The green and golden bell frog (Litoria aurea) is an Australian hylid, which was once common with a relatively continuous distribution. Historically, this distribution extended from northern New South Wales (NSW), as far as Ballina, to East Gippsland in Victoria; with inland populations as far west as Bathurst and Tumut. Today the species is reported to have disappeared from 80% of its former range and remaining populations are mostly fragmented and typically restricted to the coastline, extending from Yuraygir National Park (northern NSW) to East Gippsland. In this thesis, I report a comprehensive study designed to identify the phylogeographic and conservation genetic parameters of L. aurea. In doing so, I also investigate evolutionary relationships within the ???bell frog??? species group. In this study, microsatellite and mitochondrial DNA (mtDNA) markers are employed. The development of species-specific microsatellite markers and the collection of samples was a substantial component of the study. These markers and samples should prove useful for future studies of L. aurea and perhaps more generally the ???bell frogs???. Initially, a large-scale assessment of genetic structure and diversity in L. aurea using microsatellite markers was undertaken. Twenty-one locations were sampled from throughout the species range covering 1000 kilometres of the east coast of Australia. Levels of allelic diversity and heterozygosity were high (uncorrected mean alleles/locus and HE: 4.8-8.8 and 0.43-0.8 respectively) compared to other amphibian species and significant differences among sampled sites were recorded. Despite recent population declines, no sites displayed a genetic signature indicative of a population bottleneck. Significant genetic structuring (overall FST = 0.172) was detected throughout the species range, but was relatively low compared to previous amphibian studies that used microsatellites. In addition, some areas sampled within continuous habitat showed evidence of weak genetic structuring (data subset FST = 0.034). Next, relationships among extant bell frogs (Litoria aurea species-group) were investigated, using mitochondrial ND4 nucleotide sequence data. Analyses supported a clade comprised of the temperate members of the species-group, L. aurea, L. cyclorhyncha, L. moorei, and L. raniformis but failed to support the inclusion of the tropical bell frog L. dahlii in this group. Relationships among the four members of the bell frog clade correlated with geographical distribution: the south-western Australian bell frogs (L. cyclorhyncha, L. moorei) and the south-eastern Australian bell frogs (L. aurea, L. raniformis) were reciprocally monophyletic. Results also indicated that divergence of these two lineages occurred during the late Miocene, which was consistent with results of previous studies and with more general assertions that much of the major differentiation and radiation of the Australian biota predated the Quaternary. Following this, intraspecific phylogeography of L. aurea using two mitochondrial genes COI and ND4 was investigated. I examined extant populations from throughout the species??? range, sequencing 263 individuals from twenty-six locations. Recent evolutionary history, as well as the current population structure of L. aurea, was inferred from the resulting pattern of genetic variation amongst haplotypes, in conjunction with demographic and population analyses. Results indicated that there were no phylogeographic divisions within L. aurea, despite a general consensus that amphibians are highly structured. However, I did still detect significant structure amongst extant populations (FST = 0.385). Overall, patterns of haplotype relatedness, high haplotypic diversity (mean h = 0.547) relative to low nucleotide diversity (mean Pi= 0.003), and mismatch distribution analysis supported a Pleistocene expansion hypothesis with continued restricted dispersal and gene flow. Taken together, the results of this thesis indicate that L. aurea is a species with relatively weak population and phylogeographic structure compared to other amphibians. The data provide no support for the existence of distinct evolutionary lineages within L. aurea, implying that there are no historically isolated populations that should be viewed as separate evolutionarily significant units. Nevertheless, remaining populations are still significantly structured but not all populations are genetically distinct. Low phylogeographic structure, coupled with evidence for recent gene flow among many sites would permit ???well managed??? intervention to mediate gene flow amongst currently isolated populations, and I provide some guidelines for the implementation of such conservation strategies. However, there is no evidence to suggest that supplementation through artificial immigration is at this time necessary given current levels of genetic variation within populations. In the short-term, conservation management in L. aurea should focus on the protection of local populations and habitat to promote population connectivity to ensure processes that maintain adaptive diversity and evolutionary potential are conserved. Preservation of the species??? natural range and the maintenance of dense networks of suitable habitat, in conjunction with maximising local carrying capacity and reproductive output, as well as minimising known threats, are key to securing the long-term persistence of the green and golden bell frog.

The green and golden bell frog (Litoria aurea) is an Australian hylid, which was once common with a relatively continuous distribution. Historically, this distribution extended from northern New South Wales (NSW), as far as Ballina, to East Gippsland in Victoria; with inland populations as far west as Bathurst and Tumut. Today the species is reported to have disappeared from 80% of its former range and remaining populations are mostly fragmented and typically restricted to the coastline, extending from Yuraygir National Park (northern NSW) to East Gippsland. In this thesis, I report a comprehensive study designed to identify the phylogeographic and conservation genetic parameters of L. aurea. In doing so, I also investigate evolutionary relationships within the ???bell frog??? species group. In this study, microsatellite and mitochondrial DNA (mtDNA) markers are employed. The development of species-specific microsatellite markers and the collection of samples was a substantial component of the study. These markers and samples should prove useful for future studies of L. aurea and perhaps more generally the ???bell frogs???. Initially, a large-scale assessment of genetic structure and diversity in L. aurea using microsatellite markers was undertaken. Twenty-one locations were sampled from throughout the species range covering 1000 kilometres of the east coast of Australia. Levels of allelic diversity and heterozygosity were high (uncorrected mean alleles/locus and HE: 4.8-8.8 and 0.43-0.8 respectively) compared to other amphibian species and significant differences among sampled sites were recorded. Despite recent population declines, no sites displayed a genetic signature indicative of a population bottleneck. Significant genetic structuring (overall FST = 0.172) was detected throughout the species range, but was relatively low compared to previous amphibian studies that used microsatellites. In addition, some areas sampled within continuous habitat showed evidence of weak genetic structuring (data subset FST = 0.034). Next, relationships among extant bell frogs (Litoria aurea species-group) were investigated, using mitochondrial ND4 nucleotide sequence data. Analyses supported a clade comprised of the temperate members of the species-group, L. aurea, L. cyclorhyncha, L. moorei, and L. raniformis but failed to support the inclusion of the tropical bell frog L. dahlii in this group. Relationships among the four members of the bell frog clade correlated with geographical distribution: the south-western Australian bell frogs (L. cyclorhyncha, L. moorei) and the south-eastern Australian bell frogs (L. aurea, L. raniformis) were reciprocally monophyletic. Results also indicated that divergence of these two lineages occurred during the late Miocene, which was consistent with results of previous studies and with more general assertions that much of the major differentiation and radiation of the Australian biota predated the Quaternary. Following this, intraspecific phylogeography of L. aurea using two mitochondrial genes COI and ND4 was investigated. I examined extant populations from throughout the species??? range, sequencing 263 individuals from twenty-six locations. Recent evolutionary history, as well as the current population structure of L. aurea, was inferred from the resulting pattern of genetic variation amongst haplotypes, in conjunction with demographic and population analyses. Results indicated that there were no phylogeographic divisions within L. aurea, despite a general consensus that amphibians are highly structured. However, I did still detect significant structure amongst extant populations (FST = 0.385). Overall, patterns of haplotype relatedness, high haplotypic diversity (mean h = 0.547) relative to low nucleotide diversity (mean Pi= 0.003), and mismatch distribution analysis supported a Pleistocene expansion hypothesis with continued restricted dispersal and gene flow. Taken together, the results of this thesis indicate that L. aurea is a species with relatively weak population and phylogeographic structure compared to other amphibians. The data provide no support for the existence of distinct evolutionary lineages within L. aurea, implying that there are no historically isolated populations that should be viewed as separate evolutionarily significant units. Nevertheless, remaining populations are still significantly structured but not all populations are genetically distinct. Low phylogeographic structure, coupled with evidence for recent gene flow among many sites would permit ???well managed??? intervention to mediate gene flow amongst currently isolated populations, and I provide some guidelines for the implementation of such conservation strategies. However, there is no evidence to suggest that supplementation through artificial immigration is at this time necessary given current levels of genetic variation within populations. In the short-term, conservation management in L. aurea should focus on the protection of local populations and habitat to promote population connectivity to ensure processes that maintain adaptive diversity and evolutionary potential are conserved. Preservation of the species??? natural range and the maintenance of dense networks of suitable habitat, in conjunction with maximising local carrying capacity and reproductive output, as well as minimising known threats, are key to securing the long-term persistence of the green and golden bell frog.

Thesis (PhD.)--University of Canberra, 2007. / Includes bibliography (p. 152-176)

Variation in advertisement call structure of whistling frogs, Litoria ewingi, was studied among breeding sites in the vicinity of Christchurch on the East coast and Harihari on the West coast of the South Island. Males vocalized in breeding choruses throughout the year and females moved toward and appeared to evaluate calls of territorial males before initiating amplexus. Acoustic interactions between neighbours led to modification of call structure and timing, and wrestling matches between males were frequent. I quantified call structure for 1623 calls of 168 individuals using 24 call parameters. Most temporal characteristics were significantly correlated with the caller's body temperature and dominant frequency was negatively correlated with body size. These results are consistent with the prediction of temperature-dependent metabolic rates in a poikilotherm's nervous system. I compared call structure among the study populations using both multivariate and univariate analyses. Advertisement call structure showed highly significant variation among populations for most call variables. The greatest difference occurred between the West and East coast populations, and a clinal trend occurred in the East coast populations on a microgeographic scale. Variation among East coast populations in some variables was comparable to the scale of variation between coasts. These results suggest that whistling frogs exhibit natal philopatry. I obtained repeated recordings for 21 individuals and examined variation in call structure within and among individuals using nested multivariate analyses of variance. Highly significant variation occurred both within and among individuals for most call characters, but certain call characters were more variable among individuals. This suggests that whistling frog advertisement calls are individually distinctive.

Two Australian frogs were introduced to New Zealand over 100 years ago. Since their introduction they have become widespread and well established with Litoria ewingii being more prevalent in alpine and cooler areas of New Zealand, while Litoria raniformis is found in more temperate coastal areas. Very little physiological data exists for these frogs and aside from their distribution not much is known about them in New Zealand. Thus the effects of thermal acclimation and temperature change on respiration and locomotion were examined in these two exotic frogs. The more terrestrial and alpine dwelling L. ewingii was found to be able to thermally acclimate its respiration performance, where respiration was highest when acclimation temperature matched test temperature. It was also able to thermally acclimate its locomotory performance, jumping significantly further at lower temperatures, however, acclimation to high temperatures impacted its jump performance at cold temperatures. The frog L. raniformis was found to only be able to thermally acclimate its respiration and locomotion to high temperatures, as performance at low temperatures was often poor. The data shows that L. ewingii is a cold temperate frog rather than a warm habitat frog, while L. raniformis is an animal adapted to warm temperatures. From this we can begin to see the effect that temperature has on the physiology of these two exotic frogs and the major role that temperature may be playing in driving the differences seen in the distribution of these two species in New Zealand.

In frogs, part of the important immune defence system of their skin is the secretion of antimicrobial peptides from granular glands. This study investigated the immune function of skin peptides in protection against bacterial pathogens associated with infectious bacterial dermatosepticemia under a number of environmental conditions and at certain stages of the life cycle of frogs. The natural peptide mixture of skin peptides was collected from skin secretions of three semi-aquatic Litoria frog species L. aurea, L. raniformis and L. ewingii and aquatic Xenopus laevis and assayed for activity against the bacterial pathogens: Aeromonas hydrophila, Chryseobacterium meningosepticum, Citrobacter freundii, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa and Serratia liquefaciens. The peptide mixtures of three frog species Xenopus laevis, Litoria aurea and Litoria raniformis showed activity against C. freundii, C. meningosepticum, K. pneumoniae and P. aeruginosa in vitro indicating a likely protective function. One Litoria species, L. ewingii, had a peptide mixture that did not have activity against any pathogen. Subsequently, in experimental exposure of animals to the pathogen K. pneumoniae, this species was found to be susceptible to disease while the other sympatric species L. raniformis was found to be resistant. A strong correlation was shown between composition of skin peptides and resistance to disease. A comparison of the production and activity of skin peptides from four frog species showed the aquatic X. laevis to have more effective immune defence against bacterial pathogens than three tested Litoria species. X. laevis produced significantly greater amount of bioactive peptide mixture than three tested Litoria species. Three pathogens A. hydrophila, P. mirabilis and S. liquefaciens are abundant components of the skin microbiota of healthy frogs and were found to be resistant to the peptide mixtures of all four frog species tested. It was shown that one pathogen, A. hydrophila, had the ability to secrete proteases which could inactivate skin peptides. Thus while skin peptides could function against several pathogens, some pathogens might have co-evolved to resist skin peptides. A comparison of the peptide mixtures from skin secretions of adults, metamorphs and larvae of L. ewingii using liquid chromatography-mass spectrometry analyses showed that peptide mixtures of post metamorphic animals, adults and metamorphs, had a species-specific profile that included the antimicrobial peptide uperin 7.1, while the larval peptide mixture did not contain uperin 7.1 or any other known species-specific peptide. This finding indicates the absence of a secretory mechanism that could compensate for the absence of granular glands in larvae. Analyses of the production and activity of skin peptides of L. raniformis after exposure to two different environmental stressors, low environmental temperature and pesticide carbaryl, showed that the total amount of bioactive peptide was significantly reduced which could consequently increase susceptibility to disease. Thus suppression of skin peptides could be a possible mechanism for synergism between the important stressors and pathogens in disease development.

Variation in advertisement call structure of whistling frogs, Litoria ewingi, was studied among breeding sites in the vicinity of Christchurch on the East coast and Harihari on the West coast of the South Island. Males vocalized in breeding choruses throughout the year and females moved toward and appeared to evaluate calls of territorial males before initiating amplexus. Acoustic interactions between neighbours led to modification of call structure and timing, and wrestling matches between males were frequent. I quantified call structure for 1623 calls of 168 individuals using 24 call parameters. Most temporal characteristics were significantly correlated with the caller's body temperature and dominant frequency was negatively correlated with body size. These results are consistent with the prediction of temperature-dependent metabolic rates in a poikilotherm's nervous system. I compared call structure among the study populations using both multivariate and univariate analyses. Advertisement call structure showed highly significant variation among populations for most call variables. The greatest difference occurred between the West and East coast populations, and a clinal trend occurred in the East coast populations on a microgeographic scale. Variation among East coast populations in some variables was comparable to the scale of variation between coasts. These results suggest that whistling frogs exhibit natal philopatry. I obtained repeated recordings for 21 individuals and examined variation in call structure within and among individuals using nested multivariate analyses of variance. Highly significant variation occurred both within and among individuals for most call characters, but certain call characters were more variable among individuals. This suggests that whistling frog advertisement calls are individually distinctive.

The decline and extinction of amphibian species over the past three decades is widely acknowledged as one of the greatest biodiversity crises of modem time. Providing convincing data to support hypotheses about these declines has proved difficult, which has greatly restricted the development and implementation of management actions that may prevent further amphibian declines and extinctions from occurring. In this thesis, I present research that was undertaken as part of the recovery programs for the southern corroboree frog (Pseudophryne corroboree), and the Booroolong frog (Litoria booroolongensis); two species that underwent very rapid declines in distribution and abundance during the 1980's. More specifically, I investigated potential causal factors in the declines of both species using experimental and correlative studies, and examined the mechanisms by which one threatening process (chytridiomycosis) may be causing continued decline and extinction in P. corroboree. I also examined the implications of population dynamics for monitoring L. booroolongensis, and suggest a possible monitoring strategy that may reliably facilitate the implementation of recovery objectives for this species. I also tested one possible reintroduction technique aimed at preventing the continued decline and extinction of P. corroboree populations. In Chapters 2 and 3, I present the results from a series of experiments in artificial enclosures designed to examine whether the tadpoles of L. booroolongensis are susceptible to predation by co-occurring introduced predatory fish species; brown trout (Salmo trutta), rainbow trout (Oncorhynchus mykiss), European carp (Cyprinus carpio), redfin perch (Percafluviatilis), and mosquito fish (Gambusia holbrooki). I demonstrated that the tadpoles of L. booroolongensis, and a closely related species Litoria lesueuri, were palatable to non-native trout species, but not to two native predatory fish species, Gadopsis bispinosus and Galaxias olidus. A pond breeding frog species included in this experiment, Limnodynastes tasmaniensis, was palatable to both the native and non-native fish species. In a separate experiment I also demonstrated that the tadpole of L. booroolongensis is palatable to the three other introduced fish species examined in this study; C. carpio, P. fluviatilis, and G. holbrooki. In three of the experiments, the provision of rock within enclosures as a potential refuge habitat did not afford protection to L. booroolongensis tadpoles from predation by any of the five introduced fish species examined. While all the introduced fish species tested here did consume L. booroolongensis tadpoles, the results also suggested that chemical unpalatability might afford some level of protection against some of these fish species. Firstly, the addition of alternative prey items in one of the experiments reduced the proportion of tadpoles consumed, suggesting that L. booroolongensis may not be a preferred prey item. Secondly, the proportion of tadpoles consumed varied greatly among the different fish species examined, suggesting differing levels of palatability. Overall, this study supports previous research in suggesting that chemical unpalatability may be an important strategy for the tadpoles of riverine frog species in south-eastern Australia to avoid predation by native fish species, and that this strategy is less effective against introduced fish species. While L. booroolongensis currently persists in streams inhabited by a number of introduced fish species, this study supports the likelihood that these species are having a negative impact on populations of L. booroolongensis in the wild. In Chapter 4, I present the results of a study aimed at examining potential monitoring techniques for L. booroolongensis. The results of a mark-recapture exercise demonstrated that L. booroolongensis may exhibit large fluctuations in abundance from one year to the next, and through a prospective power analysis approach, I demonstrated that it would be difficult to confidently identify population trends of interest using either indices or estimates of abundance for this species. An assessment of the capacity to identify the presence or absence of L. booroolongensis using nighttime spotlight surveys demonstrated the high detectability of this species using this technique, at both the scale of 300-meter sections of stream and individual breeding areas (typically less than 10-meters of stream). This study suggests that the monitoring objectives of the L. booroolongensis recovery program would be most effectively achieved using presence/absence surveys at different scales. In Chapter 5, I present the results of a field survey aimed at determining the current distribution and habitat requirements of L. booroolongensis in the South West Slopes region of New South Wales. Of the 163 sites I surveyed across 49 streams,I located L. booroolongensis along 77 of these sites from 27 streams. Based on population and habitat connectivity, this study identified 18 populations of L. booroolongensis that are likely to be operating as independent populations. Twelve of these populations are not represented in conservation reserves, but rather occur along streams that flow through the agricultural landscape. A broad scale habitat analysis identified a positive relationship between extent of rock structures along the stream and the occurrence of L. booroolongensis, and a negative relationship between the proportion of canopy cover and this species' occurrence. At the breeding habitat scale, this study identified a positive relationship between the presence of breeding males and; number of rock crevices in the aquatic environment, extent of emergent rocks, and proportion pool. This analysis also detected a negative relationship between occupancy and water depth. These results confirm previous work suggesting the importance of rocky stream habitats to the persistence of L. booroolongensis, but also suggest how disturbance processes, such as increasing sedimentation and weed invasion, may reduce the suitability of rocky structures as breeding sites. In Chapter 6, I investigated current levels of amphibian chytrid fungus (Batrachochytrium dendrobatidis) infection in corroboree frog populations, and used retrospective screening of museum specimens to assess the possibility that this pathogen was implicated in the initial decline of the corroboree frogs. Using histology, I did not detect any B. dendrobatidis infections in corroboree frog populations prior to their decline, however using the same technique, moderate levels of infection were detected in post-decline populations of both species. Real-time PCR screening of skin swabs identified much higher overall infection rates in post-decline populations of P. corroboree (between 44% and 59%), while significantly lower rates of infection were observed in P. pengilleyi populations (14%). These results suggest that the initial and continued decline of the corroboree frogs may well be attributed to the emergence of B. dendrobatidis in populations of these species. In Chapter 7, I investigated how B. dendrobatidis may be causing the continued decline of P. corroboree through the presence of an abundant reservoir host for this pathogen. I found that populations of adult C. signifera in sub-alpine bogs carry high B. dendrobatidis infection rates (86%), but appear unaffected by this infection. An experiment involving the release of P. corroboree tadpoles into 15 natural pools resulted in metamorphs from seven of these pools testing positive for B. dendrobatidis, with all these individuals dying soon after metamorphosis. These results support the possibility that B. dendrobatidis infection in P. corroboree populations is being facilitated by the presence of large numbers of infected C. signifera in the shared environment. Chapter 8 presents the results of a population augmentation study for P. corroboree. I investigated the extent to which increasing recruitment to metamorphosis may result in population recovery in this species. This was undertaken by harvesting eggs from the field and rearing them through to mid stage tadpoles over the winter period prior to being released back to their natal ponds in spring. While I was able to increase recruitment to metamorphosis by an average of 20 percent, this did not result in a noticeable influence on the subsequent adult population size, as both manipulated and non-manipulated sites declined over the course of this study by an average of 80 percent. I observed a positive relationship between natural recruitment to a late tadpole stage and subsequent adult male population size, however there was considerable variation associated with this relationship. The relationship between recruitment and subsequent population size at the augmentation sites was consistent with the relationship observed at the non-manipulated sites. These results suggest that recruitment to metamorphosis may not be the most important life stage restricting the population recovery of P. corroboree, but that mortality during post-metamorphic stages may be more important in regulating current population size. Hence, further attempts to use captive rearing to increase P. corroboree populations in the wild should focus on the release of post-metamorphic frogs. Overall, this thesis demonstrates the value of quantitative research to the implementation and progress of threatened species recovery programs. While this research will specifically contribute to the recovery programs for L. booroolongensis and P. corroboree, it more broadly contributes to the understanding and capacity to respond to the concerning levels of amphibian extinctions currently occurring throughout the world.

Surfactants are one of the more ubiquitous contaminants in aquatic systems. Their importance as toxic components of pesticide formulations has, however, been largely overlooked. Amphibians particularly, as inhabitants of shallow, temporary and often lentic aquatic environments may be at risk from exposure to these chemicals when they enter aquatic systems. This thesis presents data on the toxicity of surfactants to amphibians. Several experimental exposures were conducted with embryo-larval, tadpole and adult developmental stages of the Australian species- Crinia insignifera, Helcioporus eyrei, Limnodynastes dorsalis and Litoria moorei and the exotic species- Bufo marinus and Xenopus laevis. Animals were variously exposed to glyphosate formulations that contain a high proportion of nonionic surfactants, or commercial pesticide wetting agents (alcohol alkoxylate and nonylphenol ethoxylate (NPE) surfactants). Feeding stage tadpoles of C.insignifera, H. eyrei, L. dorsalis and L. moorei were exposed to three commercial glyphosate formulations, glyphosate isopropylamine and glyphosate acid in static-renewal acute toxicity tests. The 48-h LC50 values for Roundup Herbicide (MON 2139) tested against tadpoles of C. insignifera, H. eyrei, L. dorsalis and L. moorei ranged between 8.1 and 32.2 mg/L (2.9 and 11.6 mg/L glyphosate acid equivalent (ae)), while the 48-h LC50 values for Roundup Herbicide tested against adult and newly metamorphosed C. insignifera ranged from 137-144 mg/L (49.4-51.8 mg/L ae). / Touchdown Herbicide (4 LC-E) tested against tadpoles of C. insignifera, H. eyrei, L. dorsalis and L. moorei was slightly less toxic than Roundup with 48-h LC50 values ranging between 27.3 and 48.7 mg/L (9.0 and 16.1 mg/L ae). Roundup Biactive (MON 77920) was practically non-toxic to tadpoles of the same four species producing 48-h LC50 values of 911 mg/L (328 mg/L ae) for L. moorei and >1000 mg/L (>360 mg/L ae) for C. insignifera, H. eyrei and L. dorsalis. Glyphosate isopropylamine was practically non-toxic producing no mortality amongst tadpoles of any of the four species over 48 h, at concentrations between 503 and 684 mg/L (343 and 466 mg/L ae). The toxicity of technical grade glyphosate acid (48-h LC50, 81.2-121 mg/L) is likely to be due to acid intolerance. Feeding stage tadpoles of B. marinus, X laevis, C. insignifera, H.eyrei, L. dorsalis and L. moorei were exposed to NPE and alcohol alkoxylate in static renewal acute toxicity tests. All species exhibited non-specific narcosis following exposure to both these surfactants. The 48-h EC50 values for NPE ranged between 1.1 mg/L (mild narcosis) and 12.1 mg/L (full narcosis). The 48-h EC50 values for alcohol alkoxylate ranged between 5.3 mg/L (mild narcosis) and 25.4 mg/L (full narcosis). Xenopus laevis was the most sensitive species tested. The sensitivity of the other five species was size dependent with larger species displaying greater tolerance. Replicate acute toxicity tests with B. marinus exposed to NPE at 30 degrees celsius over 96 hours indicated that the narcotic effects were not particularly time dependant. / The mean 24, 48, 72 and 96-h EC50 (mild narcosis) were 3.6, 3.7, 3.5 and 3.5 mg/L respectively. The mean 24, 48, 72, and 96-h EC50 (full narcosis) values were 4.0, 4.1, 4.2 and 4.0 respectively. Acute toxicity tests with B. marinus exposed to NPE at 30 degrees celsius under conditions of low dissolved oxygen (0.8-2.3 mg/L) produced a two to threefold increase in toxicity. The 12-h EC50 values ranged from 1.4 to 2.2 mg/L. The embryotoxicity of NPE was determined in X. laevis, L. adelaidensis and C. insignifera using a Frog Embryo Teratogenesis assay-Xenopus (FETAX). The 96-h LC50, EC50 and MCIG (LOEC) values for X. laevis were 3.9 to 5.4 mg/L, 2.8 to 4.6 mg/L and 1.0 to 3.0 mg/L respectively. The 140-h LC50, ECSO and MCIG values for L. adelaidensis were 9.2 mg/L, 8.8 mg/L and 5.1 to 6.0 ing/L respectively. The 134-h LC50, EC50 and MCIG values for C. insignifera were 6.4 mg/L, 4.5 mg/L and 4.0 mg/L respectively. Teratogenicity indices for the three species ranged between 1.0 and 1.6 indicating either no or low teratogenicity. Xenopus laevis was the more sensitive of the three species and the only species that displayed indisputable terata. The acute toxicity data indicated that the amphibian species tested were of similar sensitivity to fish and some invertebrates. Developmental retardation and oestrogenic effects following exposure to nonylphenol ethoxylate were indicated by sublethal toxicity tests. Crinia insignifera embryos were exposed during early embryogenesis to sublethal concentrations of NPE. / Exposure to NPE did not affect either weight nor size (snout-vent length) at metamorphosis. Exposure to 5.0mg/L NPE resulted in a significant delay in the time required to reach metamorphosis. Also, exposure to 3.0 mg/L NPE for the first 6 days of embryonic development or exposure to 5.0 mg/L NPE from day 2 to day 6 resulted in a statistically significant predominance in the female phenotype amongst metamorphosing froglets. Exposure for the first five days to 1.5 ing/L or 3.0 mg/L NPE had no effect on sex ratio. The results indicated that exposure to NPEs has endocrine disruptive effects in this species and that a narrow window of susceptibility exists for the induction of predominantly female phenotype. This study has also followed the degradation of a mixture of NPE oligomers and the concomitant formation of individual oligomers in static die-away tests with and without illumination in freshwater. Over 33 days in darkness there was a progressive and complete loss of long chain oligomers (NPEO(subscript)8-17), transient increases and subsequent loss of short to medium chain oligomers (NPE0(subscript)4-7), and large persistent increases (approximately 1000%) in short chain oligomers (NPE0(subscript)1-3). In the presence of illumination, biodegradation was retarded and heterotrophic bacterial proliferation was inhibited. After 33 days there was complete loss of long chain oligomers (NPE0(subscript)9-17), incomplete loss of medium chain oligomers (NPE0(subscript)6.8) and increases in short chain oligomers (NPE0(subscript)1-5). / This thesis discusses the importance of persistent metabolites of NPE degradation as it pertains to the habitat, developmental time frame and ecology of amphibians. Degradation of NPE is likely to occur over a time frame that is longer than that required for complete embryogenesis and metamorphosis of many species of amphibians, and may easily encompass those critical stages of development during which oestrogenic metabolites can affect development.