Using Chironomidae to assess water and sediment quality Submitted by Kallie Rose Townsend Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy January 2013 Department of Zoology Faculty of Science The University of Melbourne Printed on archival quality paper I Abstract Biological assessments with Chironomidae (chironomids) are often used to provide information on the causes and effects of aquatic pollution. However, these applications are constrained by several factors, some of which were investigated in this thesis. Laboratory toxicity test results are used to determine toxicity in the field, but these are ecologically irrelevant and often fail to account for the influence of complicating factors in the field. Two such factors are food limitation, potentially caused by an indirect effect of pollution, and the exposure of previous generations to stressors. The effect of food limitation was investigated in a multigenerational laboratory experiment with Chironomus tepperi . Responses to food limitation were similar to toxicity, with reduced survival, reproduction and increased development times as food became limited. The offspring of food limited parents were also affected. There was evidence that the quality of these offspring were compromised, even when offspring were raised under standard food conditions. Males and females responded differently, with greater developmental delays seen in female offspring. There is also a scarcity of data regarding the effects of various pollutants on different chironomid species, particularly for Australian chironomids, under ecologically relevant conditions. In field-based microcosm experiments the effects of 17 α- ethinylestradiol (EE2), tebufenozide and diuron were investigated. High nominal concentrations (10 µg L -1) of EE2 consistently induced a novel antennal deformity in Procladius species larvae, but no effects on assemblages, abundances or development rate were observed except for an increased number of taxa at lower concentrations. Similarly, aside from increasing the number of taxa at the lowest nominal concentration of 0.004 mg L -1, diuron had no effect on assemblages or species. Tebufenozide had no effect on these parameters. Another constraint is an inadequate understanding and insufficient use of alternative endpoints, such as sex ratio skewing and adult responses in situ . Experimental studies showed that sex ratio skewing was stressor specific, occurring after exposure to II tebufenozide and diuron but not EE2 or food limitation. It was also species specific; tebufenozide and diuron caused skewing in different species. There was evidence that skewing was consistent, with the feminised sex ratios in Procladius villosimanus caused by diuron similar to that in P. paludicola in a previous study. In addition to sex ratios, other adult endpoints such as abundance and diversity are also useful for measuring impairment, although these are rarely including in biomonitoring studies. To measure adult responses in situ a method was developed using yellow sticky traps. The method was successfully able to detect differences between sites based on the composition and sex ratios of the chironomid fauna captured. Some of these biological parameters could be related to zinc pollution in sediments. The results of these studies demonstrate the value of considering factors aside from toxicity, continued pollutant testing and the use of alternative endpoints in bioassessments with chironomids. Multigenerational testing and measuring sex ratios should be routinely included in studies with chironomids and, along with adult biomonitoring, represent useful additions to current approaches that need further investigation. III Declaration This is to certify that: i. the thesis comprises only my original work towards the PhD except where indicated in the preface, ii. due acknowledgement has been made in the text to all other material used, iii. the thesis is fewer than 100 000 words in length, exclusive of tables, maps, bibliographies and appendices. Kallie Rose Townsend 15 th January 2013 IV Preface The experimental results of this thesis are presented in five self-contained chapters (Chapters 2 to 6). Chapter 5, A method for biomonitoring with adult Chironomidae, has been submitted to Environmental Monitoring and Assessment and is under review. This paper is co- authored with V. J. Pettigrove and A. A. Hoffmann. However, the contents are my own work and co-authorship represents a supervisory role of the other authors, in addition to providing scientific advice and site selection, assistance with statistical analyses, and reviewing the manuscript before submission. Chapter 4 is presented as the substantially unchanged paper submitted as: Townsend, K. R., Pettigrove, V. J. and Hoffmann, A. A. (2012). Food limitation in Chironomus tepperi : Effects on survival, sex ratios and development across two generations. Ecotoxicology and Environmental Safety 84 , 1 – 8. The contents of this paper are my own work and co-authorship represents a supervisory role, in addition to advice for experimental design and statistical analyses, and revision of the paper prior to submission. V Acknowledgements This thesis is the result of the hard work, sacrifices and support of many people throughout my candidature. While people who contributed to specific parts of an experiment are acknowledged in those chapters, in this space I want to express my thanks to those who have helped me get this far. Firstly, I thank my supervisors Ary Hoffmann and Vin Pettigrove for giving me inspiration, instruction and correction when I needed it. Without them this thesis would not exist (alternatively, it would be filled with very lengthy paragraphs and poor statistics). I also want to acknowledge the effort of people from CAPIM and CESAR who gave me advice, helped me with field work and made me laugh. I especially thank Sara Hoskin for being there while I went through the trauma of Starvation Creek, Bryant Gagliardi (I owe him a lot for making him dig up the dam bank in the 40 °C heat), Daniel MacMahon who was dragged out on a lot of field work expeditions, Dave Sharley for encouraging me to get this finished, two anonymous reviewers who gave me helpful comments on this thesis, and to Emily Thomson and Cindy Halliwell. Thanks also to Nancy Endersby and others at Bio21, the Department of Zoology and the University of Melbourne who have been involved in various parts of this process. Thanks to Mum, Dad, Kate and Haydn for tolerating my absences, loving me unconditionally and for believing I could get this done. Similarly, I thank my dear friends who supported me during this time, especially Amy Chung, Sam Rawson and Alicia Wong. A very special thanks goes to Stephen Tan, who has loved me through a very ugly, trying period of my life, and thank you dear God that somehow I have made it when two years ago I was ready to quit forever! Finally, I wish to acknowledge the University of Melbourne and Melbourne Water Corporation for providing a scholarship, and the Melbourne Water Corporation, the Victorian Department of Business and Innovation through CAPIM, and the Australian Research Council through their Fellowship scheme for funding this research. VI Contents Chapter 1: Introduction ..............................................................................................1 Freshwater ecosystems...............................................................................................1 Threats to freshwater ecosystems ..............................................................................2 Assessing aquatic ecosystem pollution......................................................................7 Biological assessments with macroinvertebrates.....................................................15 Outline of study........................................................................................................19 References................................................................................................................22 Chapter 2: The effects of an environmental estrogen, 17 α-ethinylestradiol, on Chironomidae .............................................................................................................41 Abstract....................................................................................................................41 Introduction..............................................................................................................42 Materials and methods .............................................................................................45 Results......................................................................................................................51 Discussion................................................................................................................60 Acknowledgements..................................................................................................67 References................................................................................................................67 Figures......................................................................................................................79 Tables.......................................................................................................................87 Chapter 3: The effects of tebufenozide and diuron on Chironomidae .................96 Abstract....................................................................................................................96