EFFECTS OF FUNGICIDES ON AUSTRALIAN AMPHIPODS AND ORGANIC MATTER BREAKDOWN IN AQUATIC ENVIRONMENTS Submitted by Hung Thi Hong Vu Submitted in fulfillment of the degree of Doctor of Philosophy February 2017 School of BioSciences Faculty of Science The University of Melbourne ABSTRACT Fungicides are used widely in agriculture to control fungal diseases and increase crop yield. After application, fungicides may be transported off site via air, soil and water to ground and surface waters therefore have the potential to contaminate freshwater and marine/estuarine environments. However, relatively little is known about their potential effects on aquatic ecosystems. Amphipods are important in ecosystem service as they help with nutrient recycling through the decomposition of organic matter. The aim of this thesis is to investigate the effects of common fungicides on biological responses in two Australian amphipod species, Allorchestes compressa and Austrochiltonia subtenuis, through a combination of single and mixture laboratory experiments. In addition a field experiment investigated the effects of fungicides on organic matter breakdown. In laboratory studies, juveniles of the marine amphipod A. compressa and the freshwater amphipod A.subtenuis were chronically exposed to two commonly used fungicides, Filan® (active ingredient boscalid) and Systhane™ (active ingredient myclobutanil) at environmentally relevant concentrations. A wide range of endpoints that encompass different levels of biological organization were measured including survival, growth, reproduction, and energy reserves (lipid, glycogen, and protein content). Long term interaction effects of fungicides Filan® and Systhane™ on mature amphipod A. subtenuis was also investigated to evaluate how the results of mixture studies vary between endpoints and to determine suitable endpoints for mixture toxicity studies. In the field study, leaves and cotton strips were deployed at 26 sites, 24 study and 2 reference sites, in an intensive agricultural region in south-eastern Australia to investigate the effects of fungicides and other anthropogenic stressors on organic matter breakdown. Leaves and cotton strips were deployed at the sites for a three week period and repeated twice in winter and spring. Breakdown rates of leaf and cotton at studied sites were compared to that of the reference site two which has similar altitude to the study sites to determine the effects of fungicides and other stressors on functional stream health. Pesticide concentration and physico-chemical parameters of sites were monitored during the study. The relationship between organic matter breakdown rates and environmental variables was investigated. Page 1 of 120 Laboratory results demonstrated that Filan® and Systhane™ caused significantly adverse effects on survival, growth, reproduction, and energy reserves of both species of amphipod at environmentally realistic concentrations. Female amphipods were more sensitive to fungicides than males in terms of growth. Reproduction was the most sensitive endpoint and most affected by fungicide exposure. The effects of fungicide mixtures on A.subtenuis were endpoint-dependent and antagonistic effects were observed only on reproduction. Field data showed that organic matter breakdown rate was significantly correlated with pesticide concentrations and nutrients but leaf breakdown was also strongly impacted by temperature. Leaf and cotton degraded differently but both indicated the same results on functional stream health for majority of the sites. This thesis provides the first evidence of the effects of common fungicides on survival, growth, reproduction, and energy reserves of two Australian amphipods at environmentally relevant concentrations. The results suggest that fungicide pollution could affect the viability of amphipod populations in the natural environments that consequently could cause cascading effects on the ecosystem. This is also the first study to investigate individual relationships between different pesticide groups with organic matter breakdown in a field environment. The results of this study emphasize the importance of considering the long-term effects of fungicides in risk assessments for aquatic ecosystems and contribute to the literature of fungicide toxicity on aquatic environments. Page 2 of 120 DECLARATION This is to certify that: i. The thesis comprises only my original work towards the PhD ii. Due acknowledgement has been made in the text to all other material used iii. The thesis is less than 100,000 words in length, excluding tables, maps, bibliographies and appendices Hung Thi Hong Vu February 2017 Page 3 of 120 PREFACE This thesis comprises one introduction chapter (Chapter 1), three scientific papers (Chapters 2, 3, and 4), one manuscript (Chapter 5), and one discussion chapter (Chapter 6). Chapter 1 - The literature review Chapter 2 Hung T. Vu, Michael J. Keough, Sara M. Long, and Vincent J. Pettigrove. 2016. Effects of the boscalid fungicide Filan® on the marine amphipod Allorchestes compressa at environmentally relevant concentrations. Environmental Toxicology and Chemistry 35:1130-1137. The major research of this publication is my own work. Other co-authors provide scientific advice, training in laboratory and data analysis techniques, and reviewing the manuscript before submission. Chapter 3 Hung T. Vu, Michael J. Keough, Sara M. Long, and Vincent J. Pettigrove. 2017. Effects of two commonly used fungicides on the amphipod Austrochiltonia subtenuis. Environmental Toxicology and Chemistry 36:720-726. The major research of this publication is my own work. Other co-authors provide scientific advice, training in laboratory and data analysis techniques, and reviewing the manuscript before submission. Chapter 4 Hung T. Vu, Michael J. Keough, Sara M. Long, and Vincent J. Pettigrove. 2017. Toxicological effects of fungicide mixtures on the amphipod Austrochiltonia subtenuis. Environmental Toxicology and Chemistry. DOI: 10.1002/etc.3809. Page 4 of 120 The major research of this manuscript is my own work. Other co-authors provide scientific advice, training in laboratory and data analysis techniques, and reviewing the manuscript before submission. Chapter 5 Hung T. Vu, Jackie H. Myers, Simon M. Sharp, Claudette R. Kellar, Sara M. Long, Michael J. Keough, and Vincent J. Pettigrove (in preparation). Can organic matter decomposition indicate the effects of multiple anthropogenic stressors on functional stream health? The research of this manuscript was incorporated in the Western Port Toxicant Study Stage 3 project and some of the results were also reported in “Myers, JH., Sharley, D., Sharp, S., Vu, H., Long, S., and Pettigrove, V. (2016), Final Report Western Port Toxicant Study Stage 3 – Pesticide Sourcing Study and Aquatic Flora and Fauna Assessment, Centre for Aquatic Pollution Identification and Management, Technical Report No. 63A, University of Melbourne, Victoria, Australia.” In this manuscript, the nature and extent of authors’ contributions to the work were the following: Name Nature of contribution Extent of contribution (%) for student authors only Hung T. Vu Field work, sample processing, data 60% analysis and interpretation of results, contribution to project design, and preparation of manuscript Jackie H. Myers Project manager, assistance with field N/A work, and review of data and manuscript Simon M. Sharp Pesticides and water chemistry analysis, N/A assistance with field work Claudette R. Kellar Macroinvertebrate identification and N/A Page 5 of 120 review of manuscript Sara M. Long Scientific advice and review of N/A manuscript Michael J. Keough Scientific advice and review of data and N/A manuscript Vincent J. Pettigrove Scientific advice and review of data and N/A manuscript Chapter 6 – General discussion Page 6 of 120 ACKNOWLEDGMENTS I would like to express my special gratitude to my supervisors Associate Professor Vincent Pettigrove, Professor Michael Keough, and Dr Sara Long. Thank you for your great support, guidance, and encouragement throughout my PhD candidature. I am grateful for many hours, in and out of working time, of discussion and advice you provided me. It has been a great honor and pleasure to be your student. I would like to thank all the CAPIM staff and students for helping me during my candidature. In particular, I would like to thank Jackie Myers, Dave Sharley, Katy Jeppe, Claudette Kellar, Cameron Amos, Steve Marshall, Rebecca Brown, Daniel MacMahon, Simon Sharp, Pat Bonney, Rhianna Boyle, Jessica French, and Tyler Mehler for their help with field work. I would like to thank Allyson O’Brien for providing comments and helping improve the second chapter. I would like to thank Peter Symes and Therese Turner, Royal Botanic Gardens Melbourne who provided support and assistance with collecting Pomaderris aspera leaves using in my laboratory studies. I would also like to thank my parent, my parent in-law, my two sisters, and my brother and his wife for their support and assistance with taking care of my children while I was busy with my study. I dedicate this PhD thesis to my husband and our two beloved children. Thank you for your accompanies during this challenging but interesting journey. Thank you for joining me in happy moments and cheering me up when I was feeling down. Page 7 of 120 The Melbourne International Research Scholarship (MIRS) and the Centre of Aquatic Pollution Identification and Management (CAPIM) funded this study. Extra funding was gained through Melbourne Water, Holsworth