A RE-EVALUATION OF THE SKELETAL ABNORMALITIES IN FROGS IN THE ADELAIDE HILLS REGION By Steven J. Walker Department of Environmental Biolo gy University of Adelaide A thesis submitted to the University of Adelaide for the Degree of Master of Science. October 2000 Gontents Abstract 111 Declaration iv Acknowledgments v Section One - Field SurveY 1. lntroduction 1 1.1 Disease 1 .2 Habitat Degradation 2 1.3 Global Warming 2 1.4 Ultraviolet Radiation J 1.5 Pollution 3 1.6 Aims 7 2, Materials and Methods 8 2.1 Collection Sites 9 2.2 Collecting Methods 10 2.3 Fixation, Preservation and Storage 1t 2.4 Examination and Alizarin Preparation 1,2 2.5 Deformity Classification 12 2.6 Statistical Analysis r4 3. Results 15 3.1 Catalogue of Deformities 15 3.2 Collection Summary 62 3.3 Statistical Analysis 70 4. Discussion 73 4.1 Abnormalities 73 4.2 Brooks 1979 74 4.3 lnjuries 75 4.4 Recent Discoveries of Abnormal Frogs 75 Section Two - Toxicological Research 5. Gavan - Background 77 5.1 Survey SummarY 78 6. lmpromptu SPawning 80 6.1 Methods 80 6.2 Results 80 6.3 Discussion 82 7. Sediment Bioassay 1 - Collected Sediment 83 7.1 Methods 83 7.2 Results 84 7.3 Discussion 91 8. Sediment Bioassay 2-Prepared Sediment 92 8.1 Methods 92 8.2 Results 93 8.3 Discussion 105 9. Gonclusions 109 9.1 lncidence of Abnormality 109 9.2 Parasites and Cysts 110 9.3 lnjuries ll0 9.4 Future Directions ll1 10. Appendix 1 - Metamorphosis of frogs from lmpromptu Cavan spawning 112 1 1 . Appen dix 2 - Preparation of Moderately Hard Synthetic Freshwater 115 l2"Appendix 3 - Sediment Bioassay 1 metamorphs 116 l3.Appendix 4 - Sediment Bioassay 2 metamorphs 1t7 14. Appendix 5 - Presence of cysts in deformed frogs collected during the field survey 121 15. References 122 Abstract This study has shown that abnormal frogs representing ten species can be found in a range of habitats in the Mt Lofty Ranges, Flinders Ranges and the South East of South Australia. There was no signitìcant difference in the incidence of abnormality between the Flinclers Ranges, which has little or no pollution, and the Mt Lofty Ranges. However incidence of abnormality in frogs is associated with land use in the Mt Lofty Ranges. The incidence of injury in frogs was much higher than has generally been reported but was similar to the study of Brooks (1979).Incidence of injury did not vary significantly between different land use areas or geographic regions. Most sites within the Adelaide Hills and surrounds had low levels of abnormality but an exception was high levels in two species at a pond in an industrial area. Further investigation found that the levels of various heavy metals in the sediment were above those considered desirabl dlreshwater systems. ^ Eggs that were laid by frogs collected from the polluted site and reared under laboratory conditions produced very low levels of abnormal frogs, well within allowable limits, This observation suggests that pollutants are not accumulated and transferred to successive generations. Abnormal frogs collected from the same location did not successfully reproduce. There was a reduction in the survival of tadpoles reared in sediment collected from the polluted site, but no difference in survival was detected when tadpoles were reared in artificial sediment that had similar heavy metal levels. There was however an increase in developmental problems with increasing metal concentration: o Crinia signifera took longer to reach metamorphosis. o Litoria ewingi also took longer to metamorphose and furthermore attained a larger size at metamorphosis. Although no statistical analyses could be undertaken there appeared to be an increase in the incidence of abnormality in the polluted aquaria, but the rate of abnormality was lower than that recorded in the wild. Therefore, despite the fact that these metals can have a significant effect on growth and development, they are not solely responsible for the high incidenoe of abnormality in the field. 111 Declaration This work contains no material that has been accepted for the award of any other degree or diploma in any university or other tertiary institution. To the best of my knowledge no material previously published or written by another person has been included, except where due reference has been made in the text. I give consent to this copy of my thesis, when deposited in the University Library, being made available for loan and photocopying. rzf ro{e.ooo 1V Acknowledgments I particularly wish to thank my superuisor, Assoc/Prof. Michael Tyler, for stimulating my enthusiasm and giving support during the course of this study and for criticism of this manuscript during its preparation. I would like to thank all of the people who assisted me during the field suryey; principally Alison Beck, Brydie Hill, Colin Bailey and David Gooding. Members of the South Australian Frog and Tadpole Study Group, and the South Australian Herpetology Group also provided assistance in the field. Lynn Joosen was also extremely valuable in obtaining the support of numerous landholders in the Mt Lofty Ranges. I very much appreciate her efforts and enthusiasm. Sincere thanks are expressed to those who gave me access to their property. Thanks to Ben Smith for assistance with the toxicology experiments and looking after animals while I was away. I would like to show my appreciation to Dr Margaret Davies who reviewed the manuscript during its early stages. The quality of the text has improved as a result of her suggestions. Dr Keith'Walker gave considerable assistance in solving statistics problems, I am sincerely grateful to him. Mr Phil Kempster's advice and help with photography is much appreciated. Salisbury Council provided funds for the analysis of sediment during the toxicology experiments. For this I am most grateful. I would especially like to thank Melissa Bradbury for her support and assistance during the study, and for keeping me sane, well almost! V l lntroduction In the late 1970s numerous herpetologists began noticing declines in of amphibians in locations throughout the world (see RicharJs et ul. 1993, Tyler' 1994a, Carcy and Bryant lgg5). Every population of organisms experiences variation in size as a result of many factors including breeding success, predation, age structure and migration. However it seems unlikely that these natural processes would lead to concurrent declines of amphibians in different parts of the world. Amphibians have been on the planet about 350 million years; since the latter stages of the Devonian period (Colbert 1969, Tyler 1994a). They have survived many environmental changes ineluding ice ages and numerous likely extinetion events, including those that resulted in the extinction of the dinosaurs. They have remained relatively unchanged morphologically for about 200 million years. It follows that the sudden drop in species that are widely separated spatially and many which may have been abundant for thousands of years suggests an environmental cause; one that is conceivably the result of anthropogenic factors. ,A^ number of causal agents has been suggested for this global decline; including disease, global warming, ultraviolet radiation, habitat degradation and pollution, including acid rain (Ferraro and Burgin 1993, Richards et al. 1993, Tyler 1994a, Boyer and Grue 1995, Carey and Bryant 1995). The current consensus is that no single factor is responsible. 1"{ Disease Reichenbach-Klinke and Elkan (1965) prouia"linformation on infectious, parasitic and non- parasitic diseases of the Amphibia and Speare (1990) lists large numbers of diseases that infect the cane toað, Bufo marinus. Recent evidence suggests that a number of new pathogens may be spreading through frog populations, possibly introduced by human activities. Delvinquier (1936) has shown that the gall-bladder protozoan, Myxidium immerswm, has boen introduced into Australia with B. marinus; this protozoan has since infected native anurans. A fungus, Bøtrachochytrium dendrobatidrs (Chytridiomycota: Chytridiales), which has recently been detected in wild frogs from various parts of Australia and Central America and also in captive specimens in Australia and the USA (Berger et al. 1998, Speare et sl. 1999) has hacl a profound effect on frogs. Frog deaths through Eastern Australia have been in a manner mimicking the spread of a pathogen (Berger et al. 1998, Richards et al. 1993). It is, therefore, reasonable to conclude that disease is of major concern. The spread of new diseases will result that in a decrease in the number of individuals in a population, and it is also quite feasible many species that have quite restricted distributions may be lost altogether (Osborne 1989). 1 "2 Habitat Degradation It is well known that some species of frogs, particularly arboreal frogs from the north of Australia, are able to cohabit with humans (Tyler 1994a, Tyler and'Watson 1998). Farm dams, shallow quarries and backyard ponds are all commonly used by amphibians as breeding sites, but a large number of species suffer as a consequence of human population growth and habitat loss. Land clearance for housing and farming is directly responsible for removing a large amount of riparian habitat (Osbome 1989, Ferraro and Burgin 1993, Richards et al' 1993, Thoms 1998). A decrease in native scrubland and particularly the modification of natural waterways into concrete-lined stormwater drains removes necessary breeding and sheltering sites. This action also highly modifies the natural water regimes (Environment Protection Agency l99S) which may include reduced inundation of the floodplains, common breeding sites for many species. A reduction in habitat size also leads to an inability to colonise new areas; exposing frog populations to sudden pressures, such as drought and disease which would further increase the decline and reduce genetic diversity in the population (Osborne 1989, Fenaro and Burgin 1993). Increased erosion as a result of land clearance further degrades riparian vegetation (Ferraro and Burgin 1993, Thoms 1998) and the build-up of soil particles and silt in waterways may have a profound effect on the aquatic flora and fauna.