Chronic exposure to fluoxetine (Prozac) causes developmental delays in Rana pipiens larvae.

Selective serotonin reuptake inhibitors (SSRIs), such as fluoxetine, are among the many pharmaceuticals detected in aquatic ecosystems. Although the acute effects of SSRIs on select organisms have been reported, little is understood about the chronic effects of these drugs on amphibians, which are particularly sensitive to environmental pollutants. Serotonin plays important roles in many physiological functions, including a wide array of developmental processes. Exposure to SSRIs during development may cause developmental complications in a variety of organisms, but little is known about the degree of exposure necessary to cause deleterious effects. Here, we sought to gain a better understanding of the effects of SSRIs on amphibian development by use of a combined laboratory and outdoor mesocosm study. Tadpoles in a laboratory setting were exposed to a low (0.029 µg/L) and a high (0.29 µg/L) concentration of the common SSRI fluoxetine from stages 21 and 22 through completion of metamorphosis. Tadpoles in outdoor mesocosms were exposed to fluoxetine concentrations ranging from 0.1 to 0.3 µg/L. Exposed tadpoles in the laboratory showed delayed development compared with controls when stage was assessed throughout the experiment. Control tadpoles also gained weight faster than treatment tadpoles, which may be explained by reduced food intake. Mesocosm tadpoles exhibited similar trends, but no significant differences were detected. These results indicate that ecologically relevant levels of fluoxetine may cause developmental delays in amphibians.

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 (0.21W/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 (0.22W/m(2)) and/or OP (0.01nM or 10nM) 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.

Exposure to coal combustion residues during metamorphosis elevates corticosterone content and adversely affects oral morphology, growth, and development in Rana sphenocephala.

Coal combustion residues (CCRs) are documented to negatively impact oral morphology, growth, and development in larval amphibians. It is currently unclear what physiological mechanisms may mediate these effects. Corticosterone, a glucocorticoid hormone, is a likely mediator because when administered exogenously it, like CCRs, also negatively influences oral morphology, growth, and development in larval amphibians. In an attempt to identify if corticosterone mediates these effects, we raised larval Southern Leopard Frogs, Rana sphenocephala, on either sand or CCR substrate and documented effects of sediment type on whole body corticosterone, oral morphology, and time to and mass at key metamorphic stages. Coal combustion residue treated tadpoles contained significantly more corticosterone than controls throughout metamorphosis. However, significantly more oral abnormalities occurred early in metamorphosis when differences in corticosterone levels between treatments were minimal. Overall, CCR-treated tadpoles took significantly more time to transition between key stages and gained less mass between stages than controls, but these differences between treatments decreased during later stages when corticosterone differences between treatments were greatest. Our results suggest endogenous increase in corticosterone content and its influence on oral morphology, growth and development is more complex than previously thought.

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 (approximately 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.

Assessment of the risk of solar ultraviolet radiation to amphibians. II. In situ characterzation of exposure in amphibian habitats.

Ultraviolet B (UVB) radiation has been hypothesized as a potential cause of amphibian population declines and increased incidence of malformations. Realistic studies documenting UV irradiance or dose have rarely been conducted in wetlands used by amphibians. Our data indicates that 99% of UVB is attenuated in the top 5-20 cm of wetlands in our study region (northern Minnesota and Wisconsin). Furthermore, vegetation and other habitat features have substantial impacts on local UVB irradiance levels and dose. UVB attenuation in the water columns of our wetlands is controlled by the specific absorption of dissolved organic carbon (DOC), and consequently, UVB attenuation is best predicted by simple laboratory absorbance measurements such as bulk water color (absorbance at 440 nm) or wavelength-specific absorbance coefficients. Seasonal data indicate thatthe UVB absorption by early and mid-season DOC is higher than that of late summer and fall DOC, suggesting increased protection from UVB during the potentially sensitive stages of amphibian development. In addition to dissolved components, our model indicates that suspended solids play a small role in UVB attenuation in our wetlands but apparently only at high concentrations. Models predicting UV attenuation in wetlands should be used cautiously and should consider temporal variability, given the volatility and dynamic nature of water column characteristics in wetlands. Organism behavior is a critical but poorly understood phenomenon that must be addressed for development of an accurate UV exposure risk model for amphibians.

Hindlimb deformities (ectromelia, ectrodactyly) in free-living anurans from agricultural habitats.

High prevalences of hindlimb deformities were recorded in wild-caught green frogs (Rana clamitans), northern leopard frogs (Rana pipiens), American toads, (Bufo americanus), and bullfrogs (Rana catesbeiana) from agricultural sites exposed to pesticide runoff in the St. Lawrence River Valley of Québec, Canada, between July and September 1992 and 1993. Of 853 metamorphosing anurans examined in 14 farmland habitats, 106 (12%; range 0 to 69%) had severe degrees of ectromelia and ectrodactyly, compared to only two (0.7%; range 0 to 7.7%) of 271 in 12 control sites. However, the variation in the proportion of deformities among sites was too large to conclude that there was a significant difference between control and pesticide-exposed habitats. Clinical signs varied and were characterized by segmental hypoplasia or agenesis of affected limbs. Conspicuous abnormalities interfered with swimming and hopping, and likely constituted a survival handicap. Because of circumstances and the frequency of these malformations in nine distinct habitats, and in three different species from one of our study sites, we propose a teratogenic action of exogenous factors. Despite the fact that many biotic and abiotic agents are potentially harmful to limb development, agricultural contaminants were suspected as primary aggressors. Thus, clinical examination and frequency of deformities in anurans might be an economical screening tool to assess ecosystem health and the presence of environmental contaminants.