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. Ctogging of feeding and respiratory systems and decreased food availability, due to limited photosynthesis as light penetration decreases, have a profound effect on many aquatic organisms, including frogs"
1.3 Global Warming Tyler (1994b) predicted that global warming is unlikely to be responsible for frog declines in
A,ustralia, but may in fact improve conditions for many species of arnphibians in northern Australia" A rise in sea surface temperature of a few degrees Celsius will lead to a higher frequency of cyclones, or of their severity, and the consequent movement of moisture inland to the drier regions of Australia. It is unlikely that increased water availability will be detrimental to arid zone species. Almost 40Yo of known frog species from the wettest regions in Australia are fossorial and many of these species also oocur in more arid zones"
2 An increase in noxious atmospheric gases associated with the combustion of fossil fuels would result in a greater possibility of acid rain, but the global warming itself is unlikely to be a factor in the global decline of amphibians.
1 .4 Ultraviolet Radlatio¡'¡ Associated with global warming is the partial loss of the ozone layer and this may have significant effectsËthe amount of ultraviolet radiation (UV) reaching the earth's surface. UV damage is potentially a major problem to amphibians because, unlike higher vertebrates, they lack hair or feathers to act as a UV filter. Blaustein (1994) has further suggested that synergistic effects of UV and acid rain can contribute to the death of amphibians, especially in the egg and tadpole stages. Absorption of UV energy by proteins and DNA in cells can result in the alteration of biochemical processes. These malfunctions are especially problematic when they are associated with DNA. If the genetic information is interpreted incorrectly during cell replication mutations and ultimately death may occur (Blaustein (1994)). However, despite increases in the amount of UV radiation in parts of the world (for example the western
United States) there are many locations where there has been no change in the radiation levels
(MeDonald 1999).
1.5 Follution
Pollution is a likely cause of major amphibian decline and the one that has attracted the most public attention. Follutants, which include a wide range of items such as synthetic chemical waste, petrochemicals and hydrocarbons, minerals and organie material, heary metals and radioactive waste, thermal pollutants and household refuse may enter tho aquatic environment in a variety of ways. Major sources of pollutants include discharges from industrial activities (point source), agricultural runoff (diffuse source), stormwater runoff (both point source and diffuse source) and direct dumping of lifier and other wastes (Connell 1974, Environment
Protection Authority 1998, NWQMS 1999). Tyler (pers. comm.) reports that three trash racks installed within tributaries to the River Tonens in Adelaide had captured 250 tonnes of debris in 1998. Of this approximately 80% comprised organic matter and the remaining 20o/o assorted liter. It is anticipated that the addition of another three racks installed in 1999 will collect an additional 300 tonnes per year; totalling 550 tonnes of debris entering the River
Torrens each year. These contaminants can have a number of different impaets on frogs. This may result in the direct death of organisms, or they may act in sub-lethal ways causing the animals to be more susceptible to other elements (Connell 1974, Cooke 1981, Carey and
Bryant 1995, Hecnar 1 995). 1.5"1 Physical Effects of Foltutants High levels of nutrients, for example fertiliser runoff and waste organic matter, in water promote the growth and reprocluction of cyanohacteria which are responsible for algal blooms (Connell 1974, Environment Protection Authority 1998). These algal blooms reduce light penetration and the resulting reduction in photosynthesis, combined with the depletion of oxygen when large numbers of cyanobacteria die and decompose, ultimately leads to the death of many aquatic organisms. The biological activity of bacteria can also have an effect on sediment chemistry that may alter the bioavailability of pollutants in the sediment (NWQMS ßgg). As mentioned previously suspended particles, including cyanobacteria, can also block the gills and feeding apparatus of aquatic organisms and, perhaps more importantly, they can produce highly toxic chemicals which are responsible for large scale deaths of aquatic organisms (Environmental Protection Authority 1998, NViQMS 1999). Large proportions of nutrients entering waterways are released as a result of agricultural practices (Connell 1974, Environment Protection Authority 199S) and many agricultural chemicals oontain surfactants.
Surfactants are spreading (wetting) agents that are used to increase the efficiency of the active ingredient. In other words, the surfactant in a herbicide enables the droplets of herbicide to disperse and form a complete film over the entire surface of a leaf, and thereby improve its activity. Frogs have a very loose, permeable skin that is a major respiratory organ. V/hen they are exposed to a substance containing a surfactant it is assumed to inhibit gas exchange and lead to asphyxiation. ln somo cases the surfactant may be more toxic than the active compound it spreads. For example, Glyphosate is used as a herbicide with high activity on virtually all plants and is broken down easily by soil bacteria (National Registration Authority for Agricuitural and Veterinary Chemicals 1996a). However, glyphosate is generally used with a surfactant additive that is highly toxic to frogs (Bidwell and Gomie 1995, National Registration Authority for Agricultural and Veterinary Chemicals I996a). As a result of this and other highly publicised concerns the safety levels have been revised. The Australian Federal Government decided that ehanges to the labelling should be made, outlining restrictions to its use around waterways from June 30th 1997 (National Registration Authority for Agricultural and Veterinary Chemioals tr996a,b, T'yler 1997).
Large amounts of oil and other petroleum products enter the system from stormwater runoff, especially from roads (Connell 1974). Oil slicks on the surfaee of the water also prevent oxygen exchange with the air and along with the toxic effects they can have a huge impact on the aquatie environment.
4 1"5"2 Chennical Effects of Pollutants Historically, accounts of abnormal amphibians have usually been little more than brief dcscriptive notes (Bishop and Hamilton 1947 , Hebard and Bntnson 1963) or anecclotal records (Ouellet et al 1997, Reaser and Johnson 1997), commonly reported in the popular media or held in museum collections. There have been exceptions to this (Sessions and Ruth 1990) and it is now commonly aceepted that frogs are extremely sensitive to aquatic pollutants, many of which may induce abnormality (Tyler I994a, Boyer and Grue 1995, Ouellet et al 1997), possibly with a similar action to thalidomide. Thalidomide produced horrendous abnormalities in human foetuses during a eritical period in pregnancy (Tyler 1996).
r Frogs are the highest!äfof life that lays a naked egg in water (Tyler 1996). Hence, unlike birds and some reptiles, they do not have a hard protective coating around the developing embryo. The frog egg is surrounded only by a number of thin membranes within a jelly layer which serves to minimise damage from physical contact, predation, temperature shock and some diseases (Holmes 1927) but the jelly does not act as a very efficient buffer (Ferraro and Burgin 1993)" V/hen the egg is first laid the jelly layer is highly hygroscopic, causing it to swell many times its original size. As the water is absorbed any dissolved pollutants may eome directly into contact with the developing embryo; causing disruption to normal growth and development (Read and Tyler 1994, Guillette 1994, Carey and Bryant 1995, Hecnar 1995,
Rowe et al. 1996, Tyler 1996).
If disturbed tadpoles may reduce movement to avoid predation, howevet even very low eonoentrations of ehemicals can signifieantly ehange their activity levels (Abbasi and Soni
1984, Ferraro and Burgin 1993). For example, 0.1ppm DDT may induce hyperactivity, muscle spasms and even death in tadpoles (Ferraro and Burgin 1993, Tyler I994a). If a tadpole increases its activity as a result of pollution it will be much more easily seen and consumed by predators. There is also a high chance of bioaccumulation of pollutants (Guillette 1994). This is very well represented by the use of DDT in Canada" DDT was widely used in mosquito control from the late 1930s until 1967 (Russell and Hecnar 1996). A study in a National Park was undertaken in 1995 to record the levels of DDE (a breakdown product of DDT) in two species of frogs. An analysis of water quality from before 1900 showed that levels were only
0.006 ppm, much lower that the 0.1 ppm required to induce hyperactivity. However the 1995 levels in Rana clamitans were 5 ppm (50 times greater than 0.1 ppm) and in Pseudauis
5 crucifer 47 ppm (470 times higher) 20 years after DDT use had officially ended (Russell and Flecnar 1996)"
Followi¡g chance discovcrics of the effects of plastic compounds on cell cultures during breast cancer research (Soto et al. l99l), further studies have shown that approximately three dozen chemicals, including DDT and DDE, which are used in the production of many plastics have properties which make them mimic sex hormones. These chemicals alter the ratio of male to female hormones and as such cause embryos to grow with increased female characteristics or reduced male characteristics. After observations of a 90%o decline in alligators from Lake Apopka between 1980 and 1984, a study of a population in Florida demonstrated that alligator eggs normally hatch with20o/o being female and 80% being male. Lake Apopka was exposed to an enorrnous spill of dicofol (modified DDT) in 1980' Following this,75Yo of eggs did not hatch and,of those which did surviver600/o of the males had reduced penises and testicles (some as small as 25o/o normal size, others weÍe entirely laeking) and20Yo of hatchlings were intersex. The intersex hatchlings are alligators that have both rudimentary ovaries and testes. The level of oestrogen normally present in newborn alligators is 10,000 times lower than the level of DDE measured in many eggs. levels of these chemicals in the water rwere very low. The males from this lake had oestrogen to testosterone ratios similar to females from clean lakes and had a greatly reduced reproductive output; many were sterile. Some females had notably elevated oestrogen levels and many produced as many as 5 or 6 eggs in a single follicle. It is especially interesting to consider that this decline in alligators and the abnormal morphological and reproductive state of these, and other, animals corresponds with the first reports of the worldwide amphibian decline (Guillette 1,994).
1.5.2.1 Skeletal Abnormalities of Frogs Even in perfectly clean water one would expect to find some individuals which were abnormal as a result of,natural genetic factors (Martinez et al. 1992, Read and Tyler 1994, Tyler 1994a)" Every population of organisms includes some individuals that vary in siight ways from the normal pattem. Examples include a slight variation in the length of each leg, or eye colour, on the same person. In general all members of a species follow the particular body organisation of that species. Recordings of frogs from near pristine sites from around the world (Tyler and Crook 1930) showed that the incidence of abnormality ranged approximately 0 - 3Yo, but generally less than 2Yo. Thercfore if we assume that 3%o is the maximum normal incidence of abnormaiity in an undisturbed area it is of conoern if any location has an incidence significantly higher than 3%.
6 In an investigation of natural levels of abnormalities in South Australian frogs, so as to dehne criteria for biological monitoring, Brooks (1979) surveyed a number of locations in and around thc Mt Lofty Ranges. The inoidence of abnormality appeared to be correlated with the use of chemicals, especially agricultural fertilisers and pesticides, and abnormalities could be induced in developing frogs when exposed to these pollutants.
1.6 Aims
The present study aims to: . examine the incidence of abnormality and injury in frogs of the Mt Lofty Ranges and surrounds o create a comprehensive catalogue of deformities encountered in the wild o determine if, there is any observable difference in levels of abnormality between the present frog population and those studied by Brooks at the time of the first reports of global amphibian declines c attempt to replicate deformities from lab-based experiments
Implementation of these aims may show if the changes to ohemical use, both self-imposed and due to legal requirements, have made any obvious impaet on the amphibian fauna of the region"
In her study in the Adelaide Hills Brooks (1979) was limited by the fact that sampling was undertaken for only a few months. As such there is the possibility that seasonal effects may have affected her sampling. I-ow frog numbers during the short period she had to collect may have redueed her sample sizes and, as a result, incorrect conclusions may have unwittingly been drawn from her data. It is hoped that in testing over a period of four years bias will be removed. It is further intended that a greater number of sites will be available for sampling over this time period.
7 2 Materials and Methods
Between April l6th 1995 ¿ncl April 2nd 1998 fiekl surveys werc unclertaken to ascertain the incidence of abnormality of frogs in the Mt Lofty Ranges and adjacent plains (see map, figure
1). In addition specimens were collected because of the opportunity to visit the South East of South Australia, Kangaroo Island, the Flinders Ranges and the Gammon Ranges. Although beyond the scope of this investigation these additional sites provided beneficial comparative data.
Figure 1. Regional distribution of field survey collection sites.
(Detailed distribution of sites within regions can be seen in chapter 3)
'.t
8 2.1 Collection Sites Where possible the sites of collection were those visited by Brooks (1979) complemented with others visited as a result of fortuitous circumstances. The classification of sites of collection principally reflected lantl use. The groupings of the sites and the particular activitics that may affect the environmental parameters of those sites are as follows:
2"1"'! Golfcourses Ç*t^ Golfcourses are heavily managed areas that commonly have large open spaces and are free s{ weeds" To maintain this weed free environment herbicide and pesticide use is high. Although they are highly visited and very disturbed areas the inclusion of heavily vegetated water traps in many of these golfcourses provide a habitat in which frogs may be found'
2"1"2lndustria! Industrial estates are usually associated with artificially constructed stormwater drains, which are often modified creeks. This association entails a highly reduced habitat, increased water flow and the presence of large quantities of rubbish, liquid waste, and other pollutants.
Industrial estates are often located alongside busy roadways, which may lead to an input of petrochemicals that have originally leaked from passing vehicles. The presence of traffic and often a lack of ground cover reduce the possibility of migration between sites.
2 "'l "3 I ntensive Ag niculture Intensive agriculture ("FIeavy Agriculture" sensu Brooks (1979)) includes market gardens, orchards and vineyards, as well as the farming of wheat and other crops" These areas are characterised by low diversity of plant species and greatly reduced natural vegetation as a result of land clearance. Intensive use of chemicals to control invertebrate and other pests and the use of fertilisers is a common practice. These areas rnay be established in high rainfall areas but are also supplemented by watering systems.
2.1.4 Light Agriculture Light Agriculture refers to agricultural sites that have a low use of chemicals. These include the farming of beef and dairy cattle, sheep, horses and "organic" crops. These regions often have extreme land clearance and the presence of grazing stock can cause trampling of the riparian zone around creeks and dams. This disturbance often results in severe erosion of creeks with a corresponding lack of riparian vegetation and ground cover. It is quite common for blackberries to be found growing in these disturbed sites. The presence of dams in many 9 agricultural areas, often with large quantities of water plants, may provide abundant habitat for frogs (Tyler and'Watson 1998).
2.1"6 Scrub Scrub may refer to either of two different types of habitat that may not be easily distinguishable: land in pristine condition or previously disturbed areas that have been revegetated. Scrub sites are commonly found in a relatively undisturbed condition and are characterised by diverse flora and fauna, which may include introduced species, unimpeded water flow in creeks and low maintenance by humans.
2.1.6 Suburban Suburban and urban creeks are characterised by extremely high disturbance. The conversion of these creeks into stormwater drains has resulted in a deficient riparian zone and an increase in water flow. Large quantities of silt and rubbish which are carried in the water often aocumulate but provide only a poor habitat for aquatic organisms. As with industrial sites, close proximity to roads may result in an influx of hydrocarbons.
2"1"V Wetlands The recent establishment of many artificial wetlands has provided habitat for numerous species of aquatic and semi-aquatic organisms. An abundance of vegetation provides large numbers of sheltering sites. These wetlands typically have permanent water, much of which comes from stormwater. Often wetlands are constructed in areas that would not ordinarily have habitats of this type.
2"2 Çollecting Methods During periods of little or no rainfall frogs aggregate around any points of moisture, particularly under rocks in a creek bed, and therefore it is most plausible that the collection of frogs at these sites would represent a large proportion of the frog population. Conversely during wet weather the movement of frogs is not restricted by moist conditions and collecting a large percentage of the population is unlikely. As a result of the difficulties associated with sampling a population it was decided that a minimum of 30 frogs should be required for any statistical investigation. This follows the studies of Tyler (unpublished) who recorded a suite of physical characteristics (e.g. ratio of tibia length to snout-vent length) and determined that
30 was the minimum number of frogs required to observe the mean physical parameters for a sample of frogs from a particular population. 10 Following the suggestions of Davies (1993) frogs were loeated at a site by a thorough search which involved picking up and looking under any rocks, bark, litter and also by scouring through riparian vegetation. At some sites the lack of suitable ground cover made it extremely difficult to catch frogs by the normal method and so an alternative method was tried. This involved the use of a pitfall trap with an artif,rcial ground cover, however this trap proved unsuccessful on all occasions it was used and therefore was subsequently abandoned. In some cases frogs were detected by their vocalisation.
When a frog was found it was taken in hand and placed in a large polythene bag. If no frogs had been colleoted within a reasonable length of time the search was terminated. Each frog was thoroughly examined at the site to detect the presence of any deformity of the post-axial skeleton. Every limb and digit was assessed as was the vertebral column and head. Any major ehange to the skeletal structure of the limb results in a corresponding change in the appearance of the surrounding soft tissues and this can be seen by external examination. Skeletal deformities of the limbs and digits can easily be detected with only a small amount of experience" The incidence of injury may be underestimated because some repairs may be successful in that they completely regenerate the structure and leave no external signs of damage. The number of normal and deformed frogs collected was recorded and then all of the normal frogs were released at the site. Following Brooks (1979) all frogs regardless of species were grouped together for the analyses.
Any interesting aspects of the site were noted. For example, the presence of calling males or large amounts of litter and other pollution.
2.3 Fixation, Freseruation and Storage
Following Davies (1993) deformed frogs were euthanased by placing them in a jar containing a small amount of a 3Yo chloral hydrate solution. Post mortem the frog was then set in a suitable posture for examination, with fingers and toes positioned for optimal viewing, on paper towel soaked with 3Yo formalin in a baking tray. A further paper towel covered the frogs' dorsal surface. Once rigor mortis had occurred the frog was preserved in a 650/o solution ofethanol and stored in a glassjar.
11 2"4 Ëxamination and Alizarin Freparation
The preserved specimen was examined using a Wild M5 dissecting microscope and with the aid of a camera lucida an illustration was made of the external morpltolugy of the defouriity. Initially some photographs were taken using an attached camera, but depth of field problems resulted in poor quality and the illustrations were considered superior. Following the completion of this illustration the preserved specimen was cleared and stained employing a Double Alizarin / Alcian Blue staining techniqve sensu Hanken and Wassersug (1981) with the following modifications :
. In order to accelerate the staining process the cartilage stain was altered by increasing the
quantity of Alcian Blue from 1Omg to 100mg.
. By increasing the amount of trypsin from 1g to l0g and placing the sample in a 37oC oven the soft tissue elearing process was modified to hasten the operation.
2.5 Deform ity Glassification
Classihcation of the origin of deformities was based upon the initial external examination and then internal examination from the double stained specimen.
After an injury there first occurs a vascular response where bleeding from bone, soft tissue and vessels oause the production of a haematoma and, depending on the amount of soft tissue damage, an oedema (Hall 1965). Fhagocytic cells remove debris and the destruction of proteins and excretion of nitrogen results in the production of rejuvenated and de- differentiated cells (Sinclair 1978). Subsequent to the vascular damage there is a proliferation of blood vessels. The haematoma may be oonverted into either granulation tissue (connective tissue and capillaries), which may ossiff, or a "fibrin-lined fluid-filled joint-like eavity" (Hall tr965, Sinclair tr97E).
Following these processes bone repair occurs. The connective tissue is replaced by an accumulation of undifferentiated cells derived from the mass of inflamed tissue, including the oedema, muscle, and granulation tissue. Intercellular matrix loosely holds the bone ends together. A mass of tissue, collagen on which caloium saits are deposited, forms at the fracture point and the ensuing reorganisation of the undifferentiated cells into a provisional callus (blastema) restores the bone, cartilage and muscle (Hall 1965, Sinclair 1978). In minor injuries the callus is formed entirely of bone, however in more severe cases the central portion
t2 of the callus is composed of cartilage. It appears that the formation of oartilage allows time for the accumulation of blood vessels that are required for the formation of new bone (Hall 1965). The rate of rejuvenation is dependent on suitable dietary protein, vitamins and minerals. It also decreascs with agc and season (Sinclair 1978). The ability to regenerate a limb completely is lost progressively in the course of ontogeny (Tyler 1994a)'
Therefore deformities as a result of injury following metamotphosis may be identified by any indications of repair, which may be in the form of abnormal ossification. Injuries may also result in irregularities in the surrounding tissues, especially when the injury is recent there may be swelling or bruising. In most cases any deformity should be confined to the area of injury.
In contrast a deformity as a result of teratogenic or congenital abnormality may be suggested by an absence of localised external disturbance, and malformation of the affected bones which may take the form of complete loss of bones, fusion of bone elements and often a distorted appearance. Abnormality is not necessarily confined to any particular bone. Consequently, deformity of bones more proximal to the area of initial interest is indieative of teratogenic abnormality.
In the event of any uncertainty as to the aetiology of a deformity it was decided that erring should be on the side of caution and therefore the deformity was classified as injury.
2.5"1 Nomenclature of Abnormalities Gardiner and Hoppe (1999) comment on the diff,rculty in comparing different studies due to the wide range of terms and characters used. They give the example of a deformity they term "bony triangle" which may be classified as "phocomelia", "bony excrescences" or "anteversions" in other papers. Terms such as "luxations" and "subluxations" (Alvarez et al i995) and "lobster-claw" (Hebard 1963) have been used to describe other abnormalities in different studies. For a standard eomparison the nomenclature in this study follows that of
Tyler and Crook (1980). In particular the following terms are used: o ectrodactyly - the loss of digits or elements with a digit o syndactyly - fusion of digits - may include, but is not limited to, fusion of the bones . polydactyly - the presence of supernumerary elements or digits . hemimely - the loss of all or part of the distal portion of a limb c ectromely - the complete loss of a limb 13 . braehymely - a change in the proportions of a limb relative to the body . micropthalmia - reduction in the size of an eye . anopthalmia - absence of an eye o mandibular hypoplasia - reduction in the development of the jaw.
A complete definition of these terms, which have not been used when the deformity is the result of injury (the exception being the use of anopthalmia), can be found in Tyler (I99aa).
2.6 Statistical Analysis
Statistical analyses were performed using methods found inZar (1984). Prior to analysis, data were assessed for normality using the Anderson-Darling Statistic (Snedecor and Cochran
1e89).
2.6"1 Mt Lofty Flanges Despite several attempts at datatransformation normality could not be assumed, therefore data were analysed by non-parametric methods. Kruskal-Wallis analysis of variance (incorporating tied ranks and unequal sample sizes) was used to compare differences between land use types. If differences were detected a Tukey-type test was used to determine where those differences existed"
2"6"2 Elrosks {979 The data collected by Brooks (1979) have been re-analysed for comparative purposes using the same criteria and statistical tests used for the Mt Lofty Ranges data.
2.6.3 Mt Lofty and Flinde!"s Ranges A comparison was made between Mt Lofty and the Flinders Ranges using the Mann-Whitney
U Test, which included tied ranks (Zar 1984)"
t4 3 Results
A total of 1923 frogs (6 species) was examined in the Mt. Lofty region, 84 from the South East (5 species) and 741 from the Flinders Ranges (3 species). In total 2748 frogs from 10 species were collected. The details of frogs collected are as follows:
Family HYLIDAE Litoria ewingi(Duméril and Bibron 1841) Brown Tree Frog 618 SPECIMENS Litoria peroni (Tschudi I 838) Peron's Tree Frog 1 SPECIMEN
Litoria raniformis (Keferstein 1 867) Southern Bell Frog 8 SPECIMENS Litoria rubella (Gray 1842) Desert Tree Frog 5 SPECIMENS
Family LEPTODACTYLI DAE Crinia signifera (Girard 1853) Common Froglet 1133 SPECIMENS Crinia riparia (Littlejohn and Martin 1965) Streambank Froglet 686 SPECIMENS
Limnodynastes dumerili (Peters 1 864) Eastern Banjo Frog 22 SPECIMENS
Limnodynastes peroni (Dumêril and Bibron 1 841 ) Brown Striped Marsh Frog 22 SPECIMENS
Lim n ody n astes tasm a n iensrs Gunther 1 858 Spotted Grass Frog 253 SPECIMENS Neobatrachus picfus Peters 1864 Painted Frog 1O SPECIMENS
3"1 Catalogue of Deformities
On the basis of the previously described criteria each frog was classified as normal, abnormal or injured (or in the case of multiple deformities a combination). From this classification it was possible to determine the incidences of abnormality and injury for each location sampled.
A complete catalogue of all of the deformed frogs collected is presented in figures 2 - 135.
The catalogue has been arranged as follows: o The Mt Lofty Ranges region (including a single Kangaroo Island site) grouped by species" o The South East grouped by species. ' The Flinders Ranges grouped by site.
External and skeletal morphology is shown for all deformities. In a small number of individuals additional deformities were identified after the clearing and staining process. These are presented in the catalogue but no external morphology is shown. Scale bars
represent 1 mm.
l5 Figure 2. CS01 Crinia signifera- North q_f Monûies Bridge.
Right foot : External morphology (A) and skeletal elements (B) - abnormal.
Hemimely - abnormal formation of astragalus and calcaneum.
Complete absence of distal elements.
Figure 3. CS03 Crinia signifëra- The Paddocks.
Left foot : External morphology (A) and skeletal elements (B) - abnormal.
Syndactyly - fusion of digits IV and V.
X'igure 4. CS04 Crinia signifera- Mt Pleasant (Watly Creek).
Left foot : External morphology (A) and skeletal elements (B) - injured.
Fracture and partial repair to antepenultimate phalanx of digit IV.
Note extensive area of cartilage. cso1
A B
cs03 A B
cs04
A B
I t
T6 Figure 5. CS05 Crinia signifera- Mt Pleasant (Wally Creek).
Right hand : External morphology (A) and skeletal elements (B) - abnormal.
Gross disruption of digit III metacarpal and polydactyly involving duplication of digit IV.
Figure 6. CS06 Crinia signifera- Mt Pleasant ('Wally Creek).
Right hand : External morphology (A) and skeletal elements (B) - abnormal.
Ectrodactyly of digit I"
Syndactyly - partial fusion of digits III and IV involving basal phalanx.
Figure 7. CS07 Crinia signifera- I-enswood Memorial Park.
Right hand : External morphology (A) and skeletal elements (B) - injured.
Gross disturbance of all digits. I-ocalised partial repair" cs05
A B
cs06
a A B
I
cs07
A B
t7 Figure 8. CS08 Crinia signifera- Harrosate Picnic Grounds. Right hand : External morphology (A) and skeletal elements (B) - injured.
Fracture of penultimate phalanx on digit IV (the absence of cartilage at the site
of injury suggests recent damage : possibly caused at time of collection).
Figure 9. CS09 Crinia signifera- Harrosate Picnic Grounds.
Skull : External morphology (A) and skeletal elements (B) - injured.
Damage to anterior portion of the skull including loss of left premaxilla
Figure 10. CS10 Crinia signifera- HarraggÉe Picnic Grounds.
Left foot : Extemal morphology (A) and skeletal elements (B) - abnormal and injured.
Ectrodactyly - absence of phalanx on digit IV Loss of terminal phalanx on digit V. cs08
A B
cs09
A B
cs1 0
A B
18 Figure 11" CSll Crinia signifera- Dry Creek (Modbury Heights). Right foot : External morphology (A) and skeletal elements (B) - abnormal
Polydacffly - Y-shaped basal phalanx with absence of distal phalanges
Additional abnormalities noted after clearing and staining: (C) Osteosarcoma of left radioulna (D) Abnormal metacarpal of digit III, right hand.
Figure 12" CS12 Crinia signifera- Drv Creek (Modbury F{eiehts).
Right foot : External morphology (A) and skeletal elements (B) - injured.
Irregular basal phalanx of digit IV. Loss of distal elements. cs1 1
A B
c D
cs1 2
A B
I9 Figure 13. CS13.1 Crinia signifera- Dry Creek (Modbury Heights).
Right hand : External morphology (A) and skeletal elements (B) - injured
Fracture and repair to penultimate phalanx of digit I and metacarpal on digit II.
Figure 14. CS13.2 Crinia signifera- Ðry Creek (Modbury Fleights). Left foot : External morphology (A) and skeletal elements (B) - injured.
Fracture and repair to digit IV involving loss of terminal phalanx
Figure 15. CS14 Crinia signifera- Ðry Creek (Modbury Heights). Left eye : External morphology (A) and skeletal elements (B) - abnormal.
Sinistral micropthalmia. cs13.1
A B
cs13.2
A B
cs1 4
A B
20 Figure 16. CS15 Crinia signifera- Dry Creek (Modbury Heights). Left hand : External morphology (Ð and skeletal elements (B) - abnormal.
Polydactyly - bifid basal phalanx of digit III with additional abbreviated terminal phalarx.
Figure 17. CSf6 Crinia signifera- Fifth Creeknr St Ignatius College.
Left foot : External morphology (A) and skeletal elements (B) - injured.
Injury to soft tissue of digit V.
I i
I I
I
I
i
I
t. i
Figure 18. CS17 Crinia sígnifera- Fifth Creek nr St Ignatius College. I
I Left foot : External morphology (A) and skeletal elements (B) - abnormal. I
I
Irregular terminal and penultimate phalanges of digit IV.
r.
'.. cs1 5
a A B
cs1 6
A B
cs1 7
A B
2l Figure 19. CS18 Crinia signifera- Fifth Creek nl St Ignatius College.
Left foot : External morphology (A) and skeletal elements (B) - abnormal
Polydactyly - additional phalanx (appearance of terminal phalanx) distal to basal
phalanx of digit trV.
Figure 20. CS19 Crinia signifera- Fifth Creek nr St Ignatius College.
Right f,oot : External morphology (A) and skeletal elements (B) - abnormal.
Presence of sesamoid bones associated with mostjoints (replicated on left foot) with disruption to terminal joint of digit IV"
Figure 21. CS20.1 Crinia signifera- Fifth Creek nr S! Ignatius College" R.ight hand : External morphology (A) and skeletal elements (B) - injured.
Damage to soft tissues of digit il. cs1 I
A B
cs1 9
An Q¡
A B
cs20.1
B
22 Figure 22. CS2A"2 Crinia signifera- Fifth Creek nr St lgnatius College.
Left foot : External morphology (A) and skeletal elements (B) - injured.
Loss of terminal phalanx of digit V
Figure 23" CS21 Crinia signifera- Fifth Creek nr St Isnatius Colleee.
Left foot : External morphology (A) and skeletal elements (B) - injured.
Distortion of terminal phalanx of digit IV
Figure 24, CS22.l Crinia signifera- Fifth Creek nr St Isnatius College.
Right foot : External morphology (A) and skeletal elements (B) - injured
Loss of terminal phalanx and relocation of basal phalanx of digit IIL cs20.2
A B
cs21
A B
cs22.1
A B
23 Figure 25. C522.2 Crinia signifera- Fifth Creek nr St Ignatius Collese.
Right hand : External morphology (A) and skeletal elements (B) - injured.
I-oss of terminal phalanges of digits II and IV
Figure 26" CS23 Crinia signifera- Belair National Park.
Right leg : External morphology (A) and skeletal elements (B) - abnormal.
Ectromely - complete absence of right leg, reorientation of urostyle and ilia.
Absenoe of right side of ilium. cs22.2
A B
cs23 B A
H
24 Figure 27. CS24 Crinia signifera- Clarendon Oval.
Right eye : External morphology (A) and skeletal elements (B) - injured.
Dextral anopthalmia.
Figure 28. CS25 Crinia signifera- Clarendon Oval.
Right hand : External morphology (A) and skeletal elements (B) - injured.
tr-oss of terminal phalanx of digit IV
Additional injuries deteetod after olearing and staining:
(C) Disturbance to penultimate phalanx of digit trII, left hand.
(D) Fracture and repair to terminal phalanx of digit III, right foot cs24
A B
cs25
A B
c D
25 Figure 29. CS26 Crinia signifera- Fifth Creek nr Black Hill Flora.
Right hand : External morphology (A) and skeletal elements (B) - injured.
Fracture and repair to penultimate phalanx of digit III, basal phalanx of digit IV
Figure 30. CS27 Crinia signifera- Fifth Creek nr Black Hill Flora.
Right foot : External morphology (A) and skeletal elements (B) - injured.
Fracture and repair to digits II - V.
Note extensive areas of cartilage.
Figure 31. CS28 Crinia signifera- Fifth Creek nr Black Hill Flora.
Right foot : External morphology (A) and skeletal elements (B) - abnormal
Ectrodactyiy - absence of,terminal phalanx of digit IV" Additional skeletal elements present. cs26
A B
ÇJ
cs27
A B
cs28 ærææ-,q
A B
26 Figure 32" CS29 Crinia signifera- Fifth Creek nr Black Hill Flora.
Right foot : External morphology (A) and skeletal elements (B) - injured.
Fracture and some repair to digits II and V
Figure 33. CS30 Crinio signifera- Yundi.
Right eye : External morphology (A) and skeletal elements (B) - abnormal.
Dextral micropthalmra.
Figure 34" CS31 Crinia signifera- Cavan.
Left leg : External morphology (A) and skeletal elements (B) - abnormal
Hemimely - complete absence of limb below distorted tibiofibula. cs29
A B
cs30
A B
cs31 t-¡
A B
27 Figure 35. CS32.1 Crinia signifera- Cavan.
Left hand : External morphology (A) and skeletal elements (B) - abnormal
Irregular and reduced number of phalanges of digit IV.
Figure 36" CS32.2 Crinia signi-fera- Çavan.
Left foot : External morphology (A) and skeletal elements (B) - injured.
Fracture and repair to digits IV and V" Note extensive area of cartilage.
Figure 37. CS33 Crinia signifera- Cavan.
Left foot : Extemal morphology (A) and skeletal elements (B) - injured.
Gross deformation and syntosis of digits III - \i Note extensive area of cartilage. cs32.1
A B
cs32.2
A B
cs33
A B
28 Figure 38. CS34 Crinia signifera- Yundi 2 (Enterprise Rd).
Left foot : External morphology (A) and skeletal elements (B) - injured
Fracture and repair to digit IV involving loss of phalanges.
Figure 39" CS35.1 Crinia signifera- Yundi 2 (Enterprise Rcù
Right foot : External morphology (A) and skeletal elements (B) - injured.
Disturbance and some repair to digits [V and V
Figure 40. CS35.2 Crinia signifera- Yundi 2 (Enterprise Rdl.
Left hand : External morphology (A) and skeletal elements (B) - injured
Deformation and repair to digit IV involving all phalanges. cs34 A B
cs35.1
Æ éâ€f B ê@
cs35.2 A B
t-l
29 Figure 41. CS36 Crinia signifera- Echunga Golf Course fNorthern End)' Left hand : External morphology (Ð and skeletal elements (B) - injured.
Damage and repair to digits I - III. Note extensive area of cartilage.
Additional injury detected after clearing and staining: (C) Damage and repair to digit IV, left foot.
Figure 42. CS37 Crinia signifera- Parndana (Ka.ngaroo Island).
Left foot : External morphology (A) and skeletal elements (B) - injured.
Fracture to phalanges of digits I and IV.
Figure 43. CS38 Crinia signifera- Parndana (Kangeroo Island).
Right foot : External morphology (A) and skeletal elements (B) - injured.
Fracture to penultimate phalanx and loss of terminal phalanx of digit IV. cs36
t-l A B
c Þ-
cs37
A B
cs38
A B @up
30 Figure 44. CS39 Crinia signifera- Hawthorndene.
Right foot : External morphology (A) and skeletal elements (B) - injured.
Damage and repair to antepenultimate phalanx of digit IV with loss of distal
elements.
Figure 45. CS40 Crinia signifera- Hawthorndene.
Left leg : External morphology (A) and skeletal elements (B) - injured.
Loss of all elements below fractured tibiofibula. Note extensive area of cartilage"
Figure 46. CS41 Crinia signifera- Flawthorndene.
Right arm : External morphology (A) and skeletal elements (B) - abnormal
Hemimely - absence of all elements distal to abnormal humerus CS39 A B
cs40 A B
cs41 A B
t-l
31 Figure 47. CS42 Crinia signifera- Montacute.
Right foot : External morphology (A) and skeletal elements (B) - injured.
Fracture and repair to digits III - V
Note extensive areas of cartilage.
Figure 48. CS43 Crinia signifera- Inelewood.
Right foot : External morphology (A) and skeletal elements (B) - injured"
Fracture and repair to phalanges of digit IV
Figure 49" CS44 Crinia signifera- F'ourth Creek. Right foot : External morphology (A) and skeletal elements (B) - abnormal
Ectrodactyly - absence of terminal and penultimate phalanges with deformed antepenultimate phalanx of digit IV" cs42
t-J A B 6
cs43
A B tl
cs44
A B
32 Figure 50. CS45 Crinia signifera- Fourth Creek.
Right foot : External morphology (A) and skeletal elements (B) - abnormal
Deformation of antepenultimate and penultimate phalanges of digit IV.
Figure 51. CS46 Criniq signifera- Brukunga.
Left hand : External morphology (A) and skeletal elements (B) - injured.
Fracture and repair to phalanges of digits II and III
Figure 52. CS47 Crinia signifera- Brukunga.
Right hand : External morphology (A) and skeletal elements (B) - injured.
Fracture and repair to phalanges of digit III with some damage and repair to
carpals and radioulna. cs4 A B
cs46
ffi
A B
ê ê H cs47 A A B
55 Figure 53" CS48 Crinia signifera- Oakbank.
Right leg : External morphology (A) and skeletal elements (B) - injured.
Loss of all elements distal to astragalus/calcaneum.
Figure 54. CS49 Crinia signifera- Qakbank.
Right leg : External morphology (A) and skeletal elements (B) - abnormal.
Hemimely - absenee of elements distal to abnormal astragalus/calcaneum
Figure 55. CS50 Criniq signifera- Oakbank.
Right eye : External morphology (A) and skeletal elements (B) - injured.
Dextral anopthalmia with damage to the squamosal. cs48
A B
c 9
A B
!
CSsO
A B
34 Figure 56. CS51 Crinia signifera- Oakbank.
Right foot : External morphology (A) and skeletal elements (B) - injured.
I
Fracture and repair to metatarsal and phalanges of digits IV with some loss of
elements. Note extensive area of cartilage.
Minor damage to terminal and penultimate phalanges of digit V.
Figure 57. CS52 Crinia signifera- Oakbank.
Left foot : Extemal morphology (A) and skeletal elements (B) - injured
Dislocation of basal phalanx of digit IV.
Figure 58. CS53 Crinia signifera- V/oodside.
Left arm : External morphology (A) and skeletal elements (B) - injured,
Gross damage and repair to elements distal to humerus. css1
A B
cs52
A B
cs53
A B
35 Figure 59. CS54 Crinia signifera- V/oodside'
Left foot : External morphology (A) and skeletal elements (B) - injured.
Fracture and repair with some loss of elements of digits III and IV
Figure 60. CS55 Crinia signifera- Woodside.
Left foot : External morphology (A) and skeletal elements (B) - injured.
Loss of phalanges of digits IV and V.
Figure 61" CS56 Crinia signifera- Apex Fark.
Right foot : External morphology (A) and skeletal elements (B) - abnormal
Eetrodactyly - absence of numerous phalanges of digit IV
Figure 62. CS57 Crinia signifera- Apex Park.
Left hand : External morphology (A) and skeletal elements (B) - abnormal.
Fusion of soft tissues of digits I and II (penultimate phalanx is abbreviated and
misaligned), absence of distal elements. cs54
A B cs55
A B
cs56 B
cs57
A B
36 Figure 63. LT01 Limnodynastes tasmaniensis- The Paddocks.
Left foot : Extemal morphology (A) and skeletal elements (B) - abnormal.
Ectrodactyly - gross deformity of phalanges of digits II - V with relocation of
bone elements to region of metatarsals.
Figure 64"LT02 Limnodynastes tasmaniensis-The addocks.
Left foot : External morphology (A) and skeletal elements (B) - injured"
Loss of phalanges with repair to digit IV.
Figure 65. LT03 Limnodyt astes tasmaniensis- Meadows 2.
Right hand : External morphology (A) and skeletal elements (B) - injured.
Loss of phalanges of digit II with repair including soft tissue damage to digit III LTOl
A B
LT02
A B
LTO3
A B
¡-¡
37 Figure 66. LT05 Limnodynastes tasmaniensis- Milang.
Righr foot : Extemal morphology (A) and skeletal elements (B) - injured.
Fracture and repair to metatarsals and phalanges on digits II - IV.
Note extensive areas of cartilage.
Fi gu re 67 . LT 06 Limno dynas t e s t as m an i e ns i s - Milang.
Left foot : External morphology (A) and skeletal elements (B) - injured.
Fracture and repair to digits II - IV.
Note extensive areas of cartilage.
Figure 68. LT07 Limnodynastes tasmaniensis-
Right leg : External morphology (A) and skeletal elements (B) - abnormal.
Brachymely - reduction in the proportions of the limb including some loss of
bones. LTOS
A B
LTO6
A B t-J
ilÃ
LTOT w
A B
38 Figure 69. LT08 Limnodynastes tasmaniensis- Cavan. External morphology (A) and skeletal elements of (B) abnormal left foot (LT08.l)and (C) abnormal right leg (LT08.2).
Polydactyly - Bilateral digit I (LT08.l) and ectrodactyly - absence of phalanx of digir IV. Ectrodactyly - gross malformation of limb with large reductions in the number of
skeletal elements (LT08.2)
Figure 70. LT09 Limnadyr astes tasmaniensis- Cavan.
Left foot : External morphology (A) and skeletal elements (B) - abnormal.
Gross deformation of the limb including fusion of foot with leg, and a reduction
in the number of tarsals and digits. n ü¡
LTOS
A B
q
,#:fl\; \Þdiu q C
ç Êl AJ Ê Ã (\ üà @ LTO9 å
A B
l-J
39 Fi gure 7 l. LT l0 Limnodynaste s Íasmoni ens is - Cavan
Right leg : External morphology (A) and skeletal elements (B) - abnormal.
Hemimely - gross disturbance to the elements of the ilium and an absence of
bones in the leg.
Fi gu re 7 2. LT ll Limno dynas t e s tas m ani e ns i s - Cavan.
Right leg : External morphology (A) and skeletal elements (B) - abnormal.
Ectromely - disturbance to the development of the ilium and the leg"
Figure 7 3. LT 12 L imno dynas te s t as mani ens i s - Cavan.
Right leg : External morphology (A) and skeletal elements (B) - injured
tr-oss of leg below the femur with some repair LT1 O
l¡t l:11u A B
LT1 1
A B
r-l
LT12
A B
40 Fi gure 7 4 " LT 13 Limno dynas t e s tas mani e ns i s - Cavan. Right leg : External morphology (A) and skeletal elements (B) - abnormal.
Ectromely - disturbance to the development of the ilium and leg
Fi gu re 7 5 " LT 14 L irnno dynas t e s t as mani e ns is - Cavan. Jaw : External morphology (A) and skeletal elements (B) - abnormal
V{andibular hypoplasia.
Figure 76. LT15 Limnodynastes tasmaniensis- Cavan.
Left leg : External morphology (A) and skeletal elements (ts) - abnormal.
Hemimely - absence of many elements of the leg and foot. LT1 3
A B t-l
LT14
t
A B
LT1 5 \o A B
4 1 Fi gu re 7 7 . LT 16 l-i mno dynas t e s t as m anie n s i s - Cavan.
Right foot : External morphology (A) and skeletal elements (B) - injured.
Damage and repair to digit III with loss of terminal phalanx'
Note area of cartilage.
Figu re 7 8 " LT 17 Limno dynas te s t as manie ns i s - Cavan. Left leg : External morphology (A) and skeletal elements (B) - injured.
Loss of leg below the tibiohbula with some repair.
Figure 79. LT18 Limnodynastes tasmaniensis- Cavan.
Right foot : External morphology (A) and skeletal elements (B) - abnormal
Deformation of digit I. Ectrodactyly - missing digit III. Bifid phalanx digit IV. LT16 w h A B w
l!¡¡¡ Y/ a
BN l-t l¡l
çfi i1
LT17
A B ¡-l
LT1 8 ?Þàì* \ÉiÐ\{I A B
42 Figure 80. LT19 Limnodynastes tasmaniensis- Milang 2'
Right foot : External morphology (A) and skeletal elements (B) - injured.
Loss of phalanges of digit V with some repair to metatarsal.
Figure 81. LT20 Limnodynastes tasmaniensis- Milagg 2.
Right foot : External morphology (A) and skeletal elements (B) - injured.
Fracture and repair to phalanges of digit IV.
Figure 82"LT21 Limnodynastes tasmaniensis- Milagg 2.
Right arm : External morphology (A) and skeletal elements (B) - injured
Multiple fractures and repair to elements with complete loss of hand. LT1 9
A B
t-l
LT20
A B
LT21 t-J A B
43 Figure 83. LE01 Litoria ewingi- Yundi'
Right arms : External morphology (A) and skeletal elements (B) - abnormal
Supernumerary limbs. I - normal right arm. II additional right arm consisting of fused humerus with right (IIa) and left (IIb) hands.
Figure 84.LE,02 Litoria ewingi- Hiehercombe Golf Course.
Left hand : External morphology (A) and skeletal elements (B) - injured.
Fracture and repair to digit I
Figure 85. I-803 Litoria ewingi- Fliehercombe Golf Course.
Left hand : External morphology (A) and skeletal elements (B) - injured.
Gross disturbance to radioulna, carpals, metacarpals and phalanges with some
repair" Complete loss of digits. LEOl
I IIa
A B
IIb
LEO2
A B
t-l
LE03 A B
44 Figure 86. LE04 Litoria ewingi- Highercombe Golf Course.
Left hand : External morphology (A) and skeletal elements (B) - injured.
Loss of phalanges of digit II with repair
Figure 87. LE05 Litoria ewingi- Highercombe Golf Course.
Left hand : External morphology (A) and skeletal elements (B) - injured.
Gross disturbance to phalanges, metacarpals and carpals with some repair
Figure 88" LE06 Litoris ewingi- Norton Summit.
Left foot : External morphology (A) and skeletal elements (B) - injured.
Fracture and repair to phalanges of digits I - V LE04
A B V
#,Þ...ìi¡,=**,,r".4ffi vËarr'r. ::,.: ;lff Éq41.å
ffi LE05 ffi- A B "49
LE06 u5_rfS.r-' A- B
45 Figure 89. LP01 Limnodynastes peroni - Bool Lagoon.
Left foot. : External morphology (A) and skeletal elements (B) - injured.
Fracture and repair to phalanges of digit lV.
Figure 90. LR0l Litoria raniformis- Bool Ï-agoon.
Right foot : External morphology (A) and skeletal elements (B) - abnormal
Ectrodactyly - absence of phalanx of digit IV. deformation of antepenultimate phalanx" u"eEyf>-
LPOl
A B
LROl
A B
46 Figure 9f . BRAO1 Crinia riparia Brachina Gorge.
Left foot : External morphology (A) and skeletal elements (B) - injured.
Fracture and repair of digits I and V.
Figure 92. BRA02 Crinia riparia Brachina Gorse.
Left foot : Extemal morphology (A) and skeletal elements (B) - abnormal.
Ectrodacffly - gross disruption to digits I, II,IV and V
Figure 93" BRA03 Crinia riparia Brachina Gorge.
i
Left foot : External morphology (A) and skeletal elements (B) - injured. I
I t: l.
Fracture and repair to digit IV L i I
I I I
I
I
I i t,
l.
I
I Figure 94" BRA04 Crinia riparia Brachina Gorge. I t. I Left foot : Extemal morphology (A) and skeletal elements (B) - injured. I l.'I
I Fracture and repair to digit IV
i 1. i BRAOl
A B
BRAO2 A B
BRAO3 A B -"4 ÉøíúÍ"
BRAO4
A B I-¡
47 Figure 95" BUN01 Criniariparia Bunyeroo Goree.
Left hand : External morphology (A) and skeletal elements (B) - injured.
Orientation of metacarpal III resulting in protuberance through palm'
Figure 96. B[JN02 Crinia riparia Bunyeroo Gorge.
R.ight foot : External morphology (A) and skeletal elements (B) - injured
Soft tissue damage to digit IV
Figure 97" BUN03 Crinia riparia Bunyeroo Gorge.
Left foot : External morphology (A) and skeletal elements (B) - abnormal and injured.
Ectrodactyly - absence of phalanx on digit IV.
Fracture and repair to phalanges of digits II and V
Figure 98. BUN04 Crinia riparia Bunveroo Gorge.
Right foot : External morphology (A) and skeletal elements (B) - injured.
Fracture and repair to phalanges of digits [I - V t--¡ BUNOl d
A B
BUNO2
B
BUNO3
A B
BUNO4
A B
{$. Figure 99" BUN05 Crinia riparia Bunyeroo Gorse.
Left foot : External morphology (A) and skeletal elements (B) - injured.
Relocation of phalanges of digit IV to location of missing phalanges digit V
Figure 100. BUNO6 Crinia riparia Bunyeroo Gorge.
Left hand : External morphology (A) and skeletal elements (B) - injured.
Fracture and repair to digits II - trV.
Figure 101. BUN07 Limnodynastes tasmaniensis Bunyeroo Gorse.
R.ight foot : External morphology (A) and skeletal elements (B) - injured
Fracture and repair to penultimate phalanx of digit IV
Figure 102. BUN08 Crinia riparia Bunveroo Gorge.
Right foot : Extemal morphology (A) and skeletal elements (B) - injured.
Fracture and repair to phalanges of digits III - V BUNOS f
A B
H
A B BUN06
BUNOT A B
t-¡ BUNOS
A B
49 Figure 103. BUN09 Crinia riparia Bunyeroo Gorge.
Right foot : External morphology (A) and skeletal elements (B) - abnormal
Ectrodactyly - fusion of phalanges in digits III and IV
Figure 104. BUN1D Crinia riparia Bunyeroo Goree.
Left hand : Externai morphology (A) and skeletal elements (B) - injured
Fracture and repair to phalanges and metacarpals of digits II and IV.
Figure 105. BUNII Crinia riparia Bunyeroo Gorge.
R.ight foot : External morphology (A) and skeletal elements (B) - injured.
Fracture and repair to basal phalanx of digit IV
Figure X06. BUN12 Crinia riparia Bunyeroo Gorge.
Right hand : External morphology (A) and skeletal elements (B) - abnormal
Gross disruption to radioulna and distal elements. BUNOg A B
BUNl O A B re
BUNl 1 A B
BUNl 2 B
50 Figure 107. ERE0 l.l Limnodynas tes tas maniensls Eresunda. Left foot : External morphology (A) and skeletal elements (B) - injured.
Fracture and repair to basal phalanx of digit IV
Figure 1 08. EREO 1.2 Limnodynaste s tasmaniensls Eregunda' Left hand : External morphology (A) and skeletal elements (B) - injured
Fracture and repair to phalanges of digit IV
Figure 1 09. ERE 02.l Limnodynqste s tasmaniensls Eregunda.
R.ight hand : External morphology (A) and skeletal elements (ts) - abnormal
Bifid phalanx - replication of penultimate phalanx of digit IV EREOl .1
e A B
EREO1.2 q'--b"\fu A B % fr
EREO2.1
A B
51 Figu re 1 1 0. EREO 2.2 Limno dynas t e s t as mqniensls Ere gunda'
Right leg : External morphology (A) and skeletal elements (B) - abnormal.
Hemimely - absence of limb below grossly deformed femur
Figure 111. ERE03 l-imnodynastes tasmaniensls Eregunda.
Right foot : External morphology (A) and skeletal elements (B) - injured
Fracture and repair of metatarsals and phalanges of digits I - IV
Figure 112. ERE04 Limnodynastes tasmaniensls Eregunda.
R.ight foot : External morphology (A) and skeletal elements (B) - injured'
Fracture and repair to basal phalanges of digits IV and V EREO2.2
A B
EREO3
A B
l, -'jþ: ..r -1-l l¡r*, ;1pa"
ëR EREO4 ffi H A B
52 Figure 113. MAMDI Crinia riparia Mambray Left foot : External morphology (A) and skeletal elements (B) - abnormal
Ectrodactyly - reduction in the number of digits including absence of tarsals
and phalanges in remaining digits.
Figure 114. MAM02 Crinia riparia Mambray Creek.
Right hand : Extemal morphology (A) and skeletal elements (B) - injured.
Fracture and repair to penultimate phalanx of digit III and carpals and
nnetacarpals of digit trV" MAMOl
l-t
A B
MAMO2
A B
53 Figure 115. NIL0I Limnodynastes tasmaniensis Nildottie' Left arm : External morphology (A) and skeletal elements (B) - injured'
Fracture and repair to tibiofibula, humerus, and carpals"
Figure 116. NIL02 Limnodynastes tasmaniensis Niklqttie. Right hand : External morphology (A) and skeletal elements (B) - injured.
Fracture and repair to all carpals, and metacarpal of digit I' b NILOl
A B
Ê B ït Æ
NILO2
A B
54 Figure 117" ORAOI Crinia riparia Oratunsa' Left foot : External morphology (A) and skeletal elements (B) - injured'
Fracture and repair to elements below tibiofibula'
Figure 118" ORA02 Crinia riparia Oratunqa. Left foot : External morphology (A) and skeletal elements (B) - abnormal
Ectrodactyly - absence of phalanx of digit IV.
Figure 119. ORA03 Crinia ripariø Oratunga'
Left foot : External rnorphology (A) and skeletal elements (B) - injured'
Fracture and repair to penultimate phalanx of digit IV' ORAOl
A B
ORAO2
A B
ORAO3 ç,\ 1à çt A B TJ
55 Figure 120. ORA04 Crinia riparia Oratunga' Left foot : External morphology (A) and skeletal elements (B) - injured'
Fracture and repair to basal phalanx of digit IV
Figure 121. ORA05 Limnodynastes tasmaniensls Oratunga' Right foot : External morphology (A) and skeletal elements (B) - injured.
Fracture and repair to elements distal to and including astragalus and
calcaneum
Figure 122. ORA06 Criniø riparia Oratunga. Left foot : Extemal morphology (A) and skeletal elements (B) - injured.
Fracture of metatarsal and basal phalanges of digits IV and V.
Note absence of oartilage suggests recent injury, possibly at time of collection.
Figure 123" ORA07 Crinia riparia Oratunsa.
R.ight foot : External morphology (A) and skeletal elements (B) - injured.
Fracture and repair to phalanges of digits III and IV. ORAO4
B
-
ORAO5
B
ORAO6
B
ORAOT
B
56 Figurc 124" PAR0l.l Crinia riparia Paralana Hot Springs' Right hand : External morphology (A) and skeletal elements (B) - abnormal
Ectrodactyly - Disturbance to terminal phalanx of digit III and absence of phalanges of digit IV.
Figure 125. PAROl.2 Crinia riparia Paralana Hot Sprines. Right foot : External morphology (A) and skeletal elements (B) - abnormal
Ectrodactyly - absence of phalanx of digit IV
Figure 126. PAR02 Crinia riparia Faralana Hot Springs.
Right eye : Bxternal morphology (A) and skeletal elements (B) - injured.
Dextral anopthalmia. PAROI .1
A B
PARO1.2
A B
PARO2
A B
57 Figure 127. SNO0I Crinia riparia Snob's Hut Springs' Right foot : External morphology (A) and skeletal elements (B) - abnormal
and injured"
Ectrodactyly - absence of phalanx of digit IV
Fracture and repair to digits I and II.
Figure 128. SNO02 Crinia riparia Snob's Hut Sprinss' Left foot : External morphology (A) and skeletal elements (B) - injured.
Fracture and repair to basal phalanx of digit IV.
Figure 129. SNO03 Crinia riparia Snob's Hut Sprines. Left eye : External morphology (A) and skeletal elements (B) - injured.
Sinistral anopthalmia. SNOOl
A B
¿rl¡.i)
SNOO2
A B
t--¡
t-¡ SNOO3
A B
.:i:
, J.. :"
58 Figure 130. SNO04 Crinia riparia Snob's Hut Springs' Left arm : External morphology (A) and skeletal elements (B) - injured.
Fracture and repair to radioulna and distal elements'
Figure 131. SNO05 Crinia riparia Snob's FIut Sprines. Left hand : External morphology (A) and skeletal elements (B) - injured
Fracture and repair to basal phalanges of digits II - IV SNOO4
A B
SNOO5
A B
t--¡
19 Figure 132. WEEDI Crinia ripariø Weetootla Gorge" Left foot : External morphology (A) and skeletal elements (B) - injured'
Fracture and repair to basal phalanx of digit V
Figure 133" \ilEEAZ Crinia riparia V/eetootla Gorge' Left hand : External morphology (A) and skeletal elements (B) - injured
Fraoture and repair to carpals and metacarpals of digits I - III. WEEOl
A B
WEE02
A B
60 Figure 134. WIL0I Crinia riparia Wilkawillina Gorge' Left foot : Extemal morphology (A) and skeletal elements (B) - injured
Fracture and repair to metatarsat of digit IV
Figure 135. WIL02 Crinia riparia Wilkawillina Gorge' Right foot : External morphology (A) and skeletal elements (B) - injured
Fracture and syntosis of metatarsals of digits III -V WILOl
A B
H ä\L
WILO2
A B
6l 3.2 Gollection Summary Figures 136 - 138 show the regional distributions of the locations visited during the present study. A summary of the collections made at these sites is presented in tables I - 3. Included in these tables are the total number of frogs, the number and identification of abnormal frogs and the number and identification of injured frogs. As previously discussed each location was classed according to land use and this is also included in the tables (Flinders Ranges sites were all classed as Scrub).
X'igure 136. Distribution of field survey sites within the Mt Lofty Ranges.
¡un \ nowooo
MAN NtJM
AOELAIOE
KER
M ILAN
62 Figure 137. Distribution of field survey sites within the South East of South Australia.
¡
KINGSTON
NA 0 O RTE I
I L (F"E)"' ' Bool goon
ROBE
F
IÊ ENT Li._
Lynuood (SE)r W¡ndillo Forest (5 E) r
MoUNT GAMBIEBT il, ,
C l¡ rk's P (s E) t
63 Figure 138. Distribution of field survey sites within the Flinders Ranges and surror¡nds. t i- ir Hot :!ii .'1
LE O l¡." dorsY 0oûNs 4
I ii
64 ,l lnjury lO Location Aþnormal 0 4 0 bo Park (LittlehamPton) Suburb 0 43 2 cs56, GS57 Apex Park Wetland 0 1 Belair National Park Scrub 13 0 1 0 Black Hill PonY Club Light Agric 0 0 0 Bridgeway Hotel Suburb 2 cs46, cs47 33 0 Brukunga Light Agric 0 0 0 Carey Gully lntense Agric LT18 5 cs32, CS33, 1T12, 1T16, LT17 67 10 CS31, LTO7, LTO8, LTOg, LT1O,1T11,1T13,1T14, LTl5' Gavan lndustrial 0 2 0 Charleston Light Agric 0 4 0 Christies Creek Light Agric 2 cs24, cs25 0 Oval Light Agric 49 o 0 Cnr Burbridge Rd / West Tee 1 o 0 Cormack Rd (Wingfield) lndustrial 0 0 0 Dernancourt Suburb 21 2 12, cs13 35 3 11, CS14, CS15 Dry Creek (Modbury Heights) Suburb 'l cs36 46 0 Echunga Golf Course (Nth End) Golfcourse 0 0 Echunga Golf Course (Sth End) Golfcourse 184 3 cs26, cs27, cs29 7 1 cs28 Fifth Creek nr Black Hill Flora CF Scrub 4 cs16, cs20, cs21, cs22 96 3 cs17, cs19 Fifth Creek nr St lgnatius College Suburb 0 0 Flinders UniversitY Scrub 19 0 40 2 cs44, cs45 Fourth Creek Suburb 0 0 Gorge Rd (Cudlee Creek) Light Agric 11 o\ (,¡¡ Abnormality lD # ln¡urÞd lnjury lD Location # 0 0 0 Gumeracha Light Agric 0 o 0 Hahndorf Suburb 3 cs08, cs09, cs10 185 I cs10 Harrogate Picnic Grounds Scrub 2 cs39, cs40 49 1 GS4.I Hav'¡thomdene Light Agric 4 LEO2, LEO3, LEO4, LEOs 93 0 Highercombe Golf Course Golfcourse 0 24 0 HorsnellGullY Scrub 1 cs43 19 0 lnglewood Light Agric 0 0 Jupiter Creek Scrub 1 0 0 0 Kaurna Park Wetland 0 30 0 Kuitpo Forest (Chookarloo Trail) Scrub 1 cs07 I 0 Lenswood Memorial Park lntense Agric 0 21 0 Little Para River (Salisbury) Suburb 0 13 0 Lynton Scrub 0 7 0 Mannum Scrub 0 2 0 Meadows Light Agric 1 LTO3 18 0 Meadows 2 Light Agric 2 LTO5, LTO6 32 0 Milang Wetland 3 1T19, LT2O, LT21 58 0 Milang 2 Wetland 1 cs42 35 0 Montacute Light Agric 0 4 0 Montacute Conservation Park Scrub 0 0 0 Montacute nr Black Hill Scrub 0 27 0 Morialta Conservation Park Scrub 1 cs04 38 2 cs05, cs06 Mt Pleasant (WallY Creek) Light Agric o\ o\ Location Habitat # Frogs #Abnofmâl Abnormality lD # lnlufèd Injury lD
Mt Torrens Light Agric 7 0 0
Norton Summit Scrub 30 0 1 LE06
Norton Summit 2 Scrub 37 0 0
Norton Summit Rd / Summertown Rd Light Agric 0 0 0
Nth of Monfries Bridge Light Agric 34 1 cs01 0 4 cs48, cs50, cs51, cs52 Oakbank Light Agric 47 1 cs49
Paradise Suburb 15 0 0
Parndana (Kangaroo lsland) Scrub 39 0 2 cs37, cs38
Sandy Creek Scrub I 0 0
South Rd (Wingfield) lndustrial 26 0 0
The Paddocks Wetland 18 2 cs03, LT01 1 LTO2
Upper Sturt Scrub 131 0 0
Warren Conservation Park Scrub 0 0 0 3 cs55 Woodside Scrub 47 0 cs53, cs54,
Yundi Light Agric 96 2 cs30, LE01 0
Yundi2 (Enterprise Rd) Light Agric 55 0 2 cs35
à{ lD # lnJured lnjury lD Location Land Use # Frogs # Abnormal AbnormalitY
1 LPOI 1 LROl Bool Lagoon Wetland 43 0 Clark's Park Light Agric I 0 0 1 0 Drain L Light Agric 0 0 Lynwood Light Agric 2 0 Wandillo Forest Light Agric 30 0
. :: 11 -ti.l AbnönflâlitY lD 0 n\roona Waters 0 0 3 BRAOl,BRAO3,BRAO4 Brachina Gorge 34 1 BRAO2 10 BUNO2,BUNI03,BUNO4,BUN05, BUNO6,BUÌ{O7,BUNO8,BUN 1 O'BUN1 Bunyeroo Gorge 178 3 BUN03,BUNOg,BUN 0 Dingley Dell l3 0 0 Ëcho CamP Waterhole 34 0 3 EREOl,EREO3,EREO4 Eregunda 15 1 EREO2 1 MAMO2 Mambray Creek 34 I MAMOl 2 NtL01,NlL02 Nildottie 20 0 6 oRAO 1, O RA03,ORA04,ORA05, O R406, ORA07 Oratunga 159 1 ORAO2 2 PARO2 Paralana Hot SPrings 41 PAROl 7a 5 SNOO1,SNOO2,SNOO3,SNOO4,SNOO5 Snob's Hut SPrings SNOOl 2 WEEOl,WEEO2 Weetootla Gorge v7 0 2 ì/1/tL01,wlL02 Wilkawillina Gorge 65 0 0 Wilpena Creek I 0 o\ Ø 30. (a) Mt l-ofty Ranges
Land Use Location lnjured
Golfcourse Echunga Golf Course (Nth End) 0.00 2.17
Golfcourse Echunga Golf Course (Sth End) 0.00 0.00
Golfcourse Highercombe Golf Course 0.00 4.30
lndustrial Cavan 14.93 7.46
Light Agric Brukunga 0.00 6.06
Light Agric Clarendon Oval 0.00 4.08
Light Agrie Hawthorndene 2.04 4.08
Light Agric Montacute 0.00 2.86
Light Agric Mt Pleasant (Wally Creek) 5.26 2.63
Light Agric Nth of Monfries Bridge 2.94 0.00
Light Agric Oakbank 2.13 8.51
Light Agric Yundi 2.08 0.00
Light Agric Yundi2 (Enterprise Rd) 0.00 3.64
Scrub Harrogate Picnic Grounds 0.54 1.62
Scrub Kuitpo Forest (Chookarloo Trail) 0.00 0.00
Scrub Norton Summit 0.00 3.33
Scrub Norton Summit 2 0.00 0.00
Scrub Parndana (Kangaroo lsland) 0.00 5.26
Scrub Upper Sturt 0.00 0.00
Scrub Woodside 0.00 6.38
Suburb Dry Creek (Modbury Heights) 8.57 5.71
Suburb Fifth Creek nr St lgnatius 3.13 4.17
Suburb Fourth Creek 5.00 0.00
Wetland Apex Park 4.65 0.00
Wetland Milang 0.00 6.25
\fi/etland Milang 2 0.00 5.17
(b) South East
Land Use Percent,Abnonnal Percent lnjured
Wetland Bool Lagoon 2.33 2.33
Light Agric Wandillo Forest (SE) 0.00 0.00 I
69 (c) Flinders Ranges
Land Use Location Percent Abnormal Fercent lnjured
Scrub Brachina Gorge 2.94 8.82
Scrub Bunyeroo Gorge 1.69 5.62
Scrub Echo eamp Waterhole 0.00 0.00
Scrub Mambray Creek 2.94 2.C4
Scrub Oratunga 0.63 3.V7
Scrub Paralana Hot Springs 2.44 2.44
Scrub Snob's Hut Springs 1.37 6.85
Scrub lVeetootla Gorge 0.00 2.60
Scrub Wilkawillina Gorge 0.00 3.08
3.3 Statistical Analysis 3.3.'f Mt !-ofty Ranges ,{s stated earlier, only sites where at least thirty frogs were eolleoted have been used for statistical analysis. Therefore only those sites listed in table 4 are considered" As only one sample from an Industrial site, i.e. Cavan, was available this land use class was eliminated
from analysis.
3.3.'î.'î [ncidence of Ahnonmality
A significant difference existed between land use classes (Hr:lI.724, X2oos,q:9.488, 0"025 < P < 0.01). The T'ukey-type test revealed that the samples fell into a oontinuum with the only detectable difference being between the Scrub sites at the low end and the Suburban sites at the high end. The remaining three land use types fall within these two land use types (i.e. soruhl, golfcoursesu't, light agriculturel'2, wetlandsÍ'2, suburban2).
3.3"4"2 [neidenee of flnjury No signifieant difference existed between land use elasses (Hr:tr "336 y2oos,a:9.488) and therefore no further testing was required.
3.3.2 Brooks 1979 When analysing her held data, Brooks (1979) incorporated all of the samples, regardless of nur,¡bcc ¡ãe. Wfrite this may be statistically valid, it is biologically unrealistic; a sample size of five or six frogs is unlikely to give a true indication of the incidenoe of abnormality at a site. Therefore using the criteria applied to my own data (i.e, at least thirty frogs per sample, deformities of unknown origin classed as injuries) Brooks' data were re-analysed. Only 13 of 70 the original 39 samples (see table 5) met the criteria and of these the quarry site at Greenhill Rd had to be eliminated due to the laok of replication of land use type.
Land Use Percent Abnormal
Golfcourse Paracombe 1 4.40 5.49
Golfcourse Paracombe 2 1.85 1.85
Heavy Agric Carey Gully 1 6.67
Heavy Agric Carey Gully 2 4.76 0
Heavy Agric Carey Gully 3 0.00 4
Heavy Agric Longwood 4.08 0.00
Heavy Agric Upper Hermitage 9.68 0.00
Light Agric Clare A 0.00 0.00
Light Agric Clarendon C 0.00 1.96
Light Agric Hahndorf A 0.57 2.
Light Agric 1.39 4.1
Light Agric 2.78 0
Quarry 3.92 7.84
3.3.2.1 lncidence of Abnormality No significant differenee (H":4.269, y20.0s,2:5.99I) between land use types.
3"3"2"2 [nc¡dence of Injury
No significant difference (Hr:2.603, X2o.os,z:5.991) between land use types
3.3.3 Gomparison of Mt Lofty and Flinders Flanges s¡tes Twenty-six sites frorn the Mt Lofty Ranges (using all land use types) were compared with nine sites f,rom the F'linders Ranges for both incidence of abnormality and inoidence of injury.
3"3.3.1 Incidence of Abnormality No significant difference (U':128.5, Uo.os(z),s,ze:170) was detected in the incidence of abnormality between the Mt Lofty Ranges and the Flinders R.anges.
The data were further analysed to compare only scrub sites for both regions. ,{ significant difference (U:51, Uo.os(z),2,s:51) was detected suggesting that there was a greater incidence of abnormality in scrub sites in the Flinders Ranges than scrub sites in the Mt Lofty Ranges.
7t A comparison of non-scrub sites in the Mt Lofty Ranges with the Flinders Ranges scrub sites demonstrated no statistical difference (U'=93.5, U6.6512¡e,¡e:120) in the incidence of abnormality.
3.3.3.2 lncidence of lnjury There was no significant difference (lJ':132, IJo.os(z),g,ze:170) in the incidence of injury between the Mt Lofty Ranges and the Flinders Ranges.
Comparison of incidence of injury for only scrub sites in both regions also demonstrated no significant difference (lJ:42.5, Uo.s(z)z,s:51) as was the case for comparison of non-scrub sites in the Mt Lofty Ranges with the scrub sites in the Flinders Ranges (U:89.5, U¡.512;e"1e:120)'
72 4 Disct¡ssion
This study has confirmed that frogs exhibiting externally detectable skeletal abnormalities can be found in the Mt Lofty Ranges and surrounds. The presence of abnormal individuals is not restricted to any particular land use type, but there is an increase in the incidence of abnormality in areas that are likely to experience high levels of pollution. Difficulties in gaining access to private properties resulted in a moderately small sample size, especially with regard to intensive agricultural sites, however enough information was gathered to demonstrate a statistical difference in the incidence of skeletal abnormality of frogs in some of the different land use classes.
4.1 Abnormal¡t¡es
As may have been expected, the relatively unpolluted scrub sites had the lowest incidence of abnormality (0 - 0.54%). This is comparable to the collections made of frogs from various unpolluted sources reported by Tyler and Crook (1930) where incidence of abnormality was generally below 3Yo. Only five sites in the Mt Lofty Ranges had levels of abnormality above this normal maximum of 3%o and none of the Flinders Ranges sites had abnormality levels above 3 It is of minor interest that a statistical comparison showed scrub sites in the ìlf Flin dece S mt bÇfv ßno;".í had a higher incidence of abnormality than sites in the -F_linCers-f,arrgd Flowever this is not of great concern because, as stated previously, the recorded incidences were within supposed natural levels.
Some of the abnormalities reported in this study, and also by Brooks (1979), appear to be very mild and one may suggest that a slight reduction in the length of one digit would not have much of an impaet on the ability of a frog to survive and reproduce. However a comparison between the inoidence of abnormatity in juveniles and adults of three species of frogs by Tyler and Crook (1980) showed a significant difference. Similar reports are provided by Dodd (1993) from reports at a National Institute of, Environmental Flealth Sciences (NIEHS) workshop on amphibian deformities. it appears that abnormal individuals are removed from the population before they have a chanoe to reach maturity" For randorn selection of individuals by predation or disease one would expect the incidence of abnormality to be the
same throughout life. Therefore the presence of large numbers of abnormal frogs at a site, taking into account the probable removal of many of these, is potentially useful in biological monitoring. This is especially beneficial because measuring water quality by conventional
?2 chemical means is very expensive (Greenhouse 1976). When recording water quality from a site it is necessary to take samples frorn a number of points within that site (Thorns 1998). 'V/ater quatity may fluetuate due to differences in the biotie environment (filter fecders and plants removing some of thc pollutants) or the physical environment (differences in mixing of water, proximity to input). It is also neoessary to record over time as water quality can change quite radically with climatic conditions; drought would increase the eoncentration of chemicals in the water, rains would dilute them (Ferraro and tsurgin 1993). For example, in a retention pond at .Iabiru frogs (Cyclorsna australis and C. longipes) that bred before summer rains produced spawn of which 38"95% (total of 95 frogs eollected) developed abnormally
(Tyler and Crooke 1980). A few weeks later after heavy rains more frogs bred in the same pond and produced only 1 .6Yo abnormal offspring (305 collected).
Four of the five Mt Lofty sites that had levels of abnormality above 3Yo were from areas where high levels of chemicals may be expected: one from an industrial area, one from light agriculture, three from suburban sites, and one from a wetland. Despite the laok of replication, and theref,ore the inabiliff to include it in the statistical analyses, it appears that there is a biologically meaningful effect on the growth and development of frogs from the industrial area. The incidence of nearly l5o/o in frogs collected from Cavan was well above the expected
3o/o and requires more detailed investigation.
4"2 Braaks '1979 As indicated earlier (chapter 3.3.2) there are reasons for concern as to the validity of the claims made by Brooks (1979) regarding the distribution of abnormal frogs within the Mt
Lofty Ranges. As well as the fact that very small sample sizes were used in the analysis of the data there also appears to be a re-classification of land use types for the analysis. Initially all of the sites visited were classed as one of six land use types (Golf-eourse, Fleavy Agriculture,
Light Agriculture, Quarr/, Scrub, or Suburb) but for the analyses all of the sites were grouped as being from areas with either heavy or light agricultural practices. Brooks (page 22) stat$ "...the type of agricultural practices carried out on the land immediately surrounding the water souroe, did appear to correlate with the pattern of teratogenic occurrence that emerged." and also (page 23) "Hence the collections were categorised as being from sites where heavy or light agricultural practices were carried out on the land immediately surrounding the water source...". No analysis used to demonstrate any correlationþsprouiard. The re-analysis based on the original land use classification, and excluding those data that were not oonsidered biologically realistic, demonstrated that the assumptions made by Brooks cannot be sustained, t4 4.3 lnjunles
There was no significant difference in the incidence of injury in frogs from different regions or land use ffpes. R.ates of injury of frogs encountered in the Mt Lofty region averagecl 2.65% for all frogs, with a maximum of 7.43Yo at sites with > 29 frogs collected. For the Flinders
Ranges the average for all frogs collected was 4.65yo, with a maximum of 8.820/, at sites with > 29 frogs collected" These values are much higher than was expected. Tyler and Crook (1930) report the incidence of injury in frogs from Jabiru reaching a maximum of 0.99Yo, however these data are not based on internal examination, and Dubois (cited in Tyler and Crook (1980)) recorded incidence of injury in species of Rana from Europe with a maximum of 4.5o/o. Injuries of frogs from the Mt Lofty region collected by Brooks (1979) reached a maximurn of 7.84%o, for sites with > 29 frogs collected. It is therefore obvious from this study and that of Brooks (1979) that the rate of injury in frogs in the wild is highly variable, but can reach relatively higher levels than have been widely published.
4"4 Recent Discover;es of Abnorma¡ Frogs In August 1995, shortly after the commencement of this project, extremely large numbers of abnormal frogs were discovered by a group of school children during a field trip in Minnesota, IJSA (NARCAM 1997, Gilbert 1998, MPCA 1998, Gardner and Hoppe 1999). Follow-up surveys have shown that these large incidenees of abnormalities are occurring over much of North America, especially in the agricultural areas such as Vermont. A 1997 survey of,2l sites in Verrnont found that tr4 sites had populations of frogs with greater than 4Yo abnormality and one site had an incidence of 45o/o (Gilbert 1998). Abnormalities in these regions have ranged frorn frogs with slight deformities of the digits to more extreme cases including complete or partial replication of limbs, missing eyes, translocation of limbs and deformed jaws (Dodd
1998)"
Much research has been undertaken as a result of these f,rndings, but scientists appear to be little closer to explaining the phenomenon. Theories for the cause of the high incidence have included chemical contamination through agriculture, retinoid-compounds from wide-scale pesticide use, natural mutation, UV radiation, parasitic infection (Sessions and Ruth 1990), fungal infection, increased levels of heavy metals and even better reporting of abnormalities due to increased surveying. Whatever the cause of the abnormalities it is evident that the
elevated number of abnormal frogs is not limited to farming areas in North America. This project suggests that the incidence of abnormality in agricultural regions, areas which for a 75 Australia, may be less long time have been btamed for much of the environmental damage in suburban and industrial of a concern in South Australia than the incidence of abnormality in use in agricultural areas areas. Despite the possibility that there is alarge amount of chemical sites' Refuge (EpA 199S) these areas frequently provide large numbers of refuge and breecling
and breeding sites are commonly removed from urban areas.
led the project The extremely high incidence of abnormalities from the industrial site at Cavan in the direction of toxicological research, with the aim of determining potential causes of abnormalities at that site.
76 5 Gavan - Background
Suggestions of a problem with the pond at Cavan arose as a result of communication with a member of the SA Herpetology Group, who had collected a number of frogs at the site (J. Cloosterman pers. coÍìm.). He suggested that perhaps 5 - 10% of the frogs caught displayed some sort of abnormality. Onthis basis the site was visited. The site at Cavan (figure 139) was a large pond situated at the outlets from two drains, one from the north and one from the south, believed to come from the nearby residential and I or industrial areas. A number of large rocks and discarded material, including a small quantity of tyres and railway sleepers, provided ground cover away from the loosely vegetated banks. Typha sp. gfew in the southern end of the pond. Adjacent to the pond on the west was a multi-tracked railway line and on the east a six-laned highway (Port Wakefield Road). To the south was a brewery and a high- voltage power line ran above the pond, with a tower present at the southern end. An access track was situated on the eastern side of the pond, parallel to the highway. In spite of this there appeared to be a relatively abundant frog population, possibly due in part to the movement of individuals from a major wetland, Greenfields, a few hundred metres northeast. The pond does not completely dry in sufiimer, however the water level drops considerably from the maximum depth of about 1.3 m.
Figure 139. The survey site at Cavan (facing (a) south, (b) northeast (c) northwest)' (a)
(b) (c)
77 5.{ Survey Summary As can be seen from chapter 3 the incidence of abnormality from the initial survey was approximately l5Yo. Subsequent trips were made to the pond and on each of these occasions abnormal frogs were oollected. Results for some of these additional surveys are presented in table 6. Sampling involves the removal of abnormal frogs and the release of normal frogs, therefore repeated surveying results in a bias towards normal frogs, some of which may have been collected in previous surveys. Consequently they cannot be included in any statistical analyses. However, the large number of abnormal frogs that has been collected at this site warrants some attention (see figure 140).
Date Total,Number êolleeted NumbêrAbnormal o/o Abnormal
23t5t96 9'r x7" '[8.68
9t7t96 55 9" 16.36
19t7t96 22 5" 22.73
20/8/96 42 2* 4.76 *These frogs were not cleared and stained to test for injuries.
Sediment was sampled using a polycarbonate eorer (110 mm diameter) that allowed the sediment to be collected with minimal disturbance to surrounding sediment and without exposing the soil to the overlying water. The sediment was placed in 5L containers, residual water was decanted, and was taken back to the laboratory in insulated 'eskies'. Sediment samples were sent to the Australian Water Quality Centre (Hodgson Rd, Bolivar, South Australia) where the total metal content (mg/kg) for Cadmium, Copper, Lead and Zinc was measured by ICP-Sl (inductively coupled plasma spectroscopy). An analysis of the sediment frorn points within the Cavan site suggested that there were high levels of heavy metals (see tabte 7). Lead and Zinc levels were higher than those recommended in high effect range for sediment quality guidelines (NWQMS 1999)
Sedirnent cd (ppm) Gu (ppm) Pb (ppm) Zn (ppm)
South 1.76" 47.1 130* 210*
Norlh 7.14" 1 60* 1 1 60** 740**
North-west 7.67* 120* 430** 440**
West 3.53. 42.1 120* 165
(* Levels above recommended low effect range, ** Levels above high effect range (NWQMS reee)
78 Figure 140. Additional abnormal Limnodynøstes tasmaniens¡s collected from Cavan.
a
\
79 6 lmpromptu SPawning
On four separate occasions normal pafus of Limnodynastes tasmaniensls collected from Cavan spawned unaided ín the laboratory.
6.1 lVlethods The resulting foam nests were removed from the tanks containing the adult frogs and placed in 60L glass aquaria to which dechlorinated tap water had been added. Tanks were continuously aerated. T'he eggs were then reared through the tadpole phase until metamorphosis. trndividual foam nests were kept separately and the hatohing tadpoles were fed a combination of softly boiled lettuce and goldfish flakes until metamorphosis" Upon completion of metamorphosis each frog was examined for externally visible skeletal abnormaiities using the previously described criteria"
6"2 Results A total of 239 frogs from the four nests successfully underwent metamorphosis. Only two individuals displayed any form of skeletal abnormality, both of these abnormalities being reductions in the size and funotion of the forelimbs (figure 141). A tadpole exhibiting a condition commonly known as ''kinky tail" suffered little impairment and at metamorphosis demonstrated no visible abnormality of the skeleton; absorption of the tail following metamorphosis resulted in a norrnal frog. F'our of the tadpoles laeked skin pigmentation, metamorphosing as albino f,rogs. A eomplete list of the spawning and metamorphosis dates for each of the surviving individuals is given in appendix 1, a summary is presented in table 8"
Spawn Date Metamorphosis F SE
16/07/96 185.9 r'4.1
13/08/96 139 r
06/09/96 167.3 r- 6.3 M3.2r 4.7
80 Figure 141. Abnorrnal fi'ogs fi'orn impromptu spawning.
(a) Note: digits of the right hand are visible protluding through the spiracle (see allow)
(b)
8l 6"3 Discussion Despite the unplanned nature of the spawnings some interesting data were gathered from them. It was considered possible that the high incidence of abnormality at Cavan was the result of genetic factors, but as the abnormalitics were not limited to a single species this was not thought to be the most likely explanation. All of the eggs were reared in tap water and were subject to any impurities and additives present in the water; chlorine had been removed using a carbon filter. The filter apparently failed at one point and a large number of tadpoles died in the tanks from the 13108196 spawn. Despite this the majority of tadpoles grew and metamorphosed normally. It appeared that the normal adults from Cavan did not carry any genes predisposing their ofßpring to deformities.
It was expected that if a hereditary factor was responsible then this would have been expressed given the number of offspring produced, but more intensive genetic studies are obviously required to gain a complete understanding of the heritability of abnormalities.
A small number of abnormal frogs collected from Cavan, including a very vocal male frog lacking one krind lirnb, that were kept in the laboratory spawned on at least two occasions but the resulting eggs did not hatch. Whether this occurred because the abnormal frogs were infertile or due to other factors is unknown. Time constraints, particularly the larval lifespan af Limnodynastes tasmaniensis, prevented studies in this direction being carried further.
It is probably more important to note that the low number of abnormal offspring produced suggests that no pollutants able to affeot normal development were deposited in the eggs, even though the adults may have accumulated pollutants from the site.
82 7 Sediment Bioassay 1 - Gollected Sediment
Toxic effects of pollutants are often only measured as the amount that is required to cause the death of the embryo; aeute toxicity. This is generally done by exposing the tadpoles to high doses of chemicals that have been dissolved in water, or sometimes by a more vigorous method of directly injecting pollutants into the body cavity of animals (Tyler 1997). These sorts of tests may give us a good idea of what levels are required to kill frogs, but they will not tell us anything about long term exposure to chemicals; chronic toxicity.
Pollutants often enter the eeosystem slowly, but many may settle or become bound to sediment. Levels of these pollutants in the main body of the water are consequently low. The pollutants accumulate until the occuffence of events causing disturbance to the sediment, for example rapid water flow following a storm. The disturbance may cause these pollutants to become re-suspended but, even if re-suspension does not occur, quantities of pollutants may be absorbed by aquatie organisms feeding on or near the sediment. Therefore it may be more useful to determine the effects of polluted sediments, rather than just polluted water, on these organisms. Studies have shown that chronic exposure can lead to a reduction in fitness that will leave tadpoles more susceptible to disease or may increase ehances of predation as a result of behavioural changes (Abbasi & Soni 1984, Ferraro & Burgin 1993). This led to the speculation that there may be similar events occurring with the frogs at Cavan.
7.1 Methods
Initially sediment was collected from four spots within the Cavan site and also from a nearby site that was part of the Barker Inlet Wetland and had previously been examined as part of a study "to investigate potential contaminant effects associated with sediments" (Bidwell et al. 1997). Sediment was sampled using the proviously described methods. All of the sediment in each of the 5L containers from a site was emptied into a large tub and sieved; any obvious debris, organie matter or large organisms were removed. Two litres of the homogenised sediment was deposited in a small aquarium (175mrn V/ x 212mm D x 355rnnn tr-) to which 7L of artificially reconstituted freshwater (USEPA 1989, see appendix 2) was then added. Four replicates of the sediments were made, as well as cornmereially prepared aquarium gravel for a control. Each aquarium was randomly placed on a three-shelved stand and allowed to sit for at least one week, with aeration, before testing.
83 A total of 10 Crinia signifera eggs that were newly spawned in the laboratory were placed into each of the aquaria. Temperature, measured using a Digitron 1408-K thermocouple, pH, measured using a Hanna pHep3 pocket pH meter, and conduetivity, measured using a TPS LCS1 oonductivity meter, were reoordecl approximately every one or two days' Dissolved oxygen was measured with a YSI-518 dissolved oxygen meter and recorded weekly. The eggs were allowed to hatch and the resulting tadpoles were maintained within the aquaria until metamorphosis; equal amounts of tropical fish food flakes per tadpole were added to each tank. In the event of any evaporation from the aquaria water levels were replenished by the addition of reverse osmosis water; no solutes were added so as not to artificially increase the concentration"
7"2 Results
Less than four weeks after the experiment had commenced it was discovered that all of the tadpoles in ten of the tanks (including all sediment types) had died from unknown causes and the experirnent was therefore abandoned. No frogs had reached metamorphosis.
The experiment was restarted, but this time sediment was collected from two spots within the Cavan site and also from sites within the Barker Inlet V/etlands. Four replicates of the sediments were made, as well as an artif,rcial sediment for a control (kindly prepared by J. Gorrie, University of South Australia). The artif,rcial sediment was eomposed of 75Yo elay,
20Yo silt (2-53 prm), 25o/o fine sand (53-250 ¡rm), 39% medium sand (250-500 ¡rm) and 1% organic matter (sphagnunn naoss) by weight.
As before, the collected sediment was homogenised, and a sample of each sediment was analysed by the Australian Water Quality Centre for total heavy metal content (able 9).
Sedirnent Gd (mg/kg.dry) ,Cu tmglkg dry) Pb (mg/kg dry) Zn (mg/kg dry) Drainl (Barker lnlet) <0.200 24.5 10.0 73
Drain3 (Barker lnlet) <0.200 31.0 27.6 162
South (Cavan) 0.360 64.8 141* 136
West (Cavan) 0.200 15.4 33.9 72
(+ Level above recommended low offect range (NWQMS reee))
The protocol followed that previously outlined and at the completion of metamorphosis (this was classed as the point at which the forelimbs protruded) all frogs were examined for skeletal abnormalities using the previously defined criteria. 84 From the initial 200 eggs placed into aquaria a total of 117 survived and eompieted metamorphosis. A total of four abnormal frogs was produeed, two each from Artif,icial and
Vy'est treatments. Table 10 shows a sumrnary of surviving and abnormal frogs (see appendix 3 for a eomplete trist).
Both of the abnormal frogs from the control (Artihcial) sediment exhibited the same abnormalþ of the head. Externally the abnormality had the appearance of a swelling or blister just under the skin (figure tr42). One abnormal frog from the V/est sediment had ectrodactyly of digits IV and V on the left foot and also digit IV on the left hand (figure i43). The left foot of the other V/est frog had the morphology of a right foot. r.e. the frog had two right feet (figure 144). Unfortunately this frog died and started to decompose before being photographed"
Sedirnent
Artificial 2
Drain 1 0
Drain 3 0
South 0
West 2
85 Figure 142. Abnormal frogs from the control sediment' Sediment Bioassay 1' (arrows)' Both frogs exhibit abnormality of the head with the appearance of a bump (a)S8101
(b)s8103
86 Figure 143" Abnormal frog SB 1 02 fi'om the west sediment, Sediment Bioassay 1 (a) left foot
(b) left hand
o? Figure 144. Abnormal frog SB104 fromthe west sediment, Sediment Bioassay 1. Right foot above, left foot below.
An analysis of variance (data passed tests for normality and equal variance) to compare the rate of survival of tadpoles between each of the treatments demonstrated a significant difference (F:6.805, Foos(r),+,ro:3.01, P:0.002). A Tukey test revealed that there was a diflerence between the Cavan sites (South and West) and the non-Cavan sites (Artificial, Drainl and Drain3), but there was no detectable difference within those two groups.
No analysis could be undertaken to compare dif[erences in the incidence of abnormality due to the resulting small sample sizes and lack of variation in the recorded incidence of abnormality.
7.2.1 Water qual¡ty parameters Graphs of the temperaturo, pH, conductivity and dissolved oxygen recorded during the course of the experiment are presented in figures 145 - 148.
Analyses of variance (or Kruskal-Wallis ANOVA on ranks if normality and equal variance could not be assumed) for the mean recorded values of each parameter during the period of the bioassay were undertaken to determine if any differences existed between the treatment tanks. 88 Sediment Bioassay I Figure 145. Temperature ("c) recorded in tanks during Ambient air temperature is shown for comparison'
21 n
19 - A¡r
18 * Conlrol oÇ +Drainl g 17 +Drain3 b 't6 ---.--- Soulh t- - -- x-- - WBsl 15
14
13
12 SEBsEE EEEEEE EEEEEq++ËÉËË EEEEÈ+ÊË ÊÊËååå È s ÊË== <;-1 iÉÉÉñx RRÉÉÉÉ déÉJÉÊ ÃÑRà 0aic
Figure 146. pH recorded in tanks during sediment Bioassay l.
91 89
8,7 B5
B3
B1
7.9 å Conlrol +Drainl 77 +Drain3 -n7 5 ---.--- Soulh 7.3 ---x.--WBst
7.1
6.9
6.7
6.5
6.3
6.1 5.9 EEEEEESE EESEqEqqÊsåqq3r; EEEEEE åËääËååå 8"1ñ+töóóNr EERÑRd Dû1.
89 Oryqent (tng:Ll co ¡ducrivity rc Dksolved úe o o o o o o o P È! o o 6 'l(D o 1+Jur98 16-Junff È "Þ æ 16-J uÞ98 -¡ 1&Juft98 Õ 1&JuÞS o U) ZNUÈ98 v) ãlJutrS o. o Ê 22.Jur98 o 2-Jutg€ (D
è 2+Jun-98 2¡lJuçS o k X 2ÈJuÞ98 2FJUÞæ 0a (t) (l 2BJun98 Ft 2&'JuÞæ 1!Jur$ (lo o flJunS ùq 2-Jul-98 Ft (DÈ - 4-Jul-98 2Jul$ ¡_i È (D E o 3 a¡uræ o 4JuÈS ê Flo Þ Þ BJul-S ÈJul-S (D tf Þ lGJul-98 v) À ÈJul-S l2-Jul-S É !) lGJul-æ ,f 1{Jul-98 @ U) (h l2-Julæ a- 16-Jul-S Ào Ft 1ÞJul98 14Jul-98 iJ oq ZNul-s o (A l6-lul-98 o 72-Julfi EÉ È o lB-rul$ P 2il-Jul-98 s) u) (D v) 2flJ ul-98 ) 2ÈJut98 Ê) EÚ o - 9D (t) lllI + ll- U) iill tt 9D \o No statistical differences were found between treatments for temperature (F:0.287, P:0"882), or dissolved oxygen (F:1.719, F:0.tr98). Significant statistical differences were detected for pH (H:15"814, F:0.003 - Ðrainlr, Controlr,2, South2, West2, Drain32) and oonduotivity (I{:14.243, F:0.007 - Controln, V/est2, Southz, Drain32, Drainl2). 7.3 Discussion The artifieial sediment used for the control tanks was not entirely suitable for the experiment. The sediment did not completely settle, making the water cloudy, ,A.s a result tadpoles were not always visible and the tadpoles in one of the eontrol tanks experienced low ievels of survival, possibly as a result of the low pF{ recorded for the control tanks. nt is suggested that after the initial death of some individuals survivai of the remaining tadpoles was reduced due to a build-up of uneaten food, which had been added at the same rate as in other tanks, which subsequently fouled the water. This build-up was not visible until the tank was drained for inspection after only one metamorphosed frog could be found. Despite the problems associated with the control there still appeared to be some interesting results. Survival from the two Cavan sediments (South and West) was much lower than for the control and the two Barker Inlet Wetland sites. Supplementary to the rate of survival, the tadpoles metamorphosing from Cavan treatments appeared to be smaller than the metamorphs from the other tanks. trnitially the size of metamorphs was not measured, however the approximate average length (body plus tail) for later developing metamorphs from the non- Cavan tanks was 31 mm. Most of the rnetamorphs from the Cavan tanks had an approximate average length of only 24 mm. This suggested that there was an agent present within the sediment that not only affeeted survival, but also reduced growth' 9l 8. Sediment Bioassay 2 - Prepared Sediment on growth and survival As results from sediment Bioassay 1 suggested that there were effects on the from the Cavan sediment further investigation into the role of the heavy metals development of tadpoles was warranted. 8.1 Methods Unlike the previous experiment Sediment Bioassay 2 involved the preparation of sediment with known levels of pollutants; spiked sediment toxicity testing. Barker Inlet Drain 3 had very low levels of metals therefore this was used as the base to which heavy metals were prepared by BDH added. The metals added (table 11) were Zinc (in the form of ZnSO+;7H20 Chemicals), Copper (CICL2.2H2.O prepared by Sigma) and Lead (PbQ'trO3)2 prepared by Sigma). A generalised Cavan sediment \¡/as prepared by the addition of Copper (48 mglkg Cu**; in solution (0.4 litres distilled water). In addition a range of sediments for possible comparison with studies from the University of South Australia, School of Pharmacy were created by the addition of 3600 mglkgpb**,700 mg/kg Cu** and 2000 mglkgZn** to 0.8, 0.4 and 0.8 litres of distilled water respectively. These three solutions were diluted in series to create the hnal 200vo, I}ao/o, 50yo and 25yo concentrations (2 litres total solution) for sediments used in the test. All of the metals were dissolved in distilled water so an equal amount of water was added to the control (2 litres) and Cavan (an additional 1.6 litres) sediments. The individual sediment with solution was placed into a 20 litre drum and thoroughly mixed by rotating on a roller for a period of four hours, ensuring an even distribution of the solutions within the sediment. Two litres of sediment were then added to tanks and artificially reconstituted freshwater in the previously described manner. Sediment samples were saved for future analysis. Due to an inability to obtain eggs from frogs maintained in the laboratory the sediment remained unused for a period of approximately five weeks before newly laid eggs collected from the wild were placed into the tanks. A number of fieldtrips were undertaken during this period with the hope of supplementing the laboratory colony of frogs to increase the chance of the spawning. Despite the dry weather conditions at least 30 frogs were collected and added to colony, but they did not produce any spawn. During the delay the tanks were continuously aerated and the water level was maintained by the addition of distilled water. The methods 92 employed for the rearing of tadpoles in this test were the same as for sediment bioassay 1 and following metamorphosis the snout vent length (SVL) of each metamorph (live) was calculated using dial calipers, measuring to the nearest mm' Table 11. es of metal solutions added to sedllmenl Cu'* added Pb** added Zn** added Sediment (mg/kg) (mg/kg) (mg/ks) Control 0 0 0 Cavan 48 0 0 25o/o 43.75 225** 125 50% 87.5* 450** 250* 100% 175* 900** 500** 200% 350*" 1 800** 1 000** (*Dosages above the recommended low effect range, *+ Dosages above recommended high effect range (NWQHS 1999)). 8.2 Results The eggs originally added to the tanks were believed to be Crinict signifera, but unfortunately it soon became apparent that some were in fact Litoria ewingi eggs. It is interesting to note that a single clump of eggs which was split across two or more tanks produced both species, indicating that one or both species are in the habit of adding to egg clumps which are already present in the water. Despite the fact that eggs from two species were accidentally included in the experiment time constraints meant that the test could not be repeated; consequently the experiment was allowed to reach its conclusion. It is not known what proportion of Litoria ewingi eggs were actually included in the experiment and therefore no survival data can be given for individual species but only 144 of the original 240 eggs in the experiment developed to metamorphosis. A complete list of the surviving frogs and information relating to their growth are included in appendix 4 and summarised in tables 12 - 13. Photographs of the abnormalities are presented in f,rgure 1a9 (a)-(k). Despite all treatments being run concurrently the data for Cavan was compared with the Control separately to the 25Yo, 50yo, l00yo and 200o/o treatments, due to the fact that more than one characteristic, the addition of multiple metals, was altered in the those treatments. 93 Table 12. Crinia s,'qn,fera from Sedimen Number Mean SVL Mean Ðays to Number Treatment of Frogs *sE Metamorphos¡s i SE Abnormal Control 19 7.95 r 0.09 42.63 !O.78 Cavan 12 7.58 r 0.15 46.00 r 1.95 25o/o 23 7.35 r 0.15 50.61!2.24 50To 23 7.52 ! O.12 47.39 t 1.58 10oo/o 18 7.78 t 0.10 45.28 ! 1.42 2 200% 12 8.25 !O.48 92.25 !8.16 6 Table 13. Lifora ewnqlfrom Sediment Bioassav 2. Number Mean SVL (mm) Mean Days to Number Treatment of Frogs ISE Metamorphosis t SE Abnormal Control 3 13.67 r 0.33 59.00 12.00 Cavan 6 13.83 r 0.70 69.83 r 4.'17 50% 4 14.50 r 0.65 75.OO ! 4.92 100% 14 15.00 r 0.33 80.0013.',l0 200% '10 16.60 ! O.27 '126.70 f 5.63 J 8.2.1 Crinia signifera Data failed a normality test and could not be normalised using any transformation techniques. A Mann-Whitney Rank Sum test was used to examine aîy differences in size at metamorphosis and time to metamorphosis between the Control and Cavan sediments and a Kruskal-Wallis non-parametric analysis of variance was used for comparisons between the Control and artificial sediments. 8.2.1.1 Size At Metamorphosis - Cavan sediment No signihcant difference (T:153.00, P:0.116) was detected between the Control and Cavan treatment. 94 Figure tr 49. Abnormal Ílogs proelueed in Seeliment Bioassay 2 (a) Crinia signifera. 100%o treatment 2418199, SVL 7 rnm. Right hand digit I ectLodacfyly. (b) Crinia signifera. 100% tleatment24l8l99, SVL 8 mm Right hand digit I ectrodactyly. 95 (k) Litoria ewingi. 200Yo treatment 7 /12199 , SVL I 7 mm Ectrodacyly or syndactyly ofdigits I and II on left hand, syndactyly ofdigits III and IV 8.2.1.2 Time To Metamorphos¡s - Gavan sed¡ment No significant difference (T:233.5, P:0.096) was detected between the Control and Cavan treatment. On reflection is appears that a miscalculation was made when preparing the Cavan sediment and perhaps lead should have also been added; the metal levels in the 25o/o tteatment were more like those from the sediments previously collected at Cavan. 8.2.1.3 Size At Metamorphosis - Artificial sediments A significant difference (H:11 .777, P:0.019, dt4) was detected showing that treatment did have an influence on size at metamorphosis. Dunn's test for pairwise multiple comparisons was performed which showed that the Control was significantly different (Q:2.926, P<0.05) to the 25Vo treatment but not to the other treatments. No treatments were significantly different to each other (by increasing size: 25yor , 50yor'2, !00\or'2,200o/or'2, Control2). The tadpoles in the 25o/o hreatment were significantly smaller than the Control tadpoles and had a significantly longer tadpole stage. As the 25Yo treatment was the only one without Litoria 100 inter-specific competition a Litoria ewingi tadpoles it is possible that reduced is interesting to note that a role in the altered growth of these tadpoles. It in the cavan sediments in metamorphosis was believed to have occurred the 25o/o treatments' these sediments were similar in composition to with the result that there was no significant tanks were removed from the statistical analysis tanks for size atmetamorphosis (50%l 100o/or' difference (H: 5.758 ,P:0.124) between ' 200Yor, Controll). S.2.l.4TimeToMetamorphosis.Artificialsediments dt4) was detected demonstrating that A highly significant difference (H:42.186, P<0'01 ' Dunn's test was able to divide a number of time to metamorphosis was affected by treatment. with three separate groupings (by the treatments, but not all; treatments fell into a continuum r'' 200%\ increasing time: Controll, 1 00olo , 50Yot'2 , 25yo2 , ' signihcant difference in time to when the 25o/o dala are removed from the analysis a p <0.001) which gives only a slightly different metamorphosis is detected (H : 36.610, grouping (Controll, l}}Vor'2, 50Vo2, 200%\' 8.2.1 "5 lncidence of AbnormalitY in the Control,25vo and 50o/o As there was no variation in the incidence of abnormality For the same leason the cavan treatments these were excluded from statistical analyses. tanks where at least five frogs reached sediment was not compared with the control. only Rank sum Test, data passed metamorphosis were included in the analyses. A Mann-V/hitney compare the proportion of normal frogs in a normality test but failed an equal variance test, to difference (T:7'00' n:2' the 100% and 200o/o treatment failed to detect any significant P:0.333). 8.2.2 Litoria ewingi failed a normality test' therefore a Data for s\ze atmetamorphosis for the cavan treatment The data for time to metamorphosis Mann-V/hitney Rank sum test was used for comparisons. comparisions were made using a passed a normality test and an equal variance test, therefore metamorphosis in the artificial t-test. Data for both size at metamorphosis and time to equal variance, therefore an analysis of treatments passed a normality test and a test for variance was used to examine any differences' 101 8.2.2.1Si2e At Metamorphosis - Cavan sediment the Control and Cavan No signif,rcant difference (T:16.00, P:0.905) was detected between treatment. 8.2.2.2 Time To Metamorphosis - Cavan sediment No signilrcant difference (t:1 .737,P:0.126, dÊ7) was detected between the Control and well below the Cavan treatment. The power of the performed test was 0.225 with alpha:0.05, these findings' desired power (0.g). Therefore caution should be made with interpretations of 8.2.2.3 Size At Metamorphosis - Artificial sediment (F:8.057, Despite the small sample size for Litoria ewingi a highly significant difference p<0.001) was detected, demonstrating that size at metamorphosis was dependent on that treatment. The power of the performed test was 0.969, alpha:0.05. A Tukey test showed the size of metamorphs in the 200Yo treatment was greater than in all other groups; no divisions could be made within those groups (controll, 50yor,Io}yrr,200%1'No Litoria ewingi metamorphosed from the 25o/o treatment tanks and it is unknown how many, if any, were present at the start of the experiment. 8.2.2.4 Time To Metamorphos¡s - Artificial sediment A highly significant difference (F:33.782, P<0.001) was detected, demonstrating that there \ryas a difference in time to metamorphosis for the different treatments' Power of the performed test was 1.000 with alpha:0,05. A Tukey test revealed that the200o/o treatment was different to the other treatments; these could not be separated (1.e. Controlt,50yot,I00o/or, 200%\. 8.2.2.5 lncidence of AbnormalitY As there was no variation in the incidence of abnormality in the Control, Cavan, 50o/o and rc}% treatments no statistical analyses could be completed. 8.2.3 Water qualitY Parameters Graphs of the temperatur€, pH, conductivity and dissolved oxygen recorded during the course of the experiment are presented in figures 150 - 153' Analyses of variance for the mean recorded values of each parameter during the period of the bioassay were undertaken to determine if any differences existed between the treatment tanks. r02 4 Tempetmute fC¡ ûe PH ûq @ æ@æ @ @@@@ q 6 ! @ ã ñLùè b,\'@(! ã o o t n o (Þ ô 14-Jul-æ 14-Jul-æ UI UI '16-Jul-99 16-Jul-99 È (D i-lr 'I B-Jul-99 18-Jul-99 o (D ts1 (D 20-J ul-99 (t 20-Jul-99 ÊD B 4 o (D 22-Julæ o Z2-JulÐ o o4 24-Julg È 24-Julw o CD r-t È (D C) 26-Jul-99 26-Jul-99 o È4 CD æ-Jul-99 r æ-Jul-99 Þ- U) o- iJ a 30-Jul99 o 30-Jul-99 H 9l ¿ 'l-Aug-99 1-Aug-99 0a IA (n (D o- 3-Aug-99 3-Aug-99 a- tsl iJ 0q 5-Aug-99 (D s-Aug-99 (t (D 7-Aug-99 EÚ 7-Aug-99 o. Ê) (Þ (A 9-Aug-99 9-Aug-99 (A iJ Þ 1'l-Aug-99 1 1 -Aug-99 N) o ÊD '13-Aug-99 v) 13-Aug-99 v) 15-Aug-99 15-Aug-99 \, lî1!'"llii flrl, :t ÑJûNOÔ p Ê $ $ EE3$i=õ e $å U) ã1 Co r{ r ctiv Îy ûe 0issofu eil 0xygerì lûr gill INN m ór o ! æ € io-olÚ dèoo!@@o r.f o o o @ Èf ô (Þ '14-Jul-99 15-JuL99 UI UI(, l-) 1E-Jul99 H ô 17-J ul-99 o v) 18-Jul-99 CA a- o 1 9-Jul-99 () 20-Jul-99 oÈ 21-Jul-99 22-Julw X Itr oq 24-Jul99 (n 23-Jul-99 (D 4 CD -Jul-99 o 25-Jul-99 ) o 0a ÈB l-.r 28-Jul99 CD Þ- 27-Julæ (D 30-Jul-99 (D ÊD 29-Julw U) iJ 1-Auq-99 ;) CA Ê. 31-Jul-99 ÊD 3-Aug-99 H U) 5-Aug-99 0q 2-Aug-99 o- (A H o 7-Aug-99 Þ. 4-Aug-99 0a Ð U) 9-Aug-99 o CD 6-Aug:99 Ê- 1 1-Aug-99 EÚ CD o 8-Auq-99 U) 13-Aug-99 (h ÊD 15-Aug-99 1t-Aug-99 (tsD N) Ch ÊD rtii [J N..oñÕÕï1?T*' FSSSEãooooÀi d è A Kruskal-v/allis ANovA on ranks was used to compare the dissolved oxygen between treatments because data were not normal and had unequal variance' No statistical differences were founcl between treatments for tempcrature (F:0.608, P:0.695), or dissolved oxygen (H:7.394, P:0.193). Highly significant differences were detected fol pH (F:6.763, P:0.001 - z))yrr,l00yor'2, 2, 50o/o2,25o Control2, Cava#) and conductivity (F:326.115, P<0'0OI - 25%r, Controll, 5oYor'2, cavatf , looo/o3 ,2oo%\. The large differences in pH and conductivity between treatments are almost certainly due to the differences in the levels of heavy metals added to the sediment. 8.3 Discuss¡on V/ilbur and Collins (1973) suggest that there are two main factors determining the normal timing of metamorphosis in amphibians: 1. There is a minimum size at which amphibians are able to metamorphose. This limit is probably imposed through evolution; inhibiting metamorphosis reduces the low survival rate for small individuals. 2. Growth rate is responsible for the timing of metamorphosis. A tadpole with an extremely rapid growth rate may delay metamorphosis so that the maximum size and therefore maximum energy reserves may be reached before entering the terrestrial habitat. A slow growth ra1e may have the opposite effect and initiate metamorphosis at a smaller body size. The risk of metamorphosis at a small body size may by offbet by risks such as predation and desiccation if tadpoles remain in the water. There has been scepticism regarding the validity of these theories, which had not been tested experimentally (Dodd and Dodd 1976, Just et al I98l). Just ¿/ al (198I) comment that there is no evidence that growth or growth rate have any impact on the onset of metamorphosis and that the state of differentiation is more important. This may be mere semantics because state of differentiation is dependent on growth and metamorphosis is a very complex process involving biochemical, behavioural, physiological and structural modification. 105 Early work by Gudernatsch in l9l2 (cited in Dodd and Dodd l9'76,Fox 1983) demonstrated the effects of thyroid tissue on tadpoles. Since that time considerable attention has been drawn to endocrine involvement in metamorphosis. It is now commonly accepted that the primary process of metamorphosis is controlled through hormones, particularly triiodothyronine converted from thyroxine (Buscaglia et al 1985), produced by the tþroid gland. The production of these hormones are regulated via the hypothalamus, the hypophysis and the anterior lobes of the pituitary, which are responsible for producing thyroid stimulating hormone (Dodd and Dodd 1976, Just et al I98I, Fox 1983, Rosenkilde 1985). Growth hormones, possibly prolactin or somatotropin (Dodd and Dodd 1976, Fox 1983), and tþroxine (under the control of the pituitary and hypothalamus) are produced throughout the life of the larvae (Rosenkilde 1985). Larval tissues are able to respond to the thyroid hormones at any time during development but the growth promoting hormones may have an inhibitory effect on the tþroid hormones, thereby regulating the onset and progress of metamorphosis (Fox 1983). Interactions between tþroid hormones and growth hormones are not fully understood and appear to vary between species (Fox 1983, Rosenkilde 1985). A highly simplified model may be as follows: o Growth hormones cause growth of cells. Thyroxine is responsible for differentiation. Thyroxine is also converted to triiodothyronine but this process may be inhibited by the action of the growth hormones. o At a particular stage in development differentiation reduces the binding capacity of receptors for growth hormones; the antagonistic effect is reduced thereby increasing the action of triiodothyronine. c Increasing levels of triiodotþronine then enable metamorphosis. It appears that the elevated metal levels had a physiological effect; the result of which was the delay in the onset of metamorphosis. Crinia signifera follow the Wilbur and Collins (1973) model tha: a slow growth rate may delay metamorphosis (as in the 200% treatment) until body size falls within a specific range. Metamorphosis will then occur as quickly as possible, even if further food resources are available. Most of the Litoria ewingi also underwent metamorphosis after a short tadpole stage, within about 80 days, however tadpoles in the 200% treatment took longer and grew larger than 106 tadpoles in the other treatments, despite reaching a snout vent length at which metamorphosis should have occuned. Grigg et al. (1995) suggest that a normal SVL for L' ewingi at metamorphosis is approximately 11 - 14 mm. Cree (1984) reports that L. ewingi tadpoles introduced into New Zealand commonly reach metamorphosis after a period of 55 - 73 days at an SVL of 13 - 15 mm when reared at temperatures between 15oC and23"C. Tadpoles reared at temperatures above 25oC complete development in five weeks. It is possible that the elevated metal levels, particularly lead nitrate, inhibited the normal endocrine functions preventing the onset of metamorphosis and causing the increase in body size. Chaurasia and Kar (1999) have shown that treatment with lead nitrate results in the inhibition of thyroidal conversion of thyroxine to triiodothyronine in the catfish, Heteropneustes fossilis. Triiodothyronine has a greater control in the onset of metamorphosis than thyroxine (Buscaglia et at 1985) and this would explain the increased SVL of Litoria ewingi tadpoles in this experiment. It follows that Crinia signifera, for which size at metamorphosis was not altered, are more tolerant of lead pollution than Litoria ewingi. This result perhaps indicates that multiple species should not be grouped together, when looking for patterns relating to pollution between sites, because species can respond differently. This is supported from the studies of Cooke (1972), Bidwell and Gorrie (1995) and Hecnar (1995) who showed species differences in tolerance levels of frogs exposed to various pollutants. However, it does support the idea that different species can be used as bio-indicators of pollutants at a site. If a wide range of tests can be carried out on different species to determine the effects of known pollutants these effects can be used in situ to identifu the likely cause of environmental pollution. As a result of the small sample size, and in particular the difficulties arising from the inclusion of a second species in the initial sample, it was not possible to detect any statistically significant difference in the incidence of abnormality between the different treatment tanks for either species. However the data suggest that there is biological evidence to support the notion that moderately high levels of heavy metals produce skeletal abnormalities in developing frog embryos; nine out of the eleven abnormal frogs reaching metamorphosis in this experiment were from the200%o treatment. Although there was a number of abnormal frogs produced as a result of exposure to heavy metals the incidence of abnormality in the Cavan tanks was far below that encountered in the field. It seems likely then that there are multiple factors responsible for the high prevalence of t07 abnormal frogs at Cavan. Other factors may include electromagnetic radiation from the high- voltage power cables or various other pollutants entering through the stormwater drains. Shortly after the investigation into the possible role of heavy metals in this system had commenced major modifications were undertaken at the site. These modifications include a restructuring of the banks and reed beds such that the pond has now been split into three ponds, with dirt roads on the mounds separating the ponds (figure 154). Since these 'road works' the site has been visited three times but only one frog has been collected and no frogs have been heard calling. It remains to be seen how many frogs are still present in the area and whether the incidence of abnormality continues to be high. Figure 154. The survey site at Cavan after the modifications (facing (a) south (wide angle with slight distortion), (b) northeast, (c) northwest). ii, , :j. (b) (c) 108 9 Goncluslons 9"1 Incidence of AbnormalitY can still be This study has shown that large numbers of abnormal frogs, from three species, there was found in the Mt Lofty Ranges and metropolitan Adelaide. Brooks (1979) stated that a significant increase in the incidence of abnormality in areas of high chemical use (particularly the use of agricultural pesticides). However a reanalysis of her data using more only stringent criteria was unable to support this claim. Nevertheless the present study' despite five locations having an incidence of abnormality above acceptable limits, did detect a significant difference in incidence of abnormality between locations in areas of different land use use. The lowest frequency of abnormal frogs was found in the scrub sites, where chemical is expected to be minimal. A eomprehensive oatalogue of all deformities encountered in the Mt Lofty Ranges, and other regions, has been compiled. The majority of abnormalities are only minor reductions in the digits but a number of more severe forms include reorientation, displacement, combination, replication or omission of entire digits and limbs. A number of abnormalities of the soft tissues were also deteeted. Myriad deformed frogs have recently been recorded in populations inhabiting agricultural areas in North America. As yet there has been no widely accepted explanation for the cause of these abnormalities but general opinion seems to be that the activities of man, with particular reference to chemical use, may be the main culprit. Similarly, the high frequency of abnormality in frogs collected from Cavan during this study suggests an anthropogenic nature. Extremely high levels of heavy metals were found in sediment collected from a number of spots within the study site. The levels of oadmium, copper, lead and zinc were usually above those recommended by the National Water Quality Management Strategy (1999) guidelines and when exposed to these sediments under laboratory conditions there was a significant decrease in survival to metamorphosis of Crinia signifera tadpoles. There also appeared to be an effect on growth. Further testing with spiked sediments demonstrated that high levels of these heavy metals could significantly impair the normal processes of growth and development. I-arge numbers of abnormal frogs were produced in the treatment tanks with the highest levels of metals added. 109 may help to explain the marked species specific differences in the susceptibility to the metals experiments (using C' difference in the incidence of abnormality between the laboratory signifera and Limnodynastes signifera and Litoria ewingi) and the field survey (in which c" in C' signiferd was tasmaniensis were collected). However as the incidence of abnormality is likely that other factors much lower in the experiment than that detected in the survey it also play an imPortant role. 9.2 Parasites and CYsts Following reports by Sessions and Ruth (1990), that trematode cysts may alter the injured and abnormal development of, limb buds and produce skeletal abnormalities, all of the for evidence of frogs collected in the Mt Lofty Ranges during the field survey wore examined cyst infestation. one or Only 17 frogs had any evidence of cysts (see appendix 5)" These were generally only two cysts in the region of the head and shoulder girdle, not in the oloacal / hind limb-base that had region as reported by Sessions and Ruth. There were also a slight number of frogs massive infestation throughout the body. A small number of frogs collected from Oakbank also had woltns present. Fifteen of the l7 frogs with cysts were frogs with injuries; only two abnormal frogs had cysts, one of these (CS10) had only a slight infestation in the head region and also had an injury" The other abnormal frog (LE01) also had a small number of cysts in the head and shoulder (see 83) area. Sessions & Ruth may predict that the additional limbs in this individual figure may have been the result of mechanical disruption by the cysts on regulatory processes of developing limbs but severe infestation in the same region of other frogs did not produce abnormalities. There is no conclusive evidence to suggest that the eysts found in this str-rdy ean produee abnormalities in frogs. 9"3 Injuries The incidence of injury encountered during the Mt Lofty Ranges field survey was greater than in has been commonly published, however Brooks (1979) also reported a high rate of injuries No the same region. A high incidence of injury was also encountered in the Flinders Ranges. difference was detected between land uses or between the different geographical areas and this suggests that, despite the lack of prior reports, a high incidence of injury in not an uncommon occuffence. 110 appearance to Since the level of injury can be very high, and many injuries have a similar include a skeletal abnormalities, it is important that studies citing high rates of abnormality careful internal examination of all deformities. The use of double staining procedures, such as of those used in this study, provide an effective means of determining the aetiology deformities 9.4 Ft¡ture D¡rections A number of problems \ /ere encountered while attempting to run the sediment bioassays. Unsuitable sediment, a combination of species and unexpected mortality all had detrimental effects on the study. Future experimentation would help provide more insights into the growth and development of amphibians living in polluted habitats. Additional field surveys integrating a wider range of land use types and incorporating more industrial and intensive agricultural sites would be of great benefit. Although somewhat expensive it would be worthwhile to undertake a programme of water and sediment testing to correlate the presence of aotual pollutants with the incidence of abnormality. The state of the site at Cavan is still of concern and further investigation may help to explain the extreure number of abnormal frogs that have been collected. Abnormal frogs that were kept in the laboratory attempted to breed on a number of occasions without success. It is unknown if the inability of abnormal frogs to reproduce is a common ocourrenoe and it would be interesting to investigate this further" The heritability of deformity would also be worthy of future study. 111 AppendiX I - Metamorphosis of frogs from Impromptu Cavan spawning. Date of Spawn Date of Metamorphosis # Normal # Abnormal Days to Metamorphosis 16/07/96 3 0 135 16t07196 29t11196 1 0 136 16tO7196 o't12196 3 0 142 16tA7196 09t12t96 I 0 146 1 3/08/96 09t12196 1 0 118 16tO7196 13t12t96 2 0 150 16t07t96 16t',t2196 1 0 153 1 3/08/96 16t12196 I 0 125 16107196 18t12t96 1 0 155 16t07t96 19t12196 1 0 156 06/09/96 "t9t12196 1 0 104 16t07t96 22t12t96 2 0 159 1 3/08/96 22t12196 1 0 131 16107196 27t12t96 2 0 164 06/09/96 29t12t96 1 0 114 28/09/96 01t01t97 I 0 95 16tO7t96 o2to1t97 2 0 170 16107196 06to1197 2 0 174 16tO7196 07to1t97 1 0 175 28l09/96 08101197 4 0 102 16t07t96 o9t01t97 4 0 177 06/09/96 o9to1t97 2 0 125 28t09t96 09to1197 2 0 103 16t07t96 10101197 1 0 178 1 3/08/96 10t01t97 1 0 150 06/09/96 11t01197 1 0 127 16t07196 13t01197 1 0 181 28109/96 13t01t97 1 0 107 16t07t96 14tO1t97 6(K) 0 182 1 3/08/96 14101197 1(A) 0 154 06/09/96 14t01t97 1 0 130 16t07t96 16tO1t97 3 0 184 13/08/96 16t01t97 1(A) 0 156 28l09/96 16t01197 1 0 110 16t07196 18101197 3 0 186 13/08/96 18lO1lgV 1(A) 0 "158 28/09/96 18t01t97 4 0 112 16t07t96 20t0'U97 5 0 188 06/09/96 20101197 2 0 136 28l09/96 20t01197 3 0 114 16t07t96 22t01t97 1 0 190 06/09/96 22t01ts7 2 0 138 1 3/08/96 23t01t97 1(A) 0 163 16t07196 24t01t97 4 0 192 06/09/96 24tO1197 1 0 140 112 orphos¡s '1 118 28/09/96 24t01197 0 195 16107196 27t01197 2 0 28/09/96 27101197 I 0 121 28t09t96 27t01197 2 0 121 196 16107196 28t01t97 1 0 197 16t07196 29t01197 3 0 145 06/09/96 29t01197 4 0 123 28l09/96 29101197 1 0 '199 16107196 31101t97 5 0 147 06/09/96 311O1197 2 0 28t09196 31t01197 4 0 125 16t07196 02t02l97 4 0 201 06/09/96 02t02197 2 0 149 202 16t07196 03t02197 1 0 150 06/09/96 03102197 1 0 28tO9196 03to2l97 4 0 128 16t07t96 05102197 2 0 204 06/09/96 05t02t97 2 0 152 28t09t96 05102197 5 0 130 16/07/96 07lo2l97 6 0 206 06/09/96 07t02197 3 0 154 28/09/96 a7lo2l97 2 0 132 16t07196 08t02t97 2 0 207 28t]g196 08t02l97 1 0 133 16t07196 10t02197 5 0 209 28t09t96 10to2197 6 0 135 16tO7t96 12t12t97 2 0 211 28t09196 12t02t97 1 0 137 16107196 13102197 1 0 212 16t07196 14tO2t97 3 0 213 28l09/96 14t02197 2 0 139 16/07/96 17t42t97 1 216 28/09/96 17tO2197 4 0 142 16/07/96 19t02t97 1 0 218 28/09/9ô 19tÙ2t97 5 0 144 16107196 21t02t97 2 0 220 16t07196 23t02t97 1 0 222 06/09/96 24tÙ2t97 1 0 171 28/09/96 24t02t97 4 0 149 2Bl09/96 26t02197 3 0 151 28/09/96 27tO2197 1 0 152 06/09/96 28t02t97 1 0 175 06/09/96 01t03197 I 0 176 28/09/96 01t03t97 I 0 154 28l09/96 03/03/97 3 1 156 16t07t96 10t03197 1 0 237 28l09/96 10lo3ls7 2 0 163 113 187 06/09/96 12103197 0 165 28/09/96 12tO3197 2 0 189 06/09/96 14tO3197 1 0 167 28/09/96 't4t03t97 1 0 191 06/09/96 16103197 1 0 169 28t09196 16103197 2 0 193 06/09/96 't8to3l97 1 0 173 28t09t96 20tos197 4 0 198 06/09/96 23103197 1 0 176 28109/96 23103197 1 0 199 06/09/96 24t03197 1 0 200 06/09/96 25t03197 1 0 204 06/09/96 29103197 2 0 182 28t09t96 29lo3l97 1 0 208 06/09/96 02to4l97 1 0 186 28/09/96 o2l04l97 1 0 06/09/96 08104197 2 0 214 28/09/96 08to4l97 4 0 192 06/09/96 11104197 1 0 217 28109/96 11104197 5 0 195 06/09/96 14t04t97 3 0 220 28109196 'l4t04l97 4 0 198 (K) - Indicates a frog with 'kinky tail'. (A) 'Indicates an albino frog 1r4 Freshwater (USEPA 1989)' AppendiX 2 -Preparation of Moderately Hard Synthetic 1" Place 19 Litres of deionised water in a clean container' 2. Add l.2g MgSOa, l.g2gNaHCO¡ and 0.089 KCI 3. Aerate overnight 4. Add l.2gCaSO¿.2HzO to lL deionised water in a separate flask. Stir until well dissolved and then add to the 19I- and mix well. 5. Vigorously aerate the solution for a fuither 24Hrs. 115 Appendix 3 - Sediment Bioassay 1 metamorphs. Date Tank Treatrnent # Normal # Abnormal Comments Days to Metamorphosis 42 26tO711998 I 1 27t07t1998 1 Drain'! 2 43 43 29t07t1998 1 Drain 1 4 29t0711998 6 Drain 3 2 43 29tO7t1998 4 West 1 43 30/07/1 998 1 Drain 1 1 46 2t08t1998 3 Artificial 4 1 S8101 - head abnormality 49 2t08t1998 1 Drain 1 2 49 3/08/1998 12 Artificial 2 50 3/08/1998 3 Artifieial 5 50 3/08/1 998 11 Drain 1 5 50 3/08/1 998 16 Drain 3 I 50 3/08/1 998 I Drain 3 1 50 3/08/1 998 20 Drain 3 2 50 3/08/1 998 þ Drain 3 8 50 3/08/1998 17 South 4 50 3/08/1998 10 South 2 50 3/08/1 998 14 West 1 S8102 - left hand & foot ectrodactyly 50 6/08/1 998 12 Artificial 3 1 S8103 - head abnormality 53 6/08/1998 '18 Artifìcial 2 53 6/08/1 998 15 Drain 1 2 53 6/08/1998 11 Drain 1 1 53 6/08/1998 21 Drain'f 1 53 6/08/1 998 20 Drain 3 4 53 6/08/1 998 17 South 1 53 6/0811 998 I West I SB104 - left foot abnormality 53 6/08/1 998 19 \Â/est 2 53 8/08/1 998 12 1 55 8/08/1 998 24 1 55 8/08/1998 18 3 55 ÃÃ 8/0811 998 11 Drain 1 I tt 8/08/1 998 15 Drain 1 2 8/08/1 998 16 Drain 3 1 55 8/08/1 998 I Drain 3 I 55 ÃÂ 8/08/1998 20 Drain 3 1 8/08/1998 19 \lüest 4 ÃÃ x 1/08/1 998 12 Artifieial 2 58 1 1/08/1 998 18 Artifìcial 2 58 1 1/08/'t 998 21 Drain 1 5 58 1 1/08/1 998 15 Drain 1 1 5B 1 4/08/1 998 12 Artificial 1 61 x 4/08/1 998 24 Artificial 1 61 1 4/08/1 998 18 Artificial 1 6'1 1 4/08/1 998 11 Drain 1 1 61 1 4/08/1 998 15 Drain't I 61 14t08t1998 19 West 1 61 1 7/08/1 998 18 I 1 64 22t08t1998 15 Drain'1 1 69 2t09t1998 15 Drain ''l 2 80 116 Appendix 4- Sediment Bioassay 2 metamorphs Date Species Tank Treatment Abnormal SVL Comments Days to Metamorphosis 999 CS 4 n 40 22t08t1999 CS 12 Cavan n 8 40 22t08t1999 CS 3 Control n I 40 22t0811999 CS a Control n I 40 22t08t1999 CS 3 Control n I 40 22tO811999 CS 7 Controi n I 40 22ß8t1999 CS 3 Control n 8 40 23108/1999 CS 3 Control n 7 41 23108/1 999 CS 3 Control n I 41 23t08t1999 CS 3 Control n 8 41 24t0811999 CS 18 lOOo/o n 8 42 24t0811999 CS 13 100% n 8 42 24tO8t1999 CS 13 100% n I 42 24t08t1999 CS '13 lOOo/o v 7 digit I ectrodactyly RH 42 24tAU1999 CS 18 108o/o n I 42 24108/1999 CS '18 100% n I 42 24t0811999 rae 13 100o/o n 7 42 24t0gt1999 CS 18 1O0o/o n 7 42 24t08t1999 CS 13 100o/o v I digit I ectrodactyly on RH 42 24t0811999 CS 18 100% n I 42 24tO8t1999 CS 6 25% n 7 42 24tO311999 cs 17 25o/o n I 42 24tO8,t1999 CS 6 25% n 7 42 24108/1 999 CS 20 25% n 7 42 24t0g11999 CS 20 25% n 7 42 24t0gt1599 CS 20 25% n 7 42 2410811999 AQ 11 25% n 8 42 24108/1 999 /^e 11 25% n I 42 24t08t1999 CS 19 50% n I 42 24t08t1999 ES 15 50% n 8 42 24tO811999 ES 15 50o/o n I 42 24tOgt1999 CS '15 50o/o n I 42 24tO3t1999 Uù 4 50% n 7 42 24t08t1999 CS 4 50% n 7 42 24t08t1999 CS 15 50% n 8 42 24t08t1999 cs 2 Cavan n I 42 24t08t1999 CS 12 Cavan n 7 42 24t0g11999 CS 12 Cavan n 7 42 24t08t1599 CS 12 Cavan n 8 42 24t08t1999 (/Þ 12 Cavan n 7 42 24t08t1999 CS 12 Cavan n I 42 2410811999 CS 16 Control n I 42 24tO811999 CS 16 Control n I 42 24t08t1999 CS 16 Control n I 42 117 Date Species Tank Treatment Abnormal SVL Comments Days to Metamorphosis 24108/1999 cs 16 Control n I 42 24t08t1999 ES 16 Control n I 42 24t08t1999 CS 16 Control n I 42 26108/1999 CS 13 1O0o/o n 8 44 26t0811999 CS 18 100% n I 44 26t08t1999 CS 18 100% n 8 44 26/08/1999 CS 13 100o/a n 8 44 26/08/1 999 CS 11 25% n 8 44 26/08/1 999 CS 20 25% n 7 44 26108/1999 CS 20 25o/o n I 44 26108/1 999 r.e 19 50% n I 44 26108/1 999 CS 19 50% n I 44 26108/1999 CS 15 50% n 8 44 26/08/1 999 CS 19 50% n 8 44 26/08/1999 cs 4 50% n 7 44 26108/1999 CS 4 50o/o n 7 44 26/08/'1999 CS 19 50% n I 44 26108/1 999 CS 12 Cavan n 8 44 26108/1999 CS 7 Control n 7 44 26/08/1 999 CS 7 Control n I 44 26tO8t1999 CS 7 Control n 8 44 2/09/1 999 CS 13 100% n I 51 2/09/1 999 CS 13 100% n 8 51 2/09/1 999 CS 6 25o/o n I 51 2/09/1 999 CS o 25% n 6 51 2to9t1999 CS 6 25o/o n 7 51 2t09t1999 CS 4 50o/o n 7 injured digit lV on RF 51 2/09/'1999 CS 4 50% n 7 51 2to9t1999 CS 4 5jo/o n 7 5'l 2/09/1 999 CS 2 Cavan n 8 5'1 2/09/1999 CS 2 Cavan n 7 51 2/09/1 999 CS 2 Cavan n I 51 2/09/1 999 CS 7 Control n 8 51 3/09/1999 (.Þ 18 100% n 7 52 3/09/1 999 CS 22 200% n 6 52 3/09/1 999 CS 11 25o/o n 7 52 3/09/1 999 CS 11 25% n 7 52 3/09/1 999 CS 20 25o/o n 7 52 3/09/1 999 CS 17 25o/o n 7 injured toes 52 3/09/1 999 CS 19 50% n I 52 3/09/1 999 CS 15 50o/o n 7 52 3/09/1 999 CS 1ô Control n I 52 8/09/1 999 LE 9 Control n 14 57 8/09/1 999 LE I Control n 14 57 1 0/09/1 999 CS 17 25o/o n 8 59 1 0/09/1 999 CS 6 25o/o n 6 59 1i8 Date Species Tank TreatmeRt Abnormal SVL Gomments Days to Metamorphosis 1 0/09/1 999 15 n 6 59 '10/0911 999 LE 1 100% n 14 59 I 0/09/1 999 LE I Cavan n 13 59 14109/1999 CS 5 200% v I abnormal limbs 63 1 4/09/1 999 CS 17 25o/o n I 63 1 4/09/1 999 CS 4 50% n I 63 1 4/09/1 999 CS 21 Cavan n 7 63 1 4/09/1 999 LE 23 50o/o n 13 63 1 4/09/1 999 LE I Cavan n 12 63 14109/1999 LE I Control n 13 63 1 6/09/1 999 CS 1 lQQo/o n I 65 1 6/09/1 999 CS 14 200% v 7 abnormal limbs 65 1 6/09/1 999 LE 13 100% n 16 65 1 6/09/1 999 LE I Cavan n 13 65 20109/1 999 CS 17 25% n I 69 20109/1 999 Uù 19 50% n 7 69 20/09/1 999 LE I Cavan n 14 69 25109/1 999 CS 14 200% n 7 74 25109/1 999 CS 14 200% v 7 abnormal limbs and body 74 25109/1 999 t_E 24 100% n 14 74 25109/1999 LE 24 100% n 14 74 25109/1 999 LE 23 5Ao/o n 15 74 27t0gt1999 LE 1 100o/o n 14 76 27t09t1999 LÊ 1 100% n 14 76 2710911999 LE 23 50% n 14 76 27t09t1999 LE 21 Cavan n 14 76 28/09/1999 l_E 24 100o/o n 15 77 30/09/1999 LE 24 100% n x5 79 5/1 0/1 999 LE 1 100% n 16 84 5/1 0/1 999 LE 24 100% n 16 84 6/1 0/1 999 CS 17 25% n 7 85 8/'! 0/1999 LE 24 100o/o n 15 87 8/1 0/1 999 LE 23 50% n 16 87 B/10/'1999 LE I Cavan n 17 87 9/1 0/1 999 t_E I 100% n 17 88 '1 1/1 0/1 999 ^e 5 200% n I 90 11t10t1999 LE 1 100% n 17 90 1 5/1 0/1 999 CS 5 200% v 6 legs abnormal at joints 94 20110t1999 CS 5 200% n 10 99 20t10t1999 LE 22 200% n 16 99 26t10t1999 LE 22 200o/o n 16 105 26110t1999 LE 22 200o/o v 17 abnormal toes 105 28t10t1999 CS 5 2O0o/o n o 107 28t10t't999 LE 1 100% n 13 107 3/1 1 /1 999 CS 14 200% v 10 abnormal hind limbs 113 1 5/1 1 /1 999 LE 10 200% n 15 125 119 $grellrg-npnssr.5 19/1 1/1999 LE 22 200% v 16 abnormalhind limbs 129 23t11t1999 LE l0 200o/o n 18 133 26t11t1995 CS '14 200o/o n 11 136 26t11t1999 LE 22 200% n 17 136 30/1 1/1999 cs 14 200% v I abnormal hands and feet 140 4t12t1999 LE 10 2OOo/o n 17 144 4t12t',t999 LE 22 200% n 17 144 7t12t1999 LE 10 200% v 17 abnormal left-hand 147 (CS) - Crinia signifera (LE) - Litoria ewingi r20 AppendiX 5 - Presence of cysts in deformed frogs collected during the field survey. Frog cs07 lnjury small number of cysts throughout the body cs10 normality / lnjury slight numbers of cysts in the head and 'neck' area I lnjury small number of cysts in the head and shoulders cs49 lnjury small number of cysts in the head and shoulders cs50 lnjury few cysts in the shoulders and back cs51 lnjury large infestation in the shoulders and back cs52 lnjury a large infestation in the head, shoulders and back cs55 lnjury a single cyst in the mandible LT02 lnjury ct small number of cysts in the shoulder and 'neck' area LTO5 lnjury a small number of cysts in the head and upper body area and also in the legs LTO6 lnjury a slight infestation in the head and body LT19 lnjury small number of cysts in the head and shoulders LT2A lnjury few cysts in the jaw area LEOl ity small number of cysts in the head, shoulders and 'neck' area LEO3 lnjury small number of cysts in the head and shoulders LE04 lnjury a large number throughout the head and back LEOs lnjury a few cysts in the shoulders 121 References Abbasi, S. A" & Soni, R. (1984) Teratogenic Effects of Chromium (VI) in Environment as Evidenced by the Impact on Larvae of Amphibian Rana tigrina: Implications in the Etrvirorunental Management of Chromium. Intern J. Environmental Studies 23,131-137. Alvarez, R., Honrubia, M. P. & Herraez,M.P. (1995) Skeletal Malformations Induced by the Insecticides ZZ-Aphox and Folidol During Larval Development of Rana perezi. Arch. Environ. Contam. Toxicol. 28, 349-356. Berger, L., Speare, R., Daszak, P., Green, D. E., Cunninghaffi, A. 4., Goggin, C.L., Slocombe, R.., Ragan, M. 4., Hyatt,4.D., McDonald, K.R., Hines, H. 8., Lips, K. R., Marantelli, G. & Parkes, H. (1998) Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America" Proc. Nqtl. Acad" Sci. US.A 95,9031-9036. Bidwell, J' R. & Gorrie, J. R. (1995) Acute Toxicity of a Herbicide to Selected Frog Species. l[/estern Austrqlian Department of Environmental Protection Technical Series 79, l-9. Bidwell, J. R., Gorrie, J. R., Smith, B. P. C. & venus, T. (1997) "Biological Testing to Characterise Sediment Quality of the Barker Inlet V/etlands. Final Report". (Environment Research Group, University of South Australia, Adelaide) Bishop, D. V/. & Hamilton, R. (1947) Polydactyly and Limb Duplication Occurring Naturally in the Tiger Salamander, Ambystoma tigrinum. Science 106, 641-642. Blaustein, A. R. (1994) Amphibians in a Bad Light. What is killing the eggs of Oregon's western toad? NaL Í{ist. 1,Aß4,32-38. Boyer, R. and Grue, C. E. (1995) The Need for'Water Quality Criteria for Frogs. Environ Health Ferspect 103(4), 352-357. Brooks, J. A. (1979) "skeletal Abnormalities of South Australian Frogs. Honours Thesis,' (Department of Zoology, University of Adelaide). 122 Buscaglia, M., Leloup, J. & De Luze, A. (1985) The role and regulation of monodeiodination of thyroxine to 3,5,3'-triiodethyronine during amphibian metamorphosis. pp 273-293. In Balls, M. & Bownes, M. (Eds) "Metamorphosis". (Oxford University Press, Oxford). Carey, C. & Bryant, C. J. (1995) Possible Interrelations among Environmental Toxicants, Amphibian Development, and Decline of Amphibian Populations. Environ Health Perspect XO3(Suppl 4),13-17. Chaurasia, S. S. & Kar, A. (1999) An Oxidative Mechanism for the Inhibition of Iodotþrenine S'-monodeiodinase Activity by Lead Nitrate in the Fish, Heteropneustes fossilis. Water,A,ir Soil Pollut. 111, 417 -423 " Colbert, E. H. (1969) "Evolution of the Vertebrates: A history of the Backboned Animals Through Time. 2nd Edition". (John Wiley & Sons Inc., Sydney). Connell, D" W" (1974) "Water Pollution: Causes and Effects in Australia. lst Edition" ([Jniversity of Queensland Press, St Lueia, Queensland). cooke, A. s. (1972) The Effects of DDT, Dieldrin, and2,4-D on Amphibian Spawn and Tadpoles. Enviran. Pollut. 3, 5 tr-68. Cooke, A. S. (1981) Tadpoles as Indicators of Harmful Levels of Pollution in the Field. Environ" PolluL, Ser A. 25, 123-133. Cree, A. (1984) Breeding biology, repiration, and larval development of two introduced frogs (Litoria raniformis and L. ewingi). N.Z. J. Zool. '/..1,179-188. Davies, M. (1993) Collection And Preservation Of The Anura. pp 13-14 Íe Glasby,C. J., Ross,G. J" B. & Beesley, P. L. (Eds) "Fauna of Australia. volume 2,{. Amphibia And R.eptilia". (Australian Government Publishing Serviee, Canberra). Delvinquier, B. L. J. (1986) Myxidium immersum (Protozoa : Myxosporea) of the Cane Toad, Bufo marinus,inAustralian Anura, with a Synopsis of the Genus in Amphibians" Aust. J. Zool" 34,843-853. Dodd, K" (1998) NIEHS Sponsors V/orkshop on Amphibian Deformities. Froglog26,2. 123 Dodd, M. FI. L & Dodd, J.M. (1976) The biology of metamorphosis. pp 467-5991¡z Lofts, B (Ed) "Physiology of the Amphibia. Vol. III". (Academic press, New york). Environment Protection Authority (1993) State Of The Environment Reportþr South Ausrralia I998. Ferraro, T. J. & Burgin, S. (1993) Review of environmental factors influencing the decline of Australian frogs. pp 205-218 1n Lumney,D. &- Ayers, D. (Eds) "Herpetology in Australia". (Royal Zool. Soc" ofNSV/, Mosman, NSW). Gardiner, D" M. & Hoppe, D. M. (1999) Environmentally Induced Limb Malformations in N4ink Frogs (Røna septentrionalis). J. Exp. Zool. ZB4,207-216. Gilbert, M. (1998) Frogs In A Fix: Frog Malformities in Vermont (The National Wildlife Federation, Northeast Natural Resource Center, Vermont, USA.). Greenhouse, G. (1976) The Evaluation of Toxic Effects of Chemicals in Fresh V/ater by Using Frog Embryos and Larvae. Environ. pollut.ll, 303-315. Guillette jr, L. J. (1994) Endocrine-disrupting environmental contaminants and reproduction: lessons from the study of wildlife. pp 201-207In popkin, D. R. & peddle, L. J. (Eds) "Vy'omen's Health Today: Perspectives on Current Research and Clinioal practice". (Farthenon Publ" Group, New York). Hall, M. c. (1965) Bone - Repair. pp250-2Bl In"Thelocomotor system: Functional Histology". (Charles C Thomas Publisher, Springfield, Illinois). F{anken, ^I' & V/assersug, R. (19S1) The Visible Skeleton. Funct. Photog. July/Augu st"22- 44 Hebard, W' B' & Brunson, R. B. (1963) Hind Limb Abnormalities of a Western Montana Population of the Pacific Tree Frog, Hyla regilla Baird and Girard. Copeia 1963(3), 570-572 Hecnar, S. J. (1995) Acute and Chronic Toxicity of Ammonium Nitrate Fertilizer to Amphibians From southern ontario. Envir on. Toxic ol. c hem. l4(2), 2r3 1 -2137 . \24 Holmes, S. J. (1927) "The Biology of the Frog. 4th Edition" (The Macmillan Company, New York). Just, J..I., Kraus-Just, J. & Check, D. A. (1981) Survey of Chordate Metamorphosis. pp265- 326In Gilbert, L.L &. Frieden, E. (Eds) "Metamorphosis, A Problem in Developmental Biology. 2nd Edition" (Plenum Press, New York). Martinez,L, Alvarcz, R., Herraez,L &,Herraez,P. (1992) Skeletal Malformation in Hatchery Reared Rana perezi Tadpoles. The Anatomical Record233,314-320. McDonald, K. (1999) Declining amphibians - a global perspective.I|/itdtife Australia 36(2), 22" MPCA (1998) Minnesota Pollution Control Agency. Background Information about Deþrmed Frogs in Minnesota. [Online, accessed 28May l99S]. URn, :http ://www.pca. state. mn. us/hot/frog-bg.htmt. Narcam (1997) North American Reporting Centerfor Amphibian Malfurmations. Homepage of the Northern Prairie Science center. [online, accessed 7th August lggT]. URL :http :i/www.npsc.nbs. gov/narcam/backgrnd/backgrnd. html. National Registration Authority for Agricultural ancl Veterninary Chemicals (1996) Special Review af Glyphosate. Community Brief. National Registration Authority for Agricultural and Veterninary Chemicals (1996) Chemical Review Finds In Favour Of Frogs. Media Release. National V/ater Quality Management Strategy (1999) Australian and New Zealand Guidelines þr Fresh ønd Marine Water Quality. Volume L The Guidelines" (Fublic Comment Draft July rege)" Osborne, W. S. (1989) Distribution, Relative Abundance and Conservation Status of Corroboree Frogs, Pseudophryne corroborree Moore (Anura:Myobatrachidae). Aust. Witdl Res.16,537-547. 125 Ouellet, M., Bonin, J., Rodrigue, J., DesGranges, J. &Lair, S. (1997) Hindlimb deformities (ectromelia, ectrodactyly) in free-living anurans from agricultural habitats. J. Wildt. Dis. 33, 95-104. Read, J.L" &, Tyler, M. J. (1994) Natural Levels of Abnormalities in the Trilling Frog (Neobatrachus centrah's) at the Olympic Dam Mine. Bull. Environ. Contam. Toxicol.53,25- 31" Reaser, J. K. & Johnson, P. T. (1997) Amphibian Abnormalities: a Review. Froglog 24, l Reichenbach-Klinke, H. & Elkan, E. (1965) "The Principal Diseases Of Lower Vertebrates Book II Diseases Of Amphibians. 1st Edition" (T.F.H. Publications, Inc. Ltd, Hong Kong). Richards, S. J., N4cDonald, K. R. & Alford, R. A. (1993) Declines in populations of Australia's endemic tropical rainforest frogs. Pacific Conservation Biologt 1,66-77, R.osenkilde, P. (1985) The lole of hormones in the regulation of amphibian metamorphosis pp 22I-2591n Balls, M. & Bownes, M. (Eds) "Metamorphosis".(Oxford University press, Oxford)" Rowe, c. L., Kinney, o. M., Fiori, A. p" & congdon, J" D. (1996) oral Deformities in Tadpoles (Rana catesbeiana) associated with coal ash deposition: effects on grazing ability and growth . Freshw. Biol. 36, 723-7 30. R.ussell, R.. v/. & Hecnar, s. J. (1996) The Ghost of Festicides past? Froglog 19,l Sessions, S. K. & Ruth, S. B" (1990) Explanation forNaturally Occurring Supernumerary Limbs in Amphibians. I Expt. Zool. ZS4,38-47. Sinclair, D' (1978) Growth And Repair. pp 161-185 ln "Human Growth After Birth" 3rd Edition" (Oxford Medical Publications, London) 'W. Snedecor, G. W. & Cochran, G. (1989) "statistical Methods. 8th Edition" (Iowa State University Press, Iowa). 126 Soto, A. M., Justicia, H., Wray, J. V/. & Sonnenschein, C. (1991) p-Nonyl-phenol: An Estrogenic Xenobiotic Released from "Modified" Folystyrene. Enviran. Ilealth Perspect 92, 167-173. Speare, R (1990) A Review of the Diseases of the Cane Toad, Bufo marinus, with Comments on Biological Control. Aust. Wildl. Res.17,387-4I0. Speare, R., McDonald, K. & Hohenhaus, R. (1999) Hopping Mad.Itttitdtife Austrølia36(2), 19-22. Thoms, M. C. (1998) Floodplain-wetlands: transient storage areas of sediments and pollutants. pp 205-215 In Williams, Vy'. D. (Ed) "V/etlands in a Dry Land: Understanding for Management" (Environment Australia, Biodiversity Group, canberra). Tyler, N4. J. (1994a) "Australian Frogs : A Natural History. 2nd Edition". (Reed Books, Chatswood, NSW). Tyler, M. J. (I994b) Climate change and its implications for the amphibian fauna Trans. R. ,S'oc. ,S. Aust 118,53-57. T'yler, N4. J" (1996) Frogwatch. Zoo Times l3(2),1-2 Tyler, M' J. (1997) Environmentally Friendly: A False Sense of Security? Species 29,20-21 Tyler, M" J. &, Crook, G. (1980) Frogs of the Magela Creek System, Alligator Rivers Region, Northern Territory, Australia. Report to the Office of the Supervising Scientist, Alligator Rivers Region. Tyler, M. J. & Watson, G. F. (1998) Additional habitats for frogs created by human alterations to the Australian environment. Aust. Biot. n\$,144-\46. IJSEPA (1989) "Section 7 Dilution Water, Methods for Estimating the Chronic Toxicity of Effluents and Receiving V/aters to Freshwater Organisms, 2nd Edition". (Office of Research and Development, Cincinnati, Ohio). 127 V/ilbur, H. M. & Collins, J. P. (1973) Ecological Aspects of Amphibian Metamorphosis. Nonnormal distributions of competitive ability reflect selection for facultative metamorphosis Science 182, 1305-13 14. zar, J.I{. (1984) "Biostatistical Analysis. 2nd Edition". (Prentice-Hall, sydney). 128