Endocrine disruption in teleosts and amphibians is mediated by anthropogenic and natural environmental factors: implications for risk assessment.

Environmental variation in the Anthropocene involves several factors that interfere with endocrine systems of wildlife and humans, presenting a planetary boundary of still unknown dimensions. Here, we focus on chemical compounds and other impacts of anthropogenic and natural origins that are adversely affecting reproduction and development. The main sink of these endocrine disruptors (EDs) is surface waters, where they mostly endanger aquatic vertebrates, like teleost fish and amphibians. For regulatory purposes, EDs are categorized into EATS modalities (oestrogenic, androgenic, thyroidal, steroidogenesis), only addressing endocrine systems being assessable by validated tests. However, there is evidence that non-EATS modalities-and even natural sources, such as decomposition products of plants or parasitic infections-can affect vertebrate endocrine systems. Recently, the disturbance of natural circadian light rhythms by artificial light at night (ALAN) has been identified as another ED. Reviewing the knowledge about EDs affecting teleosts and amphibians leads to implications for risk assessment. The generally accepted WHO-definition for EDs, which focuses exclusively on 'exogenous substances' and neglects parasitic infections or ALAN, seems to require some adaptation. Natural EDs have been involved in coevolutionary processes for ages without resulting in a general loss of biodiversity. Therefore, to address the 'One Health'-principle, future research and regulatory efforts should focus on minimizing anthropogenic factors for endocrine disruption. This article is part of the theme issue 'Endocrine responses to environmental variation: conceptual approaches and recent developments'.

Biochemical responses revealed in an amphibian species after exposure to a forgotten contaminant: An integrated biomarker assessment.

Vanadium is a metal whose toxicity towards terrestrial and aquatic species has been under-reported to date.. The biochemical responses of vanadium in amphibian species have not been determined. To establish the effects of vanadium (V) on exposed adult Xenopus laevis, acute and chronic exposures were conducted, and biomarker analyses were performed on liver and muscle tissues from exposed frogs. Biomarkers of exposure, such as acetylcholinesterase (AChE) and metallothioneins (MT), were analysed. Biomarkers of effect were also analysed to determine possible increases in reactive oxygen species (ROS), and the effect of the exposure on the energy balance in the organisms. These included superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), protein carbonyls (PC), malondialdehyde (MDA), and cellular energy allocation (CEA) (energy available, energy consumption, lipids, proteins and glucose). In acute exposures, the energy balances in organisms were distinctly affected, possibly due to insulin mimetic properties of V. In chronic exposures, MT, AChE, SOD, CAT and GSH responses were more pronounced. Although AChE is generally inhibited by pollutant exposure, in this study, it was stimulated. There were significant inhibitions of SOD and CAT, previously observed in frog species. PC levels increased in the highest acute exposure concentration, indicating protein damage. The IBR.v2 revealed the biochemical responses of V more effectively than traditional statistical analysis.

Risk assessment considerations for plant protection products and terrestrial life-stages of amphibians.

Some amphibians occur in agricultural landscapes during certain periods of their life cycle and consequently might be exposed to plant protection products (PPPs). While the sensitivity of aquatic life-stages is considered to be covered by the standard assessment for aquatic organisms (especially fish), the situation is less clear for terrestrial amphibian life-stages. In this paper, considerations are presented on how a risk assessment for PPPs and terrestrial life-stages of amphibians could be conducted. It discusses available information concerning the toxicity of PPPs to terrestrial amphibians, and their potential exposure to PPPs in consideration of aspects of amphibian biology. The emphasis is on avoiding additional vertebrate testing as much as possible by using exposure-driven approaches and by making use of existing vertebrate toxicity data, where appropriate. Options for toxicity testing and risk assessment are presented in a flowchart as a tiered approach, progressing from a non-testing approach, to simple worst-case laboratory testing, to extended laboratory testing, to semi-field enclosure tests and ultimately to full-scale field testing and monitoring. Suggestions are made for triggers to progress to higher tiers. Also, mitigation options to reduce the potential for exposure of terrestrial life-stages of amphibians to PPPs, if a risk were identified, are discussed. Finally, remaining uncertainties and research needs are considered by proposing a way forward (road map) for generating additional information to inform terrestrial amphibian risk assessment.

Monitoring health and reproductive status of olms (Proteus anguinus) by ultrasound.

The olm (Proteus anguinus) is a troglomorphic, neotenous amphibian with extraordinary life expectancy and unique adaptations that deserve further investigation. A low reproductive rate and habitat decline render it threatened by extinction. Establishing captive populations for maintenance and artificial breeding may one day become crucial to the species. Longitudinal, in-vivo assessment of inner organs is invaluable to our understanding of reproductive physiology, health, and behavior. Using ultrasound, we measured heart rate and assessed health and reproductive status of 13 captive olms at Zagreb Zoo. Heart rate averaged 42.9 ± 4.6 bpm (32-55 bpm), as determined via pulsed-wave Doppler at 4-12 MHz. By using frequencies of up to 70 MHz (ultrasound biomicroscopy), inner organs were visualized in detail. Assessment of the gastrointestinal tract provided insights into feeding status and digestive processes. Several subclinical pathologies were detected, including biliary sludge, subcutaneous edema, ascites, and skin lesions. Detection of skin lesions by ultrasound was more sensitive than visual adspection. Olms with ultrasonographically detected skin lesions tested positive for Saprolegnia and were treated. Three of the four affected individuals survived and subsequently tested negative for Saprolegnia. Sex was reliably determined; only one individual proved male. The reason for this extreme female-biased sex-ratio remains unknown. However, as most of the individuals were flushed from the caves by strong currents in spring, the sample may not be representative of natural populations. In female olms, different stages of ovarian follicular development were observed with diameters ranging between 0.1 and 1.1 mm. Results were confirmed by comparing ultrasound, necropsy, and histological findings of one dead specimen. In summary, ultrasound proved a valuable tool to support conservation and captive breeding programs by allowing non-invasive assessment of physiological parameters, clinical condition, and reproductive status in olms.

Stem caecilian from the Triassic of Colorado sheds light on the origins of Lissamphibia.

The origin of the limbless caecilians remains a lasting question in vertebrate evolution. Molecular phylogenies and morphology support that caecilians are the sister taxon of batrachians (frogs and salamanders), from which they diverged no later than the early Permian. Although recent efforts have discovered new, early members of the batrachian lineage, the record of pre-Cretaceous caecilians is limited to a single species, Eocaecilia micropodia The position of Eocaecilia within tetrapod phylogeny is controversial, as it already acquired the specialized morphology that characterizes modern caecilians by the Jurassic. Here, we report on a small amphibian from the Upper Triassic of Colorado, United States, with a mélange of caecilian synapomorphies and general lissamphibian plesiomorphies. We evaluated its relationships by designing an inclusive phylogenetic analysis that broadly incorporates definitive members of the modern lissamphibian orders and a diversity of extinct temnospondyl amphibians, including stereospondyls. Our results place the taxon confidently within lissamphibians but demonstrate that the diversity of Permian and Triassic stereospondyls also falls within this group. This hypothesis of caecilian origins closes a substantial morphologic and temporal gap and explains the appeal of morphology-based polyphyly hypotheses for the origins of Lissamphibia while reconciling molecular support for the group's monophyly. Stem caecilian morphology reveals a previously unrecognized stepwise acquisition of typical caecilian cranial apomorphies during the Triassic. A major implication is that many Paleozoic total group lissamphibians (i.e., higher temnospondyls, including the stereospondyl subclade) fall within crown Lissamphibia, which must have originated before 315 million years ago.

Species interactions and the effects of climate variability on a wetland amphibian metacommunity.

Disentangling the role that multiple interacting factors have on species responses to shifting climate poses a significant challenge. However, our ability to do so is of utmost importance to predict the effects of climate change on species distributions. We examined how populations of three species of wetland-breeding amphibians, which varied in life history requirements, responded to a six-year period of extremely variable precipitation. This interval was punctuated by both extensive drought and heavy precipitation and flooding, providing a natural experiment to measure community responses to environmental perturbations. We estimated occurrence dynamics using a discrete hidden Markov modeling approach that incorporated information regarding habitat state and predator-prey interactions. This approach allowed us to measure how metapopulation dynamics of each amphibian species was affected by interactions among weather, wetland hydroperiod, and co-occurrence with fish predators. The pig frog, a generalist, proved most resistant to perturbations, with both colonization and persistence being unaffected by seasonal variation in precipitation or co-occurrence with fishes. The ornate chorus frog, an ephemeral wetland specialist, responded positively to periods of drought owing to increased persistence and colonization rates during periods of low-rainfall. Low probabilities of occurrence of the ornate chorus frog in long-duration wetlands were driven by interactions with predators due to low colonization rates when fishes were present. The mole salamander was most sensitive to shifts in water availability. In our study area, this species never occurred in short-duration wetlands and persistence probabilities decreased during periods of drought. At the same time, negative effects occurred with extreme precipitation because flooding facilitated colonization of fishes to isolated wetlands and mole salamanders did not colonize wetlands once fishes were present. We demonstrate that the effects of changes in water availability depend on interactions with predators and wetland type and are influenced by the life history of each of our species. The dynamic species occurrence modeling approach we used offers promise for other systems when the goal is to disentangle the complex interactions that determine species responses to environmental variability.

A review of ecological risk assessment methods for amphibians: Comparative assessment of testing methodologies and available data.

Historically, ecological risk assessments have rarely included amphibian species, focusing preferentially on other aquatic (fish, invertebrates, algae) and terrestrial wildlife (birds and mammal) species. Often this lack of consideration is due to a paucity of toxicity data, significant variation in study design, uncertainty with regard to exposure, or a combination of all three. Productive risk assessments for amphibians are particularly challenging, given variations in complex life history strategies. Further consideration is needed for the development of useful laboratory animal models and appropriate experimental test procedures that can be effectively applied to the examination of biological response patterns. Using these standardized techniques, risk estimates can be more accurately defined to ensure adequate protection of amphibians from a variety of stress agents. Patterns in toxicity may help to ascertain whether test results from 1 amphibian group (e.g., Urodela) could be sufficiently protective of another (e.g., Anura) and/or whether some nonamphibian aquatic taxonomic groups (e.g., fish or aquatic invertebrates) may be representative of aquatic amphibian life stages. This scope is intended to be a guide in the development of methods that would yield data appropriate for ecological risk decisions applicable to amphibians. Integr Environ Assess Manag 2017;13:601-613. © 2016 SETAC.

Impact of alternative metrics on estimates of extent of occurrence for extinction risk assessment.

In International Union for Conservation of Nature (IUCN) Red List assessments, extent of occurrence (EOO) is a key measure of extinction risk. However, the way assessors estimate EOO from maps of species' distributions is inconsistent among assessments of different species and among major taxonomic groups. Assessors often estimate EOO from the area of mapped distribution, but these maps often exclude areas that are not habitat in idiosyncratic ways and are not created at the same spatial resolutions. We assessed the impact on extinction risk categories of applying different methods (minimum convex polygon, alpha hull) for estimating EOO for 21,763 species of mammals, birds, and amphibians. Overall, the percentage of threatened species requiring down listing to a lower category of threat (taking into account other Red List criteria under which they qualified) spanned 11-13% for all species combined (14-15% for mammals, 7-8% for birds, and 12-15% for amphibians). These down listings resulted from larger estimates of EOO and depended on the EOO calculation method. Using birds as an example, we found that 14% of threatened and near threatened species could require down listing based on the minimum convex polygon (MCP) approach, an approach that is now recommended by IUCN. Other metrics (such as alpha hull) had marginally smaller impacts. Our results suggest that uniformly applying the MCP approach may lead to a one-time down listing of hundreds of species but ultimately ensure consistency across assessments and realign the calculation of EOO with the theoretical basis on which the metric was founded.

Use of the NatureServe Climate Change Vulnerability Index as an Assessment Tool for Reptiles and Amphibians: Lessons Learned.

Climate change threatens biodiversity globally, yet it can be challenging to predict which species may be most vulnerable. Given the scope of the problem, it is imperative to rapidly assess vulnerability and identify actions to decrease risk. Although a variety of tools have been developed to assess climate change vulnerability, few have been evaluated with regard to their suitability for certain taxonomic groups. Due to their ectothermic physiology, low vagility, and strong association with temporary wetlands, reptiles and amphibians may be particularly vulnerable relative to other groups. Here, we evaluate use of the NatureServe Climate Change Vulnerability Index (CCVI) to assess a large suite of herpetofauna from the Sand Hills Ecoregion of the southeastern United States. Although data were frequently lacking for certain variables (e.g., phenological response to climate change, genetic variation), sufficient data were available to evaluate all 117 species. Sensitivity analyses indicated that results were highly dependent on size of assessment area and climate scenario selection. In addition, several ecological traits common in, but relatively unique to, herpetofauna are likely to contribute to their vulnerability and need special consideration during the scoring process. Despite some limitations, the NatureServe CCVI was a useful tool for screening large numbers of reptile and amphibian species. We provide general recommendations as to how the CCVI tool's application to herpetofauna can be improved through more specific guidance to the user regarding how to incorporate unique physiological and behavioral traits into scoring existing sensitivity factors and through modification to the assessment tool itself.

Identifying the world's most climate change vulnerable species: a systematic trait-based assessment of all birds, amphibians and corals.

Climate change will have far-reaching impacts on biodiversity, including increasing extinction rates. Current approaches to quantifying such impacts focus on measuring exposure to climatic change and largely ignore the biological differences between species that may significantly increase or reduce their vulnerability. To address this, we present a framework for assessing three dimensions of climate change vulnerability, namely sensitivity, exposure and adaptive capacity; this draws on species' biological traits and their modeled exposure to projected climatic changes. In the largest such assessment to date, we applied this approach to each of the world's birds, amphibians and corals (16,857 species). The resulting assessments identify the species with greatest relative vulnerability to climate change and the geographic areas in which they are concentrated, including the Amazon basin for amphibians and birds, and the central Indo-west Pacific (Coral Triangle) for corals. We found that high concentration areas for species with traits conferring highest sensitivity and lowest adaptive capacity differ from those of highly exposed species, and we identify areas where exposure-based assessments alone may over or under-estimate climate change impacts. We found that 608-851 bird (6-9%), 670-933 amphibian (11-15%), and 47-73 coral species (6-9%) are both highly climate change vulnerable and already threatened with extinction on the IUCN Red List. The remaining highly climate change vulnerable species represent new priorities for conservation. Fewer species are highly climate change vulnerable under lower IPCC SRES emissions scenarios, indicating that reducing greenhouse emissions will reduce climate change driven extinctions. Our study answers the growing call for a more biologically and ecologically inclusive approach to assessing climate change vulnerability. By facilitating independent assessment of the three dimensions of climate change vulnerability, our approach can be used to devise species and area-specific conservation interventions and indices. The priorities we identify will strengthen global strategies to mitigate climate change impacts.

Farmed areas predict the distribution of amphibian ponds in a traditional rural landscape.

BACKGROUND: Traditional rural landscapes of Eastern Europe are undergoing major changes due to agricultural intensification, land abandonment, change in agricultural practices and infrastructural development. Small man-made ponds are important yet vulnerable components of rural landscapes. Despite their important role for biodiversity, these ponds tend to be excluded from conservation strategies. METHODOLOGY/FINDINGS: Our study was conducted in a traditional rural landscape in Eastern Europe. The aim of this study is twofold: (i) to model the distribution of four major man-made pond types and (ii) to present the importance of man-made ponds for the endangered Yellow Bellied Toad (Bombina variegata) and the Common Toad (Bufo bufo). Six environmental variables were used to model pond distribution: Corine landcover, the heterogeneity of the landcover, slope, road distance, distance to closest village and the human population density. Land cover heterogeneity was the most important driver for the distribution of fishponds. Areas used for agriculture with significant areas of natural vegetation were the most important predictors for the distribution of temporary ponds. In addition, areas covered by transitional woodland and scrub were important for the open cattle ponds. Bombina variegata was found predominantly in the temporary ponds (e.g. ponds created by cattle and buffalo, dirt road ponds and concrete ponds created for livestock drinking) and Bufo bufo in fishponds. CONCLUSIONS/SIGNIFICANCE: Our Maxent models revealed that the highest probability of occurrence for amphibian ponds was in areas used as farmland. The traditional farming practices combined with a low level of infrastructure development produces a large number of amphibian ponds. The challenge is to harmonize economic development and the maintenance of high densities of ponds in these traditional rural landscapes.

The energy cost of embryonic development in fishes and amphibians, with emphasis on new data from the Australian lungfish, Neoceratodus forsteri.

The rate of oxygen consumption throughout embryonic development is used to indirectly determine the 'cost' of development, which includes both differentiation and growth. This cost is affected by temperature and the duration of incubation in anamniote fish and amphibian embryos. The influences of temperature on embryonic development rate, respiration rate and energetics were investigated in the Australian lungfish, Neoceratodus forsteri, and compared with published data. Developmental stage and oxygen consumption rate were measured until hatching, upon which wet and dry gut-free masses were determined. A measure of the cost of development, the total oxygen required to produce 1 mg of embryonic dry tissue, increased as temperature decreased. The relationship between the oxygen cost of development (C, ml mg(-1)) and dry hatchling mass (M, mg) in fishes and amphibians is described by C = 0.30 M(0.22 0.13 (95% CI)), r (2) = 0.52. The scaling exponent indicates that the cost of embryonic development increases disproportionally with increasing hatchling mass. At 15 and 20°C, N. forsteri cost of development is significantly lower than the regression mean for all species, and at 25°C is lower than the allometrically scaled data set. Unexpectedly, incubation of N. forsteri is long, despite natural development under relatively warm conditions, and may be related to a large genome size. The low cost of development may be associated with construction of a rather sluggish fish with a low capacity for aerobic metabolism. The metabolic rate is lower in N. forsteri hatchlings than in any other fishes or amphibians at the same temperature, which matches the extremely low aerobic metabolic scope of the juveniles.

Carbon nanotube ecotoxicity in amphibians: assessment of multiwalled carbon nanotubes and comparison with double-walled carbon nanotubes.

The potential impact of industrial multiwalled carbon nanotubes (MWNTs) was investigated under normalized laboratory conditions according to the International Standard micronucleus assay ISO 21427-1 for 12 days of half-static exposure to 0.1, 1, 10 and 50 mg/l of MWNTs in water. Three different end points were carried out for 12 days of exposure: mortality, growth inhibition and micronuclei induction in erythrocytes of the circulating blood of larvae. Raman spectroscopy analysis was used to study the presence of carbon nanotubes in the biological samples. Considering the high diversity of carbon nanotubes according to their different characteristics, MWNTs were analyzed in Xenopus larvae, comparatively to double-walled carbon nanotubes used in a previous study in similar conditions. Growth inhibition in larvae exposed to 50 mg/l of MWNTs was evidenced; however, no genetoxicity (micronucleus assay) was noticed, at any concentration. Carbon nanotube localization in the larvae leads to different possible hypothesis of mechanisms explaining toxicity in Xenopus.

A qualitative meta-analysis reveals consistent effects of atrazine on freshwater fish and amphibians.

OBJECTIVE: The biological effects of the herbicide atrazine on freshwater vertebrates are highly controversial. In an effort to resolve the controversy, we conducted a qualitative meta-analysis on the effects of ecologically relevant atrazine concentrations on amphibian and fish survival, behavior, metamorphic traits, infections, and immune, endocrine, and reproductive systems. DATA SOURCES: We used published, peer-reviewed research and applied strict quality criteria for inclusion of studies in the meta-analysis. DATA SYNTHESIS: We found little evidence that atrazine consistently caused direct mortality of fish or amphibians, but we found evidence that it can have indirect and sublethal effects. The relationship between atrazine concentration and timing of amphibian metamorphosis was regularly nonmonotonic, indicating that atrazine can both accelerate and delay metamorphosis. Atrazine reduced size at or near metamorphosis in 15 of 17 studies and 14 of 14 species. Atrazine elevated amphibian and fish activity in 12 of 13 studies, reduced antipredator behaviors in 6 of 7 studies, and reduced olfactory abilities for fish but not for amphibians. Atrazine was associated with a reduction in 33 of 43 immune function end points and with an increase in 13 of 16 infection end points. Atrazine altered at least one aspect of gonadal morphology in 7 of 10 studies and consistently affected gonadal function, altering spermatogenesis in 2 of 2 studies and sex hormone concentrations in 6 of 7 studies. Atrazine did not affect vitellogenin in 5 studies and increased aromatase in only 1 of 6 studies. Effects of atrazine on fish and amphibian reproductive success, sex ratios, gene frequencies, populations, and communities remain uncertain. CONCLUSIONS: Although there is much left to learn about the effects of atrazine, we identified several consistent effects of atrazine that must be weighed against any of its benefits and the costs and benefits of alternatives to atrazine use.

Statewide mapping and assessment of vernal pools: a New Jersey case study.

In 2001, the New Jersey Department of Environmental Protection (NJDEP) adopted rules specifically protecting vernal pool habitat for the first time. Vernal pools are small isolated temporary bodies of water that provide critical breeding habitat for a number of amphibian species. To implement these rules and ultimately afford vernal pools protection, the NJDEP first needed to assemble a statewide database of vernal pool locations. In response, the Rutgers University Center for Remote Sensing and Spatial Analysis (CRSSA) was funded to develop a cost effective technique to map vernal pool locations statewide. The objective of CRSSA's mapping effort was to develop a complete potential vernal pool database to be able to identify individual isolated vernal pools as well as areas of high local density, or 'hotspots'. CRSSA used visual interpretation of leaf-off color infrared digital orthophotography in a computerized GIS environment to identify and map over 13,000 potential vernal pools. Using the 1m scale imagery, we determined the minimum detectable pool size to be on the order of 0.02 ha in size. Subsequent field checking has revealed a 12% error of commission that was due to our inclination towards erring on the side of inclusion in mapping many water features as potential vernal pools. For a vernal pool to receive regulated protection, it must be 'certified' that it serves as habitat for obligate or facultative vernal pool amphibian species. To aid in these efforts, CRSSA developed an interactive internet mapping site to assist NJDEP and its citizen volunteer corps in locating and navigating to their survey areas and to facilitate the on-line submittal of survey observations.

Phorbol esters and adenosine affect the readily releasable neurotransmitter pool by different mechanisms at amphibian motor nerve endings.

Phorbol esters and adenosine have been proposed to interact at common sites downstream of calcium entry at amphibian motor nerve endings. We thus studied the actions and interactions of phorbol esters and adenosine using electrophysiological recording techniques in conjunction with both binomial statistical analysis and high-frequency stimulation at the amphibian neuromuscular junction. To begin this study, we confirmed previous observations that synchronous evoked acetylcholine (ACh) release (reflected as endplate potentials, EPPs) is well described by a simple binomial distribution. We then used binomial analysis to study the effects of the phorbol ester phorbol dibutyrate (PDBu, 100 nM) and adenosine (50 microM) on the binomial parameters n (the number of calcium charged ACh quanta available for release) and p (the average probability of release), where the mean level of evoked ACh release (m) = np. We found that PDBu increased m by increasing the parameter n whilst adenosine reduced m by reducing n; neither agent affected the parameter p. PDBu had no effect on either the potency or efficacy of the inhibition produced by adenosine. Subtle differences between these two agents were revealed by the patterns of EPPs evoked by high-frequency trains of stimuli. Phorbol esters increased ACh release during the early phase of stimulation but not during the subsequent plateau phase. The inhibitory effect of adenosine was maximal at the beginning of the train and was still present with reduced efficacy during the plateau phase. When taken together with previous findings, these present results suggest that phorbol esters increase the immediately available store of synaptic vesicles by increasing the number of primed vesicles whilst adenosine acts at a later stage of the secretory process to decrease the number of calcium-charged primed vesicles.

Ecological risk assessment and testing for endocrine disruption in the aquatic environment.

Field observations of endocrine disruption (ED) in fish and other aquatic species have contributed to a global effort to establish test methods for detecting ED effects in wildlife species. For ecological risk assessment, validated tests are needed for Amphibia, fish and aquatic invertebrates, supported by a tiered approach incorporating mechanistic data and exposure characterisation. The potential for extrapolation of ED data from mammalian to aquatic species may be limited, however, due to significant physiological differences in function and regulation of hormone systems in (aquatic) lower vertebrates and invertebrates. Presently, the OECD is considering a tiered approach for ED risk assessment, incorporating a fish 14-day screening assay (Tier 1); fish development and reproduction tests (both Tier 2); and a fish full life-cycle test (Tier 3). For detection of (anti-)oestrogens, the yolk-precursor protein vitellogenin is an ideal biomarker of exposure and functionally equivalent biomarkers are being sought for (anti-)androgens in fish. At the two higher tiers, impacts are assessed in terms of apical endpoints (e.g. development, breeding behaviour and fecundity) and also gonadal histopathology. Validation of these higher tier tests should include comparison of sensitivity of biochemical and apical endpoints to optimise the value of biomarkers for predicting adverse health effects (e.g. impaired reproduction). The specificity of future OECD fish and amphibian test guidelines for endocrine disrupters needs further consideration through inclusion of mechanistic endpoints based on state-of-the-art molecular endocrinology.

Olfactory assessment of predation risk in the aquatic environment.

The aquatic environment is well suited for the transmission of chemical information. Aquatic animals have evolved highly sensitive receptors for detecting these cues. Here, I review behavioural evidence for the use of chemical cues by aquatic animals for the assessment of predation risk. Chemical cues are released during detection, attack, capture and ingestion of prey. The nature of the cue released depends on the stage of the predation sequence in which cues are released. Predator odours, disturbance pheromones, injury-released chemical cues and dietary cues all convey chemical information to prey Prey use these cues to minimize their probability of being taken on to the next stage of the sequence. The evolution of specialized epidermal alarm substance cells in fishes in the superorder Ostariophysi represent an amplification of this general phenomenon. These cells carry a significant metabolic cost. The cost is offset by the fitness benefit of the chemical attraction of predators. Attempts of piracy by secondary predators interrupt predation events allowing prey an opportunity for escape. In conclusion, chemical cues are widely used by aquatic prey for risk assessment and this has resulted in the evolution of specialized structures among some taxa.

Nitrogen pollution: an assessment of its threat to amphibian survival.

The potential for nitrate to affect amphibian survival was evaluated by examining the areas in North America where concentrations of nitrate in water occur above amphibian toxicity thresholds. Nitrogen pollution from anthropogenic sources enters bodies of water through agricultural runoff or percolation associated with nitrogen fertilization, livestock, precipitation, and effluents from industrial and human wastes. Environmental concentrations of nitrate in watersheds throughout North America range from < 1 to > 100 mg/L. Of the 8,545 water quality samples collected from states and provinces bordering the Great Lakes, 19.8% contained nitrate concentrations exceeding those which can cause sublethal effects in amphibians. In the laboratory lethal and sublethal effects in amphibians are detected at nitrate concentrations between 2.5 and 100 mg/L. Furthermore, amphibian prey such as insects and predators of amphibians such as fish are also sensitive to these elevated levels of nitrate. From this we conclude that nitrate concentrations in some watersheds in North America are high enough to cause death and developmental anomalies in amphibians and impact other animals in aquatic ecosystems. In some situations, the use of vegetated buffer strips adjacent to water courses can reduce nitrogen contamination of surface waters. Ultimately, there is a need to reduce runoff, sewage effluent discharge, and the use of fertilizers, and to establish and enforce water quality guidelines for nitrate for the protection of aquatic organisms.

200 years of amphibian water economy: from Robert Townson to the present.

In the 1790s, Robert Townson established the main features of the water economy of terrestrial amphibians: rapid evaporative water loss in dry surroundings, 'drinking' by absorption of water through the abdominal skin pressed against moist substrates, and use of the urinary bladder as a reservoir from which water is reabsorbed on land. This knowledge was of little interest to the establishment in the first half of the nineteenth century of experimental physiology as a basic medical discipline, when frogs became models in the elucidation of general physiological processes. Townson's pioneer contributions to amphibian physiology were forgotten for 200 years (Jørgensen 1994b). During (1901) and particularly Overton (1904) restored knowledge about amphibian water economy to the level reached by Townson, but the papers had little impact on the young science of animal physiology because they primarily aimed at elucidating the transport of fluids across membranes. Frog skin remained a model preparation in such studies throughout the century. With the establishment of terrestrial ecology early in the century, the relations of animals, including amphibians, to water became a central theme. Concurrently with comparative studies of amphibian water economy in an ecological setting, the subject proceeded as an aspect of animal osmoregulation. Adolph (1920-1930) and Rey (1937a) established the highly dynamic nature of water balance in amphibians in water and on land. Their observations indicated functional links between environment, skin and kidneys, the nature of which remained to be explored. Thorson & Svihla (1943) reopened the ecological approach in a comparative study of the relations between amphibian habitat and tolerance of dehydration. By mid-century, the central themes of amphibian adaptations to terrestrial modes of life were re-established, except for the function of the bladder as a water-depot. During the following decades, a rich literature appeared, particularly focusing on adaptations of amphibians to arid environments. Thus, in the 1970s, it was found that 'waterproofing' of the highly permeable skins by means of skin secretions had evolved independently in several families of tropical arboreal frogs, and that a number of amphibians that aestivate whilst burrowed in dry soil could reduce evaporation by forming cocoons from shed strata cornea. In 1950-1970 the role of bladder urine as a water depot in terrestrial amphibians was recognized: this did not change the established view of water balance in terrestrial amphibians as alternating between dehydration on land and rehydration in response to the deficit in body water. Amphibians may, however, maintain normal water balance whether the ambient medium is water or air by means of little understood integrated mechanisms in control of cutaneous drinking behaviour, water permeability of the skin and bladder wall, and urine production.