Chronic effects of environmentally-relevant concentrations of lead in Pelophylax nigromaculata tadpoles: Threshold dose and adverse effects.

Lead (Pb) is a common heavy metal in the natural environment, but its concentration has been increasing alongside widespread industrial and agricultural development in China. The dark-spotted frog Pelophylax (formerly Rana) nigromaculata (Anura: Ranidae) is distributed across East Asia and inhabits anthropogenic habitats such as farmland. Here, P. nigromaculata tadpoles (Gosner stage 19-46) were exposed to Pb at different concentrations (0, 40, 80, 160, 320, 640 and 1280µg/L) and Pb-induced survival, metamorphosis time, development, malformations, mobility and gonad structure were monitored. The results showed that above the threshold concentration of Pb, adverse effects were obvious. As the concentration of Pb increased, the adverse effects on different traits followed different patterns: the effects on hindlimb length, survival rate, metamorphosis rate, total malformation rate, swimming speed and jumping speed largely exhibited a linear pattern; the effects on snout-vent length, body mass and forelimb length largely exhibited a bimodal pattern. Sex ratio and gonadal histology were not affected by Pb, suggesting that Pb is not strongly estrogenic in P. nigromaculata.

Gonadal abnormalities in frogs (Lithobates spp.) collected from managed wetlands in an agricultural region of Nebraska, USA.

Nebraska's Rainwater Basin (RWB) provides important wetland habitat for North American migratory birds. Concern exists that pesticide and nutrient runoff from surrounding row-crops enters wetlands degrading water quality and adversely affecting birds and wildlife. Frogs may be especially vulnerable. Plains leopard (Lithobates blairi) metamorphs from RWB wetlands with varying concentrations of pesticides were evaluated for a suite of biomarkers of exposure to endocrine active chemicals. Froglets had ovarian dysgenesis, high rates of testicular oocytes, and female-biased sex ratios however, there was no clear statistical association between pesticide concentrations and biomarkers. Data interpretation was hindered because timing and duration of exposures were unknown and due to an incomplete understanding of L. blairi sexual development. Emphasis is on describing the complex developmental biology of closely-related leopard frogs, how this understanding can explain RWB L. blairi anomalies, and the need for sampling at the appropriate life stage.

Combined effects of malathion and nitrate on early growth, abnormalities, and mortality of wood frog (Rana sylvatica) tadpoles.

Use of pesticides and other agro-chemicals adversely influence amphibians either directly by killing them or by inducing sublethal, chronic effects. Many studies have investigated the effect of mixtures of pesticides or fertilizers. We studied the combined effects of nitrate and malathion ([(dimethoxy phosphino thioyl] butanediotae) on the early growth, expression of abnormalities, and mortality of Wood Frog (Rana sylvatica) tadpoles in a laboratory experiment. Tadpoles were treated with factorial combinations of 0, 8, and 16 mg NO(3)-N l(-1) and 0, 250, 500, and 1,000 µg malathion l(-1) for a period of 14 days. Feeding behaviour, total length, mean tadpole mass, frequencies of abnormalities, and survivorship in each treatment were recorded. Malathion showed a significant negative influence on all parameters and strongly influenced the frequencies of morphological anomalies. In contrast, nitrate alone did not produce any significant effects on behavior, total length, tadpole mass, or the frequency of abnormalities during the experiment. Malathion and nitrate had an interactive effect on tadpole length and mass, but did not affect any other parameters. Our results suggest that exposure to malathion, even at relatively low concentrations can have serious negative consequences for Wood Frog tadpoles. In addition, our results also indicate that there was little synergistic interaction between malathion and nitrate exposure under laboratory conditions.

Morphological abnormalities in frogs from a rice-growing region in NSW, Australia, with investigations into pesticide exposure.

Three frog species (Limnodynastes tasmaniensis, Limnodynastes fletcheri and Litoria raniformis) were surveyed in rice bays of the Coleambally Irrigation Area (CIA), NSW, Australia, during the rice-growing seasons of 2005/2006 and 2006/2007. A total external morphological abnormality index of 7.0% was observed in frogs of the CIA (n=1,209). The types and frequencies of abnormalities were typical of reports from agricultural areas with ectrodactyly being the most common aberration. A relatively low abnormality index of 1.2% was observed in L. raniformis (n=87) compared to indices of 7.1% and 8.2% observed in L. fletcheri (n=694) and L. tasmaniensis (n=428), respectively. No conclusive evidence was found of unnaturally high rates of intersex, gonadal maldevelopment or unbalanced sex ratios in any species. Rice bay surface waters differed significantly in mean pesticide concentrations of atrazine and metolachlor on farms growing rice and corn compared to farms with rice as the sole crop. However, the similar abnormality indices observed in recent metamorphs emerging from these two farm types provided no evidence to suggest a link between larval exposure to the measured pesticides and developmental malformations.

Alterations of biochemical parameters in malformed Indian rice frogs, Rana limnocharis from Southern Taiwan.

The purpose of this study is to investigate the factors that cause malformed frogs in upstream Kaoping river (KP site) and Tungkang river (T site) of Southern Taiwan. In this experiment, the activities of monooxygenase (MO), glutathione-S-transferase (GST), acetylcholinesterase (AchE) as well as the concentration of vitellogenin (Vg) in the liver were measured. Results show that activities of MO, GST and AchE, and Vg levels in normal frogs (male/female) were 0.09 +/- 0.02/0.09+/-0.01 deltaA min(-1) mg(-1) protein, 0.12 +/- 0.04/0.13 +/- 0.04 deltaA min(-1) mg(-1) protein, 6.13 +/- 2.69/6.01 +/- 2.09 U mg(-1) protein and 0.87 +/- 0.42/2.18 +/- 0.50 microg mg(-1) protein, respectively. Activities of MO, GST and AchE, and Vg levels in malformed frogs (male/female) were 0.15 +/- 0.04/0.21 +/- 0.07 deltaA min(-1) mg(-1) protein, 0.27 +/- 0.08/0.30 +/- 0.12 deltaA min(-1) mg(-1) protein, 4.59 +/- 2.71/5.19 +/- 3.74 U mg(-1) protein and 1.46 +/- 0.61/3.15 +/- 0.88 microg mg(-1) protein, respectively in KP site, and were 0.16 +/- 0.69/0.1 +/- 80.07 deltaA min(-1) mg(-1) protein, 0.21 +/- 0.07/0.24 +/- 0.08 deltaA min(-1) mg(-1) protein, 5.13 +/- 4.58/3.94 +/- 1.33 U mg(-1) protein and 2.23 +/- 1.47/4.11 +/- 1.63 microg mg(-1) protein, respectively in T site. These results indicate that male and female malformed frogs in both rivers upstream are found with higher activities. No significant difference in AchE activity was found between normal and malformed frogs in this investigation. It is therefore reasonable to speculate that the organic chemicals released from agricultural activities are presumable the main factors that lead to the malformation of frogs.

Prevalence of malformed frogs in Kaoping and Tungkang river basins of southern Taiwan.

In this study we found many amphibians with bizarre appearances, known as malformations in Pingtung County southern Taiwan. For this investigation we collected frogs inhabiting the Kaoping and Tungkang river watersheds between February 2006 and June 2007. Among the total number of 10,909 normal frogs (i.e., anurans) collected during the investigation period, the Indian rice frogs (Rana limnocharis) account for the greatest number next is the Chinese bullfrog (Rana rugulosa). Of all the 244 captured malformed frogs, the Indian rice frog account for the greatest proportion. These malformed frogs have their main distribution in upstream areas of these two rivers. Our result indicates that the appearance rate of malformed frogs is 1.8% in the upstream reaches of the Kaoping River and 2.6%, and 0.8%, respectively in the upstream and midstream reaches of the Tungkang river. The most-commonly-found malformation is the lack of palms, followed by the lack of appendages, exostosis, and a malformed appendicular. It is, therefore, reasonable to speculate that the causes for the malformation may be related to the increased organic pollutants and agricultural chemicals used in the upstream reaches of these two rivers.

Intersex frogs concentrated in suburban and urban landscapes.

The occurrence of intersex characteristics in amphibians has been linked to pesticide exposure in the laboratory and proximity to agricultural activity within natural populations. But, overall, the ecology of amphibian intersex is poorly known and, specifically, its occurrence in many landscape types and regions is unstudied. We offer the first analysis of the frequency of amphibian intersex across a range of land covers representing the major landscape types within a region. We used remotely sensed information to characterize land cover surrounding 4774 potential sampling locations within the Connecticut River Valley. From among these, we selected 24 ponds to collect postmetamorphic green frogs (Rana clamitans) from four land cover types: undeveloped, agricultural, suburban, and urban. Collected males were preserved and, then, prepared gonadal tissue samples were screened for the presence of testicular oocytes. A total of 233 animals was examined. Thirteen percent of all male green frogs had gonads containing testicular oocytes. Sexual abnormalities were not randomly distributed among sites or landscape types. Suburban landscapes had the highest frequency of abnormalities (21%), and both suburban and urban land covers were positively associated with the presence of abnormalities within a population. There was no evidence of a positive association with agricultural land cover. Examination of amphibian intersex across a range of contexts reveals that developed landscapes may be hotspots for abnormal sexual development. This new finding suggests that other mechanisms, not previously considered, could contribute to intersex in natural amphibian populations.

Explanations for deformed frogs: plenty of research left to do (a response to Skelly and Benard).

Our recent study (Ballengeé and Sessions, 2009. J Exp Zool (Mol Dev Evol) 312B:1-10) shows that deformed frogs with missing limbs can be explained by sublethal "selective predation" by predators that are too small, or have mouthparts that are too small, to consume whole tadpoles. Skelly and Benard do not agree with our conclusions and feel that they are not well founded. Here we respond to their critique.

Intersex gonads in frogs: understanding the time course of natural development and role of endocrine disruptors.

The paucity of data on sexual development of anuran amphibians has played an important role in the recent controversy over atrazine exposure. Although some studies have demonstrated the presence of abnormal gonads in control treatments, others have not, leading to varying interpretations of the effects of atrazine exposure on sexual development. However, the timing of development varies among anuran amphibians such that, at any snapshot in time, different species may exhibit different stages of sexual differentiation. We examined three species representing each of the differentiation rates (Bufo americanus=retarded rate; Hyla versicolor=basic rate; Rana sphenocephala=accelerated rate), to examine the natural time course of sexual development along with the influence of atrazine exposure. For each species, exposure to atrazine (1, 3, 10, 30 parts per billion), 17-beta-estradiol or control water occurred throughout larval life. Gonad histology was performed at 3-week intervals during the larval period or at a juvenile stage to examine the proportion of males, females, underdeveloped testes, testicular oocytes (TO; testes with 0-30% oocytes), and ovotestes (OVTs; testes with>30% oocytes). Our results illustrate that a phase of intersex gonads (TO or OVT) is normal during R. sphenocephala sexual development, a species representing the accelerated differentiation rate. Further, intersex gonads were found in juvenile stages of B. americanus and H. versicolor, representing retarded and basic rates, respectively, suggesting that a phase of intersex may be common regardless of differentiation rate. Moreover, these data highlight the importance of longitudinal studies rather than snapshots in time.

Road proximity increases risk of skeletal abnormalities in wood frogs from National Wildlife Refuges in Alaska.

BACKGROUND: Skeletal and eye abnormalities in amphibians are not well understood, and they appear to be increasing while global populations decline. Here, we present the first study of amphibian abnormalities in Alaska. OBJECTIVE: In this study we investigated the relationship between anthropogenic influences and the probability of skeletal and eye abnormalities in Alaskan wood frogs (Rana sylvatica). METHODS: From 2000 to 2006, we examined 9,269 metamorphic wood frogs from 86 breeding sites on five National Wildlife Refuges: Arctic, Innoko, Kenai, Tetlin, and Yukon Delta. Using road proximity as a proxy for human development, we tested relationships between skeletal and eye abnormalities and anthropogenic effects. We also examined a subsample of 458 frogs for the trematode parasite Ribeiroia ondatrae, a known cause of amphibian limb abnormalities. RESULTS: Prevalence of skeletal and eye abnormalities at Alaskan refuges ranged from 1.5% to 7.9% and were as high as 20% at individual breeding sites. Proximity to roads increased the risk of skeletal abnormalities (p = 0.004) but not eye abnormalities. The only significant predictor of eye abnormalities was year sampled (p = 0.006). R. ondatrae was not detected in any Alaskan wood frogs. CONCLUSIONS: Abnormality prevalence at road-accessible sites in the Kenai and Tetlin refuges is among the highest reported in the published literature. Proximity to roads is positively correlated with risk of skeletal abnormalities in Alaskan wood frogs.

Ecology: global warming and amphibian losses.

Is global warming contributing to amphibian declines and extinctions by promoting outbreaks of the chytrid fungus Batrachochytrium dendrobatidis? Analysing patterns from the American tropics, Pounds et al. envisage a process in which a single warm year triggers die-offs in a particular area (for instance, 1987 in the case of Monteverde, Costa Rica). However, we show here that populations of two frog species in the Australian tropics experienced increasing developmental instability, which is evidence of stress, at least two years before they showed chytrid-related declines. Because the working model of Pounds et al. is incomplete, their test of the climate-linked epidemic hypothesis could be inconclusive.

Proximity to pollution sources and risk of amphibian limb malformation.

The cause of limb deformities in wild amphibian populations remains unclear, even though the apparent increase in prevalence of this condition may have implications for human health. Few studies have simultaneously assessed the effect of multiple exposures on the risk of limb deformities. In a cross-sectional survey of 5,264 hylid and ranid metamorphs in 42 Vermont wetlands, we assessed independent risk factors for nontraumatic limb malformation. The rate of nontraumatic limb malformation varied by location from 0 to 10.2%. Analysis of a subsample did not demonstrate any evidence of infection with the parasite Ribeiroia. We used geographic information system (GIS) land-use/land-cover data to validate field observations of land use in the proximity of study wetlands. In a multiple logistic regression model that included land use as well as developmental stage, genus, and water-quality measures, proximity to agricultural land use was associated with an increased risk of limb malformation (odds ratio = 2.26; 95% confidence interval, 1.42-3.58; p < 0.001). The overall discriminant power of the statistical model was high (C = 0.79). These findings from one of the largest systematic surveys to date provide support for the role of chemical toxicants in the development of amphibian limb malformation and demonstrate the value of an epidemiologic approach to this problem.

Adverse effects of ecologically relevant dietary mercury exposure in southern leopard frog (Rana sphenocephala) larvae.

Southern leopard frog (Rana sphenocephala) larvae were exposed to experimental diets supplemented with aufwuchs from control and mercury-enriched mesocosms combined in proportions intended to mimic mercury concentrations and speciation in aufwuchs observed from aquatic systems contaminated by atmospheric deposition. Observations on rates of mortality, malformation, and larval growth and development were made for 254 d. Increased incidence of mortality, malformation, and changes in growth and development were observed at concentrations that reflect the highest concentrations expected in the amphibian diet from atmospheric deposition (1,500-3,300 ng Hg/g dry wt). The results of this study are probably more ecologically realistic than results obtained from previous studies of aqueous mercury toxicity and suggest that dietary mercury exposure in habitats contaminated primarily by atmospheric deposition has the potential to cause adverse effects in amphibian larvae.

Differences in susceptibility of various life stages of amphibians to pesticide exposure.

Pesticide exposure is discussed as one of the adverse factor that causes impairment of amphibian larval development. Susceptibility of tadpoles to pesticide exposure differs depending on the developmental stages at which individuals are contaminated. This study focused on the influence of the pyrethroid insecticide cypermethrin on the hatching success, mortality and deformities in further development, duration of metamorphosis, and growth of Rana arvalis tadpoles exposed at various life stages. Eggs were harmed significantly by exposure to different concentrations of alpha-cypermethrin dependent on exposure time. Amphibian embryos hatched with apparent abnormalities indicate insufficient protection by the jelly coat surrounding the embryo. Exposed individuals exhibited typical signs of cyano pyrethroid poisoning (twisting, writhing, abnormal swimming). During the life-stage treatments the observed morphological deformities and behavioral abnormalities lead mostly to death of the tadpoles in later development. Individuals exposed to alpha-cypermethrin in early life stages (as eggs or newly hatched tadpoles) metamorphosed earlier than in the corresponding control. However, exposure of the tadpoles throughout their whole development prolonged the metamorphosis. These metamorphs were distinguished by a much more compact physique (significant reduction in length and significant increase in weight compared to the corresponding control) so as to resist the adverse conditions. Observed effects of alpha-cypermethrin exposure on various life stages of R. arvalis tadpoles refer to the danger potential of this insecticide for amphibian development. Any kind of deformation influences the physiological fitness of the individuals (e.g. increased vulnerability to predation). Diminished hatching success, delays in metamorphosis and body growth impact negatively on the viability of amphibian populations in the long term. In combination with other anthropogenic influences (destruction or dissipation of habitats), these effects can cause the local extinction of amphibian species.

A regional survey of malformed frogs in Minnesota (USA) (Minnesota malformed frogs).

In late 1995, school children discovered malformed frogs in a south central Minnesota pond. Press coverage resulted in numerous citizen reports of frog malformation across Minnesota in 1996. After some initial site investigation, 3 affected frog sites and 4 nearby reference sites were selected for more detailed evaluation. Field biologists made 89 visits to study sites beginning spring 1997 through fall 1999 to examine the number and type of frog malformations. Over 5,100 Leopard frogs (Rana pipiens) were captured and examined at all study sites. Water elevations and associated littoral inundation were recorded from 1997-2000. Results indicate that malformation occurred at all study sites above historical background levels. Rana pipiens malformation across all sites over three seasons averaged 7.9% and ranged from 0 to 7% at reference sites and 4 to 23% at affected sites. At one northern Minnesota site, mink frog (Rana septentrionalis) malformation was 75% in 1998. A site characteristic common to the most affected sites was an elastic zone of littoral inundation. Climate driven hydrologic variation likely influenced water depth and associated breeding locations.

How trematodes cause limb deformities in amphibians.

We used trematode cyst infestation to induce limb deformities in two species of frogs of the genus Rana and compared them to deformities induced by surgical limb bud rotations. The specific deformities produced by both treatments closely resemble those of wild-caught deformed amphibians and are consistent with a known developmental response to disruption of the spatial organization of cells in developing limb buds. Histological analysis showed that trematode cysts cause massive disruption and abnormal cellular growth involving the limb buds of infected individuals. Our results indicate that trematode cyst infestation causes deformities in frogs by perturbation of the positional relationships of cells in developing limb buds. The crippling effects of cyst-infection on frogs may reflect complex co-evolutionary interactions among trematodes, frogs, and other hosts in the trematode's life cycle.

Developmental capacity and chromosome number in the offspring of artificially produced autotetraploids of Rana nigromaculata.

A large number of autotetraploids of Rana nigromaculata were produced in order to assess their developmental capacity and chromosome number in their offspring. An original autotetraploid male was first produced by transplanting a nucleus from an embryo (triploid) into an unfertilized egg. Next, eggs were inseminated with sperm of the autotetraploid male, then cold-treated to obtain first-, second-, and third-generation offspring. According to an investigation of the chromosome numbers of the tadpoles by the tail-tip squash method, the three generations of offspring included many tetraploids (50-80%), as well as some diploids, triploids, hexaploids and mosaics at the early tadpole stage. In addition, several percent of the second-and third-generation offspring were found to be aneuploids. Evidently, a complete set of diploid chromosomes was not precisely transferred to all of the next-generation offspring from the sperm of the artificially produced autotetraploid males. These observations suggest that there were some abnormalities in the course of spermatogenesis in the male autotetraploid frogs.

Assessment of the risk of solar ultraviolet radiation to amphibians. III. Prediction of impacts in selected northern midwestern wetlands.

Solar ultraviolet radiation, especially UVB (280-320 nm), has been hypothesized to be at least partially responsible for adverse effects (e.g., declines and malformations) in amphibian species throughout the world. Evaluation of this hypothesis has been limited by the paucity of high-quality UV dose-response data and reliable estimates of typical UV doses that occur in amphibian habitats. In this preliminary risk assessment for effects of UV radiation on amphibians, dose-response relationships quantified in outdoor experiments were compared with UV exposure estimates for 26 wetlands in northern Minnesota and Wisconsin. A comparison of wetland doses, derived from model prediction, historical data, and dissolved organic carbon (DOC) characterization, with experimental effects levels for green (R. clamitans), northern leopard (R. pipiens), and mink (R. septentrionalis) frogs indicated that the risk of mortality and malformations due to UV exposure is low for the majority of wetlands evaluated. Wetland UV dose, averaged over the entire breeding season, exceeded effects doses for mortality for all three species in two of the 26 wetlands examined and for one species in an additional wetland. On the basis of evidence that shorter term doses caused mortality in amphibian larvae, 3-day doses were also evaluated. In three of the wetlands examined, 3-day doses in excess of 85% of full sunlight (the level that appeared to trigger effects in controlled experimentation) occurred at frequencies ranging 22-100% for all three species and at frequencies ranging from 15% to 58% for R. pipiens and R. septentrionalis in three additional wetlands. Risk of malformation in R. pipiens was apparent in five of the 26 wetlands evaluated. Overall, estimated UVB doses in 21 of the wetlands never exceeded experimental effects doses for mortality or malformations. These results suggest that most amphibians are not currently at significant risk for UVB effects in northern Minnesota and Wisconsin wetlands. However, continued reduction of ozone and other global climate change effects may increase UV doses in wetlands, suggesting that the risk of UV to amphibians should continue to be monitored and studied.

Limb malformations and abnormal sex hormone concentrations in frogs.

Declines in amphibian populations, and amphibians with gross malformations, have prompted concern regarding the biological status of many anuran species. A survey of bullfrogs, Rana catesbeiana, and green frogs, Rana clamitans, conducted in central and southern New Hampshire showed malformed frogs at 81% of the sites sampled (13 of 16 sites). Brain gonadotropin-releasing hormone (GnRH) and the synthesis of androgens and estradiol, hormones essential to reproductive processes, were measured from limb-malformed and normal (no limb malformation) frogs. Normal frogs had significantly higher concentrations (nearly 3-fold) of in vitro produced androgens and of brain GnRH than malformed frogs. Because most malformations are thought to occur during development, we propose that environmental factors or endocrine-disrupting chemicals that may cause developmental abnormalities also act during early development to ultimately cause abnormally reduced GnRH and androgen production in adult frogs. The consequences of reduced GnRH and androgens on anuran reproductive behavior and population dynamics are unknown but certainly may be profound and warrant further research.