terpenes are small lipid molecules made up of isoprene units. Strictly speaking, terpenes should only be hydrocarbons. To include the other molecules, which contain other elements, the term “terpenoid” is used97. These names are often used interchangeably and “terpene” will be used here since it appears most commonly in the literature.

Chapter 2: Current Knowledge: Role of terpenes 52

Terpenes typically occur in all parts of higher plants: in mosses, liverworts, algae and lichens. Some are of insect or microbial origin. There is a huge diversity of structure with terpenes numbering over 22,000256. Some are hydrocarbons and some contain oxygen. Many are ringed, while others are open-chained. Terpenes are built from basic isoprene units (2-methyl-1, 3- butadiene) consisting of head to tail couplings of the five-carbon units257. This is the “isoprene rule”. There are some exceptions like artemesia ketone and lavandulol 258 which are not formed this way, but almost all are. Isoprene is a major volatile emitted from many forests of the world109, 259, 260 and its structure is seen in Figure 2.04.

Figure 2.04 Isoprene

CH2

H3C C CH CH2 Isoprene Formula: C5 H8 CAS registry number261 78-79-5

Classification of terpenes is based upon the number of structural isoprene units: Iridoids : contain 5 carbon atoms and are usually unstable often with a lactone or hemiacetal formation262; Monoterpenes : contain 10 carbon atoms and 2 isoprene units; Sesquiterpenes : contain 15 carbon atoms and 3 isoprene units; Sesquiterpene lactones : contain 15 carbon atoms in a ring structure; Diterpenes : contain 20 carbon atoms and 4 isoprene units; Sesterterpenes : contain 25 carbon atoms and 4 isoprene units; Triterpenes : contain 30 carbon atoms and 6 isoprene units. Although the “isoprene rule” is a convenient way of thinking about terpenes, isoprene is not the precursor of all terpenes. Nature uses “isoprene equivalents” to produce geranyl pyrophosphate which is the precursor of all monoterpenes.

Chapter 2: Current Knowledge: Role of terpenes 53

Further reaction of this substance with the isoprene equivalent, isopentenyl pyrophosphate, yields farnesyl pyrophosphate that is the precursor of all sesquiterpenes257. Triterpenes are made up of two units of 15 carbon atoms linked in the middle head-to-head.

Natural rubber is an acyclic polyterpene composed of a continuous chain of numerous units formed in this way263. Similarly, carotenoids are made up of 20 carbon units linked in this way258. There are other terpene groups such as sesterterpenes, which occur in marine animals, do not impact upon the theory presented in this thesis, and will not be dealt with here.

The terpene set of compounds contains many sub-groups based upon structure. Linear or cyclic structures set one group apart from another. Most relevant to this thesis will be the monoterpenes and the sesquiterpenes. Particularly relevant ones will be emphasised and only a few of many thousands will be mentioned here.

Monoterpenes

Noncyclic monoterpenes

These include the noncyclic monoterpenes ocimene, from sweet-peas264, and myrcene. Geraniol , linalool, and nerol belong in this group as does citronellol and the lemon-scented citral which are derivatives of geraniol258. Citral is used in household and personal products extensively because of its pleasant scent.265-269. Citral is an aldehyde and is found in large amounts in several

Australian plants, notably Corymbia citriodora270 (formerly Eucalyptus citriodora) and the lemon myrtle (Backhousia citriodora)271-274. Its structure is shown in Figure 2.05.

Chapter 2: Current Knowledge: Role of terpenes 54

Figure 2.05 Citral

OHC CH C CH 2 CH 2 CH CMe 2

Citral CAS Registry Number261: 5392-40-5 Formula: C10 H16 O

Monocyclic monoterpenes

The monocyclic monoterpenes include menthol from mint 275 and α-terpinene from many Australian trees276. An important and ubiquitous monocyclic monoterpene is limonene which is present in volatile emissions from many flowers277;278-283 . Limonene shown in Figure 2.06, is also emitted by fir

(Abies)284, oak285 (Quercus), spruce (Picea )286, 287 Eucalyptus plantations288 and oilseed rape or canola (Brassica napus)253, 289, 290 as it is called in some countries.

Figure 2.06 Limonene

CH 2

C Me

Me Limonene CAS Registry Number261: 138-86-3 Formula: C10 H16

Bicyclic monoterpenes This important group consists of several divisions according to molecular skeletal similarities: the carane, pinane, camphane and fenchane series. The carane group contains the important δ-3-carene, which has already been mentioned as an irritant in turpentine. This substance features in emissions

Chapter 2: Current Knowledge: Role of terpenes 55

from temperate forests287, 291-294, especially that of pine and spruce. It is also found in the floral volatile emissions of oilseed rape289, 290, cubeb pepper (Piper cubeba )295, cypresses (Cupressus )296, 297, pepper trees (Schinus), juniper

(Juniperus) 298and pine ( Pinus)291. This compound is seen in Figure 2.07.

Figure 2.07 Delta-3-Carene

Me Me δ-3-Carene Formula: C10 H16 CAS Registry Number261: 13466-78-9

Pinane monoterpenes include α-pinene , from juniper berries299, melaleucas300, eucalypts301-303 and pine trees292, and eucalyptol from

Eucalyptus trees270, among others. Common monoterpenes, α-pinene and β- pinene also feature strongly in the chemical profiles of many other Myrtaceae plants like manuka (Leptospermum) from New Zealand304, Australian tea trees

(Leptospermum)305, Australian bottlebrushes306 (Callistemon) and myrtles

(Backhousia)272, Thryptomene307 and many more genera308-312. These emissions frequently contain monoterpenes from the pinane group, the structure of two common members is seen in Figure 2.08.

Figure 2.08 Alpha Pinene and Beta Pinene (±)-α-Pinene Me Formula: C10 H16 Me 261 CAS Registry Number: : 80-56-8 Me (±)-β-Pinene Formula: C10 H16 261 CAS Registry Number : 127-91-3 Me CH 2 Me

Chapter 2: Current Knowledge: Role of terpenes 56

The camphane series of bicyclic monoterpenes contains camphane and the important ketone camphor, from lavender plants313. Camphor can be obtained industrially from pinene258 and is its structure is shown in Figure 2.09. The related camphene is found in many plants and trees: sage314, cherries282 , chrysanthemums315, juniper316, spruce317 and many herbs318-320. It is also found in many eucalypts321, 322 and other Myrtaceae genera in

Australia312, 323-326.

Figure 2.09 Camphor

Me Me O Me Camphor Formula: C10 H16 O CAS Registry Number261: 76-22-2

Thujane monoterpenes include thujone from Artemesia327 and umbellulone from Cupressus trees328. The famous alcoholic drink absinthe was made from wormwood ( Artemesia) until it was banned due to neurotoxic effects of thujone262. Thujene, a thujane hydrocarbon , is also in some Eucalyptus trees329, 330. Sabinene is one of four isomers of thujene isomers. It is a bicyclic monoterpene and is found in oilseed rape(Brassica napus ssp oleifera)290, bottlebrushes (Melaleuca)331, tea trees305 (Leptospermum), larch (Larix)332, beech (Betula)291, oak (Quercus)317,beech (Fagus)109,cubeb pepper trees

(Piper cubeba)295 ,spruce 286(Picea) ,geranium333 (Pelargonium), rhus

334(Rhus), and, especially, in juniper316 (Juniperus) . Sabinene is of particular interest to the models proposed in this thesis and its structure can be seen in Figure 2.10. Chrysanthemum carboxylic esters also belong to the thujane group262.

Chapter 2: Current Knowledge: Role of terpenes 57

Figure 2.10 Sabinene

Me CH 2

Sabinene Formula: C10 H16 CAS Registry Number261: 78-79-5

The fenchane series contains fenchane related compounds. They are well represented in many Australian genera. Fenchane related compounds include fenchone, isofenchone, fenchol and six types of fenchene. Fenchane compounds can be found in the Australian genera Eucalyptus301, 321, 322,

Austromyrtus325, Leptospermum323-325, 335-338 and Callitris339, 340. They are also found in Cupressus341, Thuja342, Juniperus343, Spanish myrtle (Myrtus communis) 344and several North American herbs345.

Bicyclic sesquiterpenes frequently occur with sesquiterpenes. There are many sesquiterpene structural types. Sesquiterpenes occur in cyclic formations and a number of skeletal types. Some sesquiterpene skeletons that appear frequently in chemical profiles of plants of interest in this thesis are plants are: farnesane, santalane, cadinane, cedrane, germacrane, selinane, eremophilane, aromadendrane and guaiane. There are many more258.

Sesquiterpenes

Noncyclic sesquiterpenes These include the non-cyclic sesquiterpenes such as α- and β- farnesene which are also insect alarm pheromones often found in perfumed items. Figure 2.11 shows α- farnesene; is found in the emissions from oilseed rape plants in flower 253 as well as flowers from other plants346-348. Oilseed rape is also known

Chapter 2: Current Knowledge: Role of terpenes 58

as canola and rapeseed and is associated with numerous reports of allergy and asthma253, 289, 349-353. Noncyclics include curcumene354 and zingiberene355 from tumeric and curcuma, respectively.

Figure 2.11 Alpha-Farnesene

Me 2C E E CH 2 Me Me

α-Farnesene Formula: C15 H24 Registry Number261: 502-61-4

Bicyclic sesquiterpenes Bicyclic sesquiterpenes include α-vetivone from vetiver grass and gossypol from cotton plants262. Bicylics are common in some genera and occur particularly in some of the plants from the order Pinophyta and the order Myrtales. Cadinene is a bicyclic that occurs in the following genera Juniper316, Abies 356 ( fir trees),

Pinus 357 (pines), Leptospermum358, 359 ( tea tree from Australia and manuka from N.Z.) , and in lavender flowers360. Juniper225, 361-365 , fir and pine trees are associated with allergic pollens and are implicated in asthma and hayfever225, 242.

Selinene is a bicyclic sesquiterpene that is found in many members of the Myrtaceae family, including those in Brazil366 . The oil of New Zealand Myrtaceae member, manuka (Leptospermum scoparium), is dominated by sesquiterpenes, of which selinene is one. Selinene is also known as naphthalene and its structure is seen in Figure 2.12.

Chapter 2: Current Knowledge: Role of terpenes 59

Figure 2.12 Selinene

Me H Pr-i R S R R

Selinene 261 Formula: C15 H24 CAS Registry number : 27104-12-7

Tricyclic sesquiterpenes Non-lactone tricyclic sesquiterpenes can be found in trees from the Cupressaceae, notably the juniper (Juniperus) genera which commonly contain α-cedrol and α-cedrene. These compounds are also found extensively in the cypress297, 367 genera (Cupressus). Cubeb295 pepper trees in the Piperaceae family contain large amounts of cubebene, another tricyclic. This botanical family is represented by 80 genera368 of trees in the Caribbean and

South America where asthma levels are very high7. One of the world’s worst weeds369 also contains large amounts β- cubebene370 . This weed Chromolaena odorata, Siam weed, heavily infests the islands of Micronesia (formerly the Western Caroline Islands) where asthma rates are extremely high20, 371. Myrtaceae genera Eucalyptus372, Leptospermum and Melaleuca contain tricylics α- and β- cubebene, germacrene, bicyclogermacrene, aromadendrene, longifolene, isoleptospermone359, flavesone359 and gobulol373 which can be found in many other Australian genera of the Myrtaceae 276, 308,

309, 311, 374, 375 family. Figure 2.13 shows α- and β- cubebene and the tricyclic structure. Tricyclic sesquiterpenes are important to the themes of this thesis.

Chapter 2: Current Knowledge: Role of terpenes 60

Figure 2.13 Alpha and Beta Cubebene

Formula: C15 H24

261 CAS Registry Number : 17699-14-8 α-Cubebene ↓

R R S Me R S Me H H i-Pr S S R CH 2 R R H H i-Pr

↑ β-Cubebene Formula: C15 H24

CAS Registry Number261: 13744-15-5

Within the two groups, monoterpenes and sesquiterpenes, that are of special interest here, particular components, are of importance. A number of structural chemical groups are especially relevant to the question of relationships between plants and respiratory symptoms. These will be addressed briefly.

2.2.2 Important structural groups in plant chemistry

Hydrocarbons Hydrocarbons are compounds made up of carbon and hydrogen only and are classified into sub-groups of alkane, alkanes, cycloalkanes, alkenes, alkynes and aromatic compounds376. Alkanes contain single carbon to carbon (C─C) bonds; alkenes contain at least one carbon to carbon (C═C) double bond; and alkynes contain at least one (C≡C) triple bond. They react with ozone or with nitrogen dioxide in the presence of light to produce new oxygenated products376.

Chapter 2: Current Knowledge: Role of terpenes 61

Benzene, with its characteristic carbon ring structure, is representative of aromatic hydrocarbons. These differ from the other sub-groups in that they undergo substitution reactions, not addition reactions377.

Monoterpene hydrocarbons of particular relevance to this thesis are limonene,

α-pinene, β-pinene and sabinene, all of which have the formula C10 H16.

Sesquiterpene hydrocarbons of special interest are cyperene, α-cubebene, β- elemene, δ-elemene, α-farnesene, β-farnesene, germacrene-D, humulene, α- selinene, β-selinene, viridiflorene, copaene, α-gurjunene, β-gurjunene, aromadendrene, cedrene, longifolene, and cyperene. These compounds have the formula C15 H24 and with the addition of a hydroxyl group they become alcohols.

Alcohols Alcohols are compounds that have hydroxyl (OH) groups bonded to saturated carbon atoms257. This definition does not include phenols or enols which have a different structure and will not be discussed here. Alcohols occur widely in nature and can be classified as primary, secondary or tertiary, depending upon the number of organic groups that are bonded to the hydroxyl-bearing carbon. Terpene alcohols common in plants are linalool, geraniol, farnesol, neral, globulol and patchoulol. Alcohols can be oxidized to form aldehydes or ketones.

Aldehydes, ketones and carboxylic acids These are oxidation products of alcohols, all of which possess a carbonyl group. Carbonyl compounds possess a double bonded carbon and oxygen (C = O) and they are of prime interest here. Carbonyl compounds are susceptible to nucleophilic addition reactions and have a role to play in irritant reactions which will be discussed in section 2.2.5 in relation to the oxidation of limonene.

Aldehydes are formed by the oxidation of a primary alcohol while ketones are formed by the oxidation of a secondary alcohol. Tertiary alcohols do not react

Chapter 2: Current Knowledge: Role of terpenes 62

similarly376. Aldehydes and ketones are most often pleasant odours and are used frequently in the perfume industry. Benzaldehyde, formaldehyde, citronellal are examples of aldehydes . They are often pleasantly perfumed and are associated with flowering in many plants378, 379. Acetone, carvone and camphor are ketones which are associated both with some flowering plants like chrysanthemum380 and conifers like black spruce (Picea mariana)286. Acetone occurs naturally in the buds of some conifers and production of this ketone ceases when the buds open. MacDonald and Fall 381 surmise that the acetone may have some cold protection function in the fir tree (Abies concolor).

Sometimes multiple carbonyl groups are associated with particular plants – diketones and triketones are especially associated with members of the Myrtaceae family304, 382-384 in Australia and New Zealand.

Carboxylic acids are generally weak acids and readily disassociate in water. Acetic acid (vinegar) is a commonly occurring carboxylic acid. Benzoic acid is associated with a number of plants and seems to relate to the production of esters in many floral emissions125, 385-387. Kolosova et al.387 examined the accumulation of benzoic acid in plant tissue and the related emission of related methyl benzoate ester in diurnal and nocturnal plants and found the emissions to be related to circadian rhythms in keeping with the activity of day pollinators (bees) or night pollinators (moths). Other carboxylic acids, like butanoic and propanoic, also have a role in the production of their related esters.

Esters The combination of an alcohol with a carboxylic acid forms an ester. Conversely, the breaking of the ester linkage by hydrolysis breaks the ester back into an alcohol and a carboxylic acid again. This process is important for the reactions that occur in ambient air before a thunderstorm, when atmospheric pressure drops91, 177, 388. In experiments in enzymatic esterification, ester production was shown to increase under reduced pressure

Chapter 2: Current Knowledge: Role of terpenes 63

389. This phenomenon may be related to observations of ‘gluttonous’ grazing on larkspur flowers by cattle during storms. Such feeding results in an increase in cattle deaths due to the alkaloids in the plants. In controlled studies on grazing patterns cattle were observed to increase grazing on these flowers during stormy weather390. This behaviour would be consistent with increased ester production by the plant, thus providing a sweetness that would attract the cattle. Esterification of emitted plant chemicals may be a contributor to increased asthma during thunderstorms177, 388 and will be discussed in Ch 6.

Ester formation in plants affects the pollination activities of insects391, 392 by mimicking pheromones. Plants from the Myrtaceae family are dependent upon insects and birds and animals for pollination. The bright colours and sweet odours from flowers like the Eucalypt (some genera of which are now called Corymbia) in Figure 2.14 attract a range of pollinators. Figure 2.14 shows the bud splitting open by lifting the ‘cap’ off what will grow to become a seed capsule and ‘gumnut’ (bottom right of the picture). Floral volatiles are their maximum at this stage, which is called anthesis.

Figure 2.14 Buds and flowers from Corymbia ficifolia/calophylla hybrid (cultivated specimen- formerly named Eucalyptus)

Chapter 2: Current Knowledge: Role of terpenes 64

Wind pollinated plants also produce volatiles of various types, including esters. Observation of insects on members of the daisy family (Asteraceae), for example, reveals a flurry of activity; the esters attract the bees and selected other insects are attracted to chemical blends that mimic pheromones from prospective mates.

Already, in this thesis, sesquiterpene lactones in weeds like Japanese sunflower (Tithonia diversifolia) and feverfew (Tanacetum parthenium) have been identified as allergenic. According to Polya, lactones are cyclic esters262. Sesquiterpene lactones are lipophilic, blending with oils in plants, and are present in the leaves, stems, flowers, trichomes and pollens of plants. These lactones are thought to play an important role in plant defence against herbivores. They are thought to alter feeding patterns and some inhibit insect development393.

Esters are those sweet perfumes that emanate from flowers, and are part of the distinctive odour of freshly painted rooms. Gardenias contain (Z)-3-hexenyl benzoate 394 and roses emit neryl acetate395. Esters are part of the sensory appeal of many essential oils.

2.2.3 Essential Oils

Terpenes are most familiar as components of “essential oils” which can be obtained from plants, usually by simple expression, steam distillation or solvent extraction. These oils are most often blends of terpenes although, less commonly, they are blends of aromatic compounds. Essential oils contain only the most volatile terpenes, those with fairly low molecular weight, which readily react with one another. If they were not volatile, they would be called “fixed” oils396. Some essential oils contain only a few substances, others may contain hundreds. Typical essential oils are those from Eucalyptus leaves, rose petals, pine needles, sandalwood and oil of cloves263. It is the quickly volatilizing essential oils that are responsible for the blue haze that hangs over the

Chapter 2: Current Knowledge: Role of terpenes 65

Australian bush 270 in summer. That haze originates in plants, to a large extent, trees.

2.2.3.1 Terpenes in plants

About 50 families of plants produce essential oils263. Some plant orders, like Ranunculales, are almost without monoterpenes and sesquiterpenes, while their prevalence is notable in the Magnoliales. Within the order Myrtales, only one family is noted for its monoterpenes and sesquiterpenes: Myrtaceae. Most of Australia’s native trees, including the eucalypts, belong to this family. The classic Eucalyptus odour is due to the monoterpene eucalyptol (1, 8-cineole) which is found in numerous members of Myrtaceae. Perhaps the most well known plants containing sesquiterpenes are the daisy family, Asteraceae, formerly, Compositae. Myrtaceae plants also contain substantial amounts of these, as do many other plant families. The scent of cloves is due to the sesquiterpene caryophyllene. Diterpenes are less commonly found in plants and are associated with milky sap as in Euphorbiaceae244 and resin acids in

Pinus244, 258 . The abundant triterpenes are just about restricted to the plant kingdom397. Triterpenes are not volatile and can be found in the leaf waxes of

Eucalyptus leaves398 , Prunus399 and Jacaranda400 as well as many other plants. Sesterterpenes are found in fungi and sponges.

Some terpenes are so common that they are contained in hundreds of plant groupings; others so rare that they are found in only one single plant397. Essential oils accumulate in all types of plant organs: flowers (rose); leaves (peppermint); bark (cinnamon); wood (sandalwood); rhizomes (ginger); fruits (anise); seeds (nutmeg) and even some roots (vetiver).

Oil yields It is the relative distribution of particular terpenes, and total oil yield that determines the feasibility for commercial essential oil crops. Some plants are grown for the whole oil, while others are grown so that components of the oil may be extracted. Eucalyptus globulus produces oil used commonly as an

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inhalant and in antiseptics, the dominant ingredient being 1,8-cineole (eucalyptol)397. This oil is typically used whole. Eucalyptus citriodora produces oil with 65-85% citronellal (lemon scent) making it a prime plantation candidate for use in the perfume industry, particularly270. The citronellal is extracted from the oil and used as an isolate. It is used in many household applications as an additive and is of considerable importance to the issues raised later in this thesis. It is also found in many other Australian shrubs and trees especially in Eucalyptus, Melaleuca, Leptospermum and Backhousia genera.273, 305, 401-403.

Oil production Levels of essential oil in a plant are usually quite low, less than 5%. A level of 15%, as found in the flower bud of the clove, is considered very high396. Essential oil source plants vary enormously from peppermint to sunflowers, from pepper to citrus, from cedar wood to Eucalyptus. Eucalyptus oil is produced in a number of countries including Australia but it is the developing economies of Brazil, China and South Africa that have entered this market in Australian trees enthusiastically404.

Oils may be extracted from suitable oil-producing plants by one of four means: solvent extraction, steam distillation, hydro-distillation and super-critical extraction, which utilizes carbon dioxide under high pressure405. The type of distillation can affect the composition of Eucalyptus oils406 so in drawing conclusions about fresh leaf oils, there is need to take account of this. Oils can be produced from the flowers, leaves, root, bark or seeds of plants. The primary markets of essential oils are the flavour and fragrance industries. Recently increased interest in essential oils has come from the complementary medicine market, especially in the form of aromatherapy and associated products.

Terpenes vary enormously in their distribution among plant groupings and their relative quantities in the plants. To illustrate, the leaves of the camphor laurel (Cinnamonum camphora (L.) Sieb. Var. linaloolifera) provide most of its oil from

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just one chemical. This species from Vietnam contains 91% linalool and 16 other constituents make up the remaining 9%407. This introduced handsome garden tree has become a ‘weed’ in Australia. In contrast, Brophy 273 lists 64 terpene components of Eucalyptus curtisii from South-East Queensland, Australia. The most abundant is (E)-β-ocimene, which accounts for only 16% of the leaf oil. This particular terpene also occurs in sweet-peas264.

The relative qualitative and quantitative distributions of terpenes vary enormously within and across species. A visual representation of chemical diversity is shown in Figure 2.15. This figure shows the variation among members of just one Myrtaceae genus, Melaleuca300. Of particular interest is the variation in levels of the chemical compounds of members within and across species of the same genera. Later in this chapter the factors that can affect that balance will be explained.

Figure 2.15 Relative percentages of terpenes in oil of Melaleuca species.

Relative quantities of terpenes in the oil two species of one genus : Melaleuca

100 α-pinene 1,8-cineole 90 limonene linalool β-caryophyllene terpinolene 80 α-terpinene γ-terpinene 70 caryophyllene oxide E-nerolidol E-β-ocimene E-methyl cinnamate 60 2,4,6, trimethoxyisobutytrophenone spathulenol C15, H26, O 50

30 Percentage Percentage composition 20

0 Chemotype I Chemotype I I Chemotype I Chemotype I Chemotype I Chemotype II Variety1 Variety 2 Variety 3

Melaleuca Melaleuca Melaleuca Melaleuca Melaleuca Melaleuca quinquenervia quinquenervia viridiflora viridiflora viridiflora viridiflora Species & variety of Melaleuca

In Table 2.06 numerals represent the percentage of the chemical greater than 1% found present in leaf oil. Chemical formulae represent different chemicals

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that are unnamed. The varied composition between the genera and among the species again demonstrates the error of assuming chemical uniformity: some members of the same genera contain high levels of substances in the leaf oil and others, none. E. citriodora (syn Corymbia citriodora) is one such Eucalypt and has 80% citronellal in the leaf oil, while other eucalypts shown in Table 2.06 have none. Given the variations of terpenes in plants, the individual chemicals and/or the blends of those presumably occur purposefully. In understanding these arrangements, seasonal patterns of some terpenes become more predictable. Some terpenes are produced only at either flowering time or senescence. Others occur when there are neither buds nor flowers. Although there is much to learn, some specific roles for terpenes in plants have been identified.

2.2.3.2 The purpose of terpenes in plants The purpose of terpenes in plants is not absolutely clear. Some terpenes are widely believed to act as germination and growth inhibitors to other plants. The high levels of 1,8-cineole and camphor in some Australian trees are thought to discourage growth of competitors, especially in arid regions408. The Melaleuca forests of coastal Eastern Australia are an example. Similarly, in the deserts of the U.S.A. the creosote bush releases chemicals that reduce competitors for the little available water409.

As well, substantial evidence suggests that the primary role is insect related391,

408, 410-413 . Many possible roles have been suggested and recent trends indicate that terpenes are subject to metabolic processes that result in conversion to many other compounds 414 relevant to attraction and repulsion of insects. The terpene volatiles that emanate from flowers are determined by the endogenous concentration in the flower tissue414. Chemical composition changes at flowering time will be discussed in 2.2.4. since this subject has relevance for the research described later in this thesis.

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Table 2.06 Chemical variation in Eucalyptus and Melaleuca oils Common Name Botanical Name Table Ref Yellow-barked Paperbark Melaleuca nervosa A M. sp. "Laura" B Broad-leaved Paperbark M. quinquenervia Chem 2 C Weeping Paperbark M. leucadendra Chem 2b Eastern D Australia Lemon-scented Gum Eucalyptus citriodora E Cider Gum E. gunnii F Maiden's Gum E. globulus subsp. maidenii G River Red Gum E. camaldulensis H Plant Identifier A B C D E F G H Reference 300 300 300 300 270 270 270 270 α-pinene 29.4 2 7.15 9.82 1.56 β-pinene 11.3 α-phellandrene 11.7 myrcene 1.1 1.2 limonene 32.4 6.8 5.03 3.63 α-Cubebene 2 1,8-cineole 4.5 64.9 65.8 83.7 ρ-cymene 11.4 bicycloelemene 1.04 citronellal 80.1 β-caryophyllene 2.2 1.3 E-nerolidol 7.2 aromadendrene 2.2 1.7 1 1.26 alloaromadendrene 1.2 1.44 germacrene-D 1.2 isopulegol 3.41 iso-isopulegol 8.51 α-terpineol 1.8 9.9 2.81 1.36 viridiflorene 1.5 3.87 bicyclogermacrene 11.1 methyleugenol 94.6 citronellol 8.8 4.18 C15, H26, O 1.9 C15, H26, O 3.3 C15, H26, O 1.5 C15, H26, O 2.1 C15, H26, O 3.3 globulol 2.1 1.5 3.4 6 4.4 viridiflorol 6.3 3 C15, H26, O 6.4 1 spathulenol 1.1 19 C15, H26, O 29.4 1 γ-eudesmol 1.96 C15, H26, O 5 α-eudesmol 3.5

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Terpenes, pollination and insect behaviour Many oil-bearing plants depend largely on insects for pollination (entomophilous), although some plants are also animal (zoophilous) and/or bird pollinated (ornithophilous). Some are pollinated by birds, insects and animals415 and some species are more efficient pollinators than others416. Figure 2.16 shows a common pollinator of bottlebrushes, a rainbow lorikeet, about to feed on Callistemon viminalis, the stamens of which deposit pollen onto the bird which distributes it to other plants when visiting.

Figure 2.16 Callistemon viminalis and rainbow lorikeet pollinator

Unlike wind pollinated plants (anemophilous) which have light dry pollens, the pollen of oil bearing plants like eucalypts (gum trees), melaleucas (paperbark bottlebrushes), callistemons ( bottlebrushes) and leptospermums (tea trees), is heavy, sticky, and less able to be disseminated effectively by wind. In eucalypts, the oil is pumped into the anthers of the flowers and mixed with nectar, thus making the pollen oily and sticky417. Attracting insects is required for pollination and propagation of the most plant species. The mix of terpenes in nectars attracts desired pollinators418 or may repel pests391. Specific terpenes in plant emissions have particular purposes to attract and/or repel pollinators or pests and may have specific biological effects.

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Linalool, a monoterpene alcohol found in oilseed rape, melaleucas, privet and scores of other plants, seems to be a general food attractant to bees and a sexual attractant to some species of male bees only419. The monoterpene α- pinene, the sesquiterpenes β-caryophyllene, α-cubebene, α- and γ-muurolene γ-cadinene, germacrene-D have be found in the propolis of sting-less bees411. These substances are part of the resin in ‘bee-glue’ which is related to propolis allergy420. Lemon scented citral is found in the anal secretions of thrips 421 and is an effective mosquito repellent422.

Wood borers select their host on the basis of the terpenes in the host tree, with sets of chemicals signalling the appropriate target423. Pinene was common to Eucalyptus and Pinus species selected by borers in a study by Barata in 2000423, however myrcene, (E)-ocimene, and cubebene were non-host cues for the borers.

Damage by insects to many plants is marked by the emission of a similar set of chemicals. These ‘wounding chemicals’ ( hexenoic acid, hexane, hexenyl acetate and hexenal) are the same as those emitted by grass when it is mown424, 425; when caterpillars feed on Brussels sprouts; when insects feed on tomato plants426; when a potato plant is mechanically damaged427; and hexenal emission from fruit is increased when olives are pitted428.

Ursolic acid has been shown to act as an anti-feedant for green-bugs on Jacaranda400. Bornyl acetate, found in Eucalyptus429, Leptospermum305 and

Backhousia272 in Australia is also in pine oil357 and Juniperus trees316. It is a sex pheromone of the cockroach430. Male cotton boll weevils are attracted to the host plants by the emission of (+)-α-pinene which is ingested and metabolized to form other terpenes which attract the female408. Some

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sesquiterpenes are insect juvenile hormones that serve to speed growth of pupae stages, but prevent metamorphosis, thus controlling insect predation408.

Terpene oxides Much remains to be learned about plant volatiles and insect behaviour. The relationships are complex and insects do not respond to volatiles in isolation. They respond to blends of pheromones431. The minor constituents of pheromone blends have a role as modulators of the informational content of chemical signals432. Linalool is an ubiquitous alcohol with a honey like scent. It is the base attractant for many insects391, 419, 432-436. The oxides of linalool are thought to have quite different species specific effects. Linalool attracts bees effectively; oxides of linalool do not. The (S)-(+)-enantiomer of linalool is the most effective male bee attractant. The (R)-(-)-enantiomer seems to attract female bees. The oxides seem to be attractive to434 butterflies in particular434. Experiments with butterflies and floral scents showed that linalool oxide has particular attraction for some species, much more so than linalool alone434. Reactive terpenes oxidise readily and their important role in allergic/irritant reactions and will be discussed further in 2.2.5.

Plants also produce terpenes in response to herbivore attack437 as they do to control the numbers of potential competitors for nutrients.

Terpenes as growth and behaviour regulators It is thought that 1,8-cineole and camphor are emitted by arid zone plants like eucalypts and sagebrush in order to inhibit the germination of competing species408. Much more has been recorded about the role of terpenes as pheromones.

Mite infestation resulted in greater total VOC emission from apple trees infested by the phytophagous mite Panonychus ulmi Koch when compared to uninfested trees438. Terpene changes were thought to be related to cues for

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predators to locate mite infestations. A mix of sesquiterpenes, of the same composition as found in pigeon pea plants, was found to influence oviposition and orientation in flight in moths439. Trans-β- farnesene seems to be a common, and non-specific, alarm signal to insects440. Terpenes like these ones may exhibit biological activity and may have incidental effects in humans. Their roles as irritants and/or allergens will be discussed in section 2.2.5. Some functional groups of terpenes associated with biological effects can be seen in section 2.2.6 and, before that, some discussion of the distribution of terpenes in nature is timely.

2.2.4 Terpene distributions

Terpene emissions from plants contribute to the atmospheric chemical load in varying chemical composition and in varying quantities. Such variation depends upon a range of plant processes and /or environmental variables. The presence of rain or an increase in temperature will affect the quantity of terpenes detected in the air and the chemical processes that take place. For clarity, these variations will be examined briefly in terms of variations in terpene content in various plant structures, and then, external influences on terpene production in plants. Other influences on patterns of plant distribution, and thus terpene patterns, will also be mentioned. Following that the influence of anthropogenic factors will focus upon the formation of secondary aerosols as a result of pollutant terpene mixes.

2.2.4.1‘Natural’ terpene occurrences and dispersal Increased interest in pollutant effects and greenhouse gas effects on vegetation has spurred research interest in volatile emissions. Those plant emissions detected have demonstrated the variability of emission type and quantity according to plant type, location and conditions.

Different areas of the world capable of supporting similar plants are called biomes. There are two guiding principles relating to natural plant location which are attributed to von Humbolt ( 1769-1859)441: plants occur in repeatable

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communities wherever similar conditions exist ; and, the positive relationship between altitude and latitude. Climbing a mountain in the tropics is akin to moving further away from the equator, as far as plant habitats are concerned. The world biomes are set out in the map in Figure 2.17 and understanding the patterns of vegetation provides a framework for understanding terpene emissions worldwide.

Figure 2.17 Biomes of the World

Source : Geographica442 : Used with publisher’s permission

Natural volatile emissions are most obviously connected with coniferous forests: the freshness of a pine forest on a fine morning is due to the volatile emissions consisting predominantly of pinene. The great deciduous forests of the world appear as brown on the biome map, Figure 2.17, while the coniferous (needle leaf) forests are shown as light green, immediately above the brown. Such emissions consist of a range of chemicals, many of which are of interest in this thesis. Commercial rubber plantations and fields of oilseed crops have also been examined for emissions. Table 2.07 shows just some of the volatiles that have been detected in air surrounding forests and commercial crop areas.

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Table 2.07 Volatile emissions from forests

Botanical Common Emits Reference Part Name Name examined Hevea Rubber Sabinene 259 In situ leaf brasiliensis via enclosed bag measures Brassica Oilseed rape Sabinene 289 In situ napus Myrcene headspace ssp.oleifera Limonene sampling Cis-3-hexen-1-ol- acetate Picea exelsa Spruce Camphor 443 Fresh needle 1,8-cineole litter Linalool acetate examined A-cubebene Γ-cadinene Δ-cadinene longicyclene Pinus Scots pine 1,8-cineole 443 Fresh needle sylvestris Δ-3-carene litter Limonene examined Toluene Ethanol Boreal forest Coniferous A -pinene, 294 Tethered forest –from Limonene balloon above sub-arctic Δ - 3-carene forest region Isoprene Fagus Beech Sabinene 291 Headspace in sylvatica (Z)-3-hexenyl situ leaf acetate volatiles (Z)-3-hexenol

Terpene emission also occurs in varying amounts around all oil bearing plants. These have been detected from wormwood (Artemesia) 444in the desert and various woody plants in the Mediterranean region445. Padhy et al. 446collected volatiles from 51 species on tenax cartridges of which Lantana camara, Eucalyptus and Casuarina equisetifolia were determined to be the top three emitters of volatiles.

Researchers have also examined volatile constituents of many herbs including coriander and mint447-452. Many plants have been examined to determine plant

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responses to ‘wounding’ by insects or physical damage426, 453. Such responses are similar to the emissions from grass when cut425, 454. Understanding that volatiles are produced by plants, when growing or damaged, is an important addition to the current focus on pollens as the major sources of plant respiratory allergens or irritants.

Much of the chemical analysis that has been performed on plants has involved the plant leaf material and the small amount of oil that can be extracted by processes already mentioned. Less well known is that the distribution of terpenes in many plants varies according to the different plant structures and to stages of growth (ontogeny). Understanding of these variations is critical if the occurrence of respiratory symptoms, in relation to plants, is to be understood.

Seasonal variation and health While medical researchers have recognised the relationship of pollen availability with respiratory symptoms for decades, the seasonal variations of plant gaseous emissions have not been considered in this context, with one notable exception. Scottish researchers in the 1990s253, 289 demonstrated the variation of gaseous emissions of oilseed rape and proposed that the asthma symptoms experienced by people nearby were related to some of the substances emitted. Some of the volatiles detected were sabinene, myrcene; limonene and cis-3- hexen-1-ol-acetate as seen in Table 2.07. These studies recorded gaseous variations only and did not record concurrent human respiratory symptom patterns. Other studies involving analysis of plant structures provide valuable information about probable emissions from flowers.

Terpene variation in plant structures A plant in leaf only produces very different volatiles from one which is in flower. One of the few Australian studies regarding the chemical composition of Myrtaceae flowers and leaves was carried out by Joseph Brophy. He has reported a large number of analyses on Myrtaceae leaf oils, including many Eucalypt and Melaleuca analyses. Brophy examined the chemistry of the

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swamp daisy from Western Australia326. This member of Myrtaceae demonstrates the importance of α-pinene in plants from this family. The swamp daisy (Actinodium cunninghamii) has more than 91% α-pinene in its flower and 89% α-pinene in its leaf oils. Such similarity of quantity is unusual. Table 2.08 shows the variations between flower and leaf oils from any one plant for a range of genera, across a number of terpene constituents. Note that the Eucalyptus globulus has more than twice the amount of the sesquiterpene aromadendrene in its flowers compared to the leaf. This type of pattern is much more common than the uniformity shown in the swamp daisy. The patterns depend upon the species and the chemical constituent involved. When a plant may have hundreds of chemical constituents the potential for variation according to growth stage and flowering habit is extensive.

Further variation in the chemical emission from plants can occur because of environmental variations, the most obvious of which is location. Geography can also subsume a number of other variables which will be discussed later.

Location influences

Geographical location has been shown by many researchers to be an important influence on the chemistry of plants. Research utilizing several plant types has demonstrated the variation possible across several families and genera.

European Myrtaceae Investigation of variations of terpenes in the European member of the Myrtaceae family, Myrtus communis, have revealed substantial variations in chemical content. This plant naturalized in the Mediterranean following introduction in ancient Greece455 from West Asia456. Spanish myrtle oils457 contain substantial amounts of myrtenyl acetate as do Croatian, Turkish and Yugoslavian, while Greek ones do not, reported Tubersoso et al. 458in their discussion of the variations found in the same species of one genus.

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Table 2.08 Percentage composition of flowers and leaves

Terpene Botanical Name Common Name Flower Leaf Ref Myrtenyl acetate Myrtus Myrtle 35.90 23.55 344 communis 1,8-cineole Myrtus Myrtle 29.89 16.51 329, communis 330, 344 Eucalytpus Gum tree 36.95 50.30 globulus

Eucalytpus Manna tree 49.40 42.00 viminalis

(E)-2-Hexenyl Lamium Purple dead- 7.90 3.10 452 acetate purpureum nettle Limonene Rhus mysurensis Rhus 51.30 26.20 334 Alpha-pinene Rhus mysurensis Rhus 15.50 26.80 330, 334, Myrtus 344 communis Myrtle 8.18 19.35

Eucalytpus Viminalis Manna tree 6.20 27.60 326

Actinodium cunninghamii Swamp daisy 91.06 89.70

Lamium Purple dead- 452 purpureum nettle 27.60 13.70 Aromadendrene Eucalytpus Gum tree 16.57 7.23 329, globulus 330

Eucalytpus Manna tree 13.30 5.60 viminalis Globulol Eucalytpus Gum tree 3.05 3.40 329 globulus Myrcene Chrysanthemum Chrysanthemum 15.90 5.40 380 coronarium Camphor Chrysanthemum Chrysanthemum 18.60 .04 380 coronarium

Table 2.09 shows the range of chemical groups found in the essential oils in Myrtus communis from eight plants from different parts of Sicily. Tuberoso’s team examined the composition of essential oils from berries and leaves of eight M. Communis plants. They found no myrtenyl acetate in their Sicilian samples at all. The existence of esters in plants are strongly implicated in

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allergic responses253, 459-463. Such marked variation in chemical content has implications for research into allergic responses. Making assumptions about plant composition needs to be tempered with information about factors that influence that composition, some of which are geographic and affect a range of genera.

Table 2.09 Chemical variation in a European Myrtaceae species

Structural Form Plant Berry Leaf Reference Monoterpenes Myrtus 72.2- 72.1- 458 communis 89.1% 92.6%

(Essential Sesquiterpenes 1.9-5.0% 0-1.6% 458 oil 458 Oxides fraction) 8 .01-1.4% 0-.8% Esters plants 1.8- 1.9-9.5% 458 sampled in 19.1% Sicily

Geographic influences on terpene constituents in general Terpene variation associated with geography is common among plant groups and is seen in groupings quite distinct from one another. Rosemary from five different zones in Portugal was shown to vary significantly in the quantities of 10 different terpenes in the oils extracted464. Site also affected oil composition for oregano(Origanum tyttanthum)465, tansy (Tanacetum vulgare)466, tomatoes467, as well as for juniper (Juniperus communis)299. Variability has also been shown with Lippia in Zimbabwe 468 and Artemesia 469 in Tuscany. Guchu et al. 470recommend that oak be selected for wine barrels on the basis of variations in terpene content in oaks in different parts of Europe. They concluded, though, that variations in chemical content were much greater among different species of Quercus than within the one species in different locations.

Geographic variation in the constituent terpenes has also been established for Australian members of Myrtaceae. Analysis of oil from Eucalyptus globulus

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grown on the Montenegro coast and the east coast of Spain329 has demonstrated that α- pinene content ranged from 2.54% to 17.85%; globulol ranged from 0.29% to 8.02% and spathulenol ranged from 0 .12% to 4.6%.

In an investigation of terpene constituents of Melaleuca ericifolia, Brophy and Doran471 found that movement south from New South Wales to Victoria , was associated with a reduction of linalool and linalool oxides. Geography though may simply subsume a number of other variables like soil quality, altitude, temperature, light intensity and water availability. The body of information regarding terpene emission in plants is growing rapidly so only a few of the more relevant studies regarding variability will be mentioned here.

Environmental variables and terpene emission Soils Researchers from Italy investigated the effect of soil types on terpene emission in the context of improving the quality of bergamot oil472. Terpene content directly influences the ‘bouquet’ of the oil. They found that a high sand component in the soil resulted in significantly lower linalool and linalyl acetate components but higher limonene and β-pinene content. Higher clay and silt components of the soil resulted in the opposite significant effects with lower limonene and β-pinene and higher linalool and linalyl acetate.

Altitude Researchers from central Europe tracked the composition of terpenes from sage (Salvia officinalis) oil over several growth stages at two altitudes, 300m apart473. When comparing growth stages they found that the amount of the sesquiterpene alcohol viridiflorol increased at flowering time (24% at flowering and 7.8% after flowering). In contrast camphor was greatest after flowering (29%) than during flowering (6.9%). When comparing altitude, camphor after flowering remained the same at (29%) for both sites. Viridiflorol at flowering was reduced to 13 % in the second month (compared to 29%) in the higher site. Camphor production at flowering was higher (12% compared to 6.9%) for the

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higher altitude (400m). Qualitative composition was not affected by altitude but quantitative composition depended upon stage of development.

Temperature The variability of terpene emissions according to temperature is important for relationships to health measures. Traditionally the weather, and usually the temperature, is high on the list of suspected causal agents regarding symptom exacerbation. Its change is often cited anecdotally as causal in increases in respiratory symptoms, instead of the less obvious co variables. Terpene emission is strongly associated with temperature, but not uniformly so. In Eucalyptus plantations in Portugal young leaves emit more isoprene and terpenes than old leaves; both increase emissions with increasing temperature but in different diurnal patterns. Emissions decrease when temperatures approach 40°C474 .

Peñuelas found that oaks and pines exhibit variable responses to temperature and day length and suggest that other factors like water stress are probably influential in monoterpenes emission variation292. Lerdau et al. 475examined spruce and jack pines and concluded that monoterpenes emission is controlled by vapour pressure within plant tissue. Vapour pressure is controlled by ambient air temperature and terpene concentration within plant tissues.

He, Murray and Lyons476 examined the monoterpenes emissions from 15 live Eucalyptus trees and found that emissions varied by one order of magnitude ranging from a ranking of negligible to high emitters. Of the fifteen species only four species (E. globulus, E. rudis, E. saggentii and E. robusta) demonstrated strongly exponential relationships with temperature.

Kim in a 2001 conducted a one year study of loblolly and slash pines in Florida, a climate similar to S.E. Queensland. The majority of monoterpenes emitted by loblolly and slash pine was α-pinene (av 61.5%) and was highest in winter for loblolly pines. Slash pines were more sensitive to environmental temperature

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than loblolly pines. In rain simulations, slash pine emissions decreased while loblolly pine’s increased. In the fall (sic) slash pine emissions were correlated with temperature but loblolly’s were not. Unusually volatile emissions from slash pine in the spring were attributed to emissions from buds on those branches. This underlines the relevance of growth stage to emission patterns. Figure 2.18 shows a pine tree’s floral parts.

Figure 2.18 Pine tree floral structures (Flaxton, S.E. Queensland)

Rainfall, humidity and light Measurement of emitted volatiles from apple trees from just after flowering to fruiting revealed different patterns for different terpenes477. (E.E.)-α-farnesene, β-caryophyllene, limonene and β-pinene were all influenced by temperature. Some terpene emissions depended upon interactions between temperature and humidity (limonene) and β-pinene emissions increased with daily rainfall. They also found that camphene and (E)-2-hexenal and (Z)-3-hexen-1-ol were significantly negatively correlated with rainfall. These authors did not take into account issues of solar radiation, a factor known to influence terpene emissions 424, 478, 479. Some of the variation, that has been attributed to humidity variations, in the study by Vallat, could be due to light variation.

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Yatagai et al. 478in their investigation of emissions from forest trees of different genera found that light and temperature were important in influencing terpene emissions. They investigated several trees from the Cupressaceae family and found leaf oils to be most abundant in summer. Thuja and Abies (fir) showed large differences between summer and winter. The researchers from Japan showed that temperature and light influenced terpene emission. Emission was greater at 20,000 luxes than at 5,000 luxes. The stronger the intensity of light, the higher the emission. They also found that contact with another tree seedling, by rotating and touching each other’s leaves, increased terpene emission.

Using light as a stressor, Maes and Debergh433 showed that emission of sesquiterpenes from tomato plant shoots increases during continuous light but did not affect the production of monoterpenes. He et al.’s experiments with eucalypts showed that while temperature was an important determinant for terpene emission for four varieties, there were no clear relationships for those 476 four and PAR (photosynthetically active radiation) .

Holzinger found that rain or snow events following a long dry period, resulted in bursts of monoterpenes emissions by ponderosa pines in California. Such bursts were not evident when rain events followed recent rainfall480.

While terpenes vary in quality and quantity according to a range of processes related to plants and their environments, the dispersal methods are varied too. Some of the most important should be mentioned.

Dispersal mechanisms – transportation of terpenes That flowers produce perfumes or odours is to state the obvious. Smelling a rose takes in the sweet perfume that largely consists of alcohols and esters481. The range of terpenes that make up chemical profiles of a plant can run into the hundreds. Less obvious are the terpene emissions from plants that do not register via our olfactory sense. Some examples of terpene emissions

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and the variations therein have already been mentioned. Direct emission from plants occurs through floral parts as well as through other plant tissues and related processes. Some of these will be briefly mentioned.

Plant secretory structures The precise mechanism whereby plants emit volatiles is as complex as it is varied, and will not be addressed in depth here. For most of the world’s oil bearing plants volatiles are chiefly emitted through leaf or floral structures. Terpene constituent comparisons for leaves and flowers have been presented already in Table 2.08. For those members of the order Pinophyta though, emission is through needle like structures which replace leaves, and the less obvious floral structures as shown in Figure 2.18. Other plant terpene vectors are trichomes.

Trichomes Some plants have leaves which are covered in fine hairs and these trichomes seen in Figure 2.19 contain oils consisting of terpenes250, 252, 482. Trichomes can even vary in chemical composition according to whether they are sited on the upper or underside of a leaf250 . Trichomes and their existence in many weed species are of interest here, especially since they are a vector for terpenes that is independent of flowering. As a plant dies after flowering, the trichomes separate from the plant as it desiccates in a dry atmosphere, adding to the plant particles in the atmosphere. These terpene rich particles can be inhaled readily and, along with dry leaf fragments, may be responsible for allergic reactions when living plants are not present232. Of particular relevance in Queensland are trichomes associated with weeds like Parthenium hysterophorus, parthenium232, Ageratum houstonianum (bluetop)483,and

Chromolaena odorata, Siam weed484.

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Figure 2.19 ‘Bluetop’, Ageratum houstonianum, showing trichomes

Apart from trichomes, stomata are important structures in terpene distribution.

Stomata Stomata are pores found on the undersides of leaves and are essential for photosynthesis and are shown in Figure 2.20

Figure 2.20 Stomata485 ( natural size 0.055 mm )

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They are the means by which atmospheric carbon dioxide, water and energy are exchanged with a plant. Members of the Myrtaceae family and most of the world’s plants exchange gases and moisture through stomatal openings which may open and close in response to climatic conditions.

Stomatal conductance of water is very sensitive to environmental conditions and stomatal structural arrangements can vary substantially within genera486. Several researchers have now established that stomatal opening also influences the emissions of a range of terpenes487-489. In hot dry times, stomata will remain closed during the day to conserve water, opening in the cool of the afternoon instead490.

In one of the few studies specifically addressing the relationship between terpene emissions and stomata, Niinemets and Reichstein487, demonstrated the importance of foliar structural characteristics to VOC emission. In a complex model employing physico-chemical characteristics they maintain that plant emission rates can be better understood by taking into account the role of stomatal opening and closure in relation to specific chemical qualities of terpenes in plants. The authors demonstrated that volatile emission changes as a function of stomatal opening or closure. They showed that more soluble compounds such as alcohols and carboxylic acids are controlled by stomata. Specifically, they showed that large morning bursts of aldehydes were related to stomatal opening after closure during the night. The cross species references (oaks and aspens) of the same phenomena should be of interest to health researchers. The researchers state that large stomatal sensitivity to linalool and 1, 8-cineole should be expected. Both these compounds are found in large quantities in Australian trees and may reach the atmosphere via leaf oil glands and flowers as well.

Other researchers have also demonstrated that monoterpene release is related to stomatal conductance. Noe et al. investigated Pinus pinea491, especially the

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release of linalool and ocimene. These researchers found that for volatile monoterpenes like trans-β-ocimene, stomatal conductance plays a minor role. For less volatile compounds like linalool, 1, 8-cineole and camphor, the effects of stomatal conductance can be significant. Gabriel et al. investigated Holm oak, Quercus ilex 492 and found that emission of organic acids peaked around noon in line with maximal stomatal opening. They found that the emission of acetic acid was under more stomatal control than that of formic acid perhaps due to higher cuticular permeability for formic acid. Terpenes are also found in other plant tissues.

Plant tissues Floral oils may cover pollen from oil bearing species. The particular chemical blend of those oils forms part of the diet of larvae of some insects493. Oils from flowers either supplement the insect diet of nectar or replace it totally. Oil gathering bees have special structures used as ‘mops’ to gather the oil493. The structure of a Melaleuca flower, also a member of Myrtaceae, can be seen in Figure 2.21. The pollen is found on the anthers depicted in the diagram and seen in the photograph of Melaleuca viridiflora, shown earlier in Figure 2.03, where the yellow pollen is visible on the ends of the red anthers.

Beardsell et al. 494in a thorough investigation of secretory tissue in a Myrtaceae member established the particular structural relationships between oil and floral structures for Thryptomene calycina. They identified the floral nectary as the source of nectar rich in glucose and fructose and suggested that the nectar was secreted through stomates. Further, they identified five other secretory sites which are the glands at the end of the anther connectives. The glands are accessory structures of the anther. Staining revealed that the fluid was hydrophobic and consisted largely of lipids and some phenolics. The structure of the oil bearing apparatus of Thryptomene calycina is shown in Figure 2.22.

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Figure 2.21 Structure of the flower of Melaleuca leucadendra495 (With permission from the illustrator, Murray Fagg)

Figure 2.22 Thryptomene calycina 494 (Source: CSIRO Journals, with permission)

Terpenes may also be released from secretory ducts and transfusion tissues in the needles of Juniperus communis496. The berries also contain reservoirs of oils. The biblical ‘burning bush’ , Dictamnus albus, has ‘spray glands’ in the

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fruit , flowers and upper stems and these are full of monoterpenes. In the gland- free parts of the leaves and lower stems, where oil was stored in sub-epidermal vesicles, sesquiterpenes predominate Kubeczka497 et al. found. Rose sepals and petals were shown to vary in composition of VOCs too. Differences in terpene content of Myrtaceae plants have already been discussed in terms of floristic versus leaf comparisons. Myrtaceae contain many oil glands located in the leaves of many genera and a common odour from oil glands is that of eucalyptol, 1,8-cineole. Especially well known for the fresh cineole-related fragrance are the eucalypts. Documentation of the function and contents of oil glands in Melaleuca alternifolia had been addressed by List et al.498.

Seed capsules , bark, twigs, flowers and leaves all proved a source of terpenes from the manna gum, Eucalyptus viminalis330. This tree ,native to Australia and grown for its hardwood in other countries330, shows a marked range of terpene content of various parts of the tree. Table 2.10 shows the range of values for just two sesquiterpenes, from several originally reported, in the steam distilled oil from various parts of that tree. This again illustrates that taking information about the composition of leaf oils, does not reveal what volatiles the plant may be emitting at flowering. Figure 2.23 shows flowers from Eucalyptus viminalis (the same species as in Table 2.10) and dried buds on twigs from Eucalyptus tereticornis which is native to the Brisbane area.

Plant related particulates Inspection of Table 2.10 reveals that much higher levels of two sesquiterpenes are found in flower buds, seed capsules and dry bark , than in leaves. Given the amount of leaf litter generated by Eucalyptus, the potential for adding these chemicals to the existing supply of plant particles that circulate in the air , is great. Figure 2.24 shows naturally occurring leaf litter from a mixed woodland environment in the highland region of S.E. Queensland, at Flaxton. Pio’s team from Portugal 499 identified substances in respirable particles from a coastal site. They found an abundance of compounds consistent with

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epicuticular plant parts as well as hydrocarbons believed to be of vehicular origin. Some lipids and terpenes were identified as leaf wax components.

Table 2.10 Sesquiterpenes from parts of Eucalyptus viminalis

Sesquiterpene oil yield Plant part (ml / 100g) Aromadendrene Β-Eudesmol Leaves 5.60 1.80 Twigs 9.00 3.80 Capsules 18.10 14.20 Flower buds 13.30 .04 Dry bark 10.70 15.20 Living bark 8.20 14.10

Figure 2.23 Flower buds from Eucalyptus tereticornis (Forest & Kim Starr (USGS)with permission) and flowers from Eucalyptus viminalis (Morwell National Park , Victoria with permission from Ken Harris500).

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Figure 2.24 Leaf litter from mixed woodland: Eucalyptus and rainforest species in highland S.E. Queensland.

Rogge 501 et al.’s work on sources of fine organic aerosol , predates Pio’s work and provides results consistent with that research. Rogge et al. analysed fine particles from plants in a simulation representing natural leaf abrasion by wind. They harvested green and dead leaves from 62 plant species in Los Angeles including broadleaf trees, conifers, palms , shrubs, grasses and cultivated and native plants. Leaves were agitated in clean Teflon bags while a purified airstream flowed through. Fine particles less than two microns shed from the leaves were extracted and analysed by GC/MS. Table 2.11 shows some of the terpenoids found in the leaves. Camphor was the most abundant terpene found in either green or dead leaves.

Particulates from plants can act as vectors for terpenes. Logically, when fungi grow on substrates containing terpenes, there would be some uptake of terpenes and distribution into fungal spores, contributing to the set microscopic particles found in the air.

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Table 2.11 Terpenes in abraded leaf particles

Concentration of leaf abrasion products (µ/g)501 Green leaf fine Dead leaf fine particles particles Eucalyptol 233.9 49.4 Camphor 3569.6 1654.3 Citral 462.0 1.4 Borneol acetate n.d. 567.6 β -ionone 95.4 13.6 β -cadinene 60.5 8.5 Ursolic acid 1778.7 477.7

Fungi Few researchers have examined the chemical content of fungi grown naturally on plant surfaces. When pollen distributions failed to explain asthma patterns in Brisbane , three decades ago502, research attention shifted to fungi, since fungal spores were usually much smaller than pollens and could be inhaled readily502. In respiratory research in Brisbane fungal contributions to inhaled air are largely regarded as having resulted from a saprotrophic relationship hereby a fungus takes nutrients from a dead plant, usually grass503 . Fungi may also function in a mutualistic manner, or parasitically, and may change behaviour according to circumstances. Many Eucalyptus and other species from the Myrtaceae family504 trees support mycorrhizal fungi whereby the relationships are mostly beneficial between plant and fungus. Fungal spores released into the air may be either ascomycetes or basidiomycetes depending on spore formation type504.

Research most relevant to matters here was conducted by Fischer et al. 505who examined 13 airborne fungal species in a compost facility. They found that limonene was produced by 11 species; Aspergillus fumigatus produced trans-β-farnesene in high quantities and camphene, α-pinene and β –

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phelllandrene were found exclusively in this species; many volatiles were found in only a single species; and Penicillium expansum contained several sesquiterpenes including aromadendrene and germacrene. This study demonstrates that many airborne fungi consist, at least partially, of the terpenes that are found in plants. Other contributors are bacteria.

Rösecke506 et al. examined the volatile constituents of wood-rotting basidiomycetes. Linalool, α- pinene, farnesene, γ-cadinene were present in quantities ranging from 6% to 13% of the hydrodistillate measured. Californian researchers507 showed that the effect of redwood essential oils are tolerated differently according to fungal species type and host plant chemical composition, and that fungal growth is variously affected.

The fungal spores associated with Myrtaceae are many : Pisolithus tinctorius is a fungus that readily colonizes Eucalyptus species180; Cytospora chrysosperma and Fusicoccum eucalypti were asymptomatically present in flowers of Eucalyptus globulus508; and specific ecological association has been established for Eucalyptus camaldulensis509, Eucalyptus tereticornis 510 and Cryptococcus neoformans var. gattii. The latter has been identified as causal in human Cryptococcus disease. Every year more associations between Australian plants and fungi are established511-517 and as plantation growth of Myrtaceae trees increases around the world, new associations are explored for reasons usually associated with better plantation management.

Fungi are able to transform terpenes and the abundant terpene limonene is readily transformed by fungi518. Duetz et al. 518reviewed work regarding limonene transformation and noted common limonene-derived products including : perillyl alcohol, cis- and trans- carveol, carvone , perilla aldehyde and acid, limonene oxide and iso-piperitenone. Flowers of Australian plants readily produce limonene in substantial quantities and the presence of numerous species of fungi here invites a new area of enquiry: what terpenes are being

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transported from plants to fungal spores that can be inhaled? Limonene is just one component that is potentially transformed and/or vectored by fungi. Another of interest here is pinene.

In the 1960s Bhattacharyya et al.519 reported on the microbiological transformation of α-pinene by Aspergillus niger. Co-oxidations with this fungus yielded d-cis-verbenol, d-verbenone and d-trans-sobreol. Building upon those findings, Narushima et al.520 examined the transformation of α-pinene by another fungus, Pseudomonas maltophilia. The fungus accumulated limonene, borneol, camphor, perillic acid, and 2-(4-methyl-3-cyclohexenylidene) propionic acid from α-pinene in a culture broth. In separate oxygen-uptake tests, the team demonstrated that the strain readily oxidized β-pinene, limonene, α- phellandrene, 1-8cineole, α-terpineol, borneol, camphor, perillaldehyde, myrtanol, and myrtenol, as well as α-pinene. Importantly, P maltophilia would not oxidize camphene and fenchyl alcohol. Since the fungus strain did not have metabolic pathways of camphene and fenchyl alcohol in oxygen uptake tests, the borneol and camphor produced by P. maltophilia from the oxidation of α- pinene implied that camphor could be an intermediate of the degradation pathway of borneol. This fits with Newman’s 258observation that camphor can be obtained industrially this way: pinene → camphene →isoborneol →camphor.

Sesquiterpenes are of special interest also since they feature in many plants associated with respiratory symptoms, and so many Australian trees contain them.

Abraham et al. 521examined the biotransformation of bicyclic and tricyclic sesquiterpenes in two strains of fungus, Chaetomium cochlioides and Diplodia gossypina. Biotransformation efficiency and newly formed products were compared. This team came to two important conclusions: first, that oxygenated sesquiterpenes were more biotransformable than hydrocarbons; and second that the similarity between the two strains of fungus was closer for the same substrate, than for two different substrates with the same fungus.

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In indoor environments the dependence upon species type and growth media was observed by Sunesson et al.522. Two fungal species found indoors in Sweden, Penicillium commune and Paecilomyces variotii, were grown on pine wood and gypsum board plus mineral wool. Most of the compounds produced by the fungi were alcohols, ketones, ethers and terpenoid compounds. Although there were some commonly produced compounds, metabolites varied to a big extent with the media and species. For Paecilomyces variotii, on the gypsum board/mineral wool cultures, unidentified monoterpenes and sesquiterpenes were produced although no metabolites were produced on either this or the pine wood media. For Penicillium commune geosmin was the main product for cultivation on the gypsum boards while alcohols were the main product for growth on the pine wood. These observations are consistent with the dictum ‘we are what we eat’ and add another layer of complexity to understanding the effects of bioaerosols on respiratory health.

Just as fungi can be vectors for terpenes, so too are insects. Terpenes are also found in the insects and in their excrement – this natural occurrence is found in unnatural proportions in the urban environment when domestic living conditions become inviting. The existence of chemicals in insects that are also found in plants , supports the possibility of ‘priming’523 a sensitivity, perhaps in two-way directions. Perhaps plant chemicals can ‘prime’ reactions to insect faeces and vice-versa.

Domestic insect allergens: cockroaches and dust mites Bornyl acetate is a pheromone of the cockroach430. Many pheromones are to be found in the hindguts of cockroaches524. Serum IgE from patients with cockroach sensitivity has been found to bind to the gastrointestinal epithelium and contents of the intestinal tract of cockroaches152. Perhaps some relationship exists between the high levels of cockroach allergy in some parts of the world and the emissions of proximate trees.

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Bornyl acetate is produced by several species of pine trees291, 396, firs (Abies), spruces (Picea) and larch (Larix)396. Juniper (Juniperus communis) also contains α-pinene and bornyl acetate. It is also found in many Australian trees272, 305, 429, 525-528. It may be that emissions from these trees could result in ingestion of bornyl acetate directly, and secondarily because bornyl acetate it is a metabolic product of α-pinene529, which is emitted from the same trees.

Citral530, a lemon scented aldehyde, is found in the secretions from glands of house dust mites530. It is also an alarm pheromone for oribatid mites531. Dust mites, for decades now, have been recognized as potent and widespread respiratory allergens532. Both Dermatophagoides pteronyssinus and D.farinae, contain citral530.

Some of the popular plants272, 305, 533 in Australian gardens belong to Myrtaceae and contain large quantities of citral as mentioned earlier in 2.2.1. Emission of this chemical by native trees could be related to the high incidence of allergy to dust mites in Australia. As with bornyl acetate, a priming mechanism could be part of the explanation for high insect related allergy in Australia. Other vectors of terpenes are related to anthropogenic influences.

Apart form ‘natural’ exposures to terpenes, human influence has provided more opportunities for contact.

2.2.4.2 Anthropogenic terpene exposures By milling wood, household practices like oiling furniture with essential oils, aromatherapy, and using perfumed cleansers, humans have added a source of terpene exposure to their surroundings, which would not otherwise exist in the same concentrations naturally. Additionally, in the last few decades, especially, inhabitants of more economically developed countries have increased the

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environmental load of terpenes in their surrounds by means only limited by the collective imagination of the commercial world.

Exposure to wood dust, resins and fumes from sawmill environments have been carefully measured. Malmberg 534 examined the bronchial response of sawyers compared to trimmers and other workers in a lumber mill and confirmed that sawyers had higher bronchial responsiveness than the others. Teschke535 et al. measured exposure to terpenes, resin acids and dust across various species. Spruce and pine produced higher exposures than alpine fir. This study found that sawyers had a higher exposure to terpenes than other workers placed elsewhere in the yard. The importance of measuring exposure to wood chemicals as well as wood dust particles was established in a study by Demers et al.536. Humans are increasingly making use of these concentrated substances from plants in the form of essential oils.

Essential oils For centuries, Asian and Middle Eastern cultures have burned incense which is made from storax ( resin from Styrax) and enhanced by various other substances like sandalwood and cedarwood537. Respiratory symptoms are increasingly associated with practices like incense burning538-541 although some research has proved contradictory542 .

For reasons of lifestyle rather than culture, the popularity of air fresheners provides a new source of exposure to terpenes at levels and consistency not experienced in nature. Nature provides a varying profile of terpene exposure related to climate and stage of development of a plant. An electrically powered emitter of air freshener provides a steady flow of terpenes all day, every day. That consistency is used as a selling point in advertisements on television. The negative health effects have already been identified543-545 and especially impact respiratory processes.

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Since ancient time oils have been used for cosmetics and to enhance/ hide human odours. In the last few decades though, ‘Western’ countries have embraced these compounds enthusiastically. Soap has changed from one basic type to hundreds of perfumed varieties. Houses are cleaned with items ranging from fresh to sweet odours, all obtained from essential oils and/or their components. The effects of some of these constituents will be discussed in section 2.2.5.6. The potential for components of essential oils to form secondary aerosols is an important process of interest here.

Secondary Aerosols The formation of secondary aerosols in the air can affect respiratory health. Secondary organic aerosol (SOA) forms in the atmosphere when volatile parent compounds are oxidized to form low volatility products that condense to yield organic particulate matter546. When there is intense photochemical smog, 40% to 80% of the particulate organic carbon can be secondary in origin546. SOA accumulates in fine particles (less than 2.2µm) where it can affect regional air quality and negatively influence respiratory health. These aerosols can also affect world weather by scattering and absorbing radiation and by influencing cloud properties through their role as cloud condensation nuclei547.

Rules for determining common names of the many oxidation products for terpenes were set down by Larsen et al. 548 in recognition that the formal IUPAC names are long and complex. Larsen et al. list common oxidation products of several terpenes which are listed and used as examples to illustrate the naming protocols. Those common names will be used in most cases in this thesis.

Modelling estimates Much of the research into the oxidation products of terpenes has involved the most ubiquitous of those that occur naturally, notably the pinenes and limonene549-553. It should be noted that modelling other oxidation products has

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resulted in understandings that have revealed the complexities involved in predicting SOA formation.

Research in this area covers the most well known reactive intermediates in the atmosphere: the hydroxyl radical (OH), ozone (O3) and the nitrate radical (NO3), two of which were mentioned earlier in this thesis in section 2.1.4.1. Hydroxyl radicals reach their peak during the day as solar radiation is critical for its formation. Ozone is a product of photochemical air pollution (smog), as mentioned before, but is also found in trace amounts in unpolluted air and occurs at higher levels in summer. Highest concentrations of the nitrate radical 97 occur at night because NO3 is rapidly broken down by visible light . It is formed by the oxidation of NO2 and ozone, and for reasons of chemistry, is found in drier conditions. These reactive compounds are continually being formed and consumed in the troposphere, the atmospheric zone, closest to the earth97.

Given that mathematical modelling is increasingly utilized in respiratory research, some consideration of its reliability in using terpenes in modelling for SOA estimation is required. The use of mathematical models to predict the formation of SOAs has resulted in inaccuracies. The predicted formation of ketones and aldehydes from the base terpenes of β-pinene, sabinene and δ-3- carene has been modelled by Pankow et al.546. The models used under - predicted the actual formation of ketones and aldehydes from all three base terpenes546 . The researchers also modelled the formation of many carboxylic acids for each base compound.

Hallquist 549 showed that the application of mathematical models to the oxidation of terpenes by NO3 was inappropriate due to the complexity of reactions. They found that aerosol yields depended upon the amount of terpene reacted but that carbonyl and nitrate yields did not depend upon the amount of terpene converted. O’Dowd et al.554, in chamber experiments with butanol, have

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shown that there are complex feedback processes in place in the formation of aerosols from ozone and terpenes. They warn that such uncertainties make predicting climate change very difficult. Similarly, making predictions regarding respiratory health would be affected by these factors.

The 2006 investigation555 by Ren et. al. into hydroxyl (OH) and hydroperoxy radicals resulted in a satisfactory estimation for daytime OH but significant under-prediction of HO2 in the day and night. For NO above a few ppbv, the measured HO2/OH ratios grew increasingly larger than those modelled. The underestimation of HO2 that is NO dependent was suggested to be an explanation. Another source of variation was suggested to be unsaturated reactive hydrocarbons which can react with O3 and produce HOx.

Further complications in estimating oxidation products of terpenes are experienced when water is added to the equation.

Humidity and rain influences

Seinfeld et al. 556demonstrated that changes in relative humidity have very different and particular effects on different base terpenes in the formation of secondary aerosols. The more hydrophilic a compound, the more increasing relative humidity will favour its condensation into the SOA phase. Spittler557 et at in 2006 found that pinonaldehyde and endolim were the major reaction products between α-pinene and limonene and NO3. They found that water vapour strongly decreased the aerosol mass formed by NO3 and α-pinene, and that aerosol masses formed by limonene and α-pinene reflect different processes, despite the fact that both are monoterpenes. In 2006 Jonsson, Hallquist and Ljungström 552 investigated the effect of water on the initial SOA formation from gas-phase ozonolysis of limonene, δ-3-carene and α-pinene. This study demonstrated a clear dependence of oxidation of terpenes upon relative humidity that resulted in increased number and mass for all three terpenes in increased humidity conditions. The exact relationship of water to the formation of organic acids and SOAs is so far unclear. The authors suggest

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specific investigations at selected humidities to determine the mechanism for ozonolysis of monoterpenes.

These different rates of aerosol formation will further complicate understanding of the impact of different geographic environments with different blends of base compounds in the ambient air. Understanding of such complexities are not yet evident in panel studies investigating respiratory health and air pollutants. Despite the shortcomings of measurement issues, the formation of secondary aerosols deserves more attention here.

Peroxide formation In a 2005 study, Docherty et al. investigated the oxidation of some monoterpenes in a chamber environment. They concluded that a large fraction of the SOA formed from reactions of monoterpenes with ozone is composed of organic peroxides547. This team investigated β-pinene, α-pinene, δ-3-carene and sabinene – peroxides of this group have been associated with turpentine allergy, especially that of δ-3-carene 558. Karlberg et al. suggest that the susceptibility of a chemical to air oxidation needs to be considered when determining what allergens are formed by industrial products559. A considerable body of work558-561 has resulted in greater understanding of the effects of oxidation of monoterpenes on individuals, especially limonene. These effects and skin sensitization by peroxides will be examined in section 2.2.6. Such effects occurring on a global scale, because of ozone influences, invite more investigation.

Oxidation products of common terpenes from plants Later experiments investigating the oxidation of some of the same terpenes as Pankow et al.546, 556, also indicated oxidation products for limonene and alpha pinene. Table 2.12 summarizes some of the important oxidation products from terpenes commonly occurring in nature, especially in oilseed rape crops 290

(canola) and pine forests, Australian native trees including eucalypts303, leptospermums305, melaleucas 562 and callistemons 340, 563 and northern

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hemisphere forests 332, 343 and the pepper trees of the Caribbean295. Differences in vegetative emissions of particular geographic regions, and relationships to respiratory mortality patterns will be addressed in Chapter 6.

SOA formation: oxidation processes Ozone Fan et al. 564in 2003 reported on their experiments which have shown the importance of limonene and pinene in oxidized mixtures of terpenes. They exposed a mixture of 21 VOCs and 23 VOCs to ozone. The 23 VOC mix included d-limonene and α-pinene and the 21 VOC did not. The researchers also examined a mix of ozone and d-limonene and α-pinene only, and a mix of only d-limonene and α-pinene. Chemical products are shown in Table 2.13.

Table 2.12 Oxidation products from terpenes

Base terpene Acid formed Aldehyde/ketone formed α-pinene pinic acid550, 565 pinonaldehyde550, 557, pinonic acid550, 565 566 norpinonic acid 565 10-hydroxy 565 Pinalic-4-acid565 pinonaldehyde 550, 566 4-oxopinonic acid565 acetone 10-hydroxypinonic acid565

β-pinene pinic acid546, 550 nopinone550 pinonic acid546, 550 hydroxy pina ketones546 hydroxy pinonic acid546 acetone 566 δ-3-carene 3-caric acid546, 550 Hydroxy 546 3-caronic acid546, 550 caronaldehyde 546 pinic acid546 caronaldehyde sabinene sabinic acid546, 550 sabina ketones546 pinic acid546, 550 hydroxysabina ketones546 limonene keto-limononic acid567 keto-limonaldehyde567 limonic acid 567 limona ketone550 endolim557

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Table 2.13 Terpene/ozone mixes and SOA formation: Fan et al.564.

VOCs O3 + 23 VOCs O3 + 21 VOCs O3 + only Including Including (no d-limonene d-limonene d-limonene d-limonene & no α-pinene) & α-pinene & α-pinene & α-pinene formaldehyde formaldehyde formaldehyde formaldehyde acetaldehyde acetaldehyde acetaldehyde acetaldehyde propionaldehyde propionaldehyde propionaldehyde propionaldehyde glyoxal glyoxal glyoxal p-tolualdehyde p-tolualdehyde benzaldehyde benzaldehyde methyl gyloxal methyl gyloxal formic acid formic acid acetic acid acetic acid SOAs SOAs

These experiments indicate that the drivers of the SOA formation were the d- limonene and the α-pinene. Note that the formation of the aldehyde, glyoxal, is related to the inclusion of ozone in the reaction, but is not related to the inclusion of d-limonene and the α-pinene. There may be other terpenes that are capable of driving the formation of SOAs. Given the relevance of common terpenes to the formation of secondary aerosols, there is need to explore the nature of terpenes associated with an irritant or allergenic response.

Koch et al. 568established that a number of organic acids and secondary aerosol could be from the ozonolysis of terpenes without using seed aerosol. In the ozonolysis of α-pinene, β-pinene, sabinene, δ-3- carene and limonene, the filter samples of the aerosols produced were identified as methyl ester. The corresponding dicarboxylic acids of the terpenes listed were found to be the main products of the organic acid fraction.

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NOX Splittler et al. in 2006 conducted experiments with terpenes and nitrate radicals.

Using a photo reactor facility, they identified the major NO3 oxidation reaction products of α-pinene and limonene as pinonaldehyde and endolim , respectively557. The oxidation reactions led to the formation of secondary aerosols under several experimental conditions. Water vapour was found to strongly decrease the aerosol mass for NO3 and α-pinene. The researchers postulated that either the reaction mechanism involving water favours the formation of more volatile compounds, or water uptake results in an increased presence of low-volatile products in the gas phase. They concluded that the formation of new particles from monoterpenes after heavy rainfalls would be via ozonolysis.

HOx Librando and Tringali in 2005550 investigated the reaction products of OH radicals with α-pinene, β-pinene, sabinene, δ-3- carene and limonene. Pinonaldehyde was identified as a SOA from α-pinene degradation; nopinone from β-pinene, and limona ketone from limonene. As well, the following acids were identified as secondary aerosol products: pinic, pinonic, limoninic, 3-caric, 3-caronic, and sabinic.

Wisthaler demonstrated the formation of acetone when α-pinene and β-pinene are oxidised by the OH radical566. They also found secondary acetone formation from other primary oxidation products and concluded that the global acetone source relied significantly upon the oxidation of monoterpenes.

Oxidation of linalool Little research is evident regarding the oxidation of the common floral alcohol linalool. This insect attractant is produced in great quantities by some types of flowers, particularly some essential oil producing varieties like Melaleucas. Shu et al.569 in 1997 investigated the gas-phase reactions of linalool with OH and

NO3 radicals and O3. All three oxidants resulted in the formation of acetone. As well, a number of other ketones and aldehydes were formed. One of the

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oxidation products from the OH reaction was 6-methyl-5-hepten-e-one. This ketone has been identified 570 as being a germination stimulant for uredospores of Puccinia graminis var. tritici and other rust fungi. Such relationships potentially have meaning for patterns of respiratory health.

Oxidation products of terpenes add to the number of compounds in the atmosphere that have their origins in plants. Such compounds are associated with a vast range of effects some of which have been labelled irritant and some allergenic. The next section addresses some of the related issues.

2.2.5 Plant related irritants and allergens

2.2.5.1 Terpene reaction type: Irritant or allergen?

For more than fifty years the sensitizing property of δ-3-carene 571-573 has been recognized. In more recent decades this bicyclic monoterpene has been replaced in paint thinners by less sensitizing petroleum products with the consequent reduction of ‘turpentine allergy’ being the result5. Turpentine allergy is a good example of all that is confusing about terpenes and ‘allergy’. In their 1996 paper, Lear et al. described the re-emergence of turpentine allergy in the pottery industry. The problem remains: when dealing with substances that usually occur in nature as blends of chemicals, which substances are the ones likely to be causing any sensitization and what is the nature of that sensitization : irritant or allergic?

Turpentine is a yellow gum extracted by wounding pine trees. Typically it consists mainly of α-pinene, β-pinene and δ-3-carene however ,depending upon the source, it may also consist of dipentene (limonene), and/or camphene5 and/or a tri-cyclic sesquiterpene longifolene574.

By the 1990s research into terpenes and their sensitizing effects had changed direction and focussed increasingly upon the oxidation products of terpenes instead of singular chemicals like δ-3-carene in the turpentine. The likely

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problematic components were shown to be the peroxides of base chemicals, rather than the chemicals themselves: in the case of turpentine the attention was directed towards the peroxides of δ-3-carene. This interest is reflected in the research summarized in the next section dealing with oxidation of terpenes. Table 2.12 shows some of the oxidation products of δ-3-carene.

Determining what particular chemical has the capacity to sensitize becomes a problem of first separating the precise fractions of a substance, then measuring responses to those fractions and their peroxides. The argument re the exact nature of the irritant or allergic reaction is one that invites a range of views and needs further explanation.

2.2.5.2 Allergens and irritants explained The question of whether terpenes cause allergic or irritant reactions is a complex one. What is known is that many terpenes cause reactions that range from mild to dramatic. Somatic reactions range from olfactory discomfort to severe respiratory distress. To know whether a reaction is irritant or allergic can be difficult and is not always necessary or useful. To refer to reactions that may be able to be described as either an allergen or an irritant, on the basis of the apparent physical human response, the original term ‘allertant’ will be used from now on for simplicity.

Before that general descriptor is adopted here, some understanding of the difference between allergy and irritancy is useful.

2.2.5.3 Allergy acquisition The process of acquiring an allergy is a two step process. Sensitization, the first step, occurs upon exposure to an allergen. During this process allergen antigens that have been injected, inhaled, digested or otherwise absorbed via the skin are filtered through the bloodstream to the lymph nodes where the T and B cells recognise the antigen as foreign. The interaction with lymphocytes leads to the production of IgE in atopic individuals. Memory B-cells produce more specific IgE antibody when stimulated by the same allergen575. During the

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second stage, the IgE antibody adheres to blood basophils or to mast cells located in mucosal surfaces which may be in the respiratory or gastrointestinal system. An individual is then said to be ‘sensitized’ to an allergen. Sensitization in itself does not produce allergic symptoms – this occurs the next time that the individual contacts the same allergen575.

Allergens are most often regarded as being pollens or fungal spores, animal danders or ingested substances. This thesis is concerned with another type of ‘allergen’, the hapten.

Haptens A hapten is a chemical that acts as an allergen by binding to protein, usually on the skin576. This identified binding site reflects the history of hapten investigation rather than the actual range of biological binding sites-most research regarding haptens has been about ACD (allergic contact dermatitis) and skin protein haptenation by chemicals and natural substances. The reactions of chemicals with proteins form strong bonds so that a non-self antigen is formed576.

For protein haptenation to occur a chemical must be electron deficient (electrophilic). Chemicals that are electrophilic and of interest here have a polarized bond like aldehydes, ketones and amides576. Some compounds must undergo chemical modification to become haptens.

Pro-haptens Pro-haptens that must undergo modification in order to become haptens may be from several structural groups. Reactions of chemicals with light can transform a substance from a pro-hapten into a hapten that can stimulate the immune system577.

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Pro-haptens may be hydrocarbons that are oxidized to hydroperoxides like δ-3- carene , limonene, α-pinene and terpinene577. They may also be alcohols that are oxidized to aldehydes or ketones. Examples are benzyl alcohol and terpineol577. Allergic contact dermatitis from poison ivy is thought to be due to the oxidation of urushiol577. The similar process occurs with contact allergy due to colophony578.

Other hapten reactions of relevance include esters that are undergoing hydrolysis and form their corresponding alcohol and acid. Amines too are common haptens that occur especially in relationship to contact with resins, leather processing chemicals, and creams and ointments, the components of which all emanate from plants. Such reactions are very commonly causal in allergic contact dermatitis577.

Allergic contact dermatis Warshaw and Zug 579 makes the distinction between those individuals who experience an immediate IgE mediated hypersensitivity reaction such as allergic rhinitis and those who experience a delayed response from a cell- mediated hypersensitivity as in ACD. They attribute the former to hydrophilic proteinaceous pollen allergens and the latter to oil-soluble oleoresins found in the trichomes of many weed plants. The hypersensitivity due to these chemicals is delayed. Figure 2.25 shows a delayed sensitivity response to exposure to washing up liquid. The response came 24 hours after the 12 year old female child hand washed dishes for the second time in the lemon-scented popular brand liquid. Previous exposure had not occurred due to usual usage of a dishwasher, which had broken down causing the child to be relegated to the task. Several weeks elapsed before the skin returned to normal despite repeated application of cortisone based cream.

Wakem and Gaspari580 make the distinction between irritant contact dermatitis (ICD) and allergic contact dermatitis (ACD). ICD is described as a non –

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immunological, local inflammatory reaction characterized by erythema, oedema or corrosion following a single or repeated exposure to a chemical. ACD is described as cell-mediated type IV hypersensitivity reaction requiring an atopic genetic predisposition and a chemical capable of trans-epidermal absorption. In photo-contact dermatis, both types require activation by ultraviolet light580. The boundaries of what substances are capable of causing sensitivities become more blurred when substances known as ‘irritants’ are added to the mix.

2.2.5.4 Irritants Nielson in 1991 wrote extensively about airborne chemicals as sensory irritants. He stated that two mechanisms may operate whereby sensory irritants are activated: first the chemical can be physically absorbed into a receptor site as is the case for ketones, alcohols and ethers, and second, substances may become irritants by chemical reaction. Formaldehyde and acrolein react with a thiol group in the receptor. Similarly, oxidising agents like ozone react with the thiol group and activate a receptor581. Since then, more recent research has yielded more detailed understanding of the mechanisms involved in irritant reactions.

Figure 2.25 Delayed sensitivity response to washing-up liquid

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In 2006 an accepted definition of ‘irritant’ is ‘a localized pathophysiological response to a chemical involving local redness, swelling pruritus or pain582. Respiratory tract and eye irritation can also involve a direct chemosensory effect when a chemical interacts with nerve endings, nervus trigeminus582.

Reporting on the work of Alarie583 from the 1970s, Arts et al.582 recount the classifications of chemicals that are capable of stimulating nerve endings in the respiratory tract: • A sensory irritant substance is inhaled via the nose and stimulates trigeminal nerve endings that evoke a burning sensation and perhaps an accompanying cough. Olfactory receptors respond to lower concentrations of chemicals and detection and irritation are very different. Acetone can be detected at 20 to 400ppm but actual irritation of eyes occurs at 10,000 to 40,000ppm582. • A pulmonary irritant substance is inhaled and stimulates nerve endings within the lung, increasing respiratory rate and decreasing tidal volume, resulting in rapid shallow breathing. • A bronchoconstrictor when inhaled induces resistance to airflow within the airways of the lung via action on airway smooth muscle or by neural reflex. • A respiratory irritant when inhaled can act as a sensory irritant, bronchoconstrictor and pulmonary irritant. These chemicals are capable of all three actions. Highly reactive substances like formaldehyde will cause upper airway effects while less water soluble compounds like ozone affect the lower respiratory tract.

A different way of understanding irritant groups has been proposed by Wolkoff in a 2006 article regarding indoor air. He outlines four groups of organic compounds in indoor air (OCIA)584:

• Chemically stable (non-reactive) organic compounds eg octane, toluene and butanol.

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• Chemically reactive organic compounds like alkenes eg styrene and limonene. Organic compounds that form chemical bonds to receptor sites in the mucous membranes and are biologically reactive eg formaldehyde and acrolein. • Organic compounds with known toxic properties eg fungicides. Effects of this group develop as a function of exposure and duration584.

In this thesis the focus is on the effects of terpene compounds per se, as well as the effects of the oxidation of terpenes and their oxidation products when combined with ozone and nitrogen oxides.

2.2.5.6 Terpene allertants from commercial products The recorded information about the allertant effects of terpenes varies in quality and reliability. There is a great deal of anecdotal information with little or no substantiation, and a paucity of studies investigating the effects of pure terpene fractions on human respiratory symptoms. Some of the most informative of the work that has been done in the last decade has involved patch testing or studies into specific biological processes on animals. Hundreds of such studies are reference in Polya’s excellent book about biochemical targets of terpenes262. There are other informative books available 396, 585 which give an extensive overview of the effects of terpenes. Some of the information in these books is well supported by referenced fact; some is supported by references that lack scientific rigour; and some is anecdotal. Anton de Groot and Peter Frosch’s clinical review of adverse reactions to fragrances 265 provides extensive lists of the most commonly occurring fragrance allergens in the USA and Europe. Zhou et al.586 provide a sound summary of reactive metabolites of many herbal products.

Perusal of the literature regarding the effects of some terpenes and essential oils reveals one overriding conclusion: that there are no consistent patterns to be discerned when essential oils are involved. Silva et al. 587sums up the

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situation well their paper regarding the anti-inflammatory effects of various essential oils of Eucalyptus: “No consistent results were observed…. reflecting the complex nature of the oil extracts and/or the assay systems used.” Some of the quality research that has been undertaken has been done in pursuit of understanding regarding the action of the commercially popular Australian tea tree oil, and New Zealand’s manuka and kanuka oils and eucalyptus oil which was first produced in Australia but China now sets its world price588.

Tea tree, manuka and kanuka oils Investigators researching the actions of three Australasian Myrtaceae species, tea tree (Melaleuca alternifolia), manuka (Leptospermum scoparium) and kanuka (Kunzea ericoides) showed that the oils varied in their ability to alter contractual response in guinea-pig smooth muscle. Similarly, antibacterial ability against a range of bacteria varied. Lis–Balchin, Deans and Hart 589 showed the effects of different constituents of the oils: α-terpineol and terpinin-4-ol produced strong spasmolysis; α- and γ- terpinenes produced initial spasmogenic action followed by a spasmolytic action; 1,8- cineole produced a distinct rise in tone rather than contraction, unlike any other component; β- pinene and (-)α-pinene produced an initial contraction followed by spasmolysis; the (+)α-pinene produced only a spasmogenic response. The authors comment that the variation within the Myrtaceae family results in many different essential compositions with differing bioactivities.

Eucalyptus oil Another example of complexity involved in labelling essential oils as ‘helpful’ or ‘hurtful’ can be found in a cursory examination of the effects of eucalyptus oil. This oil is associated with positive emotion in Australia particularly, where it reaches almost iconic status. Such generalized product loyalty has perhaps prevented earlier investigation into its action. Many of today’s Australian adults weathered childhood cold viruses with handkerchiefs sporting a generous

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amount of eucalyptus oil. The fresh odour makes the oil a popular choice, due to the eucalyptol (1, 8-cineole) component, wherever freshness is required. The oil is used widely in household products, especially disinfectants. It is also promoted as a desirable additive to steam vaporizers590 and as an additive in washing water591, 592 to reduce dust mites, despite questionable effectiveness155.

Juergens et al.593 established that the largest quantitative component in eucalyptus oil, 1, 8-cineole could function as an anti-inflammatory in bronchial asthma. Previously in 1997, Riechelmann et al.50 established that the effects of inhaling oil mixes were detrimental to the human respiratory cells. These researchers examined the response of nasal cilia to three oils. Effective cilial beat is necessary for clearance of excessive mucus, and this can be impaired by exposure to toxics50. They examined the effects of a menthol, eucalyptus and pine oil mix, eucalyptus oil and pine needle oil. They found a dose dependent reduction of cilial beat, for all conditions. Of the three oil types, the eucalyptus resulted in the lowest concentration required to suppress ciliary beat. Measures distinguished the results of the three oil groups from a control group in which air was inhaled and ciliary beat measured. The researchers concluded that therapeutic range is limited for use of these oils, and that overdose can impair ciliary function of respiratory cells. Dose dependent application of eucalyptus and other oils was also shown by Neisen594 to affect percutaneous penetration of chemicals.

In 2005, Keinan595 investigated ozone scavenging effects of limonene and eucalyptol on sensitized rats. They found that limonene inhalation significantly prevented bronchial obstruction while eucalyptol did not cause any change of bronchoconstriction of sensitized rats. Allertant responses in association with fragrances and food additive are numerous researchers have been involved pursuing understanding of the

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relationships for many decades. Confusion regarding ‘irritant’ or ‘allergen ‘status is evident in the literature.

Fragrances and flavour additives Essential oils have long been part of life in Asia596 and the Middle East597 especially in the form of incense and perfumed oils. They are becoming increasingly popular in the North America, Europe and Australasia, as hundreds of websites advertising essential oil products attest. Allergenic fragrance chemicals are everywhere in our environment 598 from the laundry to the bedroom. The fresh scent of lemon has been used as a marketing lure since the 1960s in products like ‘lemon Fab’ to wash clothing. Today the oils are often more discernible and whether that is due to enhancers 599 added to the oil or oil components to increase olfactory appeal, or simply increased quantity, is not clear. Olfactory detection is not a reliable way of determining chemical content, and distinction between odours and irritants is likely to depend upon previous exposure, intensity of the odour and personal biases influenced by experience and knowledge582. Olfactory acuity also varies according to degree of allergy and nasal inflammation600.

The collection of varied reports regarding negative effects of terpenes and aromatic compounds from plants is similarly varied and some of that is summarized in Table 2.14. Aromatic compounds are also important and so frequently occur together with terpenes in volatile emissions from plants, and often occur together in additives and preservative mixes, as in soft drinks.

The body of anecdotal reports regarding the effects of additives is substantial and there are many hundreds of websites devoted to the subject. Regulation of food additives is problematic, within and between countries. A substance regulated by law in one country may be used freely in another and the inconsistency is clear in international guides601.

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Some terpenes that are used as food additives are also used industrially or in the home in cleansers. Limonene is one such chemical which, in Australia, is found in lollies, non-alcoholic drinks, chewing-gum, ice-cream, baked goods and puddings601. It is also used as a degreaser, hand cleaner, car wash product, furniture polish, paint stripper, dish washing liquid, laundry powder, air freshener, fabric care agent, and in cosmetics and household cleaners602 . Amounts in flavour ingredients range from .01% to 45 % limonene in the product while industrial usage reveals up to 70% pure limonene usage as paint stripper602.

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Table 2.14 Adverse effects of additives and terpenes

Substance Response Reference Respiratory allergy symptoms (upper and Acyclic terpenes, lower) more in Muslim 597 perfumes houses than non- Muslim Affects behaviour of 3 Benzoates, food yr old adversely- only 603 colouring detected by parents not staff of clinic Herbs: coriander, caraway, fennel, celery; Frequent sensitization less to garlic, onion, as detected in prick 604 chives; rare to paprika, tests saffron Additives induce cell- Sodium benzoate mediated immune 605 ( common preservative) responses for those with urticaria Link to irritability, Calcium propionate in restlessness, sleep 606 bread disturbance & inattention in children Benzoic acid, Propionic Relief of urticaria from acid, colourings, citric 607 additive free diet acid Skin test positive – Aldehydes used as indicating capability of 608 colourings allergy induction Children with atopic Additives - citric acid, skin conditions- colourings, 38 increased positive preservatives reaction Benzoic acid, sorbic acid, cinnamic acid, Non-immunological 41 and many essential contact urticaria oils. Positive responses Dyes, benzoates, after exclusion from 609 aspirin diet- multiple measures Benzoic acid, Positive patch tests in 610 cinnamaldehyde children Rubber, fragrances ‘Angry back’ syndrome 611

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Additives, aspirin and lethal old favourites Since Feingold in the 1970s there has been a lobby against food colouring, flavouring and preservative in foods on the basis that it affects behaviour in children612. Not all research is confirmatory of the effects of additive on behaviour in children – in fact most does not. Dietary compliance issues and need for accurate diagnosis are limitations to this type of research. A double blind assessment of 19 children showed that only three demonstrated intolerance to allergies despite all 19 being thought be intolerant613. Several authors believe that psychological factors are important in matters concerning reactions to food and food additives41. Some discount the role of food chemicals in food as contributory to increased atopic disease34. More consistency seems evident in cutaneous symptom results for aspirin sensitivity and other contact with particular natural products.

Aspirin sensitivity is well known 614, 615 but not common compared to allergy to dyes or benzoic acid 609 - aspirin consists of a carboxylic acid group and ester group. Sensitivity to this product is related to a number of symptoms including, asthma and urticaria614, 616, 617. These carboxylic acids are of special interest here because of their relationship to flowering plants, and the volatiles of which they frequently are part.

The medical literature contains numerous reports of single incidents of toxicity as a result of terpene ingestion or application. They involve mostly eucalyptus oil6, 618 and camphor 619-621 which are most frequently ingested by small children. The ready availability of these substances in the home is the likely reason for their disproportionate representation when compared to toxicity incidents with other terpenes. These substances have potentially lethal effects when ingested. Less lethal, but perhaps more pervasive effects, have been investigated in the last ten years especially.

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Focussed research into terpenes reactions In the last decade substantial research effort has been directed towards understanding the problematic components of some terpenes and other preservatives and food additives, and lifestyle products. Systematic research into lifestyle products has revealed that the most commonly found fragrances in cosmetics and toiletries in the USA are linalool, phenylethyl alcohol, linalyl acetate and benzyl acetate, in that order. In the Netherlands the top four are linalool, phenylethyl alcohol, benzyl acetate and limonene265. Fragrance mix is a mixture of terpenes used to determine fragrance allergy. Constituents of the mix responsible for the most adverse reactions in Europe were reported as isoeugenol, eugenol and cinnamic aldehyde265. The authors also include a list of 80 essential oils associated with adverse reactions.

Research into the specific components of plant chemicals has been limited. The ones of prime interest here are those in which particular components of essential oils have been investigated. Such examinations began with research into the sensitizing effects of tea tree oil, the oil from Melaleuca alternifolia.

Sensitizers in tea tree oil Tea tree oil (TTO) enjoys a reputation as an antibacterial 589, 622, 623 substance and is a popular addition to the household medicine cupboard. While most people can use the product comfortably624, a small percentage of users register skin irritation.

Knight and Hausen in 1994625 tested seven people who were sensitized to TTO. Of the seven who reacted to patch-testing of 1% TTO in solution: six reacted to limonene; five to alpha-terpinene and aromadendrene; two to terpinen-4-ol; and one each to p-cymene and alpha-phellandrene. D-carvone, an oxidation product of limonene, caused no reactions among the seven patients. This was contrary to de Groot and Weyland’s findings626 that

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eucalyptol was the only substance apart from the oil to cause a positive response.

Southwell, Freeman and Rubel627, three years later in 1997, set out to clarify the issue and determine the skin irritancy of varying concentrations of pure 1.8- cineole and tea tree oils of varying cineole concentrations in paraffin. None of 28 participants patch tested with 1, 8-cineole showed any irritancy at all over 15 days. The enhancement of tea tree oil with increased 1, 8-cineole was shown to be non-irritant for those not already allergic to the oil itself. Three of the 28 participants had to withdraw from the trial due to allergic responses to the oil. The response was deemed allergic rather than irritant because erythema (redness), itching and oedema (swelling) typified the dermal response. Of the three panellists, none responded to 1,8-cineole, α-pinene, β-pinene, limonene, γ-terpinene, p-cymene, α-terpineol, or terpinene-4-ol. One responded to α- terpinene. All three responded positively to a sesquiterpene hydrocarbon fraction of the oil and one of the three, responded to a sesquiterpene alcohol fraction.

By 1999 Hausen’s628, team identified oxidized TTO as being a three times stronger sensitizer. They showed that TTO kept in open and closed bottles undergoes photo-oxidation with days to months leading to the formation of degradation products deemed to be strong sensitizers. These are mainly peroxides, expoxides and endoperoxides. The opening and closing of bottles in home use facilitates the process.

Oxidation of limonene In 1991 Karlberg’s team established that air oxidation of limonene changed it from a pure substance that induced no significant allergic reaction, to a sensitizing substance561. (R)- (-)-carvone and a mixture of cis and trans isomers of (+)-limonene oxide were found to be strong sensitizers561. In 1994 they showed that autooxidation of limonene could be prevented by storage in closed bottles stored in the dark558.

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In a large study in Europe, 60% of limonene-allergic patients showed positive patch test results to limonene hydroperoxide. After a complex and rigorous testing procedure, Matura et al. 629concluded that R-(+)-limonene, containing oxidation products, is a skin sensitizer.

In 2006, Matura et al.630 showed that oxidation mixture of both R-(+) - and S-(-)-limonene are causal in contact allergy for 2.6% of dermatitis patients. In 57% of cases simultaneous reactions were found for both oxidation mixtures. They recommend screening dermatitis patients with a 3% limonene in petrolatum patch test, but note that this is not yet available.

Oxidation of linalool Baskettter et al.631 provided support for the view that the sensitizing capacity of linalool is due to the formation of reactive species (possibly expoxides) by either autooxidation or metabolic activation or both. Linalool oxide and expoxylinalool were among compounds formed in their investigation. Sköld et al.632 showed that pure linalool was not a contact allergen while air exposed linalool sensitized guinea pigs. They also demonstrated that the sensitizing potential depends upon the amount of time air exposed.

Matura et al.598 in 2005 established that oxidized linalool results in similar rates of positive patch test response (2.7%) as does oxidized limonene. The authors also examined oxidized caryophyllene and found that only 0.5% reacted positively. In 2006, Sköld et al.’s633 work indicated that oxidized caryophyllene would be likely to be a rare sensitizer compared to oxidized R- limonene and linalool.

Pinenes Asthma symptoms are common in sawmill workers dealing with wood with substantial levels of α-pinene, β-pinene and δ(3)-carene634, 635. The more ‘green’ the wood being sawn, the greater the respiratory negative effects if the

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wood consisted of α-pinene and β-pinene. Interest in the action of pinenes is increasing as the products containing these chemicals are promoted and increasingly utilized in the home. Building on the observed difference of effect of the two enantiomers of pinene on animal tissues589, Nielson et al. in

2005636, continued to explore respiratory effects in mice. This team examined (+) and (-)-α-pinene : none of the animals died during exposure or recovery from (+)-α-pinene while death occurred abruptly during exposure or recovery for (-)-α-pinene in the range 2686-5213ppm. Survival was dependent upon concentration. Sensory irritation was persistent for (+)-α-pinene, and of short duration for (-)-α-pinene. Bronchoconstriction started at about 200ppm for (+)-α- pinene and about 800ppm for (-)-α-pinene. These effects suggest overall agreement with effects on humans, according to the authors. They conclude that (-)-α-pinene has a higher systemic toxicity than (+)-α-pinene.

Citral While citral has been under investigation as a toxic substance for decades637-

641, the most systematic investigations of its action have been published since 2005.

Fragrance mix has been used for some years to patch test reactivity to terpenes and blends of terpenes. A new fragrance mix which includes citral in the mix resulted in identification of allergens that were not identified with the first fragrance mix. Apart from the synthetic perfume aldehyde Lyral, lemon scented citral was identified as the most frequent cause of contact allergic response from the application of fragrance mix II642. In 2006, Lalko and Api 643 examined the dermal sensitization of a number of plant components including basil, citronella, geranium and Litsea cubeba and particularly examined the contribution of individual components. Citral was the most potent individual component, followed by eugenol and geraniol.

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In summary, specific research regarding sensitizers in essential oil chemicals emphasises the role of sesquiterpene hydrocarbons, sesquiterpene alcohols, aldehydes, some monoterpene and not others, and the oxidation products of some monoterpenes. Substantial progress in the area of structural relationships of sensitizers has been made. The relevant part of this research will be briefly described along with the important chemical groups that relate to the investigations here.

2.2.6 Chemical structures implicated in asthma

From the information available, the following chemical groups warrant emphasis in regard to their possible relationship to respiratory symptoms in south-east Queensland. Already in section 2.2.2 important structures in terpene chemistry have been explained. Here, their particular relevance to this project will be set out.

2.2.6.1 Hydrocarbons Monoterpene hydrocarbons Already mentioned in this thesis, research to date has identified (+) and (-)-α- pinene, β-pinene, δ(3)-carene, α-terpinene and limonene as affecting respiratory health negatively and/or causing a reaction deemed to be allergic. These compounds have been identified as hydrocarbons that oxidize to hydroperoxides with sensitizing capacity as they undergo reactions with suitable nucleophiles and form adducts577. These chemicals are found in pine plantations immediately north of Brisbane and affecting the area of interest here. The monoterpenes are also found in large quantities in the native vegetation of the south-east Queensland area. All these monoterpenes are contained in plants in the Myrtaceae family, the leptospermums, eucalypts and melaleucas, and feature strongly in this enquiry.

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Sesquiterpene hydrocarbons Circumstantially, the observation by Butcher et al. in 1994253 that emissions of the acyclic sesquiterpene α-farnesene substantially increase when oilseed rape is in flower, must be afforded considerable weight in these investigations. Given the established association of fragrance allergy460, 642, 644, with its alcohol, farnesol, the base hydrocarbon, α-farnesene is of interest here.

Cyclic sesquiterpene caryophyllene is developing a profile as a compound that can mildly sensitize when oxidized633. As it is a common fragrance chemical it is of interest598. It appears in the oils of many Australian plants311, 337, 645 and from plants elsewhere in the world646-648.

Research into the sensitizing component of tea tree oil has indicated that the tricyclic sesquiterpene hydrocarbon, aromadendrene is worthy of close attention625. This view is supported by Southwell’s work identifying the sesquiterpene hydrocarbon fraction of tea tree oil as the most important allergic component627. These findings have a connection to a synthetic649 sesquiterpene, thapsigargin.

Thapsigargin is a 649 tricyclic oxygenated sesquiterpene that is used in medical research to open calcium channels650-652and induce histamine release653. As mentioned earlier, these affect the processes involved in apoptosis, and the perpetuation of inflammation. The exact relationship of depletion of calcium stores , to the apoptotic process, is still not clear although increasingly the relationship is being defined as concerned with various Bcl apoptotic factors654, 655.

Since thapsigargin is a tricyclic sesquiterpene capable of influencing calcium channel opening, it may be that the many other tricyclic sesquiterpenes that are found in some plants may also be affecting processes related to apoptosis.

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Since a hallmark of asthma is decreased apoptosis, these tricylics are of special interest here, as are the alcohols.

2.2.6.2 Alcohols Monoterpene alcohols Terpinen-4-ol is found in many Australasian plants from Myrtaceae563, 656-660 and it occurs in many others that have an association with respiratory symptom exacerbation297, 316, 341, 469, 496, 661, 662. Its associations make it a chemical of interest here, despite studies that attest to its anti-inflammatory profile469, 659,

663, 664. Recent lack of agreement regarding its anti-bacterial qualities665, 666 contributes to the usefulness of clarifying information regarding its effects.

Linalool, a ubiquitous monoterpene tertiary alcohol, is the substance that attracts bees to flowers. It is susceptible to oxidation in the presence of air632 and when this occurs, several hydroperoxides are formed. These hydroperoxides that are formed in the auto-oxidation process decompose to secondary oxidation products : alcohols, ketones, aldehydes and polymeric materials632. Linalool in its pure state is regarded as an irritant, on the basis that no ‘structural alert’ exists which would class it as an allergen632. Bickers et al.’s667 benign conclusion regarding the safety of linalool was based mostly upon dermal studies. In one of the very few references to inhalation research examining the effects of linalool, Letizia et al. 668related an old study in which mice were exposed to a range of concentrations of nebulized linalool. Marked respiratory depression was observed. In a separate experiment mouse inhalation of linalool, via a tracheal cannula or through the nose only, resulted in a marked decrease in respiratory rate for the nose breathing mouse only. If hydrolysis is involved with the sensory irritation response, as reported by Nielsen581, and if carboxyesterases in the mouse nose can modify some chemicals to modify irritancy, as in humans, this process may explain the different response in the mouse.

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The sensitizing capacities of the oxides of linalool have already been mentioned in the previous section.

Eugenol 669, 670 and geraniol 481, 671 are also produced by plants and these sweet alcohols are also extensively used in fragrances672-674 .

As well as monoterpene alcohols, sesquiterpene alcohols are of interest too. In 2006 they were identified as sensitizing agents by Lalko and Api643 in their investigation of the dermal sensitization potential of some essential oils. Eugenol is a stronger sensitizer that geraniol and both are graded as weak senstizers643.

Sesquiterpene alcohols Sesquiterpene alcohols too are found in many Australian plants675-683 and in plants worldwide296, 297, 367, 684, 685.

Farnesol is an acyclic sesquiterpene alcohol that occurs extensively in nature276, 336, 358, 686 and in commercial fragrance269, 644, 687-689 and hygiene products.

Southwell627 identified the sesquiterpene alcohol fraction as being an allergic component of tea tree oil, albeit in only one out of three people allergic to tea tree oil. Given that the sesquiterpene hydrocarbons have a profile in allergy and sensitization, the alcohols, by association, are of interest here as they are produced in large amounts by some flowers. Along with alcohols, esters are found in abundance in flowers and fragrance products.

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2.2.6.3 Esters Esters including the sesquiterpene lactones enjoy an established profile in allergy research and have a bearing on the issues in this thesis.

Sesquiterpene lactones

As mentioned earlier in section 2.2.4.1, the presence of sesquiterpene lactones is notable in many plants associated with asthma, contact dermatitis and hay fever. These compounds are most readily associated with the Asteraceae family. Such a weed is found on the Blackall Range of S.E. Queensland, the Japanese sunflower (Tithonia diversifolia) contains at least three different sesquiterpene lactones690-693. It blooms prolifically in late autumn and early winter.

Lactones are usually bicyclic or tricyclic and many have a reactive methylene 262 (=CH2) substituent . Although this group seems important in sensitization , many sesquiterpene lactones lack this group579. Other unsaturated centres such as cyclopentenone ring or epoxy groups may be the key loci for sensitization reactions694. Many hundreds of lactones have been identified in the Asteraceae family alone695. They occur in other plant families like Apiaceae,

Lauraceae and Magnoliaceae696 but focal interest from medical research has been on the Asteraceae and to a lesser extent, the herb family, Labiatae697.

Sesquiterpene lactones are lipophilic, blending with oils in plants, and are present in the leaves, stems, flowers, trichomes and pollens of plants. Trichomes, the hairlike structures on many members of the Asteraceae (daises and many weeds) and Lamiaceae (herbs), contain the greatest concentration of sesquiterpene lactones579. Earlier in this chapter, Figure 2.19 showed the furry- looking trichomes on a common Australian weed that thrives in warmer areas, known as ‘bluetop’, or Ageratum houstonianum. Appearances are deceiving, for

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when this plant dries out after summer the trichomes take on a quality more like tiny chards of glass, rather than fur. They are readily transported by wind, and can be easily inhaled. This plant is a member of the Asteraceae family and has been identified as a possible source of allergic pollen in Brisbane since the 1960s698. This frequently seen opportunistic weed is closely related to one of the great problem weeds of the world484, 699, Chromolaena odorata, Siam weed. This weed is advancing south from North Queensland and will be discussed in chapter 7. Both of these weeds particularly colonise disturbed ground.

Non- lactone esters As mentioned in section 2.2.2, the combination of an alcohol with a carboxylic acid forms an ester. Given the extensive distribution of these two groups in plants and insects 421, 700-703 and the anthropogenic contributions, ester formation is prevalent. Australian trees produce copious amount of alcohols and some produce large amounts of carboxylic acids276. Esters are widely distributed in Myrtaceae272, 305, 358, 457, 704-706 and other families275, 450, 707, 708 . The oil from Eucalytpus sp. nov. aff. Campanulata contains the ester E-methyl cinnamate to the extent of 95%. This is an extreme example of ester composition in leaf oil. A related compound, octyl methoxycinnamate is a causal agent in photoallergic contact dermatitis709. Other esters are well known for the ability to sensitize skin461. Effects of linalool esters have been investigated to some extent and found to have a low order of acute toxicity and only slight sensitizing effects on skin for some people667 based on investigations prior to 2003. None of the investigations included in Bickers et al.’s667 review of linalool esters effects included inhalation studies.

2.2.6.4 Aldehydes and ketones Aldehydes and ketones feature in a number of investigations into regarding indoor air quality; formaldehyde has been recognized as a threat to respiratory health for more than 25 years62, 710-714. They are of great interest not only because of their ubiquitous production by flowering plants380, 715-719 , but also

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because of their extensive use in the production of chemicals, dyes, food additives, detergents and fragrances720. Potential for exposure is high. Aldehydes are extremely reactive and nucleophilic groups in proteins and nucleic acids are the most likely binding sites to effect toxicity, skin sensitization, mutagenicity and carcinogenicity720. There is an extensive body of research regarding the effects of aldehydes721-725. Benzaldehyde is one that is well known726-729.

Benzaldehyde Benzaldehyde occurs freely in nature and is used extensively in fragrances. A 2006 review 730 of its effects concluded that it is not a sensitizer, although there is evidence of its irritating effects in number of studies, especially on membranes of animals used experimentally. It was supported for use in fragrances at current recommended levels. For fish though, benzaldehyde aldehyde is deemed toxic720. This anomaly underlines a feature of some aldehydes: their solubility. Citral is an another aldehyde that is very soluble.

Citral Citral is used widely to flavour food and in many household and industrial applications. It is a common ingredient in lemon flavoured soft drinks731. In the context of contact dermatitis, it has been identified, for more than twenty years577.269, 642, 643, as a substance capable of sensitizing. Considerable work on the structure of the reactive part of the compound has been undertaken by Patlewicz268, 732, 733 and its action is well understood.

In view of the numerous applications of this pleasant lemon-scented substance269, 642, 643, 734, 735 and the frequency of dermal reactions to it, this substance is of interest here. Some popular Australian plants like Backhousia citriodora , have been heavily promoted for inclusion into urban gardens736-739 contain large amounts of citral271. The spread of this aldehyde into gardens,

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into the diet in flavourings608, 740 and homes in the form of hygiene products266 and even marking pens741, makes this is an important aldehyde. Figure 2.26 shows Backhousia citriodora. The small tree on the left is nine years old and shows the dense flowering so typical of this strongly perfumed plant. The dog indicates the scale. Adjacent is the floral cluster with a pen showing the scale. Note the brown discolouration of the senescing flower which lingers on the tree for a time before dropping to the ground.

Figure 2.26 Backhousia citriodora

Given that aldehydes are so pervasive that babies are being exposed to multiple aldehyde groups, and other problematic compounds, via the emissions from their disposable nappies742, it is important to determine the contribution of this group to negative effects on respiratory symptoms. Finally, the action of some terpenes from the bicyclic monoterpene group are potentially very important to the processes involved in asthma.

Apoptosis and terpenes Some aldehydes and ketones have been shown to affect apoptosis and the usual result, when applied to a variety of cell types, is an increase in programmed cell death79, 82, 83, 743.

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There are contrary results too. Richael in 2001 prevented apoptotic cell death in a plant by introducing a known caspase inhibitor, synthetic262 aldehyde, acetyl-asp-glu-val-asp-aldehyde (Ac-DEVD-CHO)78. In another study, the diketone, 2,5-hexanedione, was shown to affect the balance of calcium within cells in motor neurons744 and in spermatogenic cells655.

Facilitating apoptosis is inconsistent with the perpetuation of asthma, which is associated with reduced apoptosis and thus the maintenance of inflammation. The ability to affect calcium balance is related to apoptosis as discussed already. Chemical groups that affect central apoptotic processes are of great interest here especially because there are some exceptions to the modes of influence. Substances in juniper oil provide some interesting paths to explore.

Juniper oil and apoptosis Juniper oil consists largely of S(-)-α-pinene745, 746 the isomer of pinene deemed the most toxic636. In a study by Na et al.747 examined the effect of juniper oil on apoptosis in human brain astrocytes. Apoptosis can be induced by heat- shock. Oxidative stress plays a role in apoptosis so juniper oil was selected for its expected antioxidative effect. Heat shocked-induced apoptosis of these cells was blocked by pre-treatment with juniper oil. Heat shock induces apoptosis through caspase-3 activation747. In section 2.1.3.3, caspase 3 was identified as one of the ‘executioners’ in the apoptotic process. The composition of the oil is not stated by Na et al. 747and so the chemicals in that blend are not known. Na et al. say that the oil was purchased from Tisserand in the United Kingdom. In 2006, a visit to the Tisserand website, which enables purchasing of juniper oil, revealed that the pure oil for sale is made from juniper berries. If Na et al. used oil made from berries then the composition of the oil might well be alpha-pinene (29.17%) and beta-pinene (17.84%), sabinene (13.55%), limonene (5.52%), and mircene (sic) (0.33%) as determined by Pepeljnjak et al. in 2005746. Earlier, others had noted the presence of sesquiterpenes in juniper oil and stated that the main components

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were monoterpene hydrocarbons, oxygenated monoterpenes and sesquiterpenes748.

The profile of juniper oil is so important to the arguments proffered in this thesis that more detail is needed. Juniper oil exhibits a chemical profile that shares similarities with oilseed rape, with many herbs 327, 749, 750 and forest trees 357,

751, 752 associated with respiratory symptoms, and with many plants from

Australasia305, 331, 679.

In 2003, Angioni et al. 298examined the chemical composition of ripe and unripe berries and leaves from a number of species of Juniperus. Table 2.15 shows some of the terpenes of interest here and the variation in berries and leaves from Juniperus communis, the same species from which oils were extracted and then used in the Na et al. experiments.

Table 2.15 Components (%) of leaf and berry oils from Juniperus communis298

Juniperus communis leaves unripe berries ripe berries monoterpenes α- pinene 6.41 52.91 52.26 sabinene 61.09 13.73 5.58 limonene 2.50 3.81 3.11 sesquiterpene D-germacrene 0.75 6.57 6.69

The importance of the different distributions of these terpenes in the oils from different parts of the plants relates to the possible connection with action of the active chemicals. First though, Nakano 753 et al.’s work regarding camphor toxicity and apoptosis is relevant. Camphor toxicity and apoptosis

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The induction of apoptosis was also the subject of investigations by Nakano et al.753. That team sought to evaluate the cytotoxic activity and apoptotic ability of 22 β-diketones. Beta-diketones are formed from the oxidation of secondary alcohols and they contain two carbonyl units. Most cytotoxic in their study, was 3-formyl chromone followed by the two camphor ketones: (-)-3-(triflouroacetyl) camphor and then its entantiomer (+)-3-(triflouroacetyl) camphor. Further, Nakano’s team , investigated whether 3-formyl chromone, (-)-3-(triflouroacetyl) camphor and curcumin could activate caspase 3 , 8 and 9 , As mentioned in section 2.1.3.3, caspase 3 is an apoptotic executioner and caspase 8 and 9 are apoptotic initiators72. They found that activation of caspase-3, 8 and 9 was effected by 3-formyl chromone and curcumin but not by (-)-3-(triflouroacetyl) camphor. This set of experiments establishes that a terpenoid diketone can be tumor-specifically cytotoxic, and not apoptotic.

Applying this finding to asthma and environmental plant emissions, it is important to remember Rogge’s work on atmospheric plant particles, described in section 2.2.4.1, in which that team established that camphor was the most abundant terpene in dead or green abraded leaf particles. Camphor is found in the camphor laurel and its relatives754 which thrive in Queensland, cypresses297, lavender755 , and from herbs like mugwort, thyme and sage756-

759.

It is known that (+)-alpha-Pinene is structurally related to (+)-camphor760 and camphor occurs naturally in a fairly pure state258 , as does pinene. Camphor has been described as a ketone but behaves in an abnormal manner, according to Simonsen’s writings from decades ago761. To recap, camphor is derived from pinene. Pinene is a bicyclic monoterpene hydrocarbon of the pinane series258. Returning to the juniper oil studies of

Na747: if the juniper oil in that project was extracted from berries, Table 2.15 shows that the levels of α- pinene and D-germacrene are much higher in the berry oil than oil extracted from leaves. Conversely, if Na et al.’s oil was from leaves, then sabinene should be at higher levels than in the berries.

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Sabinene is a bicyclic monoterpene hydrocarbon from the thujane series258. The other monoterpenes in that series, the thujenes, enjoy an established relationship with the occurrence of respiratory symptoms via plants that abound in areas with high asthma occurrence295, 297, 316, 717, 761, 762. The fact that these bicyclics are found frequently in Australian trees329, 705, 763, 764 makes them even more relevant to the investigations here.

A 2006 article by Chiappini et al.765 has shown that the ozonolysis of sabinene gives rise to sabinaketone and sabinic acid, among other chemicals. This ketone that also derives from a bicyclic monoterpene, as does camphor, may also affect apoptosis. If so, then both pinenes and sabinene may be especially important in the maintenance of bronchial inflammation. Oilseed rape flowering ,clearly associated with asthma as already mentioned, has been shown to emit substantial quantities of limonene, β-myrcene, cis-3 hexen-1-ol acetate, linalool, sabinene and lesser amounts of acetone and farnesene253, 289. In this association, the connection with pinenes is not obvious since pinene emission at flowering is minimal. When it is realized that both sabinene and pinenes are both bicyclic monoterpenes, the relationship comes into focus and paves the way for particular attention in this thesis.

The possibility that a significant contribution to asthmatic response is due to the effects of inflammatory substances coupled with anti-apoptotic substances emanating from plants is an important path to explore. The investigations set out in the following chapters will shed more light on relationships to respiratory symptoms of some of the terpenes discussed so far.

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3. Study objectives

3.1 Summary of current knowledge Natural and anthropogenic sources of terpenes have been examined in Chapter 2. Some of those understandings are particularly relevant to this project. Some terpenes are especially likely to be related to respiratory symptoms and may be asthma triggers.

3.1.1 Terpenes as asthma triggers

Profiles of terpenes from oils of Myrtaceae trees suggest likely substances that might be emitted from trees growing in the areas of interest. Comparisons between oils from flowers and oils from leaves reveal that substances emitted from flowers vary substantially qualitatively and quantitatively as shown in Table 2.08. Plants in flower produce a different set of substances from when they are just in leaf. Those substances when emitted from flowers as gases may play an important role in triggering seasonal asthma.

Chemical structures implicated in asthma were explained in section 2.2.6. The most important groups for purposes here are: monoterpenes, oxidized monoterpenes, sesquiterpenes, monoterpene and sesquiterpene alcohols, esters, carboxylic acids, aldehydes and ketones.

Processes that maintain asthma are likely to be affected by aldehydes and ketones which may play a part in inhibiting apoptosis and therefore maintaining inflammation. The inflammation may be triggered by sesquiterpene hydrocarbons, sesquiterpene lactones, and other esters or oxidised monoterpenes both of which are capable of sensitization. The complexity of such relationships is also reflected in a number of paradoxical observations regarding pollen and respiratory symptoms.

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3.1.2 Pollination problems and paradoxes

For decades 698 now researchers have sought to understand the occurrence of respiratory symptoms in terms of pollen incidence. The incidence of pollen drop and its distribution in the atmosphere has been able to explain much of the seasonal nature of hay fever and asthma. There is also a lot left to explain.

Pollens Counting pollens is a relatively easy, though time-consuming, way of keeping track of the flowering of various grasses, trees and shrubs. When copious amounts of pollen are seen in pollen traps, they are usually from wind – pollinated (anemophilous) plants. Species that are wind-pollinated are the source of the vast majority of pollen allergens242. Although exceptions do exist, wind-pollinated plants produce little or no nectar or aromatic compounds, and the pollen grains are fairly dry. Grass (Poaceae), ragweed (Ambrosia), plantain (Plantago) and dock (Rumex) are wind-disseminated pollens associated with allergic respiratory illness766. Among common tree-pollens associated with allergic reactions are oak (Quercus), ash (Fraxinus), plane tree (Platanus) and cypress (Cupressaceae). These are light, buoyant pollens shed in great amounts and readily blown about by wind.

Understanding pollination modes is useful in underlining the fact that there are a great many plants that may be flowering at any given time. The degree to which this is detected in pollen traps does not necessarily reflect the extent of that flowering. In Australia, which has a vast number of flowering native trees, frequently there are no representative pollens in pollen traps at all (personal observation). Pollen counting via pollen traps, like the Burkhard spore and pollen trap is skewed towards detecting wind-dispersed pollens. Reports in the literature tend to reflect the incidence of pollens that are trapped in significant numbers. It is easy to dismiss the flowering of plants that produce no easily quantifiable method of monitoring their flowering. Perhaps the counting of open blossoms, on a branch, over time, may be a useful addition to pollen counts.

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The ease with which wind-disseminated pollens are detected and recorded, has led to a concentration on those pollens concerning respiratory illness. It may not be coincidence that the most investigated are also very visible, for example pine and grass pollens, and able to be counted with relative ease. Reliance upon correlations between such pollens and symptoms has often proved unsatisfactory. In their comprehensive guide to allergenic pollens of North America, Lewis, Vinay and Zenger conclude that it is fallacious to ignore all zoophilous species as potentially causal in allergy due to inhaled pollens242. Anomalies concerning pollen and allergic symptoms can be found in the literature.

Pollen reactions Measuring pollen reactions also is problematic in the light of results of the work by Casanovas et al.767. Comparing olive allergy reactions in two geographic areas, Casanovas et al. found that those with a higher exposure to olive pollen, demonstrated higher levels of specific antibodies, but significantly smaller wheal sizes, than a similar population from another area, where olive pollen is not so copious. There is a great discrepancy between the results of skin prick tests (low cutaneous reactivity associated with high allergenic environmental load) and the levels of specific IgE to the olive pollen.

In a study when 12 participants inhaled ragweed pollen, not one registered a bronchial response to inhalation of whole pollen grains. When pollens were ground to a size of one to eight microns, six out of seven participants registered a fall of 35% in airway conductance. They concluded that small-particle allergens other than ragweed pollen should be considered in most cases of fall (sic) seasonal asthma768.

Skin-testing to detect Cupressaceae allergy was found to be less than accurate by Mari et al.769. That team concluded that commercial extracts of Cupressus arizonica were more reliable than C. sempervirens in detecting cypress allergy.

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Where is the pollen?

Pollens have little consistent relationship to autumn asthma in Australia28, 502,

503. In South-East Queensland, asthma consistently peaks in autumn with a lesser peak in spring. Pollens explain some of the spring phenomenon, but there is little or no relation to autumn asthma. As yet, it is not clear why. Other bioaerosols do not explain the problem either170. Bass 770 noted that the autumn asthma in northern NSW coincides with the drop of ragweed (Ambrosia) and Tibouchina pollen. Associations were drawn from a self-report questionnaire 770 carried out two months after the plants in question flowered. More research is required to clarify this relationship.

Elsewhere pollen relationships to symptoms are similarly clouded. Despite widespread reports of respiratory symptoms in areas surrounding oilseed rape fields in the United Kingdom, little pollen is detected. People, who complain of symptoms from rape allergy, are more than not likely to be atopic and yet they rarely exhibit positive skin tests to the plant. The plant is mainly insect-pollinated and pollen grains can rarely be found away from the immediate vicinity of a field352.

Subiza et al. 771 found that asthma and rhinitis symptoms correlated with appearance of plane tree (Platanus) pollen in the Madrid atmosphere. They noted that, in a few days, participants in their research could change from complete good health, to experiencing severe symptoms. In contrast, at the end of season, symptoms persisted for a week when the pollen count had already fallen. This phenomenon may be explainable in terms of “lag” where symptoms take some time to develop772. It may also be explainable in terms of apoptosis: inflammation fails to resolve immediately because of inadequate induction of ‘cell death’ of the inflammatory cells773. Alternately, could allergens in the form of volatile emissions still be being emitted from the trees after pollen drop?

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Common chemicals? Several plant pollens are cross-reactive. Some of these are: olive (Olea) and privet (Ligustrum); birch (Betula) and alder (Alnus) 242; birch and oilseed rape

(Brassica napus)774; and bottlebrush (Melaleuca) and grass (Bahia)775.

Warshaw and Zug579, in their coverage of sesquiterpene lactone dermatitis, suggest that terpenes are the common substances in some cross-reactive responses. In the United Kingdom, those few who test positive to oilseed rape, (despite many who claim to experience symptoms when it is flowering), have invariably been also allergic to grass pollen. Is allergy to grass pollen due to cross-reactivity with common substances rather than to sensitization to a specific grass-only allergen?

Despite the low probability that individuals can become sensitized to ragweed (Ambrosia artemisiifolia), in Germany and Austria, high levels of positive RAST results to ragweed are evident. Ragweed is rare in these areas. It is thought that the positive responses are due to cross-reactivity with the frequently occurring mugwort (Artemesia vulgaris)776. Is this cross-reactivity due to common chemicals?

Discrete allergens or simply common enzymes? The nature of the relationship, if any, between identified allergens familiar in medicine and the terpene synthases is not clear. The molecular weights of common allergens are very similar to the molecular weights of terpene enzymes.

Allergens are typically described in the medical literature with a short name to stand for an enzyme that encoded a protein. For example Der p 15 were isolated, encoding mature proteins of 58.8 and 61.4 kDa in case of dust mite allergen777. Plant allergens have names like Bet v 1 ( a birch allergen)778. They have molecular weights like : Timothy grass pollen proteins of >/=60 kDa779; mung bean homolog of birch allergen Bet v 1 had a mol. wt. of 16.2

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kDa780; fungus Beauveria bassiana has a molecular mass of 35 kDa781;

German cockroach allergen is 23 kDa782.

Sweeney et al in 1994775 investigated and compared allergic responses to Melaleuca leucadendra ( weeping melaleuca ) , Callistemon citrinus (bottlebrush) and Paspalum notatum (bahia grass). Sera from allergic individuals bound IgE at the molecular weight range of 32 to 70 kDa for Melaleuca and Callistemon. The bands associated with bahia were between 27- 57 kDa. Of those tested, 81% who reacted skin-test positive to at least one of the pollens, were also positive to the other two. Perhaps terpene catalysing enzymes are connected with this phenomenon.

Terpenes enzymes catalyse the conversion of monoterpenes and sesquiterpenes in plants and fungi. Some familiar terpenes are represented in the following enzymes of interest: Linalool synthase is an enzyme that uses geranyl pyrophosphate as a substrate and catalyses the formation of linalool783. Linalool is that honey scent found in many plants and is an acyclic monoterpene that has a molecular weight of 76 kDa. Trans, trans-a-farnesene synthase is the enzyme that catalyses the conversion of farnesyl pyrophosphate to a-farnesene784. It has a molecular weight of 108 kDa. Pinene, myrcene and pinene synthases are between 71 and 73 kDa785. In Norway spruce the monoterpene synthases have associated molecular weights of 50 and 59 kDa. The sesquiterpene synthases d-selinene synthase and g-humulene synthase synthesize 34 and 52 total sesquiterpenes, respectively with molecular weight of 67 kDa for both356.

This thesis will not be a forum for investigating plant enzymes. Given the ability of enzymes to catalyse many tens of monoterpenes and sesquiterpenes per enzyme, perhaps the complexities of cross–reactive allergens can be addressed by others with that framework.

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3.2 Rationale for the investigation Asthma afflicts approximately 2,000,000 Australians who reported asthma as a current long-term condition in 2005. Just over 50 % of hospital separations for asthma are accounted for by children under 14 years of age786. Arguably, South-east Queensland has the highest incidence of asthma in Australia with 22-26% of the population reported being ever diagnosed with asthma787. This high level has been noted for several decades26, 170. Until now the focus on plant-associated causes of asthma has centred upon pollens771, 772, 788. There is a long history of pollen investigation in Queensland698. All references consulted concentrate on anemophilous pollen as opposed to entomophilous pollen. The Asthma Foundation of Queensland offers the following advice in its pamphlet "The Low Allergen Garden" under the heading "Golden Rules”. It reads:

Choose bird or insect pollinated plants rather than wind pollinated plants. Insect pollinated plants produce relatively small amounts of pollen in comparison with wind pollinated plants, which produce larger volumes of pollen grains. These pollen grains are picked up by the wind and may be inhaled. Most native trees are bird or insect pollinated and produce nectar to attract birds which in turn may reduce garden pests222.

This statement reflects the questionable use of logic and is based on the common belief that the wind-pollinated pollen is more copious, therefore is likely to be the source of the problem. It is undeniable that many wind-pollinated plants produce pollen associated with asthma771, 789. No research has been found reporting the investigation of the effect of native pollens or plants on asthma, or any other respiratory symptoms. At best there are anecdotal reports of single cases of reactions790, 791. There are some articles which report the quantifying of Eucalyptus pollen9, 11, 235, 792, 793 but only one specifically measuring a Myrtaceae pollen’s relationship to symptom scores. In a Turkish

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study in 2004, Eucalyptus pollen was shown to be significantly related to allergy symptom scores in allergic children792. (All the reports investigating Eucalyptus pollen, just referenced, were published after this project began.)

Conversely, there are many more studies which have examined the relationships between wind-born pollens and potential for allergic symptoms239,

792, 794-797. It may be the investigation of wind-pollinated plants in Australia has continued largely as a result of tradition based on medical research undertaken in Europe and the United States of America, places where these plants predominate. Until Atlintas et al. 792examined allergic symptom scores in terms of pollen, of which Eucalyptus was one of a group investigated, references to Myrtaceae pollen and allergy were scarce. The only reference found prior to that referring to Australian trees being associated with respiratory symptoms, comes from an obscure overseas publication242. Subsequently Spanish researchers suggested that the Eucalyptus pollen in Spain had ‘allergenic potential’8.

Somehow, Australian native trees escaped adequate investigation as sources of allergens. It may be that Australian native trees, the vast majority of which are insect pollinated, are a major source of emissions related to asthma, in this country and New Zealand, where asthma rates are similar to Australia’s210. Further, it could be that the pollen associated with these trees is a minor source of allergens and those volatile emissions, in the form of terpenes, contribute to a substantial extent. This thinking represents a major departure from research directions so far. This relates to Australian urban and rural areas differently because of human usage patterns and disturbance of the natural vegetation and the impact of garden and urban landscaping.

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Urban development

South-east Queensland developed more recently than older centres like Sydney, Melbourne, Adelaide and Hobart. Brisbane and Perth experienced strong growth in recent decades. Brisbane urban development boomed in the 1970s and 1980s and probably has more urban Myrtaceae plantings than nearly every other capital city. Adelaide and Perth have substantial natural occurrences and introduced plantings too.

Myrtaceae is a botanical family group that includes all the Eucalypts, Melaleucas, Callistemons and Leptospermums. Figure 3.01 shows some of the more recognizable members of the Myrtaceae family. Note that most genera of special interest here belong to the Leptospermum alliance. The complete list of Myrtaceae genera is shown in Appendix B.

The vast majority we know as bottlebrushes and gum trees. Combined with the proximity to the Wetlands where Melaleuca quinquenervia and Callistemon salignus abound, Brisbane has a lot of bottlebrushes. Figure 3.02 shows the distribution of Melaleuca quinquenervia as recorded by the Queensland Herbarium798. This tree is native to coastal South-east Queensland and the far northern coast of New South Wales. Anecdotal information about the increased incidence of asthma on the Redcliffe Peninsula compared with older areas of Brisbane, together with observation of patterns of vegetation in Brisbane, led to investigation of possible causes of this alleged phenomenon. As stated in Table 2.02, Redcliffe City registered higher allergy rates than other local government sectors in 1995, three years before the start of this project.

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ORDER MYRTALES

FAMILY MYRTACEAE

SUBFAMILY SUBFAMILY MYRTOIDEAE LEPTOSPERMOIDEAE

HETEROPYXIS Heteropyxis ALLIANCE

BACKHOUSIA Backhousia Choricarpia ALLIANCE

EUCALYPTOPSIS Eucalyptopsis Allosyncarpia ALLIANCE

Angophora EUCALYPTUS Eucalyptus ALLIANCE Corymbia

METROSIDEROS Xanthostemon Metrosideros ALLIANCE

CHAMELAUCIUM Baeckea Calytrix ALLIANCE

LEPTOSPERMUM Leptospermum Kunzea ALLIANCE Asteromyrtus Callistemon Melaleuca

Figure 3.01 The Myrtaceae family showing some popular garden genera

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Figure 3.02 Distribution of Melaleuca quinquenervia798

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Visual inspection during prolonged driving excursions around Brisbane revealed that there are differing concentrations of Myrtaceae trees in and around Brisbane, and that these concentrations of urban plantings correspond to the commencement of urban development. Those areas settled before 1970 for example, Windsor and Ascot, exhibit fewer native trees in gardens and streets than those settled after, like Kenmore and Kippa Ring. For a time in the 1990s until recently, the native garden trend diminished in popularity somewhat and there was a shift to tropical gardens with palm planting predominating in new urban estates. The passage of time revealed that the native trees in the urban garden grew straggly and dropped a lot of leaves in guttering so aesthetic appeal diminished. In 2006, the water shortage in south-east Queensland has caused more attention to be directed towards native plants again due to their drought tolerance. Unable to supply the water to thirsty tropical species, gardeners have turned to native plants once more799, 800.

Redcliffe City is located on a peninsula and is entirely built out with no vacant land available. The city exhibits a concentration of plants belonging to Myrtaceae. The Redcliffe area was almost completely settled during the native garden boom period of the 70s and 80s, except for the older areas near the beach. Almost every street is planted with bottlebrushes and most of them are Callistemon viminalis, which is pictured in the previous chapter in Figure 2.16. Figure 3.03 shows a Redcliffe City street in Rothwell, with central road planting many Melaleuca leucadendra in leaf, interspersed with Melaleuca quinquenervia which is in flower. Figure 3.04 shows the distribution of Callistemon viminalis as recorded by the Queensland Herbarium798. This shrub is native to South-east Queensland.

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Figure 3.03 Row of Melaleuca leucadendra and Melaleuca quinquenervia planted as a road divider in Redcliffe City. (M. quinquenervia is shown in bloom.)

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Figure 3.04 Distribution of Callistemon viminalis798

It has long been recognised that asthma in the Brisbane area peaks in autumn with a smaller but substantial peak in spring170. The reason for that has escaped researchers to date. Figure 3.05 shows a schematic approximation of

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asthma incidence against the flowering of the predominant species of Melaleuca and Callistemon - bottlebrushes.

Figure 3.05 Schematic view of asthma in Brisbane against flowering of bottlebrushes

Asthma Incidence in Brisbane

*** * ** *** *** ************ ** ****** *************** Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Many Melaleucas and Callistemons either bloom twice a year or have a staggered season from autumn until spring495. The top line of asterisks refers to Melaleuca quinquenervia, the next to Melaleuca leucadendra and the bottom to Callistemon viminalis. It is important to note that Melaleuca leucadendra frequently blooms twice yearly in south-east Queensland and that that may be spring/autumn, or it may be summer/winter. This phenomenon does not seem to relate to climate or soil type as two specimens of this genus in the same locality can follow a different floral rhythm: one plant may flower in summer, then the other in autumn, then the former one in spring then the latter in summer. Each plant though, keeps to its own annual synchronicity. Flowering is six months apart. These patterns are potentially important since this very attractive tree with a weeping habit produces vast numbers of flowers. It is planted prolifically as a street tree in some places like Noosa Shire, north of Brisbane. These plants are also frequently planted in Maroochy and Caloundra

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Shires. They are natives of North Queensland. Figure 3.06 shows a Melaleuca leucadendra from Noosa, with the writer in view to show the scale. This tree is one of dozens in the street, all of which were similar in size having been planted in the early 1980s and at time of the photograph, were about 15 years old. Figure 3.07 shows Melaleuca leucadendra in Australia as recorded by the Queensland Herbarium798. This species is not native to south-east Queensland and its presence in large numbers is chiefly due to the actions of local government authorities having planted it as street trees.

Figure 3.06 Melaleuca leucadendra fully grown in street landscaping by local government authority.

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Figure 3.07 Distribution of and Melaleuca leucadendra798

Following on from previous research, the source of the problem was thought to be pollen from the trees, the writer having searched for, and not found, any evidence of investigation of this substance and its relationship to respiratory health, in the literature.

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Research revealed that the pollens from native trees are about half the size of other pollens thought to be associated with asthma. Some varieties of tea tree (Leptospermum) pollen are one-fifth the size of grass pollen. This factor alone was a sound reason for further investigation, since native pollens have potential to reach deeper into the respiratory tract because of their small size242.

At the same time the chemical components of Mytraceae leaves were investigated. The oils which are sought for the essential oil industy permeate the whole plant including the flowers270. The oils are also found in the pollen tubes and on the pollen417. It is known that pollen from particular plants is associated with asthma and this has already been mentioned in Chapter 2. Research to date has concentrated on particular allergenic protein components of specific pollens of specific species of plants. The writer proposes that pollens from essential oil plants are more likely to be problematic because of the specific chemical blends in the oils, and thus a much wider focus can be set : whole genera of plants, rather than species become objects of interest as ‘allergen’ sources. This thinking takes the focus of the problem from the micro to the macro and is not reported in the literature thus far. Since oil producing plants have heavy sticky pollen, pollinated by insects, and bound together by the oils, it appears to have been dismissed as a research target because it is believed that only light wind-disseminated pollens are likely to be problematic for respiratory health maintenance. Some trees such as pines produce a lot of essential oils and light wind disseminated pollen. Perhaps the pine pollens are not the only source of respiratory irritants or allergens.

Given the effects associated with the components of the oils from plants, as already reported in Chapter 2, what effect would inhalation of pollens containing these oils have? No mention of these pollens containing essential oils is discernable in the literature. Would the effect be the same for those allergic to Eucalyptus pollen when compared to those who are not? As noted in the

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previous chapter, asthma or airway hyperresponsiveness has its base in the allergic response and AHR increases with the degree of atopy. What is the prevalence of Eucalyptus allergy in Brisbane in people with asthma compared to those without? What is the relationship of lung function in people with asthma, to the flowering of native trees in the greater Brisbane area?

In a bid to understand the likely prevalence of particular Myrtaceae oil components , and hence the chemical targets of interest in asthma, it was necessary to understand the role of the chemical components of the plants. The results of those investigations revealed that the role of terpenes seems to be a dual one : for the benefit of the plant and the insects that pollinate it. As mentioned in Chapter 2, terpenes are thought to be pheromones and can atttract, or repel particular insects; some are growth regulators and control the life cycles of pollinating insects; some contribute to growth and dormancy cycles in the plants. Others control and/or retard the growth of fungi introduced into the plant; some have a role in inhibiting the germination of competing plants408.

Eucalyptus and tea tree oils are well known anti-fungal agents270.

Fungi and Myrtaceae plants Typically, leaves for the essential oil content are not harvested at flowering time since the anti-fungal components are at their lowest levels then270, 300. The reason seems to be that fungi is introduced into or onto the plants by insects during pollination and the fungi provide an extra food source for the insects. Fungi levels increase from this time. There exists a symbiotic relationship between the fungi and the plants - the plants attract more insects, the insects and fungi take nutrients from the rich supply of sugars in the flowers and the plants are efficiently pollinated801. At flowering time, the plants produce large quantities of limonene. After flowering, the plant needs to reduce the numbers of fungi in order to protect the health of the plant, and so produces higher quantities of anti-fungal terpenes like cineole. Limonene decreases. High levels of limonene seem to be only associated with flowering. When levels of limonene are high in essential oil plants, anti-fungals like cineole and pinene are often

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lower289, 802. Similarly when levels of anti-fungals are high, limonene generally is low270.

Fungal spores and respiratory symptoms Assocation between increased asthma and higher numbers of fungal spores in the air is well known. The association between increased Cladosporium spore counts and asthma in Brisbane is also well known108, 169, 503. The common belief seems to be that Cladosporium multiply on dead plants predominantly. The question of whether fungal spores grow on Myrtaceae flowers is an important one given the millions of Melaleuca quinquenervia that bloom each autumn in coastal areas of south-east Queensland. If fungi can take up nutrients from the flowers , then perhaps the fungi grown on a substrate of native tree flowers would differ in composition from fungi grown on another substrate. Further perhaps the fungi that grows on one species of flower is composed of quite different substances from that from another species of flower. There seems to be an assumption that all fungi are much the same in composition. Perhaps not all fungi are uniform in their effect when breathed in, because of differing composition. The anomaly of high Cladosporium counts being associated with asthma in some seasons, while not in others, begins to make sense if the contibution of different growth substrates is considered. If the fungi are taking up particular oil constituents then the ingestion of that fungi would be more problematic than fungi which has grown on plants containing more innocuous substances. The exploration of these questions formed the basis of Phase 1 of the project.

When Phase 1 was well underway and spore and pollen counting began, the absence of Melaleuca pollens on collection tapes was of great concern and caused the unscheduled progression to Phase 2. This extension of the project was on the basis that if the pollens could not be identified from pollen trapping, then the source of allergens/irritants associated with autumn asthma was probably gaseous, and invisible. The fact that millions of trees were in bloom

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not far from the pollen trap, at the time was indisputable. Simple logic dictated the next step in this exploratory and iterative project.

For clarity, the objectives and strategy of the project phase 1 and 2 will now be merged since some of the original objectives regarding pollen collections are no longer relevant. The second phase resulted from unsatisfactory results of Myrtaceae pollen counts in phase 1. While Myrtaceae pollens were evident on plants and could be seen on microscopic slides clearly, they were not in the air, except in tiny quantities and when they were found they were in clumps stuck together by sticky nectar. The collection of fungi and pollens was aborted at that point and Phase 2 was pursued instead. A chance comment about the problem of oilseed rape in Scotland led to investigation of an article253 which revealed gaseous emissions by the crop. The composition of those emissions was similar to that of oils from Myrtaceae. Phase 2 became a search for a relationship between respiratory symptom occurrence and a presumed gaseous source of allergen/irritant emanating from native trees.

3.3 Objectives of the current investigation The objectives of the investigations were: 3.3.1 To determine whether children with asthma exhibit different degrees of allergic reactions to commercial Myrtaceae allergen when compared to children without asthma. 3.3.2 To determine whether children from urban coastal areas differ in their skin-test responses to Myrtaceae and other allergens, when compared to children from a rural highland area. 3.3.3 To determine skin reactivity to terpenes from flowers of Myrtaceae in participants from an urban coastal area compared with a rural highland area. 3.3.4 To determine whether the flowering of various members of Myrtaceae is related to worsening asthma in south-east Queensland in an urban coastal area compared with a rural highland area.

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3.3.5 To explore the relationships among plant volatiles in ambient air and respiratory symptoms, meteorological variables, fungal spores, pollens and air pollutants in an urban coastal area. 3.3.6 To explore the relationships among plant volatiles in ambient air and respiratory symptoms, meteorological variables, fungal spores and pollens in an urban coastal compared to a rural highland area. 3.3.7 To explore whether Cladosporium fungi vary in composition according to growth substrate. 3.3.8 To explore the association between fungal growth and Myrtaceae flowering. 3.3.9 To explore the occurrence of allertant substances from plants in household and lifestyle products. 3.3.10To explore the relationship of variations of terpenes in plants according to taxonomic grouping and world patterns of asthma and respiratory diseases.

3.4 Strategy for the investigation This project was divided into two phases and, as the investigation was both exploratory and iterative in nature, the strategies involved in that sequence will be explained sequentially. Phase 1 The independent variable of greatest interest here is location. Location selection A fundamental principle underpinning this project is that the allergic responses to be detected, and the respiratory symptoms to be measured, would be affected by the plants in the environment of the participants. Those plants are determined by their location and the geographic and anthropogenic factors that impact upon it. The location then would determine which plants would be in the vicinity of the participants on a daily basis.

The project hinges on the assumption that the vegetation of the coastal flats that make up most of Brisbane and its surrounds, particularly the autumn flowering Melaleuca quinquenervia, provides a different source of potential

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allergens than that of areas that do not support that vegetation. Thus an urban coastal area will be compared with a rural highland area. Since there was not the opportunity to compare an urban highland area, the added complexity of mixing rural versus urban had to be accommodated.

The primary comparison locations selected were the Redcliffe peninsula (sea level) and the highland area surrounding Maleny ( 450m altitude), 88 kilometres away by road in a north-east direction, on the Blackall Range. The natural vegetation surrounding Redcliffe at the time was Melaleuca quinquenervia wetlands merging into mixed Eucalyptus and Melaleuca woodland with some Acacia. Of mainland local government areas in south-east Queensland , Redcliffe City experienced the least clearance of bushland, 3,608 ha compared to Maroochy Shire’s 115,130 ha803 in the period 1987-1994. The reason for that is that only a small area was available to be cleared in Redcliffe because it is almost completely urbanized. Maroochy Shire, which takes in part of the Blackall Range, is a vast area with clearing still occurring at a great rate in 2006.

Secondarily, three coastal Brisbane locations, all of which are close to the Melaleuca wetlands on the coast were chosen to contrast with the mountainous mixed forest, rural location in the mountains. The purpose in that comparison was to test the hypothesis that all three coastal patterns of allergic response would be similar to each other but would contrast with responses from Maleny children. Children from Boondall, Slacks Creek and Kippa Ring were selected as well as Maleny children. Kippa Ring, Boondall and Slacks Creek are locations with substantial and varying amounts of Myrtaceae (eucalypts and melaleucas). The Boondall wetlands covers and area of more than 1000 hectares and is located between Boondall State school and the coastline. Uncleared areas of Melaleuca bushland also occur within only two kilometres of Kippa Ring State school and close to Slacks Creek State school. Figure 3.08 shows native wattle beside a stand of Melaleuca near Kippa Ring at Deception Bay. Actual water frontage along the coastline of greater

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Brisbane is rare because of the wetland environment which is unsuitable for construction and comes with large populations of mosquitoes which breed in the estuarine environment.

The areas which support the selected urban schools vary in the occurrence of domestically planted native trees. Kippa Ring , adjacent to Redcliffe, is almost entirely planted with native trees as the area was established at the height of the native garden popularity period, the 1970s and 1980s. Boondall is an older suburb surrounded by native trees and domestically planted with exotic species used extensively in the 1940s to 1960s. Slack's Creek is a mixture of old and 1970s and 1980s housing surrounded by naturally occurring mixed eucalypt and coastal wetland trees. Figure 3.09 shows the urban footprint and the available bushland in south-east Queensland804. The arrows have been added to mark the areas just described. The pink shading denotes urban footprint and shades of green, non-urban areas. Dark green denotes Eucalyptus forest koala habitat, the flowering of which is of interest here.

Figure 3.08 Acacia concurrens (wattle) and Melaleuca quinquenervia

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Figure 3.09 Urban footprint and rural land in south-east Queensland804

Participant selection

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Children were chosen as participants to examine the extent of allergen response to a range of commercial allergens including a Myrtaceae allergen because many children are affected by allergies and asthma. The school environment also provided a convenient point of contact from which all the procedural requirements could be executed. Healthy children and children with allergies and asthma were recruited in order to compare responses. Children with ‘seasonal‘ asthma were requested to participate. Children in grades four to seven were chosen because they needed to cooperate with the process and take instruction and because 9 or 10 years of age was deemed to be the minimum age that would enable the children to participate in the respiratory diary keeping component of Phase 1. Children, and their carers, completed an ISAAC (International Study of Asthma and Allergies in Childhood) questionnaire and, on the basis of their responses, they were classified into groups reflecting their health status. Children who had recorded that they had had asthma in the last 12 months were assigned to the ‘asthma’ group and those who had never had asthma or allergic symptoms of any kind, were assigned to the ‘healthy’ group. Only children from the asthma group were asked to participate in respiratory diary keeping. While including a control group of healthy children would have been ideal, this choice was dictated by the practicalities of engaging children with no health issues, in a protracted period of respiratory health measuring and reporting, on a daily basis.

Skin testing Given the background that Australia’s native plants have been regarded as “safe” and not contributing to respiratory symptoms, it is not surprising that no record of a regional comparison of skin-prick testing (SPT) to a Myrtaceae allergen, could be found. Indeed, no record of any skin-prick testing battery that included a Myrtaceae allergen, conducted in Australia, could be found. Thus the first task identified in order to meet the objectives just stated, was to establish the extent of skin-test positive reactions to a Myrtaceae allergen. Of particular interest here were Myrtaceae allergen responses in children with asthma, the

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‘asthma’ group, compared to children without either asthma or any other respiratory or allergic symptoms, the ‘healthy’ group of children. Along with the desire to ascertain the prevalence of allergy to native plants, the opportunity to clarify some issues related to skin-prick testing, presented itself. Already the work of Sweeney et al.775 has been mentioned , in terms of cross- reactivity and possible relationships to terpene synthesizing enzymes. To explore similar relationships here, some allergens were added to the test battery with the specific intention of investigating cross-reactivity. One of those was ragweed pollen extract and the other was olive pollen extract, because neither ragweed nor olive occurs in the urban areas of northern Brisbane.

Privet and olive allergens are cross-reactive805 and for that reason the olive was included due to the large amount of privet around Maleny. In Phase 1, privet allergen could not be obtained so olive was substituted. The reason for including either olive or privet in the allergen battery was to explore the regional responses to that taxonomic group Oleaceae. Both privet (Ligustrum) and olive (Olea) belong to that family. Redcliffe is free of privet and olive and the pattern of responses in the highlands versus the coast was of interest for this reason.

A range of grass allergens together with insect, animal and fungal allergens were also included in the battery to examine the regional differences. Tea tree oil was also included in the battery, to explore the incidence of skin response to that substance. Hypotheses related to skin-prick tests: • That responses to skin- prick tests would differ between those children with asthma and those without. • That children from the three Brisbane schools would exhibit different fequency of positive response to commercial Euclayptus and commercial olive allergen (Olea), than children from Maleny. Directionally, Brisbane children would exhibit more positive response to Euclayptus and Maleny children would exhibit more positive response to Olea commercial pollen allergens.

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Respiratory diaries and bioaerosols Groups of children who had been diagnosed as having had asthma in the previous 12 months were selected, and volunteered to participate in keeping a respiratory diary. At the same time pollens and spores were to be trapped and meteorological variables were to be measured. During Phase 1 the purpose of the diaries was to establish the relationships between peak expiratory flow (PEF), other respiratory symptoms and the variables just mentioned. Of particular interest, was the occurrence of Myrtaceae pollen in relation to respiratory symptoms?

Hypothesis related to respiratory diaries: • That the pattern of asthma incidence between Brisbane and Maleny would differ.

Floral and fungi studies During this time flowers of various Myrtaceae species were collected and analysed with a view to growing fungi on them and determining the chemical composition of the flowers and any metabolites that may result from fungi grown on them. Information was also sought about the content of chemicals in flowers compared to leaves. Information about chemicals in flowers of interest was vital in order to prepare for air sampling in Phase 2. In order to look for particular chemicals, the thousands of possible terpenes had to be narrowed to just a few in order to target the search for chemicals in ambient air in Phase 2.

Hypotheses associated with floral studies: • That the terpene content of native flowers would differ subtantially from the content of the leaves. • That the chemical content of fungi grown on a substrate of Myrtaceae flowers would vary with the species of flower.

The results of Phase 1 invited further exploration and another set of comparisons and investigations was planned and subsequently executed.

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Phase 2 Location selection On the basis of Phase 1 results, highland and coastal areas were chosen once more to compare. Once again Redcliffe on the coast and Maleny on the Blackall Range were the closest centres of each of the two areas studied in Phase 2. Participant selection Experience in Phase 1 led to the choice of an older age group for Phase 2 because focus in Phase 2 was on respiratory diary keeping while in Phase 1 the focus was on responses to allergens. Respiratory diary information that was utilized from Phase 1 children was from a ‘select’ subset of the child groups. These children were conspicuously dedicated to the task and provided diary sheets that seemed to be superior in quality of recording. The participants apparently reliably recorded respiratory symptoms and peak expiratory flow (PEFR) while other children appear to have hurriedly penned a series of entries identical to one another.

Phase 2 involved adolescents and adults with the addition of one nine year old child whose parents specifically requested inclusion in the research project. The child’s own motivation to be included was high. All participants in phase 2 experienced had asthma in the previous 12 months and were recruited on the basis that their asthma was ‘seasonal’ in pattern of occurrence. Since the research projects focussed on seasonal rhythms from plants, this aspect of participant selection was very important.

Skin testing In phase 2, skin-prick testing was again performed and this time individual terpenes were included in the battery of ‘allergens’. As well, as a number of commercial allergens were also included: Melaleuca (paperbark bottlebrush), Callistemon (bottlebrush), and Ligustrum (privet). Figure 3.10 shows Melaleuca quinquenervia in flower and Figure 3.11 shows flower of the small leaf privet, Ligustrum sinsense.

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Figure 3.10 Flowers of Melaleuca quinquenervia

Figure 3.11 Flowers of small leaf privet, Ligustrum sinense

These allergen extracts were not available in phase 1 due to international supply issues. Individual terpenes of various kinds were included in the phase 2 ‘allergen’ battery . The following were included: monoterpene and sesquiterpene hydrocarbons’ oxidised monoterpenes; oxygenated

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monoterpenes; a carboxylic acid; an ester; and methanol. Methanol was included to gauge the skin response to a simple alcohol.

Respiratory diaries and bioaerosols Groups of adolescents and adults from Maleny and Redcliffe area who had been diagnosed as having had asthma in the previous 12 months were selected, and volunteered to participate in keeping a respiratory diary for phase 2 of the project. At the same time pollens and spores were to be trapped and meteorological variables were to be measured. During Phase 1 the purpose of the diaries was to establish the relationships between peak expiratory flow (PEF), other respiratory symptoms and the variables just mentioned. Of particular interest in phase 2 was the relationship of symptoms and PEF in particular to the results of air sampling. The results of ambient air sampling for terpenes, and bioaerosol sampling, were not available until the conclusion of the data collection period so participants and researcher were blind to these variables. Some meteorological variations would have been obvious to participants and the researcher during the course of the panel study.

Air sampling In phase 2 ambient air samples were collected from coastal and highland locations to investigate the relationship of terpene occurrence with respiratory symptoms and with the meteorological, air pollutant and natural bioaerosols. Because there was no pollutant collection point at the highland location the analysis of pollutants was restricted to the coastal area only.

Floral and fungi studies During phase 2 the floral and fungal studies were extended to include investigations of the substances found in privet flowers. Other native flowers were also analysed for floral emissions and tissue chemical contents. Further fungi and floral growth substrate investigations confirmed findings in phase 1. Floral emission collection prior and subsequent to a storm provided information on possible new sources of respiratory allertants.

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Cleansers and lifestyle products sampling Some popular cleansing and lifestyle products were investigated for their capacity to emit terpenes. Products included aromatherapy oils, spray air fresheners, a cream cleanser, a disinfectant and a popular mosquito repellent.

There were no specific hypotheses for phase 2 as this phase was much more exploratory in nature than phase 1. It was thought that the terpenes found in ambient air, if any, would reflect the substances that had been identified as substances associated with the flowering of particular flowers. Similarly, the air collections at each collection point in the highland and on the coast would reflect the chemical composition of the flowers in the vicinity. Prominent flowering plants from both regions had been analysed for terpene content during the floral studies. Following the logic of the project so far, the composition of the air in the highland region should have been different from that of the coast, on the basis different vegetation patterns. Further there was the expectation that patterns of terpenes in ambient air might relate to respiratory symptoms, especially PEF.

The details of the strategy of the investigation are contained in the next section concerning methodologies.

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4. Methodologies The focus of this investigation is the possible variation in allertants that may be due to differing vegetation occurring in the areas of interest on the coast and on the range. Some description of the areas of interest, especially in terms of the vegetation, is appropriate here before describing the methods employed to investigate the effects that those differences may have.

4.1 Geography and vegetation of coastal and range areas. South-east Queensland has a projected population of 2.75 million in 2006 . Brisbane City accounts for 35.9 % of that Figure, Redcliffe City 2%; Maroochy Shire 5.3%; Caboolture 2.8%; Caloundra 3.2% and Noosa 1.8%. Chapter 3, Figure 3.09 showed the land use map of south east Queensland and on that map the urban development is shown shaded in pink with the names of the urban and country centres from which participants were drawn superimposed upon the map. The two geographic areas of interest here, coastal and range, have different characteristics. Redcliffe and Maleny are the respective commercial hubs of the areas under investigation. The three other schools included in Phase 1 allergen testing were also from the coastal strip that lies between the mountain ranges and the sea.

4.1.1 Description of coastal area under investigation

Coastal land around Brisbane is almost entirely urbanized except for the areas from Deception Bay, north of Brisbane, to Caloundra and in the south the area north of Coomera to Cleveland. These areas remain vegetated with wetland plants. The land is unsuitable for building due to tidal influxes and the mosquitoes make living there uncomfortable. There are small pockets of natural bushland along the coast between Cleveland in the south and all the way to Deception Bay. Soils on the coast are sandy and do not retain moisture well. They are not very fertile.

These bushland areas contain pockets of mangrove of a number of species including Grey (Avicennia marina), Red (Aegiceras corniculatum) and the

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Mangrove Fern (Acrostichum speciosum) nearer to land. Moving away from the water line of the coast are the paperbark forests and woodlands which are dominated by tea trees, especially Melaleuca quinquenervia.

Two Eucalypts found growing in swampy coastal conditions are the Swamp Mahogany (Eucalyptus robusta) and to a lesser extent, the Forest Red Gum, Eucalyptus tereticornis. The Swamp Box (Lophostemon suaveolens) is often found with the Red Gum. The paperbarks grow as a tall or short tree and in the understorey of the drier forests; Blady Grass (Imperata cylindrica) is common. Appendix C shows the species of Eucalyptus that are found in and around the Moreton district of south-east Queensland. Moreton is the name of the pastoral district which takes in the areas under investigation.

Casuarina (Australian Pine or She-Oak) is found throughout the coastal region as a number of species are represented. Casuarina equisetifolia and Casuarina glauca are common natural coastal inhabitants. Appendix D shows the species of Casuarina that are important to this investigation. Another coastal tree from the cypress family is the White Cypress Pine, Callitris columellaris, which thrives on sandy coastal soils. Figure 4.01 shows the Glasshouse Mountains road which cuts straight through a naturally occurring Melaleuca forest north of Brisbane on the coastal flats. This part of the investigation area has not yet been developed for forestry or housing. Progressively it is being removed for housing developments as in the pictured Glasshouse Mountains area where new house blocks back onto Melaleuca forest.

The majority of land use on the coast is urban development featuring detached houses on blocks of land of greater than 500 m2. Streets in south-east Queensland feature plantings of native trees most of which belong to Myrtaceae. Only older established suburbs feature exotic plantings. Figure 4.02 shows a typical street with the Melaleuca leucadendra planted in the street by the local government authority. All three coastal schools under investigation are situated near to busy roads on which large number of commuters make their

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way to and from the city. Between Redcliffe and Maleny there are large tracts of Melaleuca forest and some dry Eucalyptus woodland. Large areas of

Figure 4.01 Melaleuca forest divided by road cutting.

plantations devoted to pine can be found between the coast and the ranges especially north of Caboolture and in the Glasshouse Mountains area. Appendix E shows the list of Pinus species cultivated. Pinus ellliotti (slash pine) has become a weed tree in the range areas and in Maroochy shire generally.

Acacia (especially Acacia concurrens) species are opportunistic and vast numbers of plants can been seen in flower at the end of winter and early spring in bushland and on roadsides all over the coast. Different but equally numerous Acacia species (especially Black Wattle, Acacia mearsii) are found in large numbers in the ranges.

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Figure 4.02 An urban street in south-east Queensland coastal suburbs

4.1.2 Description of mountain range area under investigation

The mountain range area chosen as a contrast to coastal locations was Maleny. This town was selected because it was only one hour’s driving distance from Redcliffe and yet the vegetation and topography was very different. It was also a large enough town to provide the participant numbers required. Greater Maleny’s population is about 5000 in 2006 and the town services Witta and Conondale as well as Montville and Flaxton. The elevation of Maleny is 450 metres above sea level and the contrast of the topography with that of the coastal flats can be seen in Figure 4.03. Flaxton is marginally higher than Maleny at 475 m. This picture is taken from a ridge near Maleny looking directly towards the sea and Maroochydore.

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Figure 4.03 Coastal view from range ridge at Maleny

The range area under investigation is a hilly to mountainous dairying district and supports a number of small farms which produce a variety of food including citrus fruits, macadamia nuts, avocados, organic herbs and vegetables. The soil is rich red volcanic loam which is very fertile and has substantial clay content so it retains water well. The area attracts a number of hobby farmers and small acreage occupants. There is no broad acre farming because of the hilly terrain. Historically, the region was an important producer of citrus, avocados and pineapples, on the lower slopes.

The introduction of privet to the range areas is attributed, anecdotally, to farmers desirous of establishing shade for cattle. Large-leaved privet (Ligustrum lucidum), or Glossy Privet, grows into a large shade tree and flowers in summer, from December to February inclusive. Figure 4.04 shows a composite photograph of this plant growing next to Montville school; on the top section is a close-up of the panicle shaped flower stalk and the signature large glossy leaves.

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Figure 4.04 Glossy or Large-Leaved Privet (Ligustrum lucidum)

Small-leaved privet (Ligustrum sinsense) is also widespread and it flowers in early spring. The formation of berries that are eaten by birds and transported elsewhere makes this close relative of the olive one of the most prolific plants in the ranges. The richness of the soils enables good germination and rapid growth. Figure 4.05 shows a typical Maleny scene with young small-leaf privet that has almost certainly opportunistically germinated. Olive and privet varieties that occur in south-east Queensland are shown in Appendix F.

Another obvious seasonal floral event on the Blackall Range is the flowering of Red Natal Grass (Melinis repens), which blooms in autumn, and can be seen on road verges and cleared land. This grass can be seen in Figure 4.06 and is prolific. Tithonia diversifolia ( Mexican or Japanese sunflower) blooms in May and June especially around Montville, Flaxton and Kureelpa. Figure 4.07 shows this weed on the roadside near Montville where it has naturalized.

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Figure 4.05 Maleny dairy cow with Small-Leaf Privet

The natural vegetation of the Blackall Range consists of mixed wet Eucalyptus woodland interspersed with rainforest pockets and dry woodlands in some parts. Bunya pines (Araucaria bidwillii) are native as are Piccabeen Palms (Archontophoenix cunninghamiana).

Figure 4.06 Red Natal Grass (Melinis repens)

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Figure 4.07 Japanese or Mexican sunflower (Tithonia diversifolia) on the Blackall Range

In the wet sclerophyll parts of the Blackall Range, flooded gums are common (Eucalyptus grandis). Tallowwood (Eucalyptus microcorys), Grey Gum (Eucalyptus propinqua), Forest Red Gum (E.tereticornis ), Blackbutt (E. pilularis) and E.resinifera are also found there along with Brush box (Lophostemon confertus) and dozens of other species of mixed genera. The Forest She-Oak (Casuarina torulosa) is frequently found on the Blackall Range. Melaleuca genera are not featured on the list of wet sclerophyll flora of the area as provided by Barung Landcare, a local environmental group.

In parts dry woodland predominates and Eucalyptus cloeziana, E.crebra, E.maculata, E.microcorys, E.pilularis, E.propinqua, E.racemosa, E.resinifera, E. saligna, E.siderophloia, E.tereticornis, E.tindaliae, E.trachyphloia E.umbra are found. In the dry sclerophyll Melaleuca bracheata and M.linarifolia are listed as growing on sandy or clay soils , but not on red soils on the Blackall Range. Seven species of Acacia and Callistemon salignus are listed for dry sclerophyll. Callitris species, belonging to the Cupressaceae family, are found in hilly areas as are several Acacia species which have different blooming periods.

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The most obvious difference between vegetation in the ranges, when compared to the coast is the diversity in the range. A walk into one of the many forests reveals quite a different range of plants compared to the Melaleuca forests of the coast. Figure 4.08 shows a now protected wet sclerophyll forest near Flaxton with and old Eucalyptus stump, left from logging, next to three grass trees (Xanthorrhoea macronema), a termite mound and ferns in the wet Eucalyptus woodland. Piccabeen palms (Archontophoenix cunninghamiana) are in the distance. The diversity of flora in the area is reflected in the ‘significant’ flora of Caloundra Shire, in which Maleny is located. Appendix G lists some of these special plants that occur on the range or on the coast below.

Residents on the range seem to have a preference for planting cypresses (Cupressaceae family) and pines (Pinaceae family) . Because so many people live on acreage, the space afforded them encourages the planting of these introduced species. Many people plant multiples of Australian plants too and there are examples of both types of monoculture on the Blackall Range. Figure 4.09 shows a typical line of cypress trees at Flaxton.

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Figure 4.08 Wet sclerophyll woodland at Flaxton

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Figure 4.09 Cypress ‘fence’ of acreage home at Flaxton

4.2 Procedures for allergen testing in coastal and range children

Before the testing began, the project protocols were approved by the ethics committee of Griffith University. Phase 2 protocols were also approved by the Asthma Foundation of Queensland.

Children were recruited from the four state schools of Kippa Ring, Maleny, Boondall and Slacks Creek following formal permission from the Education Department of the Queensland Government. The schools selected were all from the same socio-economic ‘band’, at determined by the Education Department. Following approval, approaches were made to the principals of the four schools individually initially by letter and then in person. A short article was written for insertion into the school newsletters requesting participant for the study.

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Child volunteers with and without respiratory and allergic symptoms were requested. Asthma sufferers were required to have seasonal asthma or intermittent asthma. Children from grade 4 to 7 ( 8 to 13 years age range) inclusive were requested.

Those wishing to participate were given an ISAAC QUESTIONNAIRE (International Study of Asthma and Allergies in Childhood) (see Appendix I), a personal details form and an informed consent form as required by Griffith University’s ethics committee.

Children at each of the four schools were tested on four separate days, one week apart. Table 4.01 shows the numbers and sex of the children recruited. Table 4.01 Numbers and sex of children recruited in phase 1 SPT

Number of children School Males Females recruited for SPTs Boondall 124 58 66 Kippa Ring 128 64 64 Slacks Creek 34 18 16 Maleny 94 40 54 All children were aged between 9 and 13 years

All children who volunteered were accepted and were assessed by the attendant respiratory physician, Dr Charles Mitchell. Spirometry was performed for each child and they were allocated an asthma status on the basis of short interview and examination with Dr Mitchell, their spirometry result and their ISAAC questionnaire. All of the children who claimed to have been diagnosed, by a doctor, as having asthma were accepted as such. Children who had experienced episodes of asthma in the previous 12 months were classified as the ‘asthma’ (n=97) group and children who had no allergic or respiratory symptoms at all were classified as ‘healthy’ ( n=107). Among the ‘asthma’ group, 63 were classified as ‘mild , 25 as ‘moderate’ and 10 as ‘severe’

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according to the number of episodes of asthma experienced in the last 12 moths, as recorded on the ISAAC form. The other participants who were not ‘healthy’ or ‘asthma’ were variously classified according to symptoms expressed on the ISAAC questionnaire (‘intermittent asthma’: n=81; ‘atopic’: n=64; and ‘dry cough only’: n=31). The issues in this thesis are related to the distinction between the asthma and healthy groups only.

All children underwent standard skin-prick testing with a battery of manufactured allergens, a positive histamine and negative saline control, and 100% tea tree oil. Allergens were dropped onto a grid drawn on the volar forearm, the skin pricked with a lancetter and the resultant wheals were measured after 10 minutes. Allergens were obtained from Bayer Corporation in Sydney. The allergens acquired were limited by availability from international suppliers. Those used for the children in Phase 1 were (as shown on the label of each): Standardized mite of Dermatophagoides farinae Cockroach Mix 6585 (American, German) Standardized cat pelt Dog hair - dander Hormodendrum cladosporioides Alternaria tenuis Johnson grass Sorghum halepense Bahia grass Paspalum notatum Ryegrass, perennial Lolium perenne Callistemon citrinus - bottlebrush Olive, Olea europea Acacia, Golden Acacia longifolia Ragweed, mix GS (giant short) Pine, Aus/SW She-oak Casuarina glauca Eucalyptus/blue gum Eucalyptus globulus

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‘Thursday Plantation’ brand tea tree oil was obtained from a pharmacy. Figure 4.10 shows the allergens being applied and the allergic response, on an adult arm.

Figure 4.10 Skin-prick test in progress

The skin wheal from the epicutaneous skin-testing was regarded as positive if a skin wheal diameter 3mm or greater was formed after 10 minutes had elapsed from the time of pricking the skin in the middle of the allergen droplet. Measurements were entered onto a form that can be seen in Appendix H. Irregular shaped wheals were calculated by taking the width and adding the length and dividing by two to obtain an estimated wheal diameter.

Following the completion of the skin testing, the children with asthma who volunteered were recruited to participate in respiratory diary completion for the ensuing spring and following autumn. Children were supplied with a peak flow meter and trained by the writer in its use. (This part of the project was later abandoned since the Myrtaceae pollens that were supposedly being collected were not apparent in the pollen traps. The project changed direction towards trapping gases and so required another panel of participants. )

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4.3 Procedures for allergen and terpene testing in coastal and Range groups of adults and adolescents in phase 2

During phase 2, allergen testing was performed again along with a set of 100% terpene substances which had been pilot tested twice on atopic and non- atopic volunteers before hand. Participants completed the ISAAC questionnaire (Appendix I) and all participants underwent spirometry and medical assessment. All confirmed that they had experienced at least one episode of asthma in the previous 12 months and all reported that their asthma was seasonal in nature. (One participant was excluded when it was discovered that their asthma was invariable and was not in fact seasonal in nature.)

No adverse reactions were encountered so the substances were included in the battery of SPTs. Table 4.02 shows the age and sex of participants in phase 2 allergen and terpene testing. Figure 4.14 shows a plot of ages of each participant for the range and coastal groups in phase 2. Table 4.02 Age range and sex of phase 2 participants

Number of participants Age range Group recruited for Males Females SPTs Kippa Ring 9-59 years 18 35 Maleny 13-62 years 9 20

Commercial allergens were again acquired from Bayer in Sydney and this time the availability of some allergens enabled the inclusion of species not used in phase 1. Figure 4.11 shows the distribution of the ages of participants for both groups. The allergens used in SPT in phase 2 were (as shown on the labels):

Standardized mite of Dermatophagoides farinae Cockroach Mix 6585 (American, German) Standardized cat pelt

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Dog hair - dander Hormodendrum cladosporioides Alternaria tenuis Johnson grass, Sorghum halepense Bahia grass, Paspalum notatum Ryegrass, perennial Lolium perenne Callistemon citrinus, bottlebrush Privet, Common, Ligustrum vulgare Acacia, Golden Acacia longifolia Ragweed, mix GS (giant short) Pine, Aus/SW She-oak, Casuarina glauca Eucalyptus/blue gum, Eucalyptus globulus Melaleuca quinquenervia.

Figure 4.11 Participants’ ages of coast and range groups of phase 2 SPT

Ages of participants phase 2 SPT

70 RANGE GROUP COAST GROUP

30 Age in years 20

0 0 1 2 3 4 5 6 7 8 9 10111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455 Participant identifier

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Some 100% terpenes and aromatic compounds were included in the SPT battery. Those were: Alpha pinene (−)-β-pinene 1, 8-cineole (R)- (+)-Limonene Methanol Bornyl acetate Caryophyllene Ursolic acid

Procedures for administering and measuring allergens and other substances were the same as for phase 1. The form used for recording wheal diameters is shown in Appendix J.

Appendix K contains product details of the compounds. The first four listed above were also oxidized and used in the SPT battery. Oxidized compounds were prepared before SPT by decanting the terpenoid substances into a sterile wide mouth beaker and allowing the beaker to stand in a storeroom of the researcher’s house, with the window open, for a period of two weeks. The oxidized terpenes were then placed in screw top glass bottles with a dropper attached, no longer accessible to the air. Methanol was included in the battery to ascertain the response to a simple alcohol.

4.4 Procedures for air sampling and respiratory diary analysis for groups of adults and adolescents

4.4.1 Participant numbers and location

A subset of the Phase 2 allergen/terpene participants were asked to maintain respiratory diaries while air sampling was performed in the two areas of interest. No reward of any kind was offered. Figure 4.12 shows the location of

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participants in the Flaxton and Rothwell sampling areas. Table 4.03 shows the number of participants in each group for each month of the study.

Table 4.03 Participant numbers in range and coastal groups

2000-2001 October November December April May Mean Coast group 20 17 11 14 14 15 Range group 13 13 8 8 9 10

Shelter and security issues limited the location options within the areas to be sampled. The Rothwell site was in a suburban backyard of a 1000m2 block of land, surrounded by similar blocks all with houses built thereupon. The air sampling pump was housed inside a covered shed with the Tygon tube extending outside the shed and under the roofline at head height, under cover but quite open to the ambient air. The area was shaded all day. Within 5 metres was an inground swimming pool and a variety of tropical foliage plants were about 5 metres away. These plants did not produce flowers during the sampling time. Plant from adjacent houses may have produced flowers but they also had pools in the yard and not a lot of space for plants. Palms were in evidence in several adjacent properties. At the Flaxton site the sampler was open to the ambient air, positioned on an open verandah, under cover. The 10,000 m2 block with one house in a rural residential area supports a variety of trees and grass types covering the area among the trees. A natural spring fed lake was 100m away. This lake is surrounded by sedges and supports waterlilies.

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Figure 4.12 Location of participants (coast and range), air sampling sites (Redcliffe & Flaxton), meteorological sites (Brisbane Airport & Maleny) and pollutant sampling site at Deception Bay.

Air Sampling

FLAXTON ▲Maroochydore Meteorological Data Site

Maleny

N ↑ To Pollution Monitoring Brisbane

Air Sampling N REDCLIFFE ↑

Meteorological 0 30kmData Site

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4.4.2 Airspora

Airspora were sampled with a Burkhard 7 Day Volumetric trap from Burkhard Manufacturing Company, Rickmansworth, and Hertfordshire, UK. The orifice was situated at 2 metres above ground level to approximate head height. Collections tapes were changed weekly and they were divided into daily strips and mounted upon microscopic slides, stained with Calberla’s solution and examined under a light microscope. Counts were made using a 40X objective by counting a strip 100µm wide from the middle of the slide in a lengthwise transverse 903. Daily levels representing mid-morning to mid-morning were recorded. Missing airspora information from 20 days at Rothwell and 38 at Flaxton was the result of equipment failure.

Fungal taxa selected for quantification were : Cladosporium, Alternaria and ‘other fungi’ which was all other fungi. Pollens counted were Myrtaceae (any genera), Pinus (pine), Gramineae( grass pollens), Acacia (wattle), Casuarina (she-oak), Asteraceae and ‘other pollen’ which was any other pollens.

4.4.3 Air pollution

Air pollution was monitored at Deception Bay only since the Environmental Protection Authority of Queensland which operates the monitoring site, does not have a monitoring station on the Blackall Range. EPA recorded level for oxides of nitrogen (NOX), particles less than 10 microns ( PM10), and ozone

(O3) were accessed for statistical analysis for the Coast group. For the Coast group only, the pollution variables measured were: daily means for PM10, NO and NO2 and O3.

For the Range group, ozone test strips were used on each air sample day, but at no time did the disposable indicator test-strip change colour. The interpretation was therefore that on no test day was there enough ozone on the range to register on the test strip (colour could be induced in the city so the

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strips were not faulty). The Range group’s analysis did not include pollutants for the reason that there was no pollution data to access.

4.4.4 Meteorological variables

For the Coast group meteorological information was accessed from the Bureau of Meteorology. Readings were taken from Brisbane airport. The relationship of the meteorological monitoring sites to the sampling sites is shown on Figure 4.12. The airport site was the closest collections site to the sampler.

On the range, meteorological information was supplied by Maleny weather station. The monitoring site was located in Maleny town. Weather in Maleny is usually representative of the weather at Flaxton, where a number of the participants lived. Weather varies markedly between the coastal areas and the range areas with the range enjoying cooler temperatures year round, by up to five degrees Celsius.

The following variables were measured: daily means for atmospheric pressure, temperature, precipitation and wind speed; thunder ( heard or not heard) and relative humidity. Thunder was converted to a dummy variable for analysis. Relative humidity was not entered into the analysis for the Range group.

4.4.5 Air sampling and respiratory diaries

Each morning the participants were asked to measure their peak flow upon waking and to record it . They were also asked to make a number of ratings about the previous day’s symptoms. The participants did not know which days would be sample days. They were required to complete the diary every day. See Appendix L for the diary sheet.

Air samples were collected via a Tenax tube inserted into tygon tubing connected to an SKC Universal Sample Pump ( all items available from Airmet, Brisbane). Tenax TA 20/35 Mesh 100mg tubes were used (SKC 226-123). The tube with Tenax attached was then taped with duct tape to the outside wall

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under cover, in each setting. The tubing was taped so that there were no kinks in the tubing to impede flow to the pump.

Since sampling would occur in the summer, allowance was made for ‘breakthrough’ of terpenes as the volume expands with increased temperature. Trial sampling enabled the limits to be set to avoid ‘breakthrough’ in the hot weather during summer. To avoid this, the sample level was set very low at only 550ml per minute i.e. approximately one twentieth of the human rate of breathing in air. (Much pilot testing with several grades of Tenax and a range of pump rates and sampling times occurred before this rate was determined as suitable. The pump was calibrated before it was set for each sample with a 5% tolerance deemed acceptable. Appendix M shows the sampling pump.

Hours of sampling were always from 7am to 7am with the aim that the sample would end just as participants would be taking their morning medication, having just completed their respiratory diary. Air samples were taken three times per week, always once on Sunday in order to sample a day least affected by industrial and weekday traffic patterns. Usually sampling occurred on a Tuesday and Thursday as well, and sometimes it was Tuesday and Friday. Each sample was at least 48 hours after the previous one and some were 72 hours.

Sampling occurred on one third of the days of the week. Comparison with two other sets of days revealed that the sample set of days were not significantly different from the other days, on a measure of SPEF for coastal participants. All possible days were divided into three sets :Set A (day 1, day 4, day 7 etc); Set B (day 2, day 5, day 8) ; Set C (day 3, day 6, day 9 etc). A post hoc analysis (Student’s t–tests) of the SPEF means from the Coast group for the sample days indicated that the set of days used did not differ significantly in the mean SPEF scores from the other two sets of readings. None of the combinations of sets of days was significantly different from any other set.

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Air sampling periods were from 1 October until 31 December 2000 (spring) and then from 1 April until 30 May 2001 (autumn).

4.5 Procedures for plant and fungi analyses

4.5.1 Observational studies of fungi and flowers

The exploratory investigations were not structured and are included here as they augment the structured investigations.

Examination of stamens Exploratory investigations into the possible fungi associated with flowers of some Myrtaceae members involved detaching flowers from the branch and then removing stamens and examining them under a light microscope. Stamens from Callistemon viminalis, Melaleuca quinquenervia were examined and floral tissue from Ageratum houstonianum. Stamens from flowers newly open, open but not discoloured and discoloured, clearly senescent ones were examined. Stamens were plucked and examined only under a cover glass.

Mould scrapings Sooty mould was scraped from an aggregation of sooty tissue that was neither floral, nor leaf, nor flower nor branch. These structures were plentiful on the trees some months after flowering. Such structures can be the result of pathogens and this may have been ‘witches broom’ a pathogen that affects many plants and produces distorted foliage. It especially affects woody plants. The structure was roughly spherical with diameter about two centimetres and appeared as a cluster of plant tissue, similar to sepals. A scraping was taken and transferred to a slide and covered with a cover glass and examined under the light microscope, and photographed.

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Thunderstorm study An opportunistic investigation into the emissions from a flower, before and after a storm, involved the wrapping of a Grevillea ‘Robyn Gordon’ and sampling with Tenax TA 20/35 Mesh 100mg for 10 minutes at pump rate of 3 litres per minute with Tenax tube in place. One sample was taken as a storm gathered strength and there were light wind gusts and light rain at 2.45 pm. Thirty minutes later after the storm had past another 10 minute sample was taken. During the ‘storm’ there was little rain but substantial rumbling of thunder. The actual sampling event and storm at Flaxton on the Blackall Range is pictured in Figure 4.13.

Figure 4.13 Sampling of Grevillea during storm build-up

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4.5.2 Procedures for fungi and flower analyses

The plant species investigated were Callistemon viminalis (common weeping red bottlebrush used extensively in gardens and in landscaping); Eucalyptus “Summer Beauty”, hybrid bred for the home garden (Eucalyptus ficifolia X E. ptychocarpa); Melaleuca Leucadendra ( weeping cream bottlebrush used extensively for landscaping); Melaleuca quinquenervia (abundantly naturally occurring cream bottlebrush in the Wetlands and coastal NSW and Queensland).

Extract from a report by others This methodology section that follows contains technical information extracted from a report by: Noel Davies, Matthew Gregory and Prof R. C. Menary, Essential Oils Research Group, Tasmanian Institute of Agricultural Research (TIAR), School of Agricultural Science, University of Tasmania Numbered headings have been added by the writer as have some plant species names, to enhance clarity, otherwise the text is as a supplied. Where the text is added by the writer, that acknowledgement will be made.

Steps in the analysis

4.5.2.1 Pre- sampling procedures Initially flowers from Callistemon viminalis flowers were hexane extracted to develop the methodology and to see if volatile compounds were present in the extracts and could therefore be analysed by GCMS (gas chromatography mass spectroscopy). The extracts were analysed by GCFID (gas chromatography flame ionization detection) using an HP1 column.

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The samples of Callistemon viminalis, Eucalyptus “Summer Beauty”, Melaleuca Leucadendra, Melaleuca quinquenervia were examined using GCMS analysis of SPME samples.

4.5.2.2 GCMS Analysis of SPME Samples The headspaces of stamen and plated specimens were sampled by unwrapping the foil packages or lifting the lid of the plates inside a zip lock plastic bag, which were then sealed and allowed to equilibrate at room temperature for 3 hours. The headspace was then sampled by piercing the bag with a SPME (solid phase micro-extraction) needle fitted with a 100µm polydimethylsiloxane coated filament. This was then protracted for 10 minutes of absorption at 20°C. The needle was then retracted, removed from the bag and desorbed into the GC injection port at 240°C for GCMS analysis using a 25m, 0.52μm, HP1 column at 15psi with a temperature program of 60 °C (for 2min), rising at 6°C/min to 240°C.

The samples of Callistemon viminalis, Eucalyptus “Summer Beauty”, Melaleuca Leucadendra, Melaleuca quinquenervia were also examined using solvent extraction. Petrie dishes of cut stamens from the listed flowers were inoculated with Cladosporium fungi prior to leaving Queensland by air express courier service, which took two days to reach the laboratory in the University of Tasmania. Matching flower samples without fungi; Cladosporium and agar plates and agar only plates were also examined by hexane extraction to determine the composition of the fungi before and after growth upon the various substrates. ‘Cladosporium only’, ‘stamens only’ and ‘agar only’ served as control comparisons for the four ‘stamens plus fungi’ conditions.

(Additional note by the writer: the agar plates were Sabouraud dextrose agar from Micro Diagnostics in New Farm.)

Cladosporium fungi were cultured from an ambient air sample taken from Flaxton, and was confirmed as Cladosporium upon arrival at the laboratory in Tasmania. The stamens and fungi and agar plates were kept for 12 days at

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25˚C. In natural condition fungal sporulation have been observed after 21 days but temperature drops in the evening substantially. To allow for the constant incubation in what would be an autumn daily temperature, the period of 12 days was selected. In each of the ‘stamens’ conditions, 30g of cut stamens were used. Figure 4.14 shows a plate of Melaleuca stamens (left) with a small Cladosporium colony, ready for the flight to Tasmania. The plate on the right shows the base Cladosporium colony from which all the fungal discs were taken. Figure 4.15 shows a Melaleuca quinquenervia flower uncut, and a flower ‘stripped’ of stamens with scissors. (Figures 4.14 and 4.15 photographs were inserted here by the writer).

Figure 4.14 Melaleuca stamens and Cladosporium plated sample and Cladosporium base culture

Figure 4.15 Melaleuca flowers before and after stripping of stamens

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For GCMS analysis the samples were resuspended in 5ml of the relevant solvent and an aliquot of this taken for injection into the GC.

GCMS analysis was performed by split injection into a Hewlett Packard 5890 GC with conditions as for the Tenax tube samples except that the starting oven temperature was 40°C.

4.5.3 GCMS Analysis Of Tenax Tube Air Samples

After arriving in Tasmania from Queensland, the tenax samples where kept stored as they arrived, wrapped in foil and sealed in screw capped glass tubes, inside a plastic box, at room temperature, until analysed.

Analysis was performed by desorption into a Hewlett Packard 5890 Series 2 gas chromatograph equipped with a cryotron, a split injection system, an HP-1 cross linked methyl silicone gum column (30m x 0.32mm with 0.54μm film thickness) and linked to a Hewlett Packard 5970 Mass Spectrometer. Desorption of the Tenax tubes was performed by replacing the normal injector liners with them, in the injector chamber when the temperature had fallen below the desorption temperature of the type of chemicals being screened for (<100°C). Carrier gas was Helium @ 3 ml/min., with a head pressure of 10 psi. The Oven temperature program was 10°C for 4 min, then 8°C/min to 280°C where it was held for 10 minutes.

Identification of compounds from the chromatograms was made with reference to the NITS MS library database. No attempt to quantify the relative abundance of the various compounds identified was made beyond a very general classification of the ion counts for each of those compounds into OH (very high), OH-H (very high to high), H (high),H-M (high to medium), M (medium), M-L (medium to low), L (low), L-T (low to trace), T (trace) and zero.

This lack of precise quantification was due to a combination of factors including: the differing saturation characteristics of different compounds in the Tenax

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tubes at varying temperatures; and a high degree of peak merging. This included the interference of compounds emanating from the Tenax packing (bleed) as well as the variation in response of the MS over the sampling time due to the need to tune the instrument regularly. This latter problem could have been overcome with the use of external standards (for each identified compound to be quantified) being run with each set of samples to calibrate the response using single ion monitoring (SIM). The purpose of this study was, however, to perform a broad survey to identify what volatile compounds of plant origin were present at the various sampling dates. Any procedure involving quantification of specific compounds would have needed to be set up at the onset of the study with the use of standards as well as a record of the temperature at each Tenax sampling date. This was not attempted, partly because not all the compounds of interest had been identified until the survey was well advanced, but also because restriction of funding available for resources and analytical assistance would have made this beyond the financial means of the project. It was considered that the results of this survey would indicate which compounds were of most interest for specific quantitative studies in any ensuing research work. End of ‘method’ from report by Noel Davies, Matthew Gregory and Prof R. C. Menary.

Additional method information by the writer

Tenax flower sampling Volatiles from the following living attached flowers were also sampled by GCMS desorption of Tenax:

Eucalyptus “Summer Beauty” Eucalyptus tereticornis Acacia concurrens Grevillea ‘Robyn Gordon’ Melaleuca quinquenervia

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Melaleuca leucadendra Xanthostemon chrysantha (Golden Penda) Leptospermum petersonii (Lemon-Scented Tea Tree) Buckinghamia celsissima (Ivory Curl Flower) Buddleja (Buddleia) davidii

They were sampled by first wrapping the flowering branch in household grade aluminium foil, Alfoil, in the field, whilst the branch remained attached to the living tree or bush. The foil was chosen because it was not reactive with the plants, it did not produce its own emissions, and it was very inexpensive, easily obtained, and extremely malleable. It was purchased from a supermarket.

Figure 4.16 Use of foil wrapping during sampling of live flowers

The use of Tedlar bags was not deemed necessary. Since the investigations were exploratory, minimal costing was an important element. Figure 4.16

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shows how flowers were wrapped during sampling and how the pump was suspended from a tree during sampling with the Tygon tubing and Tenax inserted in to the package of flowers.

The flowers were enclosed tightly to exclude as much air as possible in order to concentrate the floral emissions. The intention was to concentrate emissions rather than to exclude ambient air totally. Several flowers were wrapped and then left for 10 minutes to accumulate some emissions inside the foil packet.

Because the aim was to investigate qualitatively rather than quantitatively, the number of flowers varied somewhat amongst samples and was dependent upon ease of wrapping the foil around the flower branch. Some branches were easy to encase and others were not because of the way the flowers attached to the branch.

A Tenax tube was then inserted into the packet and the foil pressed around it to hold it in place. The pump was switched on and run for 10 minutes with a reading of 3 litres per minute showing on the pump gauge with the Tenax tube in place in the end of the Tygon tubing that was attached to the pump. After sampling, the tube was removed, wrapped in three layers of foil and twisted at each end and placed in a screw-capped glass tube. The tubes were packed in a plastic container and sent by air express to the laboratory, taking two days.

Cut flower samples Samples of Ligustrum lucidum (large leaf privet) in bud newly open and senescent flowers were cut and sent by air for analysis by SPME and extraction. The flowers were cut and wrapped in foil packet in several layers of foil, put in a box and posted by express post taking two days from Flaxton to Hobart.

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Control Tenax tubes The batches of Tenax tubes were sent to Tasmania in batches of seven in a week of ambient air sampling, and more if flower samples were also sent. As mentioned in section 4.4, air sampling for respiratory symptom matching occurred three times per week: once, on a Sunday, to counter the weekday effects of pollution; and twice during the week, at least two days apart. Two tubes (one for each location) three times a week (six in all) constituted a week of sampling.

A control tube was added to each batch. It was never exposed to ambient air, and never taken out of its capped glass tube. When the batch of six used tubes were opened and desorbed, the control tubes were treated the same and checked that it was free of terpenes or other contaminants. If the control tube was found to be contaminated, the entire week’s samples were discarded. This occurred twice in the 20 weeks of sampling and was due to a very minor contamination thought to have occurred from the laboratory where the tubes had been kept briefly during the analysis. The tubes were discarded.

Selection of which tube would be the control tube was entirely random and was chosen from each new batch of 7 tubes that returned desorbed from the laboratory in Tasmania. Tubes were purchased new and were reused after desorption with the use of the control tube serving as a measure of the effectiveness of the desorption process. Estimation of Total Ion Count An estimate of the total ion count for the quantities of volatile compounds as offered by the contracted scientific officer at the University of Tasmania are as follows. Trace = 0-20000 Low = 20000-100000 Medium = 100000-500000 High = 500000-2500000 Very high = > 2500000

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4.6 Procedures for analysis of household and lifestyle products A number of household, cleanser and lifestyle products were sampled to ascertain the content of terpenes of interest. Some of these were SPME samples and some were Tenax samples.

Aromatherapy oils and cleansers were sampled as if they were used in a home. Thus a control Tenax sample of the researcher’s kitchen/dining area was taken at the rate of 550 ml per minute with the Tenax tube in place i.e. one hundredth of the air sample that was taken for the respiratory studies. The sample area consisted of three open-plan rooms of medium size with door and windows shut. A commercially available oil burner was used in which 100 ml of water and five drops of oil was put. When vapour began to evaporate the Tenax was suspended 8cm above the oil burner and the pump turned on for 10 minutes.

All the oils were sampled in this way. They were ‘Breathe Easy’, ‘Refresh and Renew’, ‘Tranquil and Calm’, Lavender, Lemon Myrtle, Pinus Sylvestris and Tea Tree oil.

Cleansers and lifestyle items Fragrance diffusers and room fresheners, ‘Ambi Pur Lemon Freesia’ and ‘Ambi Pur Bedroom Breathe Freshly’ were set on ‘max’ level 5 for 4 hours prior to sampling in the three rooms with the doors closed except for movement in and out. Sampling occurred in winter. A sample for 10 minutes at 550ml per minute was taken after 4 hours.

A ‘No Moz’ mosquito citronella candle was sampled in the same manner. It was allowed to burn in a room for 4 hours then a 10 minute sample taken at 550 ml per minute.

Ajax creamy cleanser was sampled by squirting 5 squirts onto the kitchen bench top at normal room temperature, which was 20 degrees Celsius on that

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winter’s day. A Tenax tube was suspended 5 cm above the bench and a 10 minute sample was taken, at 550 ml per minute.

Pine-O-Cleen Glen 20 surface spray disinfectant “Country Scent” was sampled similarly by spraying an area 20 cm by 30 cm and a Tenax tube was suspended 5 cm above the bench and a 10 minute sample was taken at 550 ml per minute.

For all these samples a period of two days elapsed between each one and the house was thoroughly aired with doors and windows left open all day, for the intervening two days.

4.7 Statistical procedures

Statistical analyses were performed with SAS Version 8. Procedures used were: Spearman’s correlations, Wilcoxon Signed Ranks tests, T –tests, Principal Components Analysis, Step-Wise Linear Regression and General Linear Regression. Data were tested for homogeneity and normality and transformed where necessary. Group means were used in the analysis of participant symptom scores and peak expiratory flow measures. The latter were standardised using a z-transformation.

Raw participant-data collection Peak expiratory flow was measured on the peak flow meters obtained from Medical Developments Australia, as supplied to the Asthma Foundation of Queensland. They were distributed to participants at the beginning of the data collection period and the participants were given training in how to use them effectively. The best of three blows was to be recorded as the PEF for that particular day.

Self report measures were recorded on a form that was posted each month to the participants along with a light conversational letter aimed at encouraging participants to remain with the project. Participants were encouraged to telephone and/or email the researcher at any time. The need to omit forgotten

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entries rather than guessing was stressed. The ‘asthma score’ was a 10 point ranked scale and the remaining self-reported symptom scales were four point ranks starting from ’zero’ and extending to ’three’. Reliever and preventer medication usage was reported on a three-point categorical scale which was converted to -1, 0, and 1 for inclusion into the group mean. The scales can be seen on the participant diary report form, Appendix L.

Air samples were transformed from the original categorically quantitative ratings which were assigned during the analysis in Tasmania. The range from ‘trace’ through to ‘very high’ covered 10 categories which were allocated a numerical ranking on a 10 point scale (see Appendix N). This scale was then log10 transformed since it was thought that the log scale better approximated the quantitative increases in compounds in ambient air.

These specifications relate to the results of several investigations that will be presented in the following chapter.

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5 . Outcomes of investigations of asthma in coastal and range communities, and their flora The investigations into the relationships among coastal and range communities began with the allergen testing of children to determine the prevalence of response to Eucalyptus and to compare responses from children in the two different geographic areas. Additionally, comparisons between children with no allergies, and children with asthma were sought.

5.1 Occurrence and discussion of reactions to allergens in coastal and range schoolchildren For the four schools where children were tested with allergens, the results of positive responses are shown in Figure 5.01. Usual practice is to examine the response to skin-prick testing with commercial allergens using a 3mm wheal as the positive benchmark. A positive response here is a wheal diameter of 3mm or greater measured from all participants in each school. Many participants responded with wheals less than 3mm. These school groups included healthy children and children with allergic conditions ranging from asthma to eczema. Figure 5.01 Positive response to allergens Phase 1 for all schools

PERCENTAGE OF POSITIVE RESPONSE (≥3mm wheal) BY SCHOOL

60 Boondall %≥3mm

Kippa Ring %≥3mm 50 Slacks Creek %≥3mm

Maleny %≥3mm 40

10 PERCENTAGE OF POSITIVE RESPONSE

H A I M IA PT A IU NE IVE H DOG CAT LY R OL BA O ACACI ERNAR JOHNSON BERMUDA DUSTMITE AUST PI EUCA RAGWEED RYEGRASS ALT COCKROAC ADOSP CL ALLERGEN

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As expected, responses to dust mite and cockroach are much more frequent than for other allergens, with response to dog being the least. Considerable variation in response to some of the plant allergens is evident among the schools.

Responses from Maleny school (green columns) are less than the others for most of the allergens. Given that the Maleny school is from the same socioeconomic band as the other schools (Band B), the reason is unlikely to be socio-economically based. The make up of the groups was mixed with each school’s group consisting of children with a range of allergic issues as shown in Table 5.01. The combined schools group consisted of the following health categories, as determined from the ISAAC forms which parents completed concerning allergic symptoms.

Table 5.01 Symptom group numbers for Phase 1 children

Symptom group numbers for Phase 1 children Mild asthma 63 Moderate asthma 25 Severe Asthma 10 Atopic 64 Dry cough only 31 Intermittent asthma 81 Healthy – no symptoms at all 107 Total all children from 4 schools 380

The numbers of ‘healthy’ children were about one-third of each school group.

Table 5.02 shows the mean wheal diameter and confidence limits in which the Australian trees, Eucalyptus and Australian Pine (Casuarina) are shown to elicit a mean wheal of less than 3mm. The same is true for ragweed and dog.

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Table 5.02 Mean wheal and confidence limits of allergen tests

Lower 95% Upper 95% Variable Confidence Limit Confidence Limit Mean Std Dev

DUSTMITE 4.6003283 5.3749405 4.9876344 2.6773944 COCKROACH 3.2418697 3.8243068 3.5330882 1.7172353 DOG 1.7258010 2.4964212 2.1111111 0.9740215 CAT 2.8729378 3.7461098 3.3095238 1.4010117 CLADOSPORIUM 2.9750303 4.1426167 3.5588235 1.6731603 ALTERNARIA 3.0147690 3.9852310 3.5000000 1.7777450 ACACIA 2.6947821 3.4013718 3.0480769 1.2690108 EUCALYPT 2.5347575 3.0277425 2.7812500 1.1076368 AUST PINE 2.5212617 3.0620717 2.7916667 1.0467531 OLIVE 2.7548018 3.3361073 3.0454545 1.1823291 RAGWEED 2.0643990 2.5606010 2.3125000 0.8544315 BAHAI 4.3116535 5.6508465 4.9812500 3.0088937 RYEGRASS 3.1773670 4.4492996 3.8133333 2.7641202 JOHNSON 3.1821544 4.4902594 3.8362069 2.4874914 ………… BERMUDA 3.3522355 4.2549074 3.8035714 2.0797617

The relationships of results among the school groups is shown in Table 5.03. This table shows the difference between groups when sets of allergen response scores are compared using Wilcoxon non-parametric analyses. All response scores were used in the analysis including zero, one, and two as well as wheal measurements greater than or equal to three. Thus the entire set of scores for Eucalypt allergen from Boondall school were compared to the set of scores for Eucalypt allergen from Maleny school. This process was repeated for each combination of each of the four schools, for each allergen. Significant differences are noted in Table 5.03.

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Table 5.03 Wilcoxon non-parametric analyses of group differences

WILCOXON COMPARISONS OF SKIN TEST RESULTS BETWEEN SCHOOLS *=.05 **=.01 ***=.001

SCHOOLS DUSTMITE COCKROACH DOG CAT Maleny X Kippa Ring * (P=.04) NS ***(P=.0003) NS Maleny X Boondall **(P=.007) NS ** (P=.0085) NS Maleny X Slacks Creek NS NS NS NS Kippa Ring X Slacks Creek NS NS NS NS Kippa Ring X Boondall NS NS NS NS Boondall X Slacks Creek NS NS NS NS *=.05 **=.01 ***=.001

SCHOOLS CLADOSPORIUM ALTERNARIA ACACIA EUCALYPT Maleny X Kippa Ring NS NS NS *(P=.02) Maleny X Boondall NS NS NS *(P=.03) Maleny X Slacks Creek NS NS NS *(P=.05)

Kippa Ring X Slacks Creek NS NS NS NS Kippa Ring X Boondall NS NS *(P=.01) NS

Boondall X Slacks Creek NS NS NS NS *=.05 **=.01 ***=.001

SCHOOLS AUST PINE OLIVE RAGWEED BAHAI GRASS Maleny X Kippa Ring NS **(P=.01) **(P=.003) *(P=.02) Maleny X Boondall NS NS NS NS Maleny X Slacks Creek NS NS NS NS Kippa Ring X Slacks Creek NS NS NS NS Kippa Ring X Boondall NS NS NS NS Boondall X Slacks Creek NS NS NS NS *=.05 **=.01 ***=.001

SCHOOLS RYEGRASS JOHNSON BERMUDA N Maleny X Kippa Ring *(P=.02) NS *(P=.02) Maleny X Boondall NS NS NS MALENY =94 Maleny X Slacks Creek NS NS NS KIPPA RING=128

Kippa Ring X Slacks Creek NS NS NS BOONDALL=124 Kippa Ring X Boondall NS NS NS SLACKS CR=34

Boondall X Slacks Creek NS NS NS

The preceding chart shows that of all the allergens applied, only one separates the schools in the manner predicted. Returning to the hypotheses, the three

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coastal schools were chosen because they might have demonstrated a response different from the montane Maleny school, if proximate vegetation has any bearing on response to plant allergens.

The results show that only one allergen distinguishes Maleny from the three coastal schools and that is Eucalyptus. Such a finding lends support to the view that the existence of coastal vegetation may be related to an allertant response, at least at the skin-reactivity level when all wheal sizes are measured. Figure 5.02 shows the percentage of responses of wheal diameter greater than zero, to demonstrate the percentage number of responses that could be clinically relevant despite the usual benchmark positive rating of 3mm. Insect pollinated plants often result in a response that is smaller than some other allergens. The rating of some Asteraceae flowers806 indicating smaller responses in skin tests compared to other entomophilous plants, is consistent with the smaller mean wheal diameter of ragweed in this study. Similarly, oil bearing fruit trees like almond and apricot result in smaller mean wheal806 diameters. More research is needed before smaller wheal responses can be discounted as meaningless, especially in relation to entomophilous species.

The contribution of smaller reactions to skin-prick tests, to asthma has not been adequately investigated. Other researchers have also shown that some responses to some insect pollinated plants may be smaller than grass and insect allergens, and there are a range of reasons why wheal sizes vary196, 767,

807, 808. Thus Figure 5.02 which shows responses greater than zero, has been included to complete the information set.

These smaller than 3mm readings are relevant to the construction of Table 5.03 which shows one entire set of response scores compared with each other entire set for the four schools in turn. It is not defensible to selectively cull the sets of scores to determine scores less than 3mm and and then compare the sets of differentially culled scores, so all scores were included as measured.

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Figure 5.02 Percentage of wheals greater than zero all schools Phase 1

PERCENTAGE OF POSITIVE RESPONSE WHEAL>0mm BY SCHOOL

60 Boondall %>0mm

Kippa Ring %>0mm 50 Slacks Creek%>0mm

Maleny %>0mm 40

PERCENTAGE OF SCHOOL GROUP SCHOOL OF PERCENTAGE 10

N ED MITE ACH DOG CAT ACIA PINE CLAD OLIVE BAHAI ST AC HNSO KRO ST O C J BERMUDA DU AU EUCALYPTRAGWE RYEGRASS CO ALTERNARIA ALLERGEN

The fact that all the urban coastal schools exhibited higher positive response rates, may also be attributed to the role of traffic in increasing sensitization to allergens93, 809, 810. Maleny did not have fewer atopic children than the other schools.

The response to Eucalyptus in all schools exceeds that of dog and cat. Eucalyptus allergen response exceeds Acacia and Alternaria, except in Maleny, and is close to levels of response with some grasses. Grasses typically induce large wheals, so by examining only wheals greater than 3mm, the bias is against describing Eucalyptus responses. Given that the mean wheal diameter for Eucalyptus in this study is 2.78 mm, this is a pertinent point. Ragweed, dog and Australian pine also lose representation with mean wheal size under 3mm as seen earlier in Table 5.02.

The response to olive is interesting, especially since olive growth in Brisbane is restricted to the occasional garden specimen which typically does not flower. The Brisbane climate with its wet summers is unlike the preferred olive-growing climate of the Mediterranean with its hot dry summers. Olive allergen was

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included in the test battery here in order to determine any probable cross- reactivity. Response to olive is substantial, and contributes evidence that the responses to particular plant allergens must be interpreted with care. The result here is similar to that described by Hirschwehr et al.772 in their description of allergic responses to ragweed allergen in the absence of likely sensitization to ragweed in Germany. Mentioned in Chapter 2, ragweed is rare in Germany.

The next set of comparisons involved ‘healthy’ and ‘asthma’ groups from all schools divided into two combined groups. Healthy children with no allergic symptoms were compared with children with asthma. The results of their skin- tests are shown in Figure 5.03 and Figure 5.04.

The response to Eucalyptus (24% of asthma participants) for the ‘asthma’ group was greater (irrespective of the wheal cut-off) than all other plant, animal and fungal allergens tested, except for Bermuda grass (27% of asthma participants). Positive response to Eucalyptus was only exceeded by Bermuda grass, dust mite and cockroach mix.

The inclusion of Eucalyptus in standard skin-prick test batteries is worthy of consideration, in view of the equivalent incidence of reactions compared to commonly tested allergens like Alternaria and grasses.

Following the allergen testing with children, Phase 2 began with allergen testing of adolescents and adults as described in section 4.3.

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Figure 5.03 Children with asthma: allergen responses (wheals >0, ≥3mm)

'ASTHMA ' GROUP PERCENTAGE OF SKIN-TEST RESPONSE BY ALLERGEN TYPE AND WHEAL DIAMETER 100 % WHEAL >0mm (N=97) 90 % POSITIVE, WHEAL ≥ 3mm (N=97) 80

RESPONSE 40

10 PERCENTAGE OF 'ASTHMA' GROUP SKIN TEST SKIN GROUP 'ASTHMA' OF PERCENTAGE 0 DOG CAT OLIVE BAHAI ACACIA BERMUDA DUSTMITE JOHNSON RAGWEED EUCALYPT AUST PINE RYEGRASS ALTERNARIA COCKROACH ALLERGEN CLADOSPORIUM

Figure 5.04 ‘Healthy’ children: allergen responses (wheals>0, ≥3mm)

'HEALTHY ' GROUP PERCENTAGE OF SKIN-TEST RESPONSE BY ALLERGEN TYPE AND WHEAL DIAMETER

100 % WHEAL > 0mm N=107 90 % POSITIVE, WHEAL ≥ 3mm N=107 80

RESPONSE 40

PERCENTAGE OF 'HEALTHY' GROUP SKIN TEST SKIN GROUP 'HEALTHY' OF PERCENTAGE 0 CAT CAT DOG OLIVE BAHAI ACACIA BERMUDA DUSTMITE JOHNSON JOHNSON EUCALYPT RAGWEED AUST PINE RYEGRASS ALTERNARIA COCKROACH

CLADOSPORIUM ALLERGEN

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5.2 Occurrence and discussion of reactions to allergens and terpenes in coastal and range adults and adolescents

Figure 5.05 shows the comparison of common allergens between phase 1 and phase 2 where ‘n’ refers to the number of participants in the group. Darker shades of pink and green colours are Phase 2 participants, lighter being Phase 1. Because age of participants can affect wheal size, as mentioned earlier, responses with wheals greater than 0mm have been shown for both groups in the two areas. Phase 1 participants were all less than 12 years of age while Phase 2 participants were adolescents or adults.

Figure 5.05 Comparison of percentage of common allergens response in Phase 1 and Phase2, wheal >0

Phase 1 + 2 Comparisons Redcliffe Phase 1 n=31

Redcliffe Phase 2 n=50 100 Maleny Phase 1 n=21 90

80 Maleny Phase 2 n=27

40 Percentage of n Percentage 30

0 RYE CAT DOG BAHAI ACACIA JOHNSON DUSTMITE EUCALYPT RAGWEED AUST PINE ALTERNARIA COCKROACH CLADOSPORIUM Allergen

Some allergen responses like cat and dog, in Redcliffe, are more prevalent in the older Phase 2 group. In Maleny the older group exhibited more occurrences of cat, grass and Eucalyptus responses. Response to dust mite is remarkably uniform between and among groups. In Redcliffe, the younger group showed more ragweed responses.

Figure 5.06 shows the results of allergen skin-prick testing for all those involved in Phase 2. All participants in these groups had been diagnosed by a physician as having asthma.

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Figure 5.06 Allergen percentage responses wheal ≥ 3mm for Phase 2 participants Coast and Range groups’ skin tests.

PHASE2 PERCENTAGE OF SKIN TEST RESPONSES WHEALS 3MM OR MORE

90 Redcliffe n=53 Maleny n=29 80

PERCENTAGE OFN, RESPONSE 3MM OR MORE 10

0 CAT RYE DOG BAHAI PRIVET ACACIA JOHNSON DUSTMITE RAGWEED EUCALYPT AUST PINE MELALEUCA ALTERNARIA COCKROACH CALLISTEMON CLADOSPORIUM ALLERGEN

Figure 5.07 Terpene and methanol positive responses ≥3mm Phase 2 Coast and Range groups skin tests.

PHASE 2 PERCENTAGE OF SKIN TEST RESPONSE WHEALS 3MM OR MORE

100 Redcliffe n=53 90 Maleny n=29

70 60 50

20 10 PERCENTAGE OF N RESPONSES GREATERTHAN 3MM 0

CINEOLE LIMONENE METHANOL OX CINEOLE BETA PINENE OX LIMONENE OX OX BETA PINE URSOLIC ACID ALPHA PINENE CARYOPHYLLENE BORNYL ACETATE OX ALPHA PINENE TERPENES/CHEMICALS

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Here the responses between the Coast and Range groups are very similar. There were many reactions which fell below the 3mm threshold. They are not shown here. Notably, Eucalyptus responses are again similar to the grass allergen responses, again inviting inclusion in standard allergen batteries. Acacia allergic response occurs at a rate less than is popularly believed, as does ragweed.

The relationships of allergy to these allergens and asthma is not very clear. Matters discussed in chapter 3 highlight the problems of equating allergen skin- prick response and asthmatic responses. With a view to investigating other possible causes of allertant reactions, some terpenes from Australian plants and methanol were applied in skin-testing procedure, along with the commercial allergens. The methanol was included to ascertain the reaction to a simple alcohol, since the Melaleuca produces large quantities of alcohol. It was also included in order to gauge reactivity to a fairly benign substance. Even though positive and negative controls were employed here, the methanol was an added measure of reactivity.

There were many reactions which fell below the 3mm threshold. They are not shown here either. Substantial responses are indicated for limonene for the Coast group more than for the Range group. Alpha pinene was represented more in the positive responses in the Range group. The Coast group were much more responsive to the methanol. See Figure 5.07. Wilcoxon non-parametric test for differences between groups revealed a significant difference for oxidized limonene: the Coast group showed significantly more positive responses than the Range group (p=.02). Comparisons were made using all responses, not just the ones 3mm and over, so the chart in Figure 5.07 does not reflect those differences accurately because information has been lost in setting a positive response at 3mm or more.

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Figure 5.08 shows all skin-test wheal diameters measuring greater than zero from Phase 2. The statistically significant difference between groups for oxidized limonene is now visible.

Figure 5.08 All responses greater than 1mm to terpenes and methanol

Phase 2 Percentage of Positive Skin-Test Response >0 to Terpenes

100 Redcliffe n=53 90 Maleny n=29

20 Percentage of n Positive Response of n Positive Percentage

0 CINEOLE LIMONENE METHANOL OX CINEOLE BETA PINENE OX LIMONENE OX BETA PINE URSOLIC ACID ALPHA PINENE ALPHA CARYOPHYLLENE BORNYL ACETATE BORNYL OX ALPHA PINENE OX ALPHA Terpenes

The chemicals selected occur in native trees in both the range and coast environments. The argument here is that for the coast, the flowering en masse of the Melaleuca quinquenervia, in particular, may induce sensitivities to those chemicals. The results here show that significantly more people in the coast sample respond to one of the major oxidized components of coast trees. What type of reaction and how much chemical is required to affect the lung function, is not known. Whether or not these chemicals affect lung function in day to day living is not known. The chemical profiles and information gleaned from experiment, discussed in chapter 2, indicates that several of the compounds included in this battery may be problematic.

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The next step in the investigation was to collect volatiles in the ambient air and explore the relationships to respiratory symptoms.

5.3 Outcomes and discussion of air sampling and respiratory diary analysis in coastal and highland adults and adolescents

Interpretation of results will focus on the coastal group for several reasons: the coast is the chief area of interest where the Melaleuca quinquenervia grows and the investigation of the relationship of the flowering of this tree to respiratory symptoms was the major goal of this enquiry; there were more participants in the coastal group (mean of 15 per month); and air pollution readings were available for analysis and this was not so for the Range group. The Range group was included as a contrast to ascertain if the patterns were similar to the coast. Analysis of the range results will be cursory.

Care in interpretation of standardized mean peak flow figures is required because measures of SPEF are the opposite of all other symptom measures used here. Because lung capacity decreases with asthma, a decrease in peak flow measures, SPEF, is associated with asthma, and an decrease in health. For all other symptom measures an increase in the measure means that the symptom worsens i.e. an increase in wheeze means that wheeze worsens and health decreases.

Interpretation of symptom relationships will be most powerful for the SPEF measure since this is the only one of a continuous scale. Next most powerful is the 10-point scale used to measure asthma score. Asthma score is a subjective rating. The remaining symptom scales are four-point scales and therefore offer less range of response. The scales for preventer usage and reliever usage are only three point scales (increase, decrease or remain the same). Thus more interpretative weight will be afforded the SPEF variable because of the continuous and measured scale.

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Correlations for the Coast group The statistical significance of correlations of symptoms with all other variables measured showed the strong relationship between SPEF and NOX (p= .0001) for several lagged measures for the Coast group ( see Appendix O). A decrease in NOX was associated with increased SPEF. Conversely, an increase in NOX would be associated with a decrease in SPEF. Increase in NOX, though, is an annual and a seasonal event. This is reflected in the findings of others. In a Brisbane study investigating the associations between outdoor pollution and hospital admissions108 no overall association over the period 1987-1994, was found for NO2. There were, however, significant positive associations for asthma and respiratory disease in autumn, winter and spring.

Figure 5.09 shows the cyclic and seasonal pattern of NOX in Brisbane over ten years from 1995-2004. NOX may be a covariate of the causal phenomenon affecting autumn asthma in Brisbane, since NOX always increases in autumn and winter.

Other associations are positive i.e. high levels of temperature, wind speed; Myrtaceae pollen and Acacia pollen are associated with high SPEF and therefore better lung capacity. All these variables are likely to be seasonal associations as they all occur together in the spring sampling period.

For the ‘asthma score’ variable, decreased atmospheric pressure, and decreased alpha pinene, beta pinene, cineole, Cladosporium, Acacia and Pinus pollen appear to associated with an increased asthma score. Increased temperature is associated with increased asthma score.

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NOX all 1995-2004

0.100

NO NO2

0.050 ppm

0.000 1/01/1995 1/05/1995 1/09/1995 1/01/1996 1/05/1996 1/09/1996 1/01/1997 1/05/1997 1/09/1997 1/01/1998 1/05/1998 1/09/1998 1/01/1999 1/05/1999 1/09/1999 1/01/2000 1/05/2000 1/09/2000 1/01/2001 1/05/2001 1/09/2001 1/01/2002 1/05/2002 1/09/2002 1/01/2003 1/05/2003 1/09/2003 1/01/2004 1/05/2004 1/09/2004

DATE

Use of reliever medication was associated with other fungi (Lag 4 only) and increased linalyl acetate and linalool (both on same day and Lag 2). Preventer medication was associated with increased linalool (same day, Lag 4 and Lag 5) and linalyl acetate (Lags 4 and 5). Increased ozone (all lags ) was strongly associated with preventer usage. PM10 was associated positively on some days (same day and Lag 1) with preventer usage and with difficulty breathing (Lag1). Myrtaceae pollen was positively associated with preventer usage on two days (same and Lag1). Increased thunder was associated with preventer use on Lag 2 to Lag 5 inclusive. This relates to the very strong association between decreased atmospheric pressure (this occurs with thunder) for wheeze, difficulty breathing, cough and itchy eyes on many days.

Increased grass pollen is associated with sneezing, blocked nose and itchy nose and Asteraceae pollen was positively associated with sneezing.

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Correlations for the Range group Increased temperature, wind speed cineole and benzaldehyde are associated with increased SPEF and therefore good lung function. Decreased Myrtaceae pollen is associated with increased SPEF. Logically, the reverse is most likely true, and a relationship between decreased SPEF and increased Myrtaceae pollen on the range is a reasonable interpretation.

For asthma score on the range, positive associations with increased Myrtaceae pollen and other fungi were found (see Appendix P).

Use of reliever medication was associated strongly with increases in other fungi, as was preventer use. Reduced atmospheric pressure was strongly associated with preventer use at all lags. Alpha pinene increase was associated with difficulty breathing as was reduced precipitation.

Increased Myrtaceae pollen was associated with sneezing (same day) and blocked nose (Lag 4). Increased Casuarina pollen was associated with blocked nose (Lag 3 and Lag 4). Increased linalyl acetate was associated with runny nose (same day and Lag 4).

Determining significant contributors to symptoms Principal components analysis revealed that the volatiles detected in the air samples load differently upon the first principal factor. With the entire variable set in the analysis the result is the same: volatiles load on the first factor similarly for many of them but differently enough to warrant examining the contributions of each. Notably, benzoic acid (-.24) was orthogonal to the other volatiles. Benzaldehyde did not load to the same degree (.31) as the terpenes (.85 to .91). (See Appendix R for detail of the loadings.)

Because air sampling occurred only every three days, there are limitations upon regression analysis caused by the paucity of sample dates. This limitation

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severely limits the degrees of freedom. Modelling of terpenes is well known to be problematic and this was mentioned in Section 2.2.4.2. Given the fact that no chemical standards were employed and that the number of sample days was limited to 54 minus six discarded samples, that is 48 samples, stepwise regression was used to determine which variables would be significant in predicting various symptoms. All variables were used in an ‘all in’ model. Calculations were base on ‘same day’ (marked as ‘S’) and lags up to five days (marked as 1,2,3,4 or 5). They were not mixed i.e. independent variables were either ‘same day’ or all were ‘lag 1 day’ etc. The results for the Coast and Range are shown in Table 5.04 a-i and Table 5.05 a-h.

Because stepwise regression has a reputation for less accuracy than general linear regression811, the results of the stepwise regression were used in a general linear model to verify any significant contributions. Verification of the significance of the stepwise regression R sq will be presented following the tabulation of all the significant variables. The following pages contain all those variables which achieved significance below 0.1. The key at the bottom of each page shows the degree of significance attained. Many of the variables are not meaningful. More meaningful significant variables are shaded in grey. For example, in the first column of Table 5.04a, increasing atmospheric pressure significantly predicted increasing SPEF (p >.01≤.05 at lag 5 days; p>.05≤0.1 at lag 3 days). Lack of grey shading means that this relationship is not regarded, by the writer, as particularly noteworthy.

Again, caution is required for interpretation of SPEF which is marked yellow to alert the reader that its increase refers to a diminution of asthma symptoms. This is the opposite direction to the other variables. An increase in wheeze would occur with a decrease in SPEF when asthma severity increases. SPEF increases when a participant is healthy and decreases during the asthmatic response . When all the other symptoms increase the participant would be expected to feel more unwell.

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Table 5.04a Statistically significant predictive variables for symptoms in coastal group of participants

HIGHER LOWER COASTAL GROUP HIGHER LOWER LESS ATMOS. ATMOS. TEMP. TEMP. RAINFALL PHASE 2 PRESSURE PRESSURE DIRECTION SYMPTOM OF EFFECT ↑↓↑↓↓ ╬ S ☼3● 5 HIGHER ↑ ☼3,5 PEAKFLOW ☼1,3,5 ●1,2,3 ASTHMA ↑ ■4 RATING ☼4,3 ●S☼2 ↑ ►2 ●1 DRY COUGH ■2●1 ↑ ☼S,3 ☼4 WHEEZE ►4 ●S,4 ☼1●2 DIFFICULTY ↑ ☼2,3 ► 3■4 BREATHING ● S,3 ↑ ►2 RUNNY NOSE

↑ SNEEZING

BLOCKED ↑ ● 5 ● 3 ● 5 NOSE

↑ ● S ● 2 ►5 ☼ 5 ITCHY NOSE

↑ ITCHY EYES

RELIEVER MEDICATION ↑ ●3 ☼4 ☼2 ☼ S USAGE

PREVENTER MEDICATION ↑ ■ 2,4,5 ●S☼1,2 USAGE COASTAL GROUP ☼ p>.05≤0.1 PHASE 2 KEY ●p >.01≤.05

■ p≥.0001≤.005 Notable variable relationship ► p>.005≤.01 ╬ p< .0001

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Table 5.04b Statistically significant predictive variables for symptoms in coastal group of participants

LOWER COASTAL GROUP MORE LESS HIGHER LOWER RELATIVE THUNDER THUNDER WINDSPEED WINDSPEED PHASE 2 HUMIDITY DIRECTION SYMPTOM OF EFFECT ↑↓ ↑ ↓ ↓

HIGHER ↑ ☼5 PEAKFLOW

ASTHMA ↑ ☼1 ●1 RATING

↑ ☼5●3 DRY COUGH

↑ ●3,5 WHEEZE

DIFFICULTY ↑ ☼5 BREATHING

↑ ■ 4 RUNNY NOSE

↑ SNEEZING

BLOCKED ↑ ● 3 NOSE

↑ ►1,4 ● 2 ITCHY NOSE

↑ ● 3 ● 1 ITCHY EYES

RELIEVER MEDICATION ↑ ►2 USAGE

PREVENTER MEDICATION ↑ ●1 ●S ☼1 USAGE COASTAL GROUP ☼ p>.05≤0.1 PHASE 2 KEY ●p >.01≤.05

Notable variable relationship ► p>.005≤.01 ■ p>.0001≤.005

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Table 5.04c Statistically significant predictive variables for symptoms in coastal group of participants

COASTAL GROUP PM OZONE OZONE NO NO PHASE 2 10 2 2

DIRECTION SYMPTOM OF EFFECT ↓↑↓↑↓ ╬1 HIGHER ↑ ►2,3 PEAKFLOW ■4

ASTHMA ↑ ☼3 RATING

↑ DRY COUGH

↑ ☼3 ☼2●S WHEEZE

DIFFICULTY ↑ ☼2 ●5 ●2 BREATHING

↑ RUNNY NOSE

↑ SNEEZING

BLOCKED ↑ ● 2 NOSE

↑ ☼ 5 ● 3 ITCHY NOSE ●2,4 ☼3 ☼S ↑ ■ 5 ITCHY EYES

RELIEVER MEDICATION ↑ USAGE

PREVENTER MEDICATION ↑ ● 2,3,4,5 USAGE COASTAL GROUP ☼ p>.05≤0.1 PHASE 2 KEY ●p >.01≤.05

■ p≥.0001≤.005 Notable variable relationship ► p>.005≤.01 ╬ p< .0001

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Table 5.04d Statistically significant predictive variables for symptoms in coastal group of participants

COASTAL GROUP MYRTACEAE MYRTACEAE GRASS GRASS PINE PHASE 2 POLLEN POLLEN POLLEN POLLEN POLLEN

DIRECTION SYMPTOM OF EFFECT ↑↓↑↓↓

HIGHER ↑ ●4 ●3 PEAKFLOW

ASTHMA ↑ ►5 ●2 ☼5 RATING

↑ ☼2 ●1 DRY COUGH

↑ WHEEZE

DIFFICULTY ↑ ●4 BREATHING

↑ RUNNY NOSE

↑ ● 5 ● 1,3 SNEEZING

BLOCKED ↑ ● S NOSE

↑ ☼ 2 ITCHY NOSE

↑ ITCHY EYES

RELIEVER MEDICATION ↑ ● S,1,5 USAGE

PREVENTER MEDICATION ↑ ● 4 ●S ☼1 USAGE COASTAL GROUP ☼ p>.05≤0.1 PHASE 2 KEY ●p >.01≤.05

Notable variable relationship ► p>.005≤.01 ■ p>.0001≤.005

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Table 5.04e Statistically significant predictive variables for symptoms in coastal group of participants

COASTAL GROUP ASTER ASTER ACACIA ACACIA 'OTHER' PHASE 2 POLLEN POLLEN POLLEN POLLEN POLLENS

DIRECTION SYMPTOM OF EFFECT ↑↓↑↓↑

HIGHER ↑ ☼S,3●5 PEAKFLOW

ASTHMA ↑ ●2 RATING

↑ ●S ☼S DRY COUGH

↑ ●2 ☼1,5 WHEEZE

DIFFICULTY ↑ BREATHING

↑ RUNNY NOSE

↑ ■ 5 SNEEZING

BLOCKED ↑ ● 1 ►2 ☼ 4 NOSE

↑ ● 2 ITCHY NOSE

↑ ITCHY EYES

RELIEVER ☼S,2,3 MEDICATION ↑ ☼4 ►1 USAGE

PREVENTER MEDICATION ↑ USAGE COASTAL GROUP ☼ p>.05≤0.1 PHASE 2 KEY ●p >.01≤.05

Notable variable relationship ► p>.005≤.01 ■ p>.0001≤.005

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Table 5.04f Statistically significant predictive variables for symptoms in coastal group of participants

COASTAL GROUP 'OTHER' CLADOSP. ALTERN. ALTERN. OTHER PHASE 2 POLLENS FUNGI FUNGI FUNGI FUNGI

DIRECTION SYMPTOM OF EFFECT ↓↓↑↓↓

HIGHER ↑ ●4 PEAKFLOW

ASTHMA ↑ ► 5 RATING

↑ ► 1 ■ S,2 DRY COUGH

↑ ●S,1,2 ●2 WHEEZE

DIFFICULTY ↑ ●S BREATHING ► S ☼1 ●S ↑ ●2,3,5 RUNNY NOSE

↑ ☼ 4 ● 5 ● 2 ☼S SNEEZING

BLOCKED ↑ ● 1 ■2 NOSE ●S,3 ● 4 ● 2 ↑ ►4 ITCHY NOSE

↑ ☼4 ITCHY EYES

RELIEVER ☼ S ☼2 MEDICATION ↑ ● 1,4 USAGE

PREVENTER MEDICATION ↑ ● 3 USAGE COASTAL GROUP ☼ p>.05≤0.1 PHASE 2 KEY ●p >.01≤.05

Notable variable relationship ► p>.005≤.01 ■ p>.0001≤.005

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Table 5.04g Statistically significant predictive variables for symptoms in coastal group of participants

COASTAL GROUP ALPHA LIMONENE LIMONENE CINEOLE CINEOLE PHASE 2 PINENE

DIRECTION SYMPTOM OF EFFECT ↑↓↑↓↓

HIGHER ↑ ☼S ■ 3 PEAKFLOW

ASTHMA ↑ ☼2 ●2,4 RATING

↑ DRY COUGH

↑ WHEEZE

DIFFICULTY ↑ BREATHING

↑ ●5 RUNNY NOSE

↑ ■ S ●1 SNEEZING

BLOCKED ↑ NOSE

↑ ● 2 ☼4 ITCHY NOSE

↑ ITCHY EYES

RELIEVER MEDICATION ↑ USAGE

PREVENTER MEDICATION ↑ ● 2,3 USAGE COASTAL GROUP ☼ p>.05≤0.1 PHASE 2 KEY ●p >.01≤.05

Notable variable relationship ► p>.005≤.01 ■ p>.0001≤.005

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Table 5.04h Statistically significant predictive variables for symptoms in coastal group of participants

COASTAL GROUP BETA BETA LINALYL LINALYL LINALOOL PHASE 2 PINENE PINENE ACETATE ACETATE

DIRECTION SYMPTOM OF EFFECT ↑↓↑↑↓

HIGHER ↑ PEAKFLOW

ASTHMA ↑ ●2 ●☼ RATING

↑ ☼ 2 DRY COUGH

↑ ●S☼1,2 WHEEZE

DIFFICULTY ↑ ●4 ●4 ☼2 BREATHING

↑ ● 5 RUNNY NOSE

↑ ● 1 SNEEZING

BLOCKED ↑ NOSE

↑ ● 2 ITCHY NOSE

↑ ITCHY EYES

RELIEVER MEDICATION ↑ ■ S●2 USAGE

PREVENTER MEDICATION ↑ ● S,4 ● S USAGE COASTAL GROUP ☼ p>.05≤0.1 PHASE 2 KEY ●p >.01≤.05

Notable variable relationship ► p>.005≤.01 ■ p>.0001≤.005

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Table 5.04i Statistically significant predictive variables for symptoms in coastal group of participants

COASTAL GROUP BENZALD- BENZALD- BENZOIC CAMPHOR PHASE 2 EHYDE EHYDE ACID

DIRECTION SYMPTOM OF EFFECT ↑↑↓↑

HIGHER ↑ PEAKFLOW

ASTHMA ↑ ☼5 ☼2 RATING

↑ ■ S DRY COUGH

↑ WHEEZE

DIFFICULTY ↑ ☼2,3 BREATHING

↑ ☼ 4 RUNNY NOSE

↑ ► 4 SNEEZING

BLOCKED ↑ ● 2 ● S ☼ 2 NOSE

↑ ☼ 2 ● 4 ITCHY NOSE

↑ ITCHY EYES

RELIEVER MEDICATION ↑ USAGE

PREVENTER MEDICATION ↑ USAGE COASTAL GROUP ☼ p>.05≤0.1 PHASE 2 KEY ●p >.01≤.05

Notable variable relationship ► p>.005≤.01 ■ p>.0001≤.005

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Table 5.05a Statistically significant predictive variables for symptoms in Range group of participants

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Table 5.05b Statistically significant predictive variables for symptoms in Range group of participants

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Table 5.05c Statistically significant predictive variables for symptoms in Range group of participants

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Table 5.05d Statistically significant predictive variables for symptoms in Range group of participants

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Table 5.05e Statistically significant predictive variables for symptoms in Range group of participants

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Table 5.05f Statistically significant predictive variables for symptoms in Range group of participants

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Table 5.05g Statistically significant predictive variables for symptoms in Range group of participants

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Table 5.05h Statistically significant predictive variables for symptoms in Range group of participants

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Important predictors of SPEF Pinus and Myrtaceae pollens featured in the Coast group as being associated with lower SPEF ( Lag 3) and increased asthma score ( Lag 5 ). See Figure 5.10.

Figure 5.10 Pinus (top graph) and Myrtaceae (bottom graph) pollens and Coast SPEF

SPEF & PINUS POLLEN COUNT

2.5

COAST SPEF

2 PINE

1.5

0.5

0 MEAN SCORE SPEF/POLLEN COUNT -0.5

-1 0 7 14 21 28 35 42 49 56 63 70 77 84 91 98 105 112 119 126 133 140 147 154

OCT 1,2000 TO DAYNUMBER APRIL 1,2001 TO DEC 26, 2000 JUNE 6, 2001

SPEF & MYRTACEAE POLLEN COUNT

MYRTACEAE 39

COAST SPEF 34

9 MEAN SCOREMEAN SPEF/POLLEN COUNT

-1 0 7 14 21 28 35 42 49 56 63 70 77 84 91 98 105 112 119 126 133 140 147 154

OCT 1,2000 TO DAYNUMBER APRIL 1,2001 TO DEC 26, 2000 JUNE 6, 2001

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The further analysis of the significant predictors of the symptom variables led to numerous verifications. Optimal employment of significant predictors for SPEF in the Coast group involves Acacia, ozone, alpha pinene and Pinus pollen at Lag 3. The stepwise regression model result for the four variables is shown in Table 5.06.

Table 5.06 Partial R sq for stepwise regression on coast SPEF variable

Variable Number Partial Model Step Entered Vars In R-Square R-Square C(p) F Value Pr > F

1 OZONE3 1 0.2542 0.2542 16.7801 12.61 0.0011 2 ACACIA3 2 0.0721 0.3263 13.7764 3.85 0.0575 3 ALPHA PINENE3 3 0.1403 0.4666 6.0372 9.20 0.0045 4 PINE3 4 0.0437 0.5103 5.0000 3.04 0.0904

R sq for the model is 51%. The model is significant at p< .0001.

The model refers to an increase in Acacia and ozone and a decrease in Pinus pollen and alpha pinene, all lagged 3 days. The model significantly predicts SPEF for the Coast group.

Conversely, it may be said that a decrease in SPEF is predicted by an increase in Pinus pollen and alpha pinene at a 3 day lag. Alpha pinene accounts for 14 % of the variance in scores for coast SPEF.

The full stepwise program showing that the four variables selected accounted for the greatest partial Rsq is shown in Appendix Q . Only four variables could be selected because of the limited number of sampling days. By using 4 variables only, an allowance of approximately 10 degrees of freedom per variable has been made, and a reasonable estimate of the amount of variance explained in the symptom measure can be achieved.

The interpretation of the contribution of the terpenes to symptom score variance needs to be made with care. Principal components analysis shows that many of

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the terpenes load on the first factor (see Appendix R). Many of the terpenes occur together as seen in Figure 5.12.

In this model ozone and Acacia are possible seasonal surrogates since ozone is higher in the summer and Acacia pollen is heaviest in spring. For the purposes here, ozone and Acacia may simply be seasonal markers and may not be causal in affecting SPEF.

Inspection of the plots of volatiles, indicates that they are found in both spring and autumn, the seasons of interest here. A graph of the coast SPEF error associated with the group mean is shown in Figure 5.11 before the plots of volatiles are added.

Figure 5.11 Coast group mean SPEF error

COASTAL SPEF ERROR

2.5

1.5

0.5

SPEF ERROR SPEF -0.5

-1

-1.5

-2

-2.5 0 7 14 21 28 35 42 49 56 63 70 77 84 91 98 105 112 119 126 133 140 147 154 OCT 1,2000 DAYNUMBER APRIL 1,2001 TO TO DEC 26,2000 JUNE 6, 2001

Figure 5.12 shows the coast SPEF group mean with some of the terpenes superimposed.

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Figure 5.12 Coast SPEF, precipitation and some terpene volatiles

SPEF, PRECIPITATION TERPENES SAME DAY COASTAL REGION

5 25

4.5

4 20

3.5

3 15

2.5

COAST SPEF 2 10 BETA PINENE 1.5 CAMPHOR ALPHA PINENE

1 5 mm PRECIPITATION CINEOLE LIMONENE 0.5 LINALOOL 0 0 LINAYL ACETATE

MEAN SCORE SPEF/ LOG RANK VOLATILES LOG SCORE SPEF/ MEAN PRECIPITATION -0.5

-1 -5 0 7 14 21 28 35 42 49 56 63 70 77 84 91 98 105 112 119 126 133 140 147 154

OCT 1,2000 TO DAYNUMBER APRIL 1,2001 TO DEC 26, 2000 JUNE 6, 2001

Figure 5.12, above, shows that the volatiles occur in dry times and in wet. Precipitation is shown as the green line. Volatile emissions and storms go together. In dry times, clusters of volatiles are similarly emitted as can be seen in the figure above.

More detail regarding thunder and volatiles can be seen in Figure 5.13 which shows only days on which rain was recorded at Brisbane Airport, the closest monitoring station for the Coast group. None of the volatiles recorded occurred on a dry day. Volatiles would have been captured before the rain fell since the rain then washes the air clean. Thunder is marked with a yellow box on the x- axis. The emission of volatiles along with thunder explains the strong correlations for both groups for a range of symptoms and a drop in atmospheric pressure, which accompanies thunder ( see Appendices O and P).

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Figure 5.13 Rainy day volatile emissions

"RAIN DAYS ONLY" VOLATILES

3 100

90 2.5

80 2 70

1.5 60 COAST SPEF LIMONENE 1 50 LINALOOL LINAYL ACETATE 40 BETA PINENE 0.5 CINEOLE ALPHA PINENE MEAN WINDSPEED kph 30 CAMPHOR 0 THUNDER PRESMEAN 20 WINDMEAN MEAN SCORE/ LOG RANK OF VOLATILES LOGRANK SCORE/ MEAN -0.5 10

-1 0 0 7 14 21 28 35 42 49 56 63 70 77 84 91 98 105 112 119 126 133 140 147 154 DAYNUMBER OCT 1,2000 APRIL 1,2001 TO TO DEC 26,2000 JUNE 6, 2001

Figure 5.14 Coast group benzoic acid and benzaldehyde and SPEF

COAST BENZOIC ACID, BENZALDEHYDE , SPEF

3 BENZOIC ACID RBENZALD 2.5 COAST SPEF

1.5

0.5

LOG RANG VOLATILE/ SPEFGROUP SCORE MEAN -0.5

-1 0 7 14 21 28 35 42 49 56 63 70 77 84 91 98 105 112 119 126 133 140 147 154 OCT 1,2000 DAYNUMBER APRIL 1,2001 TO TO DEC 26,2000 JUNE 6, 2001

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Storms, rain and pressure do not emerge as significantly predictive of coastal group mean SPEF. The reason may be that the volatiles occur on dry and wet days, as is seen in Figure 5.12 which shows precipitation and volatiles.

Figure 5.14 shows the distribution of benzaldehyde and benzoic acid for the Coast group. These substances do not appear to contribute to decrease in SPEF although benzoic acid is a significant predictor of difficulty in breathing for the Coast group only.

Further analysis of the significant predictive variables for coast SPEF revealed the ozone is a seasonal surrogate, as is NOX, as the significant effects on predicting SPEF disappear in a seasonal analysis seen in Table 5.07.

Table 5.07 NOX and ozone as predictors of Coast SPEF in combined seasons and separately in spring and autumn

SOLE PREDICTOR OF COMBINED AUTUMN SPEF IN COAST SPRING ONLY SEASONS ONLY GROUP – PROC GLM

R SQ P R SQ P R SQ P

NITROGEN DIOXIDE LAG 3 .12 <.0001 .04 .05 .01 N.S

OZONE LAG 3 .26 <.0001 .03 N.S. .04 N.S.

Further tables showing the relative contributions of the particles, nitrogen dioxide and ozone for spring and autumn combined, and separately are shown in Appendices AB, AC and AD. The contribution (R2) of these three variables in predicting SPEF for the Coast group ranges from .06 in autumn to .32 in spring. When alpha pinene is added to the model the R2 increases to .26 in autumn and to .38 in spring. In spring the pinene is a covariate of pine pollen incidence. Further, there are strong seasonal differences in the predictive ability of volatiles as shown in Table 5.08. Lag 3 has been chosen because beta pinene is a significant predictor for the Coast group at that lag.

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Table 5.08 Significant predictors of SPEF for Coast air samples

SPRING COAST AUTUMN GROUP Prediction of coast SPEF Prediction of coast SPEF GLM model one predictor GLM model one predictor TERPENE DEGREES OF FREEDOM: USED AS DEGREES OF FREEDOM: Model 1; Error 21 SOLE Model 1; Error 20 R squared Significance PREDICTOR R squared Significance P = P = .06 .22 Linalool Lag 3 .13 .08

.07 .22 Linalyl .16 .06 Acetate Lag 3 .002 .82 Alpha Pinene .21 .03 Lag 3 .002 .82 Beta Pinene .21 .029 Lag 3 .009 .65 Cineole Lag 3 .08 .18

.03 .41 Camphor .08 .18 Lag 3 .016 .55 Limonene .16 .06 Lag 3

Asthma scores

When applied to the coastal group, the asthma score at Lag 5 explains most variance in the self-reported asthma score . Although Myrtaceae predicted 19% of the variance in asthma score in a stepwise regression, and the General Linear Model analysis resulted in the same R sq for a combination of variables shown in Appendix U, once more a seasonal effect is evident. Table 5.09

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shows the differences in explained variance in each season and for the combined seasons. Table 5.09 Asthma score seasonal predictors for Coast group. SOLE PREDICTOR OF ASTHMA COMBINED AUTUMN SPRING ONLY SCORE IN SEASONS ONLY GROUP – PROC GLM

R SQ P R SQ P R SQ P

MYRTACEAE LAG 5 .05 <.0001 .00009 N.S .16 <.003

COAST GROUP

Many of the predictors that are evident in stepwise analyses disappear when subjected to the scrutiny of the General Linear Model. The observations made here are not exhaustive but reflect the predictors of most import for the purposes in this thesis. The error for the group mean of the Range group is shown in Figure 5.15 and visual inspection reveals that the decline of SPEF in autumn in the Range group is not marked, as in the coastal group, seen previously in Figure 5.11.

Figure 5.15 Range group error for SPEF mean

HIGHLAND SPEF ERROR

2.5

1.5

0.5

-0.5

-1 ZSCORE/LOG OF RANK OF VOLATILE OF RANK ZSCORE/LOG -1.5

-2

-2.5 0 7 14 21 28 35 42 49 56 63 70 77 84 91 98 105 112 119 126 133 140 147 154 DAYNUMBER

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For the Range group maximal variance in SPEF is explained at Lag 1 and Lag 4. In the model for Lag 4 (significance p>.004) an increase in SPEF is associated with a decrease in beta pinene and a decrease in precipitation (see Appendix T), and increases in Cladosporium and cineole. Conversely, reduced SPEF as experienced during asthma exacerbations are associated with increased beta pinene and precipitation and reduced Cladosporium and cineole. The reduction of cineole at such times is consistent with research concerning this terpene and its protective function against airway inflammation812.

Table 5.10 Partial R-square for Range group contributing to SPEF score

Variable Number Partial Model Step Entered Vars In R-Square R-Square C(p) F Value Pr > F

1 CLADOSPORIUM 4 1 0.1147 0.1147 13.0485 4.01 0.0539 2 BETA PINENE 4 2 0.0882 0.2028 10.8605 3.32 0.0785 3 CINEOLE 4 3 0.1106 0.3134 7.6083 4.67 0.0391 4 PRECIPITATION 4 4 0.0970 0.4105 5.0000 4.61 0.0406

Table 5.10 shows that beta pinene accounts for 9% of the variance in scores of SPEF in the Range group for Lag 4. This is smaller than 14 % seen in the Lag 3 model for SPEF in the Coast group. Consistency is evident here.

Benzoic acid and Benzaldehyde In both the sampling areas these substances occurred with only a little variation. Figure 5.16 shows precipitation, mean range SPEF and terpenes for the Range group. The distribution of benzoic acid and benzaldehyde are shown in Figure 5.17.

The relationship of linalool to reliever and preventer medication usage is of interest here especially since both the Range and the Coast groups exhibit marked effects.

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Figure 5.16 Range terpenes, SPEF, thunder score & precipitation.

RANGE PRECIPITATION , THUNDER + SAME DAY VOLATILES

2.5 120

2 100

THUNDSC 1.5 ALPHA PINENE 80 BETA PINENE CINEOLE 1 CAMPHOR LIMONENE 60

MM LINALOOL 0.5 LINAYL ACETATE RANGE SPEF 40 THUNDSC

LOG OF RANK OF VOLATILE LOG OFRANK 0 PREC

20 -0.5

-1 0 0 7 14 21 28 35 42 49 56 63 70 77 84 91 98 105 112 119 126 133 140 147 154 DAYNUMBER

Figure 5.17 Benzoic acid and benzaldehyde for range area

RANGE PRECIPITATION , THUNDER + SAME DAY VOLATILES

2.5 120

2 100

1.5 80 THUNDSC 1 RANGE SPEF BENZOIC ACID 60 MM RBENZALDEHYDE 0.5 PREC

LOG OF RANKOF VOLATILE 0

20 -0.5

-1 0 0 7 14 21 28 35 42 49 56 63 70 77 84 91 98 105 112 119 126 133 140 147 154 DAYNUMBER

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Table 5.11 shows the seasonal differences in reliever medication as predicted by Lag 5 linalool for Coast and Range groups. A GLM regression using only one predictor with degrees of freedom 1 (model) and 20 (error) demonstrate the seasonal differences. For the Coast group in autumn, reliever usage is much greater with 18% of the variance of the reliever score explained. In spring it is only 3 %. Given that Melaleucas produce their flowers and large amounts of linalool in the autumn, the use of the reliever medication would support a relationship between the linalool levels in the air and the flowering of the plants.

For the Range group the division is not marked. The Range group has local growth of privet which also produces large amounts of linalool and may explain why there is not the seasonal difference. The large leaved privet blooms in summer peaking in December and January. The effect of a probable chemical dumping of DEET (diethyl toluamide) as evident in the air samples on one occasion indicates that there is a drift of volatiles that can occur in autumn. The DEET episode resulted in a substantial amount of this chemical being found in the air sample.

The nearby wetland expanse is a favourite dumping ground for chemicals. Following that DEET, event volatiles consistent with vegetation damage (hexanal and hexanoic acid) were detected in the coast sample for some days. In the following sample collected (2 days later) those chemicals were found in the range samples. Wind patterns change in winter and wind rose patterns, from the Bureau of Meteorology, indicate that volatiles may be blown up onto the range in winter and not in summer because of wind direction changes. Using this logic, the volatiles emitted by trees on the coast in the cooler months may affect range residents. See Appendices W and X for visual representation of wind directions in January and May in Brisbane. A similar analysis of preventer usage illustrates the marked seasonal differences. See Table 5.12.

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Table 5.11 Linalool seasonal effects upon reliever usage for coast and Range groups

LINALOOL COMBINED SPRING ONLY AUTUMN ONLY LAG 5 AS SOLE SEASONS PREDICTOR USING R SQ P R SQ P R SQ P PROC GLM DF 1/19 COAST RELIEVER USAGE LAG 5 .003 N.S. .03 N.S. .18 <.05 GROUP

RANGE RELIEVER USAGE LAG 5 GROUP .07 .07 .11 N.S. .10 N.S.

Table 5.12 Linalool seasonal effects upon preventer usage for Coast and Range groups

LINALOOL COMBINED SPRING ONLY AUTUMN ONLY LAG 5 AS SOLE SEASONS PREDICTOR USING R SQ P R SQ P R SQ P PROC GLM DF 1/19 COAST PREVENTER USAGE LAG 5 .10 <.03 .02 N.S. .22 <.03 GROUP

RANGE PREVENTER USAGE LAG 5 .13 .007 .14 <.07 .25 <.02 GROUP

Preventer usage is strongly seasonal for the Coast group as predicted by lag 5 linalool levels in ambient air. Overall, more variance in preventer usage is accounted for on the range although more of that group’s usage is predicted by linalool in autumn, just the same as for the Coast group. The role of volatile drift is worthy of more investigation and could be responsible for this phenomenon. The blooming of privet in summer may again be a reason explains the increased variance explained by the Range group.

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Finally the matter of whether one area experienced more volatile emission than the other was explored by a simple additive model. Of the air sample results, all the ‘low’, and ‘medium’ and ‘high’ ratings for each volatile were weighted and added together to obtain a total volatile score. Those total scores formed two sets for a t-test comparison. The result indicated that the coast set of volatiles was significantly more in quantity as defined by the additive process just described. The distribution of the total scores for combined volatiles is shown in Figure 5.18. The graph demonstrated the high levels of volatiles in the spring season for the coast area and the lower levels in all seasons for the range. The increased asthma in autumn in the coastal areas invites questions about the qualities of the volatiles in autumn rather than just the quantities. Investigations here have been based upon approximations from GCMS graphs.

The merged peaks on the graphs mask a number of chemical substances which have not been elucidated here. This matter was mentioned earlier, in chapter 4. The analysis of the privet flowers particularly demonstrates that potent allergens are also emitted at flowering time. The complexity of the chemical mixes that are part of flowering plants needs to be considered.

Figure 5.18 Total volatiles in coast and range areas.

COAST VS RANGE "TOTAL VOLATILES" SCORE

COAST 10 RANGE

6 "TOTAL VOLATILES" SCORE VOLATILES" "TOTAL

0 0 7 14 21 28 35 42 49 56 63 70 77 84 91 98 OCT 1,2000 105 112 APRIL119 1,2001126 133 TO 140 147 154 DAYNUMBER TO DEC 26,2000 JUNE 6, 2001

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5.4 Outcomes and discussion of plant and fungi analyses A number of plant analyses were undertaken for this project. Earlier flower and leaf analyses provided information that formed the basis for allergens testing in phase 2. The compounds located in the flower were also expected to be found in ambient air at flowering time. These lists forme the basis for recognition of some of the compounds detected in the GCMS graphs from ambient air sampling.

There were three main areas of enquiry for plant analyses: A) What were the chemicals in flowers compared with trees? B) Can fungi take up chemicals from a floral growth substrate? C) What volatile gases emanate from flowers?

The focus of enquiry was the plants that are most numerous in the coastal areas, either because they occur naturally or because they have been planted in suburban gardens.

5.4.1 Headspace analyses

Sealed in aluminium bags, this first batch of plants was personally couriered to the laboratory in Tasmania, so they arrived earlier than the usual courier system that took 2 days by air express. They were analysed 18 hours after picking with insertion of the SPME (solid phase micro-extraction) needle to obtain chemical constituents of the "headspace", the air surrounding the flowers. Absorption of the headspace was allowed before injection into a gas chromatograph as described in chapter 4 GCMS analysis revealed the following: Relative amounts of terpenes: H= high levels; M= medium; L= Low.

Eucalyptus “Summer Beauty” (Showy red flowered variety planted as specimen tree in gardens): caryophyllene (H), β phellandrene, bicyclogermacrene, humulene, bornyl acetate (H), alpha pinene (H), β pinene (M), 1,8 cineole (M), limonene (M), α terpinene (M), terpinene 4-ol (M), methyl eugenol (M), globulol (L), unresolved hydrocarbon (M).

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Melaleuca leucadendra (common weeping cream bottlebrush - profusely planted as a street tree): caryophyllene (H), α pinene (M), β pinene (L), 1,8 cineole (M), limonene (L), α terpinene (L), terpinene 4-ol (M), α terpineol (M), methyl eugenol (H), globulol (L), unresolved hydrocarbon (M).

Melaleuca quinquenervia (abundantly naturally occurring cream bottlebrush in the wetlands and coastal NSW and Queensland): caryophyllene (H), α pinene (H), β pinene (M), 1,8 cineole (H), limonene (M), α terpinene (L), terpinene 4-ol (M), α terpineol (H), globulol (M), unresolved hydrocarbon (L).

Callistemon viminalis(common weeping red bottlebrush- planted extensively along freeways, along streets and in gardens): caryophyllene (H), α pinene (M), β pinene (L), 1,8 cineole (H), limonene (L), α terpinene (L), terpinene 4-ol (M), α terpineol (H), globulol (L), bornyl acetate(L), unresolved hydrocarbon (L).

These results confirmed that substantial amounts of sesquiterpene alcohols (globulol) accompany the emission of monoterpenes at flowering time. Sesquiterpene alcohols were identified in section 2.2 as being a source of allertant response. The existence of large amounts of methyl eugenol in the floral emission is of concern, since this substance has a well developed profile as a fragrance allergen. The potential for oxidation of pinenes and limonene is substantial, and the facility for sensitization by those oxidation products was discussed in detail in chapter 2.

5.4.2 Flower and leaf extractions

One of the arguments advanced in this thesis is that the seasonal peak in asthma in Brisbane is due to the flowering of the Melaleuca quinquenervia on the basis that the plant emits volatiles at flowering that are quite different from when the tree is in leaf. To that end, three flower/leaf comparisons required analysis of extracts of leaves and flowers as described in chapter 4. The results

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of those analyses are shown in Table 5.13. Because the samples were taken in spring, Melaleuca quinquenervia was not in flower so the commonly planted street tree Melaleuca leucadendra was analysed instead.

The results of these comparisons show the comparatively large quantities of tricyclic sesquiterpenes that are contained in these Myrtaceae flowers. Each of these species shows at least medium levels of bicyclogermacrene, a tricyclic sesquiterpene that is found in many Australian plants from Myrtaceae.

The Melaleuca leucadendra contains several other sesquiterpenes and several more tricyclics. Its additional methyl eugenol puts in into a category of a compound likely to cause sensitization given that it also contains alpha farnesene, a recognized allergen in fragrance products.

The Eucalyptus species contains several sesquiterpenes which put it in a similar category of likely sensitizing plants. Examples of the results, shown for Eucalyptus only, are in Appendix Y. Limonene in Eucalyptus, and linalool in Callistemon provide a base for oxidation products of those compounds to reach the ambient air and the lungs of those with sensitivities.

It is noteworthy that the Melaleuca leucadendra does not contain any pinene. This observation relates to the next set of analyses which are concerned with flowers as a substrate for fungal growth.

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Table 5.13 Flower leaf extract comparisons of compounds Species/Sample Callistemon Eucalyptus Melaleuca viminalis. hybrid “Summer leucadendra Beauty” Component Flower Leaf Flower Leaf Flower Leaf Identified a pinene M - M - - - myrcene L - - - - - a phellandrene M - - - - - limonene - - L M - - 1,8 cineole H H - - - - linalool L - - - - - a terpineol H H - - - - d elemene - - M - - - a cubebene - - - - M L geranyl acetate ? L T - - - - methyl eugenol - - - - VH H a copaene - - M - - - b elemene - - M - - - a gurjunene - - M L M L b caryophyllene - - H M M M humulene - - M L L - germacrene D - - L T H M bicyclogermacrene M L H M M M a farnesene - - - - H H calamenene - - M L M M d cadinene - - L - M M cadina-1,4-diene - - - - M M ledol ? - - L - - - globulol - - H - -

5.4.3 Flower and fungi studies As described in section 4.5, Cladosporium was introduced to plates of stamens of four types of Myrtaceae. The following is an extract from a report by the analysts (see chapter 4 for detail).

“SPME and GCMS analysis of the controls revealed that β phellandrene, bicyclogermacrene and humulene were not found in the frozen specimen of Eucalypt. Levels of caryophyllene were much less for all flowers than in the

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fresh samples submitted for headspace analysis. For most of the compounds identified, infection and/or storage at room temperature, either had little effect or caused a reduction in the levels in the headspace. There were notable exceptions, specifically increases in infected flowers of: Camphor, α terpinene and the area of unresolved hydrocarbon in Callistemon viminalis. Camphor, αterpinene, C15 sesquiterpene hydrocarbon, caryophyllene, and the area or unresolved hydrocarbon in the Eucalypt. Slight camphor and the area or unresolved hydrocarbon in Melaleuca leucadendra. Camphor, α terpinene, C15 sesquiterpene hydrocarbon and the area or unresolved hydrocarbon in Melaleuca quinquenervia.

The generation of camphor was at its greatest in the Eucalypt and the Melaleuca quinquenervia. Camphor is present in Parthenium weed, a well known toxic plant232. Camphor is also a major component of the Camphor Laurel tree. The infected flowers also revealed the presence of large amounts of sitostenone (a plant steroid). All four flower species contained this substance. It was not found in the flower controls, or the control of Cladosporium grown on agar.

Chloroform extracts of fresh flowers and infected flowers After SPME sampling of the fresh flowers, a further trial was undertaken to attempt to concentrate the headspace the samples by "extracting" that headspace in order to isolate and concentrate any chemical factor that may be in airborne pollen, as well as concentrate free volatiles. The additional substance found in all samples except Callistemon, was ursolic acid. Isolation of this compound led to similar extraction of the control and infected flowers used to determine the effect of fungal inoculation. Again, ursolic acid was found in all samples including Callistemon viminalis. No trend or difference in its

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concentration between infected and control samples was observed, indicating that any effect would be independent of the growth of fungus on the flowers. Ursolic acid is a potent modifier of many biological processes associated with the inflammatory process.“

The results of the trials are shown in Table 5.14. Results show that camphor production was so slight as to be recorded as zero in the table of results. Appendix Z shows the Melaleuca quinquenervia control result and the added Cladosporium. Appendix AA shows the production of sitostenone in the Cladosporium plus Melaleuca quinquenervia stamens condition.

Since all the other flowers produce camphor another set of procedures was performed the next year to confirm the camphor result. Again no camphor was produced. In the Chapter 2 the writer mentioned the important connection between pinene and camphor: if there is no pinene and there can be no camphor. Because Melaleuca leucadendra features methyl eugenol and not pinene, there can be no basis for the formation of camphor. The second investigation into the camphorless Melaleuca leucadendra provided insights of another kind and the relevant results can be seen in Table 5.15.

In Table 5.15 the quantification of the ion counts shows that when fungi utilize all the available flower substrate, there is an apparent increase in volatiles produced. Fungal uptake of chemicals was mentioned in section 2.2.4.1.

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Table 5.14 Flower substrates for fungal growth: results of experiments

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Table 5.15 Quantification of ion count for Cladosporium fungal infection of Melaleuca leucadendra stamens

Extract Quantification

Fresh stamen Stamens + controls extract% of Cladosporium% of volatiles volatiles Component Identified TIC area detected TIC area detected 1,8-cineole 4402100 2.02% 3692526 0.58% myrcenol trace only <0.20% trace only <0.20% linalool 2341378 1.07% 3186255 0.50% α terpineol 2691491 1.23% 3490151 0.55% methyl eugenol 18548768 8.50% 45620153 7.22% α copaene trace only <0.20% trace only <0.20% β cubebene 1808784 0.83% 2386523 0.38% β caryophyllene 8348679 3.83% 12945837 2.05% linalyl acetate trace only <0.20% trace only <0.20% humulene 2113880 0.97% 2227248 0.35% alloaromadendrene 1634443 0.75% 2088022 0.33% germacrene D 6596800 3.03% 10397264 1.64% bicyclogermacrene 3538219 1.62% 6202801 0.98% δ cadinene 2135672 0.98% 2885908 0.46% globulol 31418056 14.41% 70853555 11.21% viridifloral 3220555 1.48% 7587577 1.20%

Some of the increases in quantity of the compounds listed were thought to be due to the fungus, the breakdown of the tissue of the stamens or from the agar. Some of this was from hydrocarbon peaks that were less volatile than the compounds listed and were not identified.

The fungal growth could have increased the access of the extracting solvent to the terpenes inside, due to tissue breakdown; or the increased production of some compounds could be due to the response to fungal infection. Based on the evidence, it is impossible to determine, but invites more research as does determining the role of the sitostenone formed when Cladosporium are added to the stamens. The fact that this was formed for all the stamens plus fungi conditions invites questions about the effect of this in nature. When fungal spores are formed on flowers in nature, what chemical mix is available to be

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inhaled? Does the sitostenone have anti-apoptotic role like camphor or is the substance benign in effect? These questions remain unanswered.

What is supported here is that flowers from various members of Myrtaceae provide a growth substrate that results in the formation of compounds quite distinct from the fungi alone. The formation of an unnamed sesquiterpene in large amounts, in Eucalyptus and Melaleuca quinquenervia following fungal infection provides another source of possible sensitization in flowering seasons. Sesquiterpene hydrocarbons have been identified as sensitizing fractions in several studies already discussed.

Observations of fungal spores on Myrtaceae flowers A number of observations of fungal spores and Myrtaceae flowers were made during explorations into fungal/flower associations. Prior to the experiments just described in the previous section, some systematic collections were made of newly opened flowers and repeat visits at weekly intervals to extract single stamens revealed fungal spores on several occasions.

In May after three weeks from opening, fungal spores were seen being released from the stamen of Melaleuca quinquenervia. This event was repeated twice more and occurred at the same time interval i.e. 21 days after unfurling of the new stamens. The flowers were tagged so that an adjoining stamen from the same flower could be plucked each time for inspection.

Figure 5.19 shows the result of those inspections. The photograph was taken with a 20x and 40x objectives and the Figure here is no longer to scale as the collection was not part of any formal enquiry. The two parts of the same scene are represented in the composite photograph seen in Figure 5.19. The photograph on the left is of the end of a senescent Melaleuca quinquenervia stamen and shows many fungal spores which ranged in size from 1 to 8 microns approximately. The heavily stained scene on the right is a close up

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Figure 5.19 Ascomycetes emerging from Melaleuca quinquenervia stamen

of the same scene showing the spores an the hyphae. Identification was sought by learned researchers but no sure identification was made from the photograph. The most informed of those consulted, suggested that the spores might be that of Coletotrichum acutatum, a fungus that affects flowering plants in warm temperate areas. The fungal spores are not that of Cladosporium although both varieties are ascomycetes.

The spores shown in the Figure 5.20 are familiar. Whether they belong to Aspergillus or Penicillium is not clear from the photograph. Even though the photographs are not high quality, the formation of the spores is evident. Figure 5.20 was taken from a soot scraping which was prevalent on Melaleuca quinquenervia at the time. The structure from which the scraping was taken may have been ‘witches broom’, an opportunistic pathogen of many woody plants.

Further collections took place on a random and exploratory basis and other fungi were located on the stamens of Callistemon viminalis. Figure 5.21 shows an unidentified form attached to the side of the stamen. A probable spore, its name is not known nor could a suggestion as to its identity, be made.

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Figure 5.20 Strings of spores from Melaleuca quinquenervia

Figure 5.21 Unidentified structure attached to a fresh Callistemon viminalis stamen.

This structure was well formed on a stamen that was not senescent. The red colour is the actual pigment of the stamen, Most other fungal spores occur on senescent stamens.

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5.4.4 Tenax samples of flowers

Tables 5.16 and 5.17 show the results of Tenax samples taken from a number of flowers. Of particular interest are the high levels of limonene associated with the Melaleuca species and the Eucalyptus species. Medium levels of linalool oxide from the Melaleuca quinquenervia and high levels of esters in many species indicate that all these plants would provide compounds that would result in a range of allergic symptoms.

Of special interest is the delta carene that is emitted from the lemon scented tea tree, Leptospermum petersonii. Delta carene is a bicyclic monoterpene of the carane series and its presence in tea tree flowers is potentially very important especially in New Zealand where the manuka, Leptospermum scoparium is widespread. The association of delta carene respiratory sensitivity is well known and was discussed in chapter 2. This chemical takes its place with two other bicyclic monoterpenes associated with respiratory symptoms, pinene (from the pinane series) and sabinene from the thujane series.

The high levels of methyl ester associated with the popular Ivory Curl flower (Buckinghamia) and the dimethyl benzene (xylene) in the Xanthostemon are of concern given that these are plants gaining popularity and these compounds mentioned are associated with negative respiratory symptoms.

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Table 5.16 Tenax Flower samples A

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Table 5.17 Tenax flower samples B

-cubebene = α = M-H geraniol = L benzyl alcohol = H, benzyl alcohol H, = tetrahydro-ional H, = benzyl acetate = VH, benzylVH, acetate = isocaryophyllene = L, L, = isocaryophyllene phenylethyl alcohol = VH, acetic acid estermethyl = M L,exo-2-hydroxycineole = M-H benzoic H, esteracid = methyl germacrene-D = L, = germacrene-D linaloola-copaene L, = = M, oxide 2-methyl-1-dimethyl propanoic2-methyl-1-dimethyl acid OTHERIDENTIFIED COMPOUNDS KEY TO COMPOUNDS DETECTED IN TENAX SAMPLES DETECTED IN TENAX -farnesene α -caryophyllene linalyl acetate Β COMPOUNDS IDENTIFIED ocimene β− linalool camphor acid benzoic limonene cis 159 2610 4812 3711 123456789101112 _TTM-M-LL? - - TTTL-MTT---- L-M - - M M H VH - - H - - M-H---L------pinene -pinene Acacia β α Eucalypt 1,8-cineole Melaleuca Melaleuca tereticornis concurrens benzaldehyde leucadendra quinquenervia Flower Sample Flower

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Storm samples A Tenax sample was taken before and after a storm to ascertain if volatile emission was affected by the storm. Evidence of relationships between a drop in atmospheric pressure and volatile emission was presented in chapter 2. The increase in ‘damage’ volatiles before the storms is consistent with the notion of increases emission because of pressure responses in the plant. The volatiles trapped in this instance are similar to those from cut grass. The incidence of such volatiles in this collection supports the explanation that there is a ‘rush’ of volatiles emitted when there are storms and thunder. Air samples collected during Phase 2 exhibit increased number and quantity of volatiles during stormy weather. Table 5.18 A sample of floral emissions before and after a storm

Grevillea Before Storm After Storm ‘Robyn Gordon’ linalool M-H - cis β-ocimene L L-M

benzaldehyde L-M - hexenal H L

hexanoic acid H α-phellandrene - L sabinene - T camphor - T

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5.5 Outcomes and discussion of analysis of household and personal care products

Table 5.19 Tenax desorbs from lifestyle products H = M-H geranyl acetate= L-M cedrene = M, b-farnesene = L, thyopsene = = L M,cedrene = L,thyopsene b-farnesene farnesene = H, b-cubebene = M-H,= = a-cadinene farnesene M, H, b-cubebene caryophyllene oxide = M L-M, menthyl actate = L-M, dihydrocarveol = L-M, neryl acetate = L-M = acetate neryl L-M, = dihydrocarveol L-M, = actate menthyl L-M, dihydromyrcenol = H, menthol= L, dihydrocarveol acetate= L, isocaryophyllene = M, a- = M, linalyl anthranilate = VH, citronellol = H, citronellyl formate = H, neryl actate = L-M, L-M, = actate neryl H, = formate citronellyl = H, citronellol VH, = anthranilate linalyl = M, H, = M, borneol lavandulyl = acetate H,geranyl = acetate H, b-caryophyllene= b- H-VH, acetate = M, geranyl actate = H, a-bergamotene = M, b-bisabolene = M-H = b-bisabolene M, = a-bergamotene = H, actate geranyl M, = acetate G-terpinene = M-H, myrcene = M-H, a-terpineol =M-H, terpinolene = M-H,citral = H, neryl myrcene= M, menthone = M,borneol = M, a-terpineol = M, menthol =M-H, fenchyl acetate= myrcene= b-ocimene L-M, trans = M-H, a-terpinoline = L,terpinene-4-ol = H, a-terpineol= M- a-phellandrene =a-phellandrene L, dihydromyrcenol = H, menthol = L-M,fenchyl acetate= VH, carvone= M- myrcene = M, 3-hexanol= H, a-terpinolene= L-M, neryl nitrile = H, geraniol = M, geranyl nitrile myrcene = M, p-cymene= M, = a-terpinene M-H,menthone = M-H,borneol = M, terpinene-4-ol linalyl linalyl acetate B-caryophyllene a-farnesene benzoicacid other compounds identified cis b- ocimene limonene linalool camphor L-M - VH H L - - - - dihydromyrcenol = H, benzyl acetate = M-H, citronellol = M, a Ajax Cream - - VH ------Pine-o-clean L - VH H - H - - - Breathe Easy H - M-H H H H M - - No-Moz candle L-M - M ------hexenal = M, hexanoic acid= L-M Cleanser or Air Air or Cleanser Lavender Oil ang. L H M VH M VH H H - Tranquil Calm and L M-H VH VH T M M _ - Haze-Citrus SplashHaze-Citrus L - VH H - M-H - - - =L, hydrate benzylacetate = H, sabinene Freshener Sample 1,8-cineole Refresh and RenewRefresh and - M VVH H T H M-H - - mbi-pur Lemon freesi Ambi-pur Breathe Easy VH - H H T - T - - A

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Table 5.19 shows some of the compounds found in the products listed. The list of components is not comprehensive but does show the major components in a relative description. Of special interest is the very high level of limonene found in many of the products.

The Ambi-Pur Lemon Freesia is designed to be plugged into an electricity socket for day long emission of fragrance. It emits a ‘very high’ level of limonene daily and a medium to high level of benzyl acetate which is the ester that occurred in ‘very high’ levels in the Tenax sample of wattle flowers (Acacia concurrens), shown in Figure 3.08.

Pine-0-Cleen contains high levels of ‘very high’ levels of limonene and ‘high ‘ levels of linalool along with low levels of farnesene : three substances known to be sensitizing agents. The tri-cyclic sesquiterpene cedrene is a likely link with further sensitivities.

Lavender oil is a popular choice for aromatherapy. Its range of esters, several tricyclic hydrocarbons and high linalool, limonene and medium camphor levels establish it as one of the most problematic pleasant perfumes of all time.

The No-Moz candle blends limonene with chemicals associated with wounding a plant - a very similar mix to that which results in asthma for people with allergies who cut the lawn. These chemicals are those that were found in the Tenax sample before and after the storm.

This body of evidence provides a basis for some revised models of asthma acquisition which will be addressed in an introductory manner in chapter 6.

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6. New models relating environmental variables and asthma

6.1 A chemotaxonomic model for understanding world asthma patterns

The results of investigations that have been reported here lend support to a new view of asthma occurrence and the patterns that may be associated with it throughout the world. To date the understanding of plants and their relationship to asthma occurrence has focussed on the mode of pollen dissemination. Already, in chapter 2, the reliance on wind dissemination of pollens as a reason for plant related allergic symptoms has been noted.

The information contained in chapter 2 sets out in some detail all the ways in which terpenes in plants can be disseminated : by direct emission, by transport in plant fragments, by transport in special structures like trichomes, and in pollens. A recognition that plant chemicals are dispersed in gaseous form requires that the thinking that decrees that wind-pollinated plants are the only source of allergens, needs to be reviewed.

The evidence provided in this thesis supports that which was provided in Chapter 2 in which alpha and beta pinene, linalool and limonene were identified as sources of sensitization, especially via oxidation processes. Additionally the role of sesquiterpene hydrocarbons was identified as substantial in matters of sensitization, especially in the are of fragrance chemicals.

As well as chemicals that cause sensitization or initiate inflammation, the roles of others which can exacerbate that inflammation or diminish it, are equally important. The many effects of the aldehydes and ketones fall into this category. The unusual role of camphor, as product of pinene, in apoptosis is especially relevant. While some aldehydes and ketone trigger apoptosis, camphor has been shown not to and may retard the process of programmed cell death.

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The combination of plant chemicals such as those just mentioned could result in initiation of respiratory inflammation and a failure to then resolve that inflammation via apoptotic processes. This is consistent with the processes of asthma.

If a taxonomic view of asthma relationships is taken, then the patterns of asthma occurrence reveal some common threads. Those threads are woven from groups of chemicals called bicyclic monoterpenes and tricyclic sesquiterpenes. The latter are prevalent in many plants that cause allergic reactions as discussed in chapter 2. The bicyclic monoterpene provide the base for production of ketones such as camphor and sabina ketone.

Some of the locations in the world with the highest incidence of asthma have vegetation that provides the chemical mix just mentioned. Some botanical observations about those countries follow:

Australia Some of the patterns of vegetation have already been mentioned. An additional observation relates to a study of South Australian children in 1995. The varying patterns of asthma around South Australia were quite marked. The patterns of vegetation in South Australia are likely to have considerable bearing on the matter. Vast numbers of Myrtaceae surround Adelaide and the environs. One of the highest asthma rate areas was Port Lincoln. Is it relevant that the Eucalyptus cladocalyx which contains cyanide476, 813-816 grows in the Port Lincoln area? Many of the Eucalypts that grow there exhibit very high sesquiterpene levels which would be expected to be reflected in the floral emissions and thus be inhaled.

In Victoria where an investigation into geographical differences was conducted817 no account was taken of the variety of plants in the different

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areas. The mallee scrub country and the Black Box communities, the Red River Gums, and other riparian plants like Melaleucas, all provide emissions that would affect asthma substantially.

In New South Wales a geographical comparison818 between steel cities did not mention the proximity to wetland swamps near Newcastle, nor the vast tracts of Eucalyptus maculata ( spotted gum) which contain very hight amounts of the aldehyde citral. This lemon-scented substance was mentioned in chapter 2 for its well known connection to sensitization.

New Zealand New Zealand’s incidence of asthma is similar to Australia’s. Insect pollinated plants are considered to have a role in the high levels of asthma then the patterns can be understood. New Zealand is a useful place to investigate because all the health boards are under the one centralized government. Records are well kept in comparison to other countries which may exhibit an erratic information base.

New Zealand logging has been a well established industry for more than a century because of the rich endowment of large trees with valuable timber. Planted forests account for 1.7 million hectares and most of it is radiata pine which provide a steady source of pinene and sesquiterpenes as well at flowering time especially. One third of the planted forests are in the central north island.

Figure 6.01 shows a map of high asthma areas as constructed from figures 22 about regional variations in New Zealand in 2004. The map only marks the areas that were designated as “high” rates.

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Figure 6.01 Areas of high asthma incidence in New Zealand

The areas in the North Island correspond with the forestry areas especially. Some other high incidence areas correspond to the distribution of the heath country . Leptospermum scoparium with high levels of sesquiterpenes (65%) in the leaf oil are shown in Figure 6.02.

Figure 6.02 High sesquiterpene chemotypes of Leptospermum scoparium

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Figure 6.03 shows the naturally occurring heathland in general and the long ribbon down the west coast of New Zealand has strip of health land that matches the high levels of asthma experienced in this harsh part of the country.

Figure 6.03 Heathland in New Zealand (after Specht819)

In the flower Tenax studies in this thesis the floral emissions from one variety of Leptospermum was shown to contain delta- carene, alpha-farnesene and limonene. This blend of gases would be expected to exacerbate asthma and if the manuka, Leptospermum scoparium, emits similar gases then the high levels of asthma in the heathland is understandable since that plant predominates there.

United Kingdom The problem of oilseed rape and asthma is well documented in the United Kingdom and has been mentioned in this thesis. The emissions of limonene, sabinene and farnesene are associated with the flowering of the crop. What is less well known is the extent of other plants of the Brassica genus. Oilseed rape is Brassica napus and its wild relatives are: Wild Turnip, Brown Mustard, Wild Cabbage, Hoary Mustard, and Wild Radish. They are clearly mapped by the

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Friends of the Earth and almost entirely cover the United Kingdom820. Those may be responsible for emitting a chemical blend which includes large amounts of sabinene, a bicyclic monoterpene. Farnesene and limonene could be expected at flowering time and could explain the high rates of asthma experienced in that country.

United States The worst states for asthma in the Unites States are shown on the map in Figure 6.04821. These places all are in the same regions as the great forests that contain many trees that consist of sesquiterpenes an bicyclic monoterpenes is great quantities.

Figure 6.04 Worst asthma states in the United States

The worst state of Pennsylvania contains large numbers of Northern White Pine and Southern Yellow Pine. Oak, Hemlock and Walnut are just some of the many trees associated with seasonal respiratory symptoms. The world distribution of conifers shown in Figure 6.05 provides a botanical perspective from which to view the distribution of high asthma rate centres. Conifers emit

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large quantities of pinene and sesquiterpenes. The pinene is a source of camphor as well.

Figure 6.05 World distribution of conifers

Central Asia The high rates of asthma experienced in Central Asia may be related to the juniper forests that occur there822. The very dry climate ensures that the floral emissions remain in the atmosphere for prolonged periods of time. Juniper trees are a source of pinene, sabinene and sesquiterpenes.

The Caribbean and South America These countries have large numbers of Myrtaceae growing there and the chemical profiles of the plants are similar to Australian relatives. This is because originally, Gondwana land was one land mass that linked South America and Australia.

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Problematic families of plants There are likely to be many dozens of genera that contain plants with high levels of compounds that may sensitize. In this thesis the emphasis has been on the Myrtaceae family. Figure 6.06 shows world Myrtaceae distribution.

Figure 6.06 World Myrtaceae distribution

In South America the family is as prolific as in Australasia. When the Myrtaceae of the world is considered along with the distribution of Pinophyta, the conifers, world asthma patterns can be understood more easily. Those likely to contain high levels of bicyclic monoterpenes and sesquiterpenes are chiefly contained in 70 genera from Pinophyta. The families that incorporate those are : Cupressaceae Pinaceae Arauciaceae Cephalotaxaceae Podocarpaceae Taxaceae Taxodiaceae

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6.2 Revision of the hygiene theory The matters raised in this thesis have relevance for the ‘hygiene theory’ mentioned in chapter 2. The problem may not be the cleaning but rather what the cleaning is done with.

The investigations into lifestyle and cleanser products in this thesis show that large quantities of substances that are found in plants are also in lifestyle items like cleansers, perfumes, incense etc. Figure 6.07 shows the relative amounts of limonene measured via Tenax for a range of lifestyle products and a number of floral emissions. In the chart the first four columns are from flowers. Sampling time was the same for air close to either the flowers or the product. This simple process gives an overview of the sources of sensitizers that are part of most homes.

Figure 6.07 Limonene emissions from flowers and lifestyle products

LIMONENE IN CLEANSERS & FLOWERS

4 LIMONENE RATING

E A M A RE RVI TTLE DRON A EN CALYPT SPLASH UINE D U E US ACK W AJAX CRE UCA L NE'S KITCHEN PINE O CLEEN B A QUINQ E J L E CITR NO- MOZ CANDLE LEMON TEA-T L L Z ME E M HA REFRESH & RENEW OIL

AMBI -PUR LEMON FREESIA NAME OF SAMPLE

In the last 20 years there has been an increase in the use of items like room fresheners, perfumed cleansers, incense etc. The frequently reported higher incidence of allergy in women may be due partly to their increased use of

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perfumes and spray deodorant which may be priming sensitivities. In a similar vein a new look at the ‘thunderstorm effect’ may prove informative.

Thunderstorm asthma In chapter 2 the matter of increased asthma in times of thunderstorms was discussed. These events have been attributed to the bursting of pollen grains. The investigations here have revealed another source of sensitizers with the emissions of substances already identified by others as respiratory irritants. Strong correlations with a drop in atmospheric pressure indicate the relationships between symptoms and the storms. The actual collections of volatiles on stormy days further support this. The flower study establishes the increased emission from a Grevillea before a storm relative to after.

These findings provide new information about how asthma may be triggered by environmental events. A new model of asthma acquisition based on these understandings is now proposed. This new model places terpenoid and aromatic compounds at the centre because of the complexity and intricacy of connections with so many other aspects of the environment. This model is a summary of that which was related at length in chapter 2 and provides a blueprint for the pathways in which these substances become part of the environment. For sensitive and allergic individuals those pathways often lead to sufficient exposure to result in respiratory symptoms and exacerbations of asthma.

This model incorporates the many types of products that utilize flavourings and scents from plants. It also incorporates the interactive pathways evident in some reseach regarding anthropogenic pollutants and terpenoid substances. Future research will determine how well the model fits the relatity of asthma acquisition and exacerbation.

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Figure 6.08 A new model of asthma acquisition

MODEL FOR THE ACQUISITION OF ASTHMA

METEOROLOGICAL FOOD & VARIABLES PLANTS: DRINK POLLENS, ADDITIVES & FUNGI VOLATILES, ASPIRIN SAPS, OILS, PARTICLES HYGIENE AND INSECTS COSMETIC PRODUCTS ENZYMES,

AROMATIC INDUSTRIAL AND INSECT PARTICLES FAECES: TERPENOID OUTDOORS COMPOUNDS GASEOUS AND INDOORS: POLLUTANTS INCLUDING MODIFIERS: COCKROACH GENETIC, OTHER TRIGGERS: TOBACCO SMOKE, AND DUSTMITE DIETARY, GAS HEATING, BIOLOGICAL ANIMALS, COLD AIR, EXERCISE, INFECTIONS, AND, ASTHMA FOOD ALLERGIES.

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7. Conclusions and recommendations This thesis has provided a body of evidence for the argument that insect pollinated plants may have a role in respiratory symptom exacerbation. Such a position is new and contrary to current thought.

In Australia, the growing of Myrtaceae trees and shrubs is actively encouraged as a ‘safe’ alternative to plants which may induce respiratory discomfort or allergic symptoms. The results contained herein establish that alpha and beta pinene, limonene and linalool especially are significant predictors of reduced peak flow, reliever and preventer usage and other respiratory symptoms as well. Recommendation : That the promotion of Australian plants as ‘safe’ for asthma and hay fever suffers be reassessed.

In this study allergens that do not grow in the area was included purposefully to ascertain the response with a view to highlighting the limitations of accepting skin-prick testing at face value. Olive allergen was included in the battery for the children of urban Brisbane, where no olives grow with the exception of a rare occurrence of a domestic planting that rarely flowers if it grows at all. The Melaleuca quinquenervia does grow nearby in great numbers had a chemical profile very similar to privet, which belongs to the same family as olive. Whether this is the reason for the positive responses to olive is not clear. If skin-test responses are related to the terpene synthases that are contained in these plants, then the results would be understandable and would explain the widespread positive response to olive allergen.

Recommendation : That interpretation of skin-test results for plant allergens should be cautious with due consideration to alternative sources of enzymes and cross-reactions.

The evidence for the responses to linalool in particular is relevant to the use of perfumed items as lifestyle products. Linalool is one of the most used terpenes

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in hygiene and cosmetic products. The predictive value of this variable in this work is disturbing in view of the use of spray deodorant and room fresheners. Already there is evidence from elsewhere that these substances are harmful.

Recommendation: That publicity is given to the issues related to the use of perfumed products in and around the home.

This was an exploratory study and as such operated upon a limited financial base. Future research would need to carefully cost the funding which would be required to provide the chemical sampling that would enable the use of standards. Chemical standards would have enabled precise quantification of chemicals trapped on Tenax. Not many research organizations would have the chemical libraries to carry out this exacting and complex work. This is a limitation for the future of this research and one which needs to be overcome if more is to be understood about these substances and their relationships to respiratory disease.

Recommendation: That further studies be commissioned to investigate the occurrence of terpenes in ambient air with a view to quantifying such gases. Included in this should be collection of storm-related emissions with a view to understanding this potential source of respiratory allertant.

Most importantly, and finally, the issue of terpene emissions and the establishment of plantation establishment has potential for enormous impact in the communities like China and South America where the populace has less access to medications to control asthma that may be triggered and /or exacerbated.

Recommendation: That terpene emissions from Eucalyptus and oilseed plantations are measured and that intellectual and fiscal energies be directed towards the pursuit of understanding the relationship to repiratory symptoms.

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This thesis has highlighted a new area of enquiry in the relationships of respiratory symptoms, asthma and terpenes and emissions from plants. There is a great deal of room for new questions to be asked and even more for creative ways to pursue the answers.

Appendix AD

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Q Weekend in The Courier Mail 2006;Sect. 36. 801. Martin MM. The evolution of insect-fungus associations from contact to stable symbiosis. Am Zool. 1992; 32(4):593-605. 802. Kaul VK, Vats SK. Essential oil composition of Bothriochloa pertusa and phyletic relationship in aromatic grasses. Biochem Syst Ecol. 1998; 26(3):347-356. 803. Queensland Government.State of the enviroment 2003. 2005. www.epa.qld.gov.au/environmental_management/state_of_the_environm ent. 804. Office of Urban Management. South East Queensland regional plan 2005-2026. Brisbane: Queensland Government in consultation with SEQROC; 2005. 805. Liccardi G, Russo M, Saggese M, D'Amato M, D'Amato G. Evaluation of serum specific IgE and skin responsiveness to allergenic extracts of Oleaceae pollens (Olea europaea, Fraxinus excelsior and Ligustrum vulgare) in patients with respiratory allergy. Allergol Immunopathol (Madr). 1995; 23(1):41-6. 806. Lewis WH, Vinay P. North American pollinosis due to insect-pollinated plants. Ann Allergy. 1979; 42(5):309-18. 807. Menardo JL, Bousquet J, Rodiere M, Astruc J, Michel FB. Skin test reactivity in infancy. 1985; 75(6):646-51. 808. Gottlieb DJ, Sparrow D, O'Connor GT, Weiss ST. Skin test reactivity to common aeroallergens and decline of lung function. The Normative Aging Study. 1996; 153(2):561-6. 809. Kramer U, Koch T, Ranft U, Ring J, Behrendt H. Traffic-related air pollution is associated with atopy in children living in urban areas. 2000; 11(1):64-70. 810. Behrendt H, Kasche A, Ebner von Eschenbach C, Risse U, Huss-Marp J, Ring J. Secretion of proinflammatory eicosanoid-like substances precedes allergen release from pollen grains in the initiation of allergic sensitization. 2001; 124(1-3):121-5. 811. Tabachnik B, Fidell L. Using Multivariate Statistics. Fifth ed: Pearson; 2007. 812. Juergens U, Stober M, Vetter H. Inhibition of cytokine production and arachidonic acid metabolism by eucalyptol (1.8-cineole) in human blood monocytes in vitro. Eur J Med Res. 1998; (1998 Nov 17;3(11):508-10). 813. Gleadow RM, Woodrow IE. Temporal and spatial variation in cyanogenic glycosides in Eucalyptus cladocalyx. 2000; 20(9):591-598. 814. Gleadow RM, Woodrow IE. Defense chemistry of cyanogenic Eucalyptus cladocalyx seedlings is affected by water supply. 2002; 22(13):939-45. 815. Webber JJ, Roycroft CR, Callinan JD. Cyanide poisoning of goats from sugar gums (Eucalyptus cladocalyx). 1985; 62(1):28. 816. Bignell CM, Dunlop PJ, Brophy JJ, Jackson JF. Volatile leaf oils of some southwestern and southern Australian species of the genus Eucalyptus (Series I). Part XIII. (Series I). (a) Series subulatae, (b) Series curviptera, (c) Series Contortae, (d) Series Incognitae, (e) Series Terminaliptera, (f) Series Inclusae, (g) Series Microcorythae and (h) Series Cornutae. 1996; 11(6):339-347. 817. Ansari Z, Haby MM, Henderson T, Cicuttini F, Ackland MJ. Trends and geographic variations in hospital admissions for asthma in Victoria. Opportunities for targeted interventions. 2003; 32(4):286-8. 818. Lewis PR, Hensley MJ, Wlodarczyk J, Toneguzzi RC, Westley-Wise VJ, Dunn T, et al. Outdoor air pollution and children's respiratory symptoms in the steel cities of New South Wales. 1998; 169(9):459-63. 819. Specht RL. Heathlands and related shrublands : descriptive studies. Amsterdam ; Oxford: Elsevier Scientific; 1981. 820. Friends of the Earth Scotland. GM pollution threat to wild relatives of oilseed rape. 2006. 821. U.S. worst asthma cities list 2006.http://72.14.235.104/search?q=cache:n7mK1pxAXE0J:www.asthm acapitals.com/asthma_capitals2006.pdf+asthma+and+top+100+and+am erica&hl=en&gl=au&ct=clnk&cd=3. 822. Brylski P.Central Asia transboundary biodiversity:Kyrgyz Republic, Kazakhstan, and UzbekistanType of TBPA:. World Bank.Organization. 2006. www.tbpa.net/docs/pdfs/Central_Asia.pdf+juniper+and+central+asia&hl= en.

Appendix A Seasonal variation on rainfall in Australia1.

Appendix A

Appendix B Some better known Myrtaceae subfamily Leptospermoideae genera 2

Acmena Corymbia Metrosideros Agonis Darwinia Myrtus Angophora Eucalyptus Pimenta Austromyrtus Eugenia Psidium Backhousia Feijoa (syn. Acca) Syncarpia Baeckea Homoranthus Syzygium Beaufortia Kunzea Thryptomene Callistemon Leptospermum Tristania Calothamnus Lophomyrtus Tristaniopsis Calytrix Lophostemon Ugni Chamelaucium Luma Xanthostemon Melaleuca

Appendix B

Appendix C1 Eucalypts in Moreton region of south east Queensland 3

Botanical Growth South East Name Common Name features Queensland Open forests on sandy loam or stony White Mahogany; soil; Flowers Eucalyptus Yellow spring-early acmenoides Stringybark summer Throughout region; Eucalyptus Bailey’s Flowers Moreton, Burnett; baileyana Stringybark summer Darling Downs; Wallum flats on sandy soils on coastal - lowlands; Orange Gum; Flowers Eucalyptus Bancroft’s Red spring- bancroftii Gum summer Coastal Lowlands In forest on Gympie sandy soils; Eucalyptus Messmate; Flowers N Moreton, Wide cloeziana Yellow Messmate summer Bay & Burnett In woodlands and open forests; Flowers late Narrow-leaved winter- ironbark; Narrow summer; Eucalyptus leaved Red sometimes Widespread in crebra Ironbark also autumn. region On sandy or stony soil; Eucalyptus Flowers Moreton & D curtisii Plunkett Mallee spring Downs Grey Ironbark; On poorer Eucalyptus Queensland Grey soils; Flowers drepanophylla Ironbark win summer Throughout region In moist Eucalypt forests; Moreton & Wide Eucalyptus Flooded Gum; Flowers Bay; Cultivated as grandis Rose Gum autumn ornamental

Appendix C

Appendix C2: Eucalypts in Moreton region of south east Queensland3

Botanical Growth South East Name Common Name features Queensland Deep sands; Flowers Eucalyptus summer- gummifera Red Bloodwood autumn Coastal Coarse Spotted Gum; Large Eucalyptus Leaved Spotted Flowers Moreton dist from henryi Gum summer Brisbane south In open forest Widespread in & woodlands; Moreton, Wide Eucalyptus Flwrs Bay, Burnett, E intermedia Pink Bloodwood sum/aut Darling Downs Gum Barked On sandy Eucalyptus Coolibah; Forest soils; Flowers intertexta Gum autumn-winter D Downs district In open forest; stony ridges; Flowers Eucalyptus Spotted Gum; winter-early maculata Spotted Iron Gum summer Throughout region Flowers Yarraman in Eucalyptus Yarraman spring- Moreton district melanoleuca Ironbark summer and N Burnett In woodlands and open Eucalyptus Silver-Leaved forests; Flwrs Widespread in melanophloia Ironbark summer. region Moist open forest on range of soils. Eucalyptus Flowers late Moreton & Wide microcorys Tallowwood winter-spring Bay districts In open forests on slightly saline Eucalyptus Gum Topped soils; Flowers Moreton, Wide Bay moluccana Box; Grey Box sum-aut & Burnett districts

Appendix C

Appendix C3 Eucalypts in Moreton region of south east Queensland 3

Botanical Growth South East Name Common Name features Queensland Coastal lower altitudes; Flowers Eucalyptus Queensland summer- nigra White Stringybark autumn Beerwah Widespread Grey Gum ( A Flowers Moreton, Wide Bay Eucalyptus prime structural summer- E Darling Downs & propinqua timber) autumn Burnett ; Widespread Moreton, Wide Bay Eucalyptus Flowers E Darling Downs & punctata Grey Gum summer Burnett Widespread Moreton, Wide Bay Eucalyptus Flowers E Darling Downs & punctata Grey Gum summer Burnett Sandy soils; Red Mahogany; Flowers Eucalyptus Red Stringybark; spring- Moreton & Wide resinifera Red Messmate summer Bay; Swampy Swamp Wallum Mahogany; country; Moreton & Wide Eucalyptus Swamp Flowers Bay; Cultivated as robusta Messmate autumn-winter ornamental Higher altitudes on mountain slopes; Flowers Blackall Range; Eucalyptus Sydney Blue summer- McPherson saligna Gum autumn. Range; On clay soils on flats; Flowers Eucalyptus Narrow- leaved spring-early Moreton & Darling seeana Red Gum summer Downs

Appendix C

Appendix C4 :Eucalypts in Moreton region of south east Queensland3

Botanical Growth South East Name Common Name features Queensland On sandy slopes; Eucalyptus Flowers Moreton & Wide signata Scribbly Gum winter-spring Bay On alluvial flats or other Throughout region; fertile soils; not common Blue Gum; Forest Flowers Darling Downs; Eucalyptus Red Gum; Red winter-late Sometimes tereticornis Iron Gum spring cultivated Open forest on sandy soils; Flowers Eucalyptus Carbeen; late spring- tessellaris Moreton Bay Ash summer Throughout region Extensively cult as Native of ornamental; rainforest; naturalized in Eucalyptus Flowers favourable coastal torreliana Cadagi; Cadaghi springing districts Open forest on sandy or stony soil; Flowers late Eucalyptus Brown summer- Common trachyphloia Bloodwood autumn throughout region

Appendix C

Appendix D Casuarinaceae species in South-East Queenland3

Growth Botanical Common South –East location Name Name Queensland features Casuarinaceae She-oak 9 species Fertile soil; Fertile soil; forest forest Casuarina Forest country; hills country;hills torulosa she-oak and slopes and slopes of of coastal coastal plain plain Casuarina Coastal in Coastal in equisetifolia stand Coast she- communities communities oak or sandy soil or sandy soil near the sea near the sea Casuarina Banks of Banks of River oak cunninghamiana rivers rivers Casuarina Thready- Darling inophloia barked Forests Downs she-oak Casuarina rigida High peaks High country and Granite Belt Casuarina Widely dist. Sandy or littorlais coastal plain Black she- stony soil in & forests ; oak forest sandy or country stony soil. Casuarina Forests on Darling Bull oak luehmannii poor soils Downs Casuarina Swampy Swamp Swampy land glauca land near oak near coast coast Casuarina West of cristata Darling Pure stand Downs. Belah communities Minor stands inland Burnett & Moreton .

Appendix D

Appendix E Pinaceae species in South-East Queensland3

Growth / Botanical Common South –East location Elsewhere Name Name Queensland features 3 main Pinaceae Pine 250 species species Originated in hilly coastal Radiata environment; Darling Native of pine Pinus radiata widely Downs, Monterey , Monterey cultivated in Toowoomba California pine SE Qld & Australia Poor sandy Darling soils ; Downs, Native of SE Pinus eliottii Slash pine naturalizes Maryborough, USA readily near Beerburrum plantations Pinus Caribbean Coastal From the Coastal carabaea pine plantations Caribbean Spreading leaved pine No longer Coastal SE Native of Pinus patula or Patula planted Queensland Mexico pine From Loblolly No longer Beerburrum, Pinus taeda Southern and pine planted Gympie Eastern USA

Appendix E

Appendix F Native and introduced Olea and Ligustrum naturally occurring in south-east Queensland3

Growth / South –East Botanical Name Common Name location Queensland features Olea Olive 1 species Near Australian olive rainforest; Olea paniculata Near rainforest Native olive flowers in spring

Ligustrum Privet 2 species

Near Tree privet Ligustrum rainforest, Naturalized Large leaf privet lucidum Aiton flowers early Moreton district Broadleaf privet summer Chinese privet Naturalized Ligustrum Flowers late Small-leaved Moreton and Wide sinense Lour. winter- spring privet Bay

Appendix F

Appendix G Caloundra Shire flora of local significance 4

Plants that are locally significant in the Shire of Caloundra which extends from the coast, including swamp land, and includes the highland area under investigation. ( ‘Significant’ plants are not the only plants that are found there.)

Acacia glaucocarpa Acacia o’shanesii Alpinia arundelliana (native ginger) Angiopteris evecta (King fern) Araucaria bidwillii (Bunya pine) Banksia conferta (Glasshouse banksia) Boronia (5 species) Elaeocarpus (quandong) Eucalyptus bancroftii Eucalyptus. curtsii Eucalyptus kabiana Eucalyptus robusta ( Swamp Mahogany) Guioa acutifolia Leptospermum luehmannii Leptospermum oreophyllum Leptospermum whitei Melaleuca nodosa (Prickly-leaf Paperbark) Melaleuca sieberi (Wallum Tea Tree) Podocarpus spinulosus (Shrubby Pine) Syzygium corynanthum (Sour Cherry) Syzygium crebrinerve (Purple Cherry) Tristaniopsis collina (Hill Kanuka)

Appendix G

Appendix H Skin – prick testing report form Phase 1 children

SCHOOL:

NAME : CODE: CLASS: HEIGHT:

WEIGHT:

CONSENTS: FEV PREDICTED : FEV ACTUAL : RELEVANT MEDICATIONS: NOTES:

RIGHT ARM LEFT ARM

ELBOW ELBOW

DUST MITE------ACACIA------RYEGRASS------COCKROACH------

EUCALYPT ------OLIVE------DOG HAIR------ALTERNARIA------

AUST PINE------BAHAI ------HORMO CLADO ------JOHNSON------

NEGATIVE BERMUDA------RAGWEED ------CONTROL------T T OIL------

POSITIVE CONTROL------CAT HAIR ------CALLISTEMON------WRIST WRIST

Appendix H

Appendix I ISAAC QUESTIONNAIRE Part a

ISAAC QUESTIONNAIRE (International Study of Asthma and Allergies in Childhood)

1. Have you ever had wheezing or whistling in the chest at any time in the past? Yes ( ) No ( )

IF YOU HAVE ANSWERED "NO" PLEASE SKIP TO QUESTION 6

2. Have you had wheezing or whistling in the chest in the last 12 months? Yes ( ) No ( )

IF YOU HAVE ANSWERED "NO" PLEASE SKIP TO QUESTION 6

3. How many attacks of wheezing have you had in the last 12 months?

None ( ) 1 to 3 ( ) 4 to 12 ( ) More than 12( )

4. In the last 12 months, how often, on average, has your sleep been disturbed due to wheezing? Never woken with wheezing ( ) Less than one night per week ( ) One or more nights per week ( )

Appendix I

Appendix I b ISAAC QUESTIONNAIRE

5. In the last 12 months, has wheezing ever been severe enough to limit your speech to only one or two words between breaths? Yes ( ) No ( ) 6. Have you ever had asthma ? Yes ( ) No ( )

7. In the last 12 months has your chest sounded Yes ( ) wheezy during or after exercise? No ( )

8. In the last 12 months have you had a dry cough at night, Yes ( ) apart from a cough associated with a cold or chest infection? No ( )

9. Have you ever had a problem with sneezing or a runny nose Yes ( ) or blocked nose when you DID NOT have a cold or the flu? No ( )

IF YOU ANSWERED "NO" PLEASE SKIP TO QUESTION 14

10. In the past 12 months, have you had a problem with sneezing or a runny nose or blocked nose when you DID NOT have a cold or the flu? Yes ( ) No ( )

IF YOU ANSWERED "NO" PLEASE SKIP TO QUESTION 14

11. In the past 12 months was this nose problem accompanied by itchy, watery eyes? Yes ( ) No ( )

Appendix I

Appendix I c ISAAC QUESTIONNAIRE

12. In which of the past 12 months did this nose problem occur? (Please tick any which apply)

January ( ) May ( ) September ( ) February ( ) June ( ) October ( ) March ( ) July ( ) November ( ) April ( ) August ( ) December ( )

13 In the past 12 months how much did this nose problem interfere with your daily activities. Not at all ( ) A little ( ) A moderate amount ( ) A lot ( )

14. Have you ever had hayfever ? Yes ( ) No ( )

15. Have you ever had an itchy rash which was coming and Yes ( ) going for at least six months? No ( )

IF YOU HAVE ANSWERED " NO" PLEASE SKIP TO QUESTION 21

16. Have you had this itchy rash at any time in the last 12 months? Yes ( ) No ( )

Appendix I

Appendix I d ISAAC QUESTIONNAIRE

IF YOU HAVE ANSWERED "NO" PLEASE SKIP TO QUESTION 21

17 Has this itchy rash at any time affected any of the following places: the folds of the elbows, behind the knees, in front of the ankles,

under the buttocks, or around the neck, ears or eyes? Yes ( ) No ( )

18. At what age did this itchy rash first occur? Under 2 years ( ) Age 2-4 years ( ) Age 5 or more ( )

19. Has this rash cleared completely at any time during the last 12 months? Yes ( ) No ( )

20. In the last 12 months, how often, on average, have you been kept awake at night by this itchy rash? Never in the last 12 months ( ) Less than one night per week ( ) One or more nights per week ( )

21. Have you ever had eczema? Yes ( ) No ( )

Appendix I

Appendix J Skin prick test results form for phase 2 participants

NAME: ID: HEIGHT: WEIGHT: CONSENTS: FORM OK: FEV PREDICTED: FEV ACTUAL:

RIGHT ARM LEFT ARM WRIST WRIST

COCKROACH DUSTMITE BAHIA AUST PINE

DOG EUCAL MELA UA

CAT LIMON PRIVET OXLIM

CLADOS α PINENE CALLIST OX α PINE

ALTERN β PINENE -VE CONT OX β PINE

ACACIA CINEOLE RYE OX CINE

RAGWEED + VE CONT JOHNSON BA

WRIST WRIST

TIME:

Appendix J

Appendix K Chemicals used in SPTs in phase 2 Sigma - Aldrich CAS number Sigma Aldrich ID alpha pinene 80-56-8 14,752

18172-67-3 11,208-9 (−)-β-pinene 1,8-cineole 470-82-6 C 8144 (R)-(+)-Limonene 5989-27-5 183164 Laboratory Supply Pty Ltd Tingalpa methanol 318 bornyl acetate B0526 caryophyllene C0796 ursolic acid U0065

Appendix K

Appendix L Respiratory diary sheet (part of monthly sheet)

«code» TUE WE THU FRI SAT

«first» «surname» 1 2 3 4 5

Since your last peak flow have you had: A Runny Nose ? 0 None 1 A little 2 A lot 3 Most of the time A Blocked Nose? 0 None 1 A little 2 A lot 3 Most of the time Sneezing ? 0 None 1 A little 2 A lot 3 Most of the time Coughing ? 0 None 1 A little 2 A lot 3 Most of the time Difficulty breathing? 0 None 1 A little 2 A lot 3 Most of the time Wheeze or chest 0 None 1 A little Tightness ? 2A lot 3 Most of the time Itchy Nose ? 0 None 1 A little 2 A lot 3 Most of the time Itchy Eyes ? 0 None 1 A little 2 A lot 3 Most of the time PEAK FLOW (Highest of 3 blows)

Do very early after waking Have you increased or decreased your asthma preventer medicine ? Put "I" for increased Put "S" if it stayed the same Put "D" for decreased Have you increased or decreased your asthma reliever medicine ? Put "I" for increased Put "S" if it stayed the same Put "D" for decreased How is your asthma today? Write a number between 1 and 10 '1' means very good, '10' means terrible

Appendix L

Appendix M: Air sampling equipment

Burkhard 7 –Day Volumetric Trap for spores and pollens in situ at Flaxton

SKC Universal Sample Pump PCXR8

Appendix M

Appendix N Rankings assigned to categories of volatiles in samples.

NIL 0

Trace 1 Trace-Low 2

Low 3

Low-Medium 4 Medium 5

Medium-High 6

High 7 High-Very High 8

Very High 9 Very Very High 10

Appendix N

Appendix O Significance of variable correlations for coastal group SPEF

STANDARDIZED COAST GROUP MEAN PEF SAME DAY LAG1 LAG2 LAG3 LAG4 LAG5 BENZOIC ACID BENZALDEHYDE ALPHA PINENE BETA PINENE 1,8-CINEOLE CAMPHOR LINALOOL LIMONENE LINAYL ACETATE ATMOS PRESSURE TEMPERATURE <0.0001 + 0.0001 + 0.001 + 0.004 + 0.01 + 0.004 + PRECIPITATION RELATIVE HUMIDITY 0.02 - WIND SPEED <0.0001 + 0.0001 + 0.0001 + 0.0001 + 0.002 + 0.0003 + THUNDER SCORE

PM 10 0.02 + 0.03 + 0.03 + OZONE <0.0001 + 0.0001 + 0.0001 + 0.0001 + 0.0001 + 0.0001 + NITROGEN OXIDE <0.0001 -<0.0001 - 0.0001 - 0.002 - 0.005 - 0.006 - NITROGEN DIOXIDE <0.0001 -<0.0001 -<0.0001 -<0.0001 - 0.0005 -<0.0001 - MYRTACEAE POLLEN 0.003 + 0.03 + 0.01 + 0.002 + 0.001 + 0.005 + GRASS POLLEN PINUS POLLEN 0.008 + ASTERACEAE POLLEN CASUARINA POLLEN ACACIA POLLEN 0.002 + 0.0001 + 0.0003 + 0.0004 + 0.0003 + 0.01 + OTHER POLLEN 0.05 - CLADOSPORIUM 0.02 - ALTERNARIA 0.02 - 0.0006 - 0.01 - 0.005 - 0.002 - OTHER FUNGI 0.0556 +

Appendix O

Appendix O Significance of variable correlations for coastal group asthma score

COAST GROUP ASTHMA SCORE SAME LAG1 LAG2 LAG3 LAG4 LAG5 BENZOIC ACID BENZALDEHYDE ALPHA PINENE 0.03 - BETA PINENE 0.03 - 1,8-CINEOLE 0.02 - CAMPHOR LINALOOL LIMONENE LINAYL ACETATE ATMOS PRESSURE 0.002 - 0.003 - TEMPERATURE 0.001 + 0.003 + 0.009 + 0.003 + 0.01 + 0.004 + PRECIPITATION RELATIVE HUMIDITY WIND SPEED THUNDER SCORE 0.004 - PM 10 OZONE NITROGEN OXIDE 0.01 - NITROGEN DIOXIDE MYRTACEAE POLLEN GRASS POLLEN PINUS POLLEN 0.03 - 0.0009 - ASTERACEAE POLLEN CASUARINA POLLEN ACACIA POLLEN 0.02 - OTHER POLLEN CLADOSPORIUM 0.05 - ALTERNARIA OTHER FUNGI

Appendix O

Appendix O Significance of variable correlations for coastal group reliever use score COAST GROUP RELIEVER USE SCORE SAME DAY LAG1 LAG2 LAG3 LAG4 LAG5 BENZOIC ACID BENZALDEHYDE ALPHA PINENE BETA PINENE 1,8-CINEOLE CAMPHOR LINALOOL 0.03 + 0.02 + LIMONENE LINAYL ACETATE 0.009 + 0.01 + ATMOS PRESSURE 0.009 - 0.001 - 0.007 - 0.04 - 0.02 - 0.01 - TEMPERATURE PRECIPITATION RELATIVE HUMIDITY 0.0553 - WIND SPEED THUNDER SCORE

PM 10 0.02 + OZONE NITROGEN OXIDE NITROGEN DIOXIDE MYRTACEAE POLLEN GRASS POLLEN 0.004 - 0.007 - 0.04- PINUS POLLEN 0.04 - ASTERACEAE POLLEN 0.02 - 0.02 - CASUARINA POLLEN ACACIA POLLEN 0.03 - OTHER POLLEN CLADOSPORIUM ALTERNARIA OTHER FUNGI 0.01 +

Appendix O

Appendix O Significance of variable correlations for coastal group preventer use score

COAST GROUP PREVENTER USE SCORE SAME DAY LAG1 LAG2 LAG3 LAG4 LAG5 BENZOIC ACID BENZALDEHYDE ALPHA PINENE BETA PINENE 1,8-CINEOLE CAMPHOR LINALOOL 0.01 + 0.01 + 0.02 + LIMONENE LINAYL ACETATE 0.02 + 0.04 + ATMOS PRESSURE <0.0001 - <0.0001 - <0.0001 - <0.0001 - <0.0001 - <0.0001 - TEMPERATURE PRECIPITATION RELATIVE HUMIDITY 0.03 - 0.003 - WIND SPEED 0.02 + 0.03 + THUNDER SCORE 0.04 + 0.04 + 0.02 + 0.04 +

PM 10 0.02 + 0.04 + 0.04 + OZONE 0.004 + 0.004 + 0.0003 + 0.0001 + <0.0001 + <0.0001 + NITROGEN OXIDE 0.03 - 0.009 - NITROGEN DIOXIDE <0.0001 - 0.0007 - 0.0012 - 0.0007 - 0.0005 - 0.0002 - MYRTACEAE POLLEN 0.03 + 0.04 + GRASS POLLEN 0.02 - PINUS POLLEN ASTERACEAE POLLEN 0.02 - 0.05 - 0.03 - CASUARINA POLLEN ACACIA POLLEN 0.04 + OTHER POLLEN 0.008 - 0.001 - 0.03 - 0.006 - 0.009 - 0.001 - CLADOSPORIUM ALTERNARIA 0.01 - 0.05 - 0.03 - OTHER FUNGI

Appendix O

Appendix O Significance of variable correlations for coastal group wheeze

COAST GROUP WHEEZE SAME DAY LAG1 LAG2 LAG3 LAG4 LAG5 BENZOIC ACID BENZALDEHYDE ALPHA PINENE BETA PINENE 1,8-CINEOLE CAMPHOR LINALOOL LIMONENE LINAYL ACETATE 0.04 + ATMOS PRESSURE 0.0009 - 0.0002 - 0.0002 - 0.0004 - 0.003 - 0.009 - TEMPERATURE 0.05 + PRECIPITATION 0.002 - 0.02 - RELATIVE HUMIDITY 0.03 - 0.009 - 0.04 - WIND SPEED THUNDER SCORE 0.03 +

PM 10 0.03 + OZONE NITROGEN OXIDE NITROGEN DIOXIDE MYRTACEAE POLLEN 0.04 - GRASS POLLEN PINUS POLLEN 0.04 - ASTERACEAE POLLEN 0.04 - 0.05 - CASUARINA POLLEN ACACIA POLLEN OTHER POLLEN 0.01 - CLADOSPORIUM 0.007 - 0.006 - ALTERNARIA OTHER FUNGI

Appendix O

Appendix O Significance of variable correlations for coastal group difficulty breathing

COAST GROUP DIFFICULTY BREATHING SAME DAY LAG1 LAG2 LAG3 LAG4 LAG5 BENZOIC ACID 0.03 + 0.04 + BENZALDEHYDE ALPHA PINENE BETA PINENE 1,8-CINEOLE 0.02 - CAMPHOR LINALOOL LIMONENE 0.03 - LINAYL ACETATE ATMOS PRESSURE 0.002 - 0.0005 - 0.002 - 0.0001 - 0.002 - 0.006 - TEMPERATURE 0.004 + 0.002 + 0.007 + 0.001 + <0.0001 + 0.001 + PRECIPITATION 0.02 - RELATIVE HUMIDITY 0.0009 - 0.008 - WIND SPEED THUNDER SCORE 0.003 -

PM 10 0.005 + OZONE NITROGEN OXIDE 0.05 - NITROGEN DIOXIDE MYRTACEAE POLLEN GRASS POLLEN PINUS POLLEN 0.01 - 0.006 - ASTERACEAE POLLEN CASUARINA POLLEN ACACIA POLLEN OTHER POLLEN CLADOSPORIUM <0.0001 - 0.001 - 0.04 - 0.03 - ALTERNARIA 0.004 - 0.03 - OTHER FUNGI

Appendix O

Appendix O Significance of variable correlations for coastal group coughing

COAST GROUP COUGHING SAME DAY LAG1 LAG2 LAG3 LAG4 LAG5 BENZOIC ACID BENZALDEHYDE 0.01 + ALPHA PINENE BETA PINENE 1,8-CINEOLE CAMPHOR LINALOOL LIMONENE LINAYL ACETATE 0.01 + 0.05 + ATMOS PRESSURE 0.005 - 0.001 - 0.0005- 0.001 - TEMPERATURE PRECIPITATION RELATIVE HUMIDITY WIND SPEED THUNDER SCORE 0.05 + PM 10 OZONE NITROGEN OXIDE NITROGEN DIOXIDE MYRTACEAE POLLEN GRASS POLLEN PINUS POLLEN 0.03 - ASTERACEAE POLLEN 0.05 - CASUARINA POLLEN ACACIA POLLEN OTHER POLLEN 0.005 - CLADOSPORIUM 0.03 - ALTERNARIA 0.002 - OTHER FUNGI

Appendix O

Appendix O Significance of variable correlations for coastal group itchy nose

COAST GROUP ITCHY NOSE SAME DAY LAG1 LAG2 LAG3 LAG4 LAG5 BENZOIC ACID 0.04 + 0.03 + 0.02 + BENZALDEHYDE 0.02 - 0.01 - ALPHA PINENE BETA PINENE 1,8-CINEOLE CAMPHOR LINALOOL LIMONENE LINAYL ACETATE ATMOSPHERIC PRESSURE <0.0001 - <0.0001 - 0.02 - TEMPERATURE <0.0001 + <0.0001 + <0.0001 + <0.0001 + <0.0001 +<0.0001 + PRECIPITATION 0.05 - 0.03 - RELATIVE HUMIDITY 0.03 - 0.03 - 0.02 - 0.005 - WIND SPEED 0.03 + 0.004 + 0.0001 + 0.0008 + 0.01 + 0.02 + THUNDER SCORE 0.0555 -

PM 10 0.04 + OZONE 0.008 + 0.01 + 0.005 + 0.0003 + 0.0006 + 0.0003 + NITROGEN OXIDE 0.0003 - 0.001 - 0.0004 - 0.0007 - 0.03 - NITROGEN DIOXIDE 0.001 - 0.004 - 0.002 - 0.004 - 0.04 - 0.002 - MYRTACEAE POLLEN GRASS POLLEN PINUS POLLEN ASTERACEAE POLLEN CASUARINA POLLEN ACACIA POLLEN 0.01 + OTHER POLLEN 0.01 - 0.02 - 0.01 - CLADOSPORIUM 0.001 - 0.001 - 0.02 - 0.0001 - 0.005 - 0.04 - ALTERNARIA 0.04 - 0.03 - 0.04 - 0.0006 - 0.007- OTHER FUNGI

Appendix O

Appendix O Significance of variable correlations for coastal group sneezing

COAST GROUP SNEEZING SAME DAY LAG1 LAG2 LAG3 LAG4 LAG5 BENZOIC ACID BENZALDEHYDE 0.0006 - ALPHA PINENE 0.03 - BETA PINENE 0.03 - 1,8-CINEOLE 0.002 - 0.002 - CAMPHOR 0.02 - 0.04 - LINALOOL LIMONENE 0.005 - 0.02 - LINAYL ACETATE ATMOS PRESSURE TEMPERATURE PRECIPITATION RELATIVE HUMIDITY WIND SPEED THUNDER SCORE 0.03 -

PM 10 0.03 - 0.01 - 0.05 - 0.04 - OZONE 0.005 - 0.006 - NITROGEN OXIDE NITROGEN DIOXIDE MYRTACEAE POLLEN GRASS POLLEN 0.007 + PINUS POLLEN 0.01 - 0.02 - ASTERACEAE POLLEN 0.05 + CASUARINA POLLEN ACACIA POLLEN 0.004 - 0.04 - OTHER POLLEN CLADOSPORIUM 0.01 - 0.02 - 0.05 - 0.008 - ALTERNARIA OTHER FUNGI

Appendix O

Appendix O Significance of variable correlations for coastal group blocked nose

COAST GROUP BLOCKED NOSE SAME DAY LAG1 LAG2 LAG3 LAG4 LAG5 BENZOIC ACID BENZALDEHYDE ALPHA PINENE BETA PINENE 1,8-CINEOLE CAMPHOR LINALOOL LIMONENE LINAYL ACETATE 0.05 + ATMOS PRESSURE 0.03 - 0.03 - TEMPERATURE PRECIPITATION 0.02 - 0.02 - RELATIVE HUMIDITY 0.002 - WIND SPEED THUNDER SCORE

PM 10 OZONE NITROGEN OXIDE NITROGEN DIOXIDE MYRTACEAE POLLEN 0.02 - GRASS POLLEN 0.01 + PINUS POLLEN ASTERACEAE POLLEN 0.02 + CASUARINA POLLEN ACACIA POLLEN OTHER POLLEN 0.009 - CLADOSPORIUM ALTERNARIA 0.02 - OTHER FUNGI

Appendix O

Appendix O Significance of variable correlations for coastal group runny nose

COAST GROUP RUNNY NOSE SAME DAY LAG1 LAG2 LAG3 LAG4 LAG5 BENZOIC ACID 0.04 + BENZALDEHYDE 0.05 - 0.05 - ALPHA PINENE BETA PINENE 1,8-CINEOLE CAMPHOR LINALOOL LIMONENE 0.03 - LINAYL ACETATE ATMOS PRESSURE TEMPERATURE .01 + 0.03 + 0.007 + 0.04 + 0.01 + 0.005 + PRECIPITATION 0.007 - 0.002 - < 0.0001 - 0.0009 - < 0.0001 - 0.0004 - RELATIVE HUMIDITY 0.004 - 0.007 - 0.002 - <0.0001 - WIND SPEED THUNDER SCORE 0.01 - 0.02 - 0.003 - 0.003 -

PM 10 0.02 + OZONE NITROGEN OXIDE NITROGEN DIOXIDE MYRTACEAE POLLEN GRASS POLLEN 0.05 + PINUS POLLEN ASTERACEAE POLLEN CASUARINA POLLEN ACACIA POLLEN OTHER POLLEN CLADOSPORIUM <0.0001 - <0.0001 - 0.0008 - <0.0001 - 0.0005 - <0.0001 - ALTERNARIA OTHER FUNGI 0.05 -

Appendix O

Appendix P Significance of variable correlations for Range group

STANDARDIZED RANGE GROUP MEAN PEF SAME DAY LAG1 LAG2 LAG3 LAG4 LAG5 BENZOIC ACID BENZALDEHYDE 0.01 + ALPHA PINENE BETA PINENE 1,8-CINEOLE 0.05 + CAMPHOR LINALOOL LIMONENE LINAYL ACETATE ATMOS PRESSURE TEMPERATURE 0.02 +0.03 +0.01 + PRECIPITATION RELATIVE HUMIDITY WIND SPEED 0.04 + 0.04 + THUNDER SCORE 0.02 +

AIR POLLUTANTS NOT MEASURED DUE TO LACK OF PROXIMATE AIR SAMPLING STATION

MYRTACEAE POLLEN 0.002 - 0.002 - GRASS POLLEN PINUS POLLEN ASTERACEAE POLLEN NO POLLENS RECORDED CASUARINA POLLEN ACACIA POLLEN NO POLLENS RECORDED OTHER POLLEN CLADOSPORIUM 0.001 + ALTERNARIA 0.005 + OTHER FUNGI SPEF

Appendix P

Appendix P Significance of variable correlations for Range group asthma score

RANGE GROUP ASTHMA SCORE SAME DAY LAG1 LAG2 LAG3 LAG4 LAG5 BENZOIC ACID 0.004 - 0.04 - 0.009 - 0.02- BENZALDEHYDE 0.03 - 0.01 - 0.002 - ALPHA PINENE BETA PINENE 1,8-CINEOLE 0.05 - CAMPHOR 0.03 - 0.04 - 0.02 - 0.02 - 0.01 - LINALOOL LIMONENE LINAYL ACETATE ATMOS PRESSURE TEMPERATURE 0.02 - 0.01 - 0.03 - 0.04 - 0.01 - 0.0558 - PRECIPITATION 0.02 - 0.03 - RELATIVE HUMIDITY NOT MEASURED WIND SPEED 0.0002 - 0.005 - 0.02 - 0.004 - 0.002 - 0.004 - THUNDER SCORE

AIR POLLUTANTS NOT MEASURED DUE TO LACK OF PROXIMATE AIR SAMPLING STATION

MYRTACEAE POLLEN 0.005 + GRASS POLLEN PINUS POLLEN ASTERACEAE POLLEN NO POLLENS RECORDED CASUARINA POLLEN ACACIA POLLEN NO POLLENS RECORDED OTHER POLLEN CLADOSPORIUM 0.008 - 0.008 - 0.01 - 0.002 - 0.03 - ALTERNARIA OTHER FUNGI 0.02 + 0.01 +

Appendix P

Appendix P Significance of variable correlations for Range group reliever use score

RANGE GROUP RELIEVER USE SCORE SAME DAY LAG1 LAG2 LAG3 LAG4 LAG5 BENZOIC ACID 0.01 - 0.006 - 0.02 - BENZALDEHYDE ALPHA PINENE BETA PINENE 1,8-CINEOLE 0.03 - 0.04 - CAMPHOR LINALOOL 0.02 + LIMONENE LINAYL ACETATE 0.05 + ATMOS PRESSURE TEMPERATURE PRECIPITATION RELATIVE HUMIDITY NOT MEASURED WIND SPEED <0.0001 - 0.0003 - 0.0002 - <0.0001 - <0.0001 - <0.0001 - THUNDER SCORE

AIR POLLUTANTS NOT MEASURED DUE TO LACK OF PROXIMATE AIR SAMPLING STATION

MYRTACEAE POLLEN GRASS POLLEN PINUS POLLEN ASTERACEAE POLLEN NO POLLENS RECORDED CASUARINA POLLEN ACACIA POLLEN NO POLLENS RECORDED OTHER POLLEN 0.02 - 0.01 - 0.001 - 0.04 - 0.04 - CLADOSPORIUM 0.03 - 0.02 - ALTERNARIA OTHER FUNGI 0.009 + 0.003 + 0.0003 + <0.0001 + 0.0002 +

Appendix P

Appendix P Significance of variable correlations for Range group preventer use score

RANGE GROUP PREVENTER USE SCORE SAME DAY LAG1 LAG2 LAG3 LAG4 LAG5 BENZOIC ACID 0.004 - 0.004 - 0.03 - BENZALDEHYDE ALPHA PINENE BETA PINENE 1,8-CINEOLE CAMPHOR LINALOOL 0.01 + 0.003 + LIMONENE LINAYL ACETATE 0.04 + ATMOS PRESSURE 0.0002 - 0.002 - 0.002 - 0.0006 - 0.0005 - 0.0001 - TEMPERATURE 0.01 + PRECIPITATION RELATIVE HUMIDITY NOT MEASURED WIND SPEED 0.04 - 0.02 - 0.01 - THUNDER SCORE

AIR POLLUTANTS NOT MEASURED DUE TO LACK OF PROXIMATE AIR SAMPLING STATION

MYRTACEAE POLLEN 0.04 - 0.02 - GRASS POLLEN 0.003- 0.008 - PINUS POLLEN ASTERACEAE POLLEN NO POLLENS RECORDED CASUARINA POLLEN ACACIA POLLEN NO POLLENS RECORDED OTHER POLLEN 0.004 - 0.0001 - 0.006 - 0.006 - 0.004 - CLADOSPORIUM ALTERNARIA 0.0009 - <0.0001 - 0.0006 - 0.0002 - 0.01 - 0.001 - OTHER FUNGI 0.0005 + 0.001 + 0.0003 + 0.003 + 0.0002 + 0.0002 +

Appendix P

Appendix P Significance of variable correlations for Range group wheeze RANGE GROUP WHEEZE SAME DAY LAG1 LAG2 LAG3 LAG4 LAG5 BENZOIC ACID 0.04 - 0.02 - BENZALDEHYDE 0.02 - <0.0001 - ALPHA PINENE BETA PINENE 1,8-CINEOLE 0.02 - CAMPHOR 0.03 - LINALOOL LIMONENE LINAYL ACETATE ATMOS PRESSURE TEMPERATURE PRECIPITATION <0.0001 - 0.0003 - 0.01 - 0.001 - 0.0009 - 0.003 - RELATIVE HUMIDITY NOT MEASURED WIND SPEED 0.02 - 0.001 - 0.0001 - 0.001 - 0.001 - 0.0001 - THUNDER SCORE 0.03 - 0.04 -

AIR POLLUTANTS NOT MEASURED DUE TO LACK OF PROXIMATE AIR SAMPLING STATION

MYRTACEAE POLLEN GRASS POLLEN PINUS POLLEN ASTERACEAE POLLEN NO POLLENS RECORDED CASUARINA POLLEN ACACIA POLLEN NO POLLENS RECORDED OTHER POLLEN 0.05 - 0.04 - CLADOSPORIUM 0.03 - 0.001 - 0.003 - 0.006 - ALTERNARIA OTHER FUNGI 0.03 - 0.03 -

Appendix P

Appendix P Significance of variable correlations for Range group difficulty breathing RANGE GROUP DIFFICULTY BREATHING SAME DAY LAG1 LAG2 LAG3 LAG4 LAG5 BENZOIC ACID 0.05 - BENZALDEHYDE 0.03 - 0.04 - ALPHA PINENE 0.05 + BETA PINENE 1,8-CINEOLE CAMPHOR LINALOOL LIMONENE LINAYL ACETATE ATMOS PRESSURE 0.03 - TEMPERATURE PRECIPITATION <0.0001 - 0.0009 - 0.02 - 0.0001 - 0.0001 - 0.002 - RELATIVE HUMIDITY NOT MEASURED WIND SPEED 0.04 - 0.008 - THUNDER SCORE 0.04 -

AIR POLLUTANTS NOT MEASURED DUE TO LACK OF PROXIMATE AIR SAMPLING STATION

MYRTACEAE POLLEN GRASS POLLEN PINUS POLLEN ASTERACEAE POLLEN NO POLLENS RECORDED CASUARINA POLLEN ACACIA POLLEN NO POLLENS RECORDED OTHER POLLEN CLADOSPORIUM ALTERNARIA OTHER FUNGI 0.05 -

Appendix P

Appendix P Significance of variable correlations for Range group coughing

RANGE GROUP COUGHING SAME DAY LAG1 LAG2 LAG3 LAG4 LAG5 BENZOIC ACID 0.04 - BENZALDEHYDE 0.02 - 0.02 - ALPHA PINENE BETA PINENE 1,8-CINEOLE 0.01 - 0.05 - 0.005 - CAMPHOR 0.004 - LINALOOL LIMONENE LINAYL ACETATE ATMOS PRESSURE 0.05 + 0.002 - TEMPERATURE 0.02 - 0.05 - 0.04 - 0.008 - PRECIPITATION 0.04 - RELATIVE HUMIDITY NOT MEASURED WIND SPEED 0.001 - 0.05 - 0.003 - <0.0001 - 0.0002 - THUNDER SCORE

AIR POLLUTANTS NOT MEASURED DUE TO LACK OF PROXIMATE AIR SAMPLING STATION

MYRTACEAE POLLEN 0.03 + GRASS POLLEN 0.05 - PINUS POLLEN ASTERACEAE POLLEN NO POLLENS RECORDED CASUARINA POLLEN ACACIA POLLEN NO POLLENS RECORDED OTHER POLLEN 0.05 - 0.03 - 0.05 - CLADOSPORIUM <0.0001 - 0.0004 - 0.05 - 0.001 - <0.0001 - 0.0001 - ALTERNARIA OTHER FUNGI

Appendix P

Appendix P Significance of variable correlations for Range group itchy eyes

RANGE GROUP ITCHY EYES SAME DAY LAG1 LAG2 LAG3 LAG4 LAG5 BENZOIC ACID BENZALDEHYDE ALPHA PINENE BETA PINENE 1,8-CINEOLE 0.03 - CAMPHOR LINALOOL 0.05 + 0.01 + LIMONENE LINAYL ACETATE ATMOS PRESSURE 0.04 - 0.01 - 0.02 - 0.007 - 0.02 - TEMPERATURE PRECIPITATION 0.004 - RELATIVE HUMIDITY NOT MEASURED WIND SPEED 0.005 - 0.009 - THUNDER SCORE

AIR POLLUTANTS NOT MEASURED DUE TO LACK OF PROXIMATE AIR SAMPLING STATION

MYRTACEAE POLLEN 0.02 + GRASS POLLEN PINUS POLLEN ASTERACEAE POLLEN NO POLLENS RECORDED CASUARINA POLLEN ACACIA POLLEN NO POLLENS RECORDED OTHER POLLEN CLADOSPORIUM ALTERNARIA OTHER FUNGI 0.003 + 0.03 + 0.003 + 0.0001 + 0.0004 +

Appendix P

Appendix P Significance of variable correlations for Range group itchy nose

RANGE GROUP ITCHY NOSE SAME DAY LAG1 LAG2 LAG3 LAG4 LAG5 BENZOIC ACID 0.03 - BENZALDEHYDE 0.004 - 0.02 - 0.003 - ALPHA PINENE BETA PINENE 1,8-CINEOLE 0.02 - CAMPHOR LINALOOL LIMONENE 0.03 - LINAYL ACETATE ATMOSPHERIC PRESSURE TEMPERATURE PRECIPITATION 0.04 - RELATIVE HUMIDITY NOT MEASURED WIND SPEED THUNDER SCORE

AIR POLLUTANTS NOT MEASURED DUE TO LACK OF PROXIMATE AIR SAMPLING STATION

MYRTACEAE POLLEN GRASS POLLEN PINUS POLLEN ASTERACEAE POLLEN NO POLLENS RECORDED CASUARINA POLLEN ACACIA POLLEN NO POLLENS RECORDED OTHER POLLEN CLADOSPORIUM ALTERNARIA OTHER FUNGI 0.04 + 0.009 +

Appendix P

Appendix P Significance of variable correlations for Range group sneezing

RANGE GROUP SNEEZING SAME DAY LAG1 LAG2 LAG3 LAG4 LAG5 BENZOIC ACID BENZALDEHYDE ALPHA PINENE 0.05 + 0.04 + BETA PINENE 0.05 + 0.04 + 1,8-CINEOLE CAMPHOR 0.01 + LINALOOL 0.03 + 0.05 + 0.006 + LIMONENE LINAYL ACETATE 0.03 + 0.03 + 0.04 + ATMOS PRESSURE 0.02 - 0.02 - 0.01 - 0.009 - TEMPERATURE PRECIPITATION 0.001 - 0.007 - 0.02 - 0.04 - RELATIVE HUMIDITY NOT MEASURED WIND SPEED 0.03 - THUNDER SCORE

AIR POLLUTANTS NOT MEASURED DUE TO LACK OF PROXIMATE AIR SAMPLING STATION

MYRTACEAE POLLEN 0.05 + GRASS POLLEN PINUS POLLEN ASTERACEAE POLLEN NO POLLENS RECORDED CASUARINA POLLEN ACACIA POLLEN NO POLLENS RECORDED OTHER POLLEN CLADOSPORIUM ALTERNARIA OTHER FUNGI

Appendix P

Appendix P Significance of variable correlations for Range group blocked nose

RANGE GROUP BLOCKED NOSE SAME DAY LAG1 LAG2 LAG3 LAG4 LAG5 BENZOIC ACID BENZALDEHYDE 0.02 - ALPHA PINENE BETA PINENE 1,8-CINEOLE CAMPHOR LINALOOL 0.01 + 0.02 + LIMONENE LINAYL ACETATE 0.04 + ATMOS PRESSURE 0.05 - 0.02 - 0.01 - 0.01 - 0.009 - 0.02 - TEMPERATURE PRECIPITATION 0.0002 - <0.0001 - 0.0008 - <0.0001 - 0.0003 - 0.02 - RELATIVE HUMIDITY NOT MEASURED WIND SPEED 0.02 - 0.002 - 0.001 - 0.001 - 0.002 - 0.013 - THUNDER SCORE 0.04 -

AIR POLLUTANTS NOT MEASURED DUE TO LACK OF PROXIMATE AIR SAMPLING STATION

MYRTACEAE POLLEN 0.04 + GRASS POLLEN 0.04 - 0.009 - 0.03 - PINUS POLLEN ASTERACEAE POLLEN NO POLLENS RECORDED CASUARINA POLLEN 0.04 + 0.01 + ACACIA POLLEN NO POLLENS RECORDED OTHER POLLEN CLADOSPORIUM 0.05 + 0.04 + ALTERNARIA OTHER FUNGI 0.004 -

Appendix P

Appendix P Significance of variable correlations for Range group runny nose

RANGE GROUP RUNNY NOSE SAME DAY LAG1 LAG2 LAG3 LAG4 LAG5 BENZOIC ACID BENZALDEHYDE 0.05 - ALPHA PINENE BETA PINENE 1,8-CINEOLE CAMPHOR LINALOOL LIMONENE LINAYL ACETATE 0.05 + 0.03 + ATMOS PRESSURE TEMPERATURE 0.001 - <0.0001 - <0.0001 - 0.0008 - 0.04 - PRECIPITATION 0.007 - RELATIVE HUMIDITY NOT MEASURED WIND SPEED THUNDER SCORE 0.01 +

AIR POLLUTANTS NOT MEASURED DUE TO LACK OF PROXIMATE AIR SAMPLING STATION

MYRTACEAE POLLEN GRASS POLLEN PINUS POLLEN ASTERACEAE POLLEN NO POLLENS RECORDED CASUARINA POLLEN ACACIA POLLEN NO POLLENS RECORDED OTHER POLLEN CLADOSPORIUM ALTERNARIA OTHER FUNGI

Appendix P

Appendix Q Program and results for stepwise regression lag 3 Coast group SPEF

Program for stepwise regression for Coast group lag 3

*proc reg data=Callterp; model CZPEAKM=PRESMEA3 TEMPMEA3 PREC3 WINDMEA3 THUNDSC3 PMTEN3 OZONE3 NDIOX3 RHUM3 MYRTAC3 GRASS3 PINE3 ASTER3 CASUAR3ACACIA3OTHPOL3CLAD3 ALTERN3 OTHFUN3 RBENZC3 RLIMON3 RBENZAL3 RALPHI3 RBETAP3 RCINE3 RCAMPH3 RLINAC3 RLINAL3 /selection = stepwise slstay=0.1 slentry=0.1;run;*

Results The SAS System 12 17:58 Thursday, December 14, 2006

The REG Procedure Model: MODEL1 Dependent Variable: CZPEAKM

Stepwise Selection: Step 6

Parameter Standard Variable Estimate Error Type II SS F Value Pr > F

Intercept -22.63576 9.28206 0.22644 5.95 0.0205 PRESMEA3 0.02141 0.00910 0.21096 5.54 0.0249 TEMPMEA3 0.03441 0.01604 0.17516 4.60 0.0397 OZONE3 17.20172 6.48845 0.26761 7.03 0.0124 PINE3 -0.40381 0.18492 0.18156 4.77 0.0364 ACACIA3 0.25153 0.06092 0.64898 17.04 0.0002 RALPHI3 -0.30191 0.09423 0.39083 10.26 0.0031

Bounds on condition number: 3.2145, 65.354 ------All variables left in the model are significant at the 0.1000 level. Summary of Stepwise Selection Variable Variable Number Partial Model Step Entered Removed Vars In R-Square R-Square C(p) F Value Pr > F

1 OZONE3 1 0.2542 0.2542 53.4249 12.61 0.0011 2 ACACIA3 2 0.0721 0.3263 46.8801 3.85 0.0575 3 RALPHI3 3 0.1403 0.4666 32.2485 9.20 0.0045 4 TEMPMEA3 4 0.0540 0.5206 27.8445 3.83 0.0586 5 PRESMEA3 5 0.0438 0.5644 24.6519 3.32 0.0776 6 PINE3 6 0.0565 0.6209 19.9531 4.77 0.0364

Appendix Q

Appendix R Principal Components analysis of volatiles in coastal analysis

The SAS System 21:21 Saturday, December 16, 2006 The FACTOR Procedure Initial Factor Method: Principal Components Prior Communality Estimates: ONE Eigenvalues of the Correlation Matrix: Total = 9 Average = 1 Eigenvalue Difference Proportion Cumulative 1 5.60271678 4.56192745 0.6225 0.6225 2 1.04078933 0.12636354 0.1156 0.7382 3 0.91442579 0.31969840 0.1016 0.8398 4 0.59472739 0.03024590 0.0661 0.9059 5 0.56448149 0.40493301 0.0627 0.9686 6 0.15954848 0.08506827 0.0177 0.9863 7 0.07448021 0.04171564 0.0083 0.9946 8 0.03276456 0.01669858 0.0036 0.9982 9 0.01606598 0.0018 1.0000 9 factors will be retained by the NFACTOR criterion. The FACTOR Procedure Initial Factor Method: Principal Components Scree Plot of Eigenvalues ‚ E 6 ˆ i ‚ 1 g ‚ e ‚ n 4 ˆ v ‚ a ‚ l ‚ u 2 ˆ e ‚ s ‚ 2 3 ‚ 4 5 0 ˆ 6 7 8 9

Šƒƒƒˆƒƒƒƒƒƒˆƒƒƒƒƒƒˆƒƒƒƒƒƒˆƒƒƒƒƒƒˆƒƒƒƒƒƒˆƒƒƒƒƒƒˆƒƒƒƒƒƒˆƒƒƒƒƒƒˆƒƒƒƒƒƒˆƒƒ ƒ 0 1 2 3 4 5 6 7 8 Factor Pattern Factor1 Factor2 Factor3 Factor4 RBENZOIC -0.24653 0.88049 -0.31604 0.12316 RBENZALD 0.31857 0.33357 0.87681 -0.07056 `RALPHAPI 0.87756 0.14063 -0.00614 0.29941 RBETAPIN 0.88334 0.09907 0.03634 0.31754 RCINEOLE 0.86491 -0.12053 -0.05540 0.23046 RCAMPHOR 0.91180 -0.15601 -0.06690 -0.10443 RLIMON 0.88836 -0.16729 -0.05551 0.09840 RLINALOO 0.88667 0.18911 -0.11985 -0.34954 RLINACET 0.85747 0.14849 -0.13931 -0.43384

Appendix R

Appendix S General Linear Model Regression Coast group SPEF

The SAS System The GLM Procedure

Number of observations 154

NOTE: Due to missing values, only 39 observations can be used in this analysis.

Dependent Variable: CZPEAKM

Sum of Source DF Squares Mean Square F Value Pr > F

Model 4 1.64000068 0.41000017 8.86 <.0001

Error 34 1.57371309 0.04628568

Corrected Total 38 3.21371378

R-Square Coeff Var Root MSE CZPEAKM Mean

0.510313 218.0491 0.215141 0.098666 Source DF Type I SS Mean Square F Value Pr > F RALPHI3 1 0.01860575 0.01860575 0.40 0.5303 OZONE3 1 0.99302696 0.99302696 21.45 <.0001

Dependent Variable: CZPEAKM Source DF Type I SS Mean Square F Value Pr > F

ACACIA3 1 0.48778900 0.48778900 10.54 0.0026 PINE3 1 0.14057897 0.14057897 3.04 0.0904

Source DF Type III SS Mean Square F Value Pr > F RALPHI3 1 0.55053471 0.55053471 11.89 0.0015 OZONE3 1 0.39446654 0.39446654 8.52 0.0062 ACACIA3 1 0.55595565 0.55595565 12.01 0.0015 PINE3 1 0.14057897 0.14057897 3.04 0.0904

Standard Parameter Estimate Error t Value Pr > |t|

Intercept -0.21418180 0.11498259 -1.86 0.0712 RALPHI3 -0.35237247 0.10217225 -3.45 0.0015 OZONE3 19.61881885 6.72033761 2.92 0.0062 ACACIA3 0.23137544 0.06676064 3.47 0.0015 PINE3 -0.34485901 0.19788128 -1.74 0.0904

Appendix S

Appendix T Programs used in SAS for determining predictive capabilities of variables.

This program performs a stepwise regression for coast SPEF using the four variables that were identified as significant in predicting SPEF for the Coast group. proc reg data=CALLTERP; model CZPEAKM= OZONE3 PINE3 ACACIA3 RALPHI3 /selection = stepwise;run;

This program performs a stepwise regression for coast SPEF using the four variables that were identified as significant in predicting SPEF for the Range group. proc reg data=hallterp; model HZPEAKM= CLAD4 RBETAP4 RCINE4 PREC4/selection = stepwise slstay=0.1 slentry=0.1;run

This program performs a GLM regression for a single predictive variable

PROC GLM DATA=HIGHAUT; MODEL HRELIEVM=RLINAL5; RUN ;

Appendix T

Appendix U Range stepwise regression for Lag 4 using significant predictors of SPEF

Stepwise Selection: Step 4 The SAS System 7

The REG Procedure Model: MODEL1 Dependent Variable: HZPEAKM

Stepwise Selection: Step 4

Variable PREC4 Entered: R-Square = 0.4105 and C(p) = 5.0000

Analysis of Variance

Sum of Mean Source DF Squares Square F Value Pr > F

Model 4 1.04860 0.26215 4.87 0.0041 Error 28 1.50614 0.05379 Corrected Total 32 2.55474

Stepwise Selection: Step 4 Parameter Standard Variable Estimate Error Type II SS F Value Pr > F

Intercept -0.28814 0.06545 1.04255 19.38 0.0001 CLAD4 0.00025873 0.00011802 0.25853 4.81 0.0368 RBETAP4 -0.53286 0.15327 0.65012 12.09 0.0017 RCINE4 0.30507 0.10812 0.42827 7.96 0.0087 PREC4 -0.00464 0.00216 0.24788 4.61 0.0406

Bounds on condition number: 2.252, 27.16 ------Summary of Stepwise Selection

Variable Variable Number Partial Model Step Entered Removed Vars In R-Square R-Square C(p) F Value Pr > F

1 CLAD4 1 0.1147 0.1147 13.0485 4.01 0.0539 2 RBETAP4 2 0.0882 0.2028 10.8605 3.32 0.0785 3 RCINE4 3 0.1106 0.3134 7.6083 4.67 0.0391 4 PREC4 4 0.0970 0.4105 5.0000 4.61 0.0406

Appendix U

Appendix V General Linear Model results for Asthma Score for coastal group

The GLM Procedure

Number of observations 154

NOTE: Due to missing values, only 40 observations can be used in this analysis.

The SAS System 27

The GLM Procedure Dependent Variable: CASTHMAM Sum of Source DF Squares Mean Square F Value Pr > F

Model 5 3.04161922 0.60832384 8.56 <.0001

Error 34 2.41611114 0.07106209

Corrected Total 39 5.45773035 R-Square Coeff Var Root MSE CASTHMAM Mean 0.557305 10.29440 0.266575 2.589512 Source DF Type I SS Mean Square F Value Pr > F

MYRTAC5 1 1.05024410 1.05024410 14.78 0.0005 ALTERN5 1 0.43556548 0.43556548 6.13 0.0184 TEMPMEA5 1 0.88513326 0.88513326 12.46 0.0012 RBENZAL5 1 0.44300833 0.44300833 6.23 0.0175 PINE5 1 0.22766805 0.22766805 3.20 0.0824

Source DF Type III SS Mean Square F Value Pr > F

MYRTAC5 1 1.08633399 1.08633399 15.29 0.0004 ALTERN5 1 0.56638541 0.56638541 7.97 0.0079 TEMPMEA5 1 1.18738121 1.18738121 16.71 0.0003 RBENZAL5 1 0.56362611 0.56362611 7.93 0.0080 PINE5 1 0.22766805 0.22766805 3.20 0.0824

Dependent Variable: CASTHMAM Standard Parameter Estimate Error t Value Pr > |t|

Intercept 0.0599849919 0.57721447 0.10 0.9178 MYRTAC5 0.0264601374 0.00676752 3.91 0.0004 ALTERN5 0.0522094866 0.01849321 2.82 0.0079 TEMPMEA5 0.0898056098 0.02196986 4.09 0.0003 RBENZAL5 0.4232050943 0.15027071 2.82 0.0080 PINE5 -.3040340609 0.16985964 -1.79 0.0824

This is the same R-square result as the stepwise regression with the same set of variables.

AppendixV

Appendix W Wind Rose information for January in Brisbane Source: Bureau of Meteorology, Aust Govt

Appendix W

Appendix X Wind rose for Brisbane May

Appendix X

Appendix Y Flower and leaf extract example: Eucalyptus

Appendix Y

Appendix Z GCMS of SPME sampled M. quinquenervia stamens

Appendix Z

Appendix AA Methanol extracts of Melaleuca quinquenervia showing sitostenone

Appendix AA

Appendix AB Regression models predicting SPEF in COAST group for spring and autumn

Appendix AB

Appendix AC Regression models predicting SPEF in COAST group for spring

Appendix AC

Appendix AD Regression models predicting SPEF in COAST group for autumn

Appendix AD