Rana Pipiens Tadpoles Research Articles

The effects of two short-chain perfluoroalkyl carboxylic acids (PFCAs) on northern leopard frog (Rana pipiens) tadpole development

Short-chain perfluoroalkyl carboxylic acids (PFCAs) have been detected in the environment globally. The presence and persistence of these compounds in the environment may lead to chronic wildlife exposure. We used northern leopard frog (Rana pipiens) tadpoles to investigate the chronic toxicity and the bioconcentration of two short-chain PFCAs, perfluorobutanoic acid (PFBA) and perfluorohexanoic acid (PFHxA). We exposed Gosner stage 25 tadpoles to PFBA and PFHxA (as individual chemicals) at nominal concentrations of 0.1, 1, 10, 100, and 1000 µg/L for 43–46 days. Tadpoles exposed to 0.1 to 100 µg/L of PFBA and PFHxA had significantly higher mean snout-to-vent lengths, mean masses, and scaled mass indexes than control tadpoles. These results indicate that exposure to short-chain PFCAs influences tadpole growth. Further investigation into the mechanism(s) causing the observed changes in tadpole growth is warranted. We observed a significantly higher proportion of males in the PFBA 1 µg/L treatment group, however further histological analyses are required to confirm visual sex identification before making concrete conclusions on the effects of PFCAs on amphibian sex ratios. PFBA concentrations in tissues were higher than PFHxA concentrations; a pattern that contrasts with previously published studies using fish, suggesting potential differences between taxa in PFBA and PFHxA bioconcentration. Bioconcentration factors were <10 L/kg wet weight, indicating low bioconcentration potential in tadpoles. Our results suggest that PFBA and PFHxA may have effects at environmentally-relevant concentrations (0.1–10 µg/L) and further investigation is required before these compounds can be deemed a “safe” alternative to their long-chain counterparts.

Can predators stabilize host-parasite interactions? Changes in aquatic predator identity alter amphibian responses and parasite abundance across life stages.

The role of parasites can change depending on the food web community. Predators, for instance, can amplify or dilute parasite effects on their hosts. Likewise, exposure to parasites or predators at one life stage can have long-term consequences on individual performance and survival, which can influence population and disease dynamics. To understand how predators affect amphibian parasite infections across life stages, we manipulated exposure of northern leopard frog (Rana pipiens) tadpoles to three predators (crayfish [Orconectes rusticus], bluegill [Lepomis macrochirus], or mosquitofish [Gambusia affinis]) and to trematode parasites (Echinostoma spp.) in mesocosms and followed juveniles in outdoor terrestrial enclosures through overwintering. Parasites and predators both had strong impacts on metamorphosis with bluegill and parasites individually reducing metamorph survival. However, when fish were present, the negative effects of parasites on survival was not apparent, likely because fish altered community composition via increased algal food resources. Bluegill also reduced snail abundance, which could explain reduced abundance of parasites in surviving metamorphs. Bluegill and parasite exposure increased mass at metamorphosis, which increased metamorph jumping, swimming, and feeding performance, suggesting that larger frogs would experience better terrestrial survival. Effects on size at metamorphosis persisted in the terrestrial environment but did not influence overwintering survival. Based on our results, we constructed stage-structured population models to evaluate the lethal and sublethal effects of bluegill and parasites on population dynamics. Our models suggested that positive effects of bluegill and parasites on body size may have greater effects on population growth than the direct effects of mortality. This study illustrates how predators can alter the outcome of parasitic infections and highlights the need for long-term experiments that investigate how changes in host-parasite systems alter population dynamics. We show that some predators reduce parasite effects and have indirect positive effects on surviving individuals potentially increasing host population persistence.

Multi-species Aquatic Toxicity Assessment of 1-Methyl-3-Nitroguanidine (MeNQ).

The Army is replacing traditional munitions with insensitive munitions (IM) resistant to accidental detonation. The aquatic toxicity of 1-methyl-3-nitroguanidine (MeNQ), which is being assessed for potential use in IM formulations, remains largely untested. The present study fills a number of critical data gaps for MeNQ aquatic toxicity by evaluating effects across two vertebrate and five invertebrate species. Specifically, responses in larval Pimephales promelas, Rana pipiens tadpoles, Chironomus dilutus, Lumbriculus variegatus, Hydra littoralis, Hyalella azteca, and Daphnia pulex were assessed in MeNQ exposures across various acute, subchronic, and chronic bioassays. Overall, survival was unaffected in most of the MeNQ exposures where significant lethal effects were only observed in D. pulex, H. littoralis, and C. dilutus and only at concentrations ≥ 2186mg/L. Significant sublethal effects on growth were observed for C. dilutus at 903mg/L and H. azteca at 1098mg/L in 10-d assays. Significantly decreased reproduction was observed at 2775mg/L for H. azteca in a chronic 35-d assay and at 174mg/L for D. pulex in the 11-d three-brood assay representing a sublethal effect one order of magnitude more sensitive than the effective lethal concentration for D. pulex (2987mg/L). Degradation of MeNQ in ultraviolet light (UV) greatly increased toxicity to D. pulex. Specifically, exposure to a MeNQ solution that was completely UV-degraded prior to D. pulex exposures resulted in an 11-d LC50 of 6.1mg/L and a 50% reduction in reproduction at 3.125mg/L, based on the original MeNQ parent-compound concentrations.

Cascading effects of insecticides and road salt on wetland communities.

Novel stressors introduced by human activities increasingly threaten freshwater ecosystems. The annual application of more than 2.3 billion kg of pesticide active ingredient and 22 billion kg of road salt has led to the contamination of temperate waterways. While pesticides and road salt are known to cause direct and indirect effects in aquatic communities, their possible interactive effects remain widely unknown. Using outdoor mesocosms, we created wetland communities consisting of zooplankton, phytoplankton, periphyton, and leopard frog (Rana pipiens) tadpoles. We evaluated the toxic effects of six broad-spectrum insecticides from three families (neonicotinoids: thiamethoxam, imidacloprid; organophosphates: chlorpyrifos, malathion; pyrethroids: cypermethrin, permethrin), as well as the potentially interactive effects of four of these insecticides with three concentrations of road salt (NaCl; 44, 160, 1600 Cl- mg/L). Organophosphate exposure decreased zooplankton abundance, elevated phytoplankton biomass, and reduced tadpole mass whereas exposure to neonicotinoids and pyrethroids decreased zooplankton abundance but had no significant effect on phytoplankton abundance or tadpole mass. While organophosphates decreased zooplankton abundance at all salt concentrations, effects on phytoplankton abundance and tadpole mass were dependent upon salt concentration. In contrast, while pyrethroids had no effects in the absence of salt, they decreased zooplankton and phytoplankton density under increased salt concentrations. Our results highlight the importance of multiple-stressor research under natural conditions. As human activities continue to imperil freshwater systems, it is vital to move beyond single-stressor experiments that exclude potentially interactive effects of chemical contaminants.

Genomic investigations of acute munitions exposures on the health and skin microbiome composition of leopard frog (Rana pipiens) tadpoles

Natural communities of microbes inhabiting amphibian skin, the skin microbiome, are critical to supporting amphibian health and disease resistance. To enable the pro-active health assessment and management of amphibians on Army installations and beyond, we investigated the effects of acute (96h) munitions exposures to Rana pipiens (leopard frog) tadpoles and the associated skin microbiome, integrated with RNAseq-based transcriptomic responses in the tadpole host. Tadpoles were exposed to the legacy munition 2,4,6-trinitrotoluene (TNT), the new insensitive munition (IM) formulation, IMX-101, and the IM constituents nitroguinidine (NQ) and 1-methyl-3-nitroguanidine (MeNQ). The 96h LC50 values and 95% confidence intervals were 2.6 (2.4, 2.8) for ΣTNT and 68.2 (62.9, 73.9) for IMX-101, respectively. The NQ and MeNQ exposures caused no significant impacts on survival in 96h exposures even at maximum exposure levels of 3560 and 5285 mg/L, respectively. However, NQ and MeNQ, as well as TNT and IMX-101 exposures, all elicited changes in the tadpole skin microbiome profile, as evidenced by significantly increased relative proportions of the Proteobacteria with increasing exposure concentrations, and significantly decreased alpha-diversity in the NQ exposure. The potential for direct effects of munitions exposure on the skin microbiome were observed including increased abundance of munitions-tolerant phylogenetic groups, in addition to possible indirect effects on microbial flora where transcriptional responses suggestive of changes in skin mucus-layer properties, antimicrobial peptide production, and innate immune factors were observed in the tadpole host. Additional insights into the tadpole host's transcriptional response to munitions exposures indicated that TNT and IMX-101 exposures significantly enriched transcriptional expression within type-I and type-II xenobiotic metabolism pathways, where dose-responsive increases in expression were observed. Significant enrichment and increased transcriptional expression of heme and iron binding functions in the TNT exposures served as likely indicators of known mechanisms of TNT toxicity including hemolytic anemia and methemoglobinemia. The significant enrichment and dose-responsive decrease in transcriptional expression of cell cycle pathways in the IMX-101 exposures was consistent with previous observations in fish, while significant enrichment of immune-related function in response to NQ exposure were consistent with potential immune suppression at the highest NQ exposure concentration. Finally, the MeNQ exposures elicited significantly decreased transcriptional expression of keratin 16, type I, a gene likely involved in keratinization processes in amphibian skin. Overall, munitions showed the potential to alter tadpole skin microbiome composition and affect transcriptional profiles in the amphibian host, some suggestive of potential impacts on host health and immune status relevant to disease susceptibility.

Effects of Environmental Contaminants at Great Bay National Wildlife Refuge on Anuran Development, Gonadal Histology, and Reproductive Steroidogenesis: A Comparison of In Situ and Laboratory Exposures.

Previous monitoring at Great Bay National Wildlife Refuge (NWR), Newington, New Hampshire documented high prevalence of amphibian malformations at sites contaminated with potential endocrine active compounds. In the present study, a combination of in situ and laboratory experiments were used to determine whether contaminants present in the sites affect amphibian growth and reproductive development. Wood frog (Rana sylvatica) tadpoles were exposed in situ at four sites (Ferry Way, Beaver Pond, Lower Peverly, and Stubbs Pond) at Great Bay NWR and northern leopard frog (Rana pipiens) tadpoles were exposed in the lab to sediments collected from three sites (Beaver Pond, Ferry Way, Stubbs Pond) at Great Bay NWR as well as a positive (estradiol) and negative control. High mortality was observed at Stubbs Pond and extended larval period at Beaver Pond in the in situ exposure. Only three malformations were noted in the lab experiment, whereas there was a 63% prevalence of rounded femurs in Beaver Pond metamorphs in the in situ exposure. Only 2.4% (5 of 207) of R. sylvatica metamorphs exhibited abnormal reproductive development, whereas intersex metamorphs occurred in treatments and controls in the lab experiment at rates as high as 26%. Reproductive development was more advanced and estradiol to androgen ratios reduced in male metamorphs from Beaver Pond in both the in situ and lab exposures. DDT, PCBs, and PAHs were detected in sediments at Great Bay NWR at concentrations that exceed regulatory or guidance values, with concentrations of PAHs being highest at Lower Peverly Pond and DDT highest at Stubbs Pond. The effects on anuran development may be attributable to the primary contaminants-DDT and PCBs-acting on the thyroid and gonadal axes.

Uptake and Depuration of Four Per/Polyfluoroalkyl Substances (PFASS) in Northern Leopard Frog Rana pipiens Tadpoles

Per/polyfluoroalkyl substances (PFASs) are persistent organic contaminants that are ubiquitous in surface waters. To date, their effects on aquatic systems, especially amphibians, are poorly understood. We examined the uptake and depuration of perfluorooctanesulfonate (PFOS), perfluorohexanesulfonate (PFHxS), perfluorooctanoic acid (PFOA), and 6:2 fluorotelomer sulfonate (6:2 FTS) in northern leopard frog (Rana pipiens) tadpoles. Whole-body concentrations were examined every 10 d during constant aqueous exposure to targeted concentrations of 10, 100, and 1000 μg/L for 40 d and for 30 d during depuration. Effects of PFAS exposure on length and development were also examined. Rapid uptake led to steady state concentrations by 10 d for most exposures. PFOS accumulated to the highest levels with whole-body bioconcentration factor (BCF) values at 40 d ranging from 19.6 to 119.3. The remaining PFASs were not found to bioconcentrate (BCF < 1.0 at 40 d). Furthermore, some BCF values decreased during the exposure ...

Toxicity of the conventional energetics TNT and RDX relative to new insensitive munitions constituents DNAN and NTO in Rana pipiens tadpoles.

An initiative within the US military is targeting the replacement of traditional munitions constituents with insensitive munitions to reduce risk of accidental detonation. The purpose of the present study was to comparatively assess toxicity of the traditional munitions constituents 2,4,6-trinitrotoluene (TNT) and 1,3,5-trinitroperhydro-1,3,5-triazine (RDX) with the new insensitive munitions constituents 2,4-dinitroanisole (DNAN) and 3-nitro-1,2,4-triazol-5-one (NTO). The following exposure durations were performed with Rana pipiens (leopard frog) tadpoles: TNT and DNAN, 96 h and 28 d; RDX, 10 d and 28 d; NTO, 28 d. The 96-h 50% lethal concentration (LC50) values and 95% confidence intervals for TNT and DNAN were 4.4 mg/L (4.2 mg/L, 4. 7 mg/L) and 24.3 mg/L (21.3 mg/L, 27.6 mg/L), respectively. No significant impacts on survival were observed in the 10-d exposure to RDX up to 25.3 mg/L. Effects on tadpole swimming distance were observed with a lowest-observed-effect concentration (LOEC) of 5.9 mg/L RDX. In the 28-d exposures, the LOECs for survival for TNT, DNAN, and NTO were 0.003 mg/L, 2.4 mg/L, and 5.0 mg/L, respectively. No significant mortality was observed in the RDX chronic 28-d exposure up to the highest treatment level tested of 28.0 mg/L. Neither tadpole developmental stage nor growth was significantly affected in any of the 28-d exposures. Rana pipiens were very sensitive to chronic TNT exposure, with an LOEC 3 orders of magnitude lower than those for insensitive munitions constituents DNAN and NTO.

Carbaryl Concentration Gradients in Realistic Environments and Their Influence on Our Understanding of the Tadpole Food Web

Although exposure to pesticides has been correlated with amphibian declines, the mechanism of their role remains enigmatic. Declines have been associated with sublethal exposure, but few outdoor studies have evaluated impacts of low pesticide concentrations. Understanding the effects of a range of pesticide concentrations on amphibians in outdoor mesocosms provides a framework for both direct and indirect effects of exposure. Indirect effects are challenging to glean from lab studies, which typically lack a food web. Our design tested direct and indirect effects of exposure to the insecticide carbaryl on the American toad (Bufo americanus) and the northern leopard frog (Rana pipiens) tadpole survival and growth. We evaluated the effects of five concentrations (2.0, 0.2, 0.02, 0.002, and 0 mg carbaryl/l). Specifically, we predicted a threshold effect in which carbaryl concentrations great enough to reduce zooplankton abundance would have negative indirect effects on tadpoles, but the degree of these effects would not be concentration-specific. Similarly, we predicted that lower carbaryl concentrations (where zooplankton abundance was not reduced) would not differ in effect from controls. We did not observe a threshold effect or any negative effect on tadpoles. The highest carbaryl concentration expedited the time to metamorphosis in the northern leopard frogs by ~4 days. The toads were unaffected by any concentration of carbaryl. Despite significant reductions in zooplankton abundance and increases in phytoplankton abundance, periphyton abundance was unaffected by carbaryl. Taken together, these results suggest that despite evidence from studies using single concentrations of pesticides, trophic cascade models do not sufficiently explain sublethal effects on larval amphibians.

The Effects of Wastewater Effluent on Sexual Differentiation and Gene Expression in the American Bullfrog

The Effects of Wastewater Effluent on Sexual Differentiation and Gene Expression in the American Bullfrog

Interactions of environmental stressors impact survival and development of parasitized larval amphibians

Infected hosts are exposed to many environmental stressors that must be taken into account in order to determine the importance of disease, as various combinations can interact in unpredictable ways. Here, northern leopard frog (Rana pipiens) tadpoles, a species in decline, were exposed to stressors singly or in combination. Stressors included infection by Echinostoma trivolvis (a trematode parasite), exposure to predator chemical cues (larval dragonflies), and exposure to varying concentrations of the herbicide atrazine. Parasitism decreased survival only in combination with exposure to 3 microg/L atrazine, with a negative interaction observed for mass as well. Similarly, a negative interaction of parasitism and predation on survival occurred. However, atrazine exposure alone negatively affected the survival, mass, and developmental stage of tadpoles. These results indicate that certain stressor combinations are particularly deleterious for young parasitized tadpoles. Notably, very common low-intensity parasite infection can be particularly harmful in certain situations. Such negative impacts on larval amphibians in certain scenarios may contribute to ongoing amphibian population declines, emphasizing that the combination of environmental stressors must be considered when evaluating the general role of disease in species extinctions.

Context-dependent effects of ranaviral infection on northern leopard frog life history traits.

Pathogens have important effects on host life-history traits, but the magnitude of these effects is often strongly context-dependent. The outcome of an interaction between a host and an infectious agent is often associated with the level of stress experienced by the host. Ranavirus causes disease and mortality in amphibian populations in various locations around the world, but most known cases of ranaviral infection have occurred in North America and the United Kingdom. While Ranavirus virulence has been investigated, the outcome of Ranavirus infection has seldom been related to the host environment. In a factorial experiment, we exposed Northern leopard frog (Lithobates pipiens, formerly Rana pipiens) tadpoles to different concentrations of Ranavirus and investigated the effect of host density on certain life-history traits, namely survival, growth rate, developmental stage and number of days from virus exposure to death. Our results suggest a prominent role of density in driving the direction of the interaction between L. pipiens tadpoles and Ranavirus. We showed that increasing animal holding density is detrimental for host fitness as mortality rate is higher, day of death earlier, development longer and growth rate significantly lower in high-density tanks. We observed a linear increase of detrimental effects when Ranavirus doses increased in low-density conditions, with control tadpoles having a significantly higher overall relative fitness. However, this pattern was no longer observed in high-density conditions, where the effects of increasing Ranavirus dose were limited. Infected and control animals fitness were consequently similar. We speculate that the host may eventually diverts the energy required for a metabolic/immune response triggered by the infection (i.e., direct costs of the infection) to better cope with the increase in environmental “stress” associated with high density (i.e., indirect benefits of the infection). Our results illustrate how the net fitness of organisms may be shaped by ecological context and emphasize the necessity of examining the direct/indirect costs and benefits balance to fully understand host-pathogen interactions.

Chronic, dietary polybrominated diphenyl ether exposure affects survival, growth, and development of Rana pipiens tadpoles

Levels of polybrominated diphenyl ethers (PBDEs) in the environment have been increasing rapidly over the past two decades; however, the toxicology of these compounds to aquatic organisms is poorly understood. Because amphibians play a role in both aquatic and terrestrial food webs, and are currently undergoing worldwide population declines, it is of interest to determine how PBDEs may affect amphibian health. This is the first study that reports chronic, dietary effects of environmentally relevant levels (7-277 ng/g wet food) of PBDEs in amphibians throughout larval development. Beginning at the free-swimming stage (Gosner Stage [GS] 25), Rana pipiens tadpoles were orally exposed to a technical pentabromodiphenyl ether mixture (DE-71) through metamorphic climax (GS 42). On exposure day 43, a subset of tadpoles was removed for body residue analysis. Sum PBDEs in whole-body tissue correlated linearly to dietary concentrations with BDE-99 represented as the highest contributing congener in both diet and tissue. Survival among all treatments compared to the control was decreased by DE-71 exposure. Further, growth and development were delayed in all but the highest treatment, perhaps indicating greater PBDE tolerance among those individuals that survived the highest treatment. Time to metamorphic climax was delayed, on average, 22 to 36 d in DE-71-treated tadpoles compared to control tadpoles. Additionally, size at metamorphosis was smaller in the highest treatment, suggesting that individuals that survived and metamorphosed similarly to the controls did so with a trade-off in size. At environmentally relevant levels, PBDEs induced mortality as well as sublethal effects on developing tadpoles through dietary exposure.

Assessment of thyroid system disruption in Rana pipiens tadpoles chronically exposed to UVB radiation and 4- tert-octylphenol

Many studies have considered recent increases in ultraviolet B radiation (UVBR) and endocrine disrupting chemicals polluting the environment as possible contributing factors to the reduction in amphibian populations. It has been demonstrated that exposure of amphibians to estrogenic chemicals or UVBR can affect the timing of larval development and metamorphosis. However, amphibians in the wild are exposed to multiple environmental stressors simultaneously. Therefore, our study examines the effects of UVBR and the estrogenic chemical 4- tert-octylphenol (OP), alone and in combination, on the thyroid system of Rana pipiens tadpoles, which is the main regulator of amphibian metamorphosis. Results demonstrate that thyroid gland histomorphology measurements in Gosner stage 31 tadpoles continuously exposed to UVBR (0.21 W/m 2) were not different than those measured in animals from the control group. In a separate experiment, tadpoles exposed to environmentally relevant levels of UVBR (0.22 W/m 2) and/or OP (0.01 nM or 10 nM) exhibited significantly delayed development starting from Gosner stage 29, given that fewer tadpoles developed past stage 29 in these groups. In addition, significantly fewer UVBR-treated tadpoles developed past stage 34 and metamorphosed. Samples were collected from stages 29 and 34 tadpoles for gene expression analysis in tail tissue and measurements of T3 (triiodothyronine) whole body levels (minus tail). UVBR and/or OP exposure did not affect T3 levels in stages 29 and 34 tadpoles. However, a decrease in deiodinase type 2 (D2) or increase in deiodinase type 3 (D3) mRNA levels was observed in groups of tadpoles with slowed developmental rates at those developmental stages. Given that D2 activates and D3 inactivates thyroid hormones (TH), UVBR/OP mediated disruptions in development are likely caused by dysfunctions in the localized metabolism of THs through alterations in the expression of these enzymes in peripheral tissues. This is the first study to our knowledge reporting a potential thyroid-based mechanism of action for the developmental delays in amphibians exposed to UVBR and/or OP.

Chronic Exposure of Rana pipiens Tadpoles to UVB Radiation and the Estrogenic Chemical 4-tert-octylphenol

While adverse effects of exposure to ultraviolet B radiation (UVBR) and environmental pollutants on amphibians have been documented, few studies examined the effects of interactions between environmental stressors on amphibian health. Here the impacts of chronic exposure to UVBR and the estrogenic chemical 4-tert-octylphenol (OP), alone and in combination, on the survival, development, growth, and metamorphosis of Rana pipiens (northern leopard frog) tadpoles are evaluated. Tadpoles were exposed to environmentally relevant levels of OP (0.01 or 10 nM), with and without exposure to UVBR (∼0.22 W/m2). After 8 mo of exposure, animals were transferred to a clean-water grow-out system for 3 additional mo. There was no effect of treatments on the weights of animals at wk 42 or on the age, snout–vent length (SVL), and weights of tadpoles at forelimb emergence (FLE). However, tadpoles exposed to UVBR had markedly delayed development and significantly fewer reached metamorphosis compared to control animals. Although exposure to UVBR and OP together produced the highest incidence of mortality and developmental anomalies, UVBR alone was sufficient to produce significant mortality of tadpoles, and exposure to UVBR or OP alone induced a significant increase in malformations and abnormalities. The malformations observed consisted mainly of spinal curvature. Our results show that chronic exposure to environmental levels of UVBR or OP alone exerts lethal and/or sublethal effects on R. pipiens tadpoles.

Multiple stressor effects of herbicide, pH, and food on wetland zooplankton and a larval amphibian

Interactions of herbicides and natural environmental stressors such as pH and food availability are poorly understood. We tested a chemical formulation of triclopyr (Release ®) at environmentally relevant test concentrations (0.25 and 0.50 mg L −1) in combination with two levels of pH (pH 5.5 and 7.5), and two levels of food availability (high and low). Population level effects of each stressor alone and in combination with the others were investigated using Simocephalus vetulus, a zooplankton species, and Rana pipiens tadpoles (Gosner stage 25), both common to forest ponds and wetlands. Herbicide treatments resulted in significant decreases in survival of both test species as well as reproduction and development time for S. vetulus at levels 5–10× below predicted worst case environmental concentrations (2.6 mg L −1). This laboratory study demonstrates a probable risk of toxic effects of Release ® herbicide which may be significantly increased by low food availability and by low pH at environmentally relevant concentrations.

Exposures to Estradiol, Ethinylestradiol and Octylphenol Affect Survival and Growth of Rana pipiens and Rana sylvatica Tadpoles

Endocrine disrupting compounds (EDCs) are often detected in the aquatic environment and can negatively affect the health of wildlife populations. However, little is known about the sensitivity of native amphibians to EDCs. Wood frogs (Rana sylvatica) and Northern leopard frogs (Rana pipiens) were exposed to three estrogenic EDCs: estradiol (E2), ethinylestradiol (EE2), and 4-tert-octylphenol (OP). In addition, R. pipiens were exposed during two developmental stages (Gosner stages 26 and 36) to examine life-stage differences in sensitivity. Tadpoles were exposed for 2 wk to 8 nominal concentrations (0.25 μM–10 μM) of each compound. Individual mortality was recorded during the exposure period, while body weight was measured at the end of 2 wk. LC50 values were calculated, and differences in body weight between vehicle control and exposed groups were assessed. Rank order toxicity of the compounds for both R. pipiens stages and both species was OP > EE2 > E2. Gosner stage 26 tadpoles were more sensitive (LC50: E2 [5.57 μM], EE2 [3.01 μM], OP [1.36 μM]) to all three compounds when compared to stage 36 tadpoles (LC50: E2 [>10 μM], EE2 [4.17 μM], OP [2.80 μM]). Interspecies comparisons revealed R. sylvatica tadpoles (LC50: E2 [2.50 μM], EE2 [1.89 μM], OP [0.74 μM]) as being more sensitive to the three compounds than R. pipiens (LC50: E2 [4.56 μM], EE2 [2.75 μM], OP [1.42 μM]). Xenoestrogen exposure also affected tadpole body weight which may have long-term adverse effects on the rate of metamorphosis. These results provide toxicological data needed for assessing sublethal effects of estrogenic compounds on amphibian development and suggest that environmental levels of OP may pose a serious risk to the health of amphibian populations.

Direct and interactive effects of ecologically relevant concentrations of organic wastewater contaminants on Rana pipiens tadpoles.

Organic wastewater contaminants (OWCs) have recently been documented in several aquatic ecosystems. We examined the effects of three OWCs (caffeine, acetaminophen, and triclosan) on the activity level, startle response, survivorship, and growth of northern leopard frog (Rana pipiens) tadpoles. We conducted two experiments: one on the effects of acetaminophen and triclosan alone and in combination, and the other on the effects of caffeine and acetaminophen alone and in combination. In both experiments acetaminophen had no effect on activity, startle response, survivorship, or final mass. In experiment 1, tadpoles exposed to triclosan were less active than controls. Tadpoles exposed to the highest triclosan concentration had lower startle response and survivorship than in the other concentrations and in the control. Tadpoles exposed to the intermediate triclosan concentrations were heavier than in other treatments, but not in the control. In experiment 2, tadpoles exposed to the highest caffeine concentration were the most active. Acetaminophen increased the effect of caffeine on activity. More tadpoles exposed to caffeine exhibited a startle response than did control tadpoles. Survivorship was 100% for all caffeine treatments. There was a tendency (P = 0.06) for tadpoles exposed to higher concentrations of caffeine to be smaller. In general, the interactive effects were not important. Our results show that ecologically relevant levels of some OWCs can have both behavioral and physiological effects on R. pipiens tadpoles. Thus, amphibians may be affected by exposure to levels of OWCs that appear to be widespread in aquatic ecosystems.

Effects of Sublethal Copper Exposure on Behavior and Growth of Rana pipiens Tadpoles

This research is designed to test how sublethal exposure to copper affects tadpole predator-escape behavior and how quickly tadpoles recover. After exposure, tadpoles were separated. Escape behavior was recorded for two-thirds of exposed tadpoles while one-third of the exposed population was measured weekly to determine growth and recovery. Control tadpoles were consumed within 15 minutes whereas those exposed to higher concentrations were consumed at a slower rate, which does not support the hypotheses. Although the rate of predation was lower, tadpoles exposed to higher Cu concentrations were on average, 1.47 cm in total body length. Those exposed to 0.93 mg/L averaged 0.86 cm. After being placed into clean water, treatment tadpoles recovered after 20 days.

Influence of Ribeiroia ondatrae (Trematoda: Digenea) infection on limb development and survival of northern leopard frogs (Rana pipiens): effects of host stage and parasite-exposure level

Recent evidence suggests that infection by larvae of the trematode Ribeiroia ondatrae accounts for a significant proportion of limb malformations currently observed in amphibian populations of North America. However, the effects of R. ondatrae infection on northern leopard frogs (Rana pipiens), one of the species most frequently reported with malformations, have not been adequately explored. Moreover, the risk factors associated with R. ondatrae-induced malformations have not been clearly identified. We examined the effects of timing of infection on tadpole survival and limb development. Rana pipiens tadpoles were individually exposed to R. ondatrae cercariae at the pre-limb-bud (Gosner stages 24 and 25), limb-bud (Gosner stages 27 and 28), or paddle (Gosner stages 31–33) stages of development and monitored through metamorphosis. The effects of infection were stage-specific. Infections acquired at the pre-limb-bud stage resulted in a high mortality rate (47.5–97.5%), whereas tadpoles infected at the limb-bud stage displayed a high malformation rate (16% overall), and the magnitude of effects increased with the level of exposure to cercariae. In contrast, infections acquired at the paddle stage had no effect on limb development or tadpole survival, which suggests that the timing of R. ondatrae infection in relation to the stage structure of tadpole populations in the wild is an important determinant of the degree to which populations are affected by R. ondatrae.