Although amphibian sex determination is genetic, it can be manipulated by exogenous hormone exposure during sexual differentiation. The timing of sexual differentiation varies among anuran amphibians such that species may or may not be a tadpole during this period, and therefore, may or may not be exposed to aquatic contaminants. Estrogenic contamination is present in amphibian habitats worldwide. We examined three species with varying somatic and ovarian developmental rates to assess their susceptibility to estrogenic contaminants. American toads (Bufo americanus), gray treefrogs (Hyla versicolor), and Southern leopard frogs (Ranasphenocephala) were exposed as larvae to 17-beta-estradiol (10(-7)M), three concentrations of a widespread herbicide (1, 3, 30 ppb atrazine), or a solvent control (ethanol). Somatic and ovarian developmental stages as well as time to metamorphosis were recorded. Toads and treefrogs were examined at three weeks and metamorphosis, while leopard frogs were examined at three, six, and nine weeks as well as at metamorphosis. Our results demonstrate that each species displays heterochronic somatic and ovarian development. Further, the more rapid of the two rates determines the susceptibility to estrogenic contaminants. These results suggests that amphibians with shorter larval periods, and therefore quicker somatic developmental rates (i.e. American toads, gray treefrogs), are more susceptible to somatic treatment effects (i.e. prolonged time to metamorphosis) due to estrogenic contaminants. Moreover, the results suggest that amphibians with relatively rapid ovarian development (i.e. Southern leopard frogs) are more susceptible to gonadal treatment effects caused by estrogenic contaminants.
"For instance, although the current study provides the first report of an alteration of developmental rate by a concentration of atrazine as low as 1 mg/L, seven studies have previously examined atrazine concentrations of this range; all of them failing to detect an effect. If sample size is considered, however, it emerges that only two of these seven studies were well-replicated studies (Carr et al., 2003; Kloas et al., 2009), the five other studies being based on much smaller sample sizes (Storrs and Semlitsch, 2008; Oka et al., 2008; Williams and Semlitsch, 2010; Spolyarich et al., 2010; Langlois et al., 2010). If these five studies are disregarded because the absence of effect may be due to the low power of the analysis, the weight-of-evidence towards an absence of effect shifts considerably from 7:1 to 2:1. "
[Show abstract][Hide abstract] ABSTRACT: Despite of the various studies reporting on the subject, anticipating the impacts of the widely-used herbicide atrazine on anuran tadpoles metamorphosis remains complex as increases or decreases of larval period duration are almost as frequently reported as an absence of effect. The aim of the present study was to examine the effects of environmentally-relevant concentrations of atrazine (0.1, 1, 10, 100, and 1000μg/L) on the timings of metamorphosis and body size at metamorphosis in the common South American toad, Rhinella arenarum (Anura: bufonidae). None of the atrazine concentrations tested significantly altered survival. Low atrazine concentrations in the range of 1-100μg/L were found to accelerate developmental rate in a non-monotonic U-shaped concentration-response relationship. This observed acceleration of the metamorphic process occurred entirely between stages 25 and 39; treated tadpoles proceeding through metamorphosis as control animals beyond this point. Together with proceeding through metamorphosis at a faster rate, tadpoles exposed to atrazine concentrations in the range of 1-100μg/L furthermore transformed into significantly larger metamorphs than controls, the concentration-response curve taking the form of an inverted U in this case. The no observed effect concentration (NOEC) was 0.1μg atrazine/L for both size at metamorphosis and timings of metamorphosis. Tadpoles exposed to 100μg/L 17β-estradiol presented the exact same alterations of developmental rate and body size as those treated with 1, 10 and 100μg/L of atrazine. Elements of the experimental design that facilitated the detection of alterations of metamorphosis at low concentrations of atrazine are discussed, together with the ecological significance of those findings.
"L. sylvaticus is considered common throughout much of its range, but it is not generally used in toxicological studies that focus on the effects of endocrine active chemicals. Other native species of North American frogs in the family Ranidae, like green frogs (Lithobates clamitans) (McDaniel et al., 2008; Park and Kidd, 2005), bullfrogs (Lithobates catesbeianus ) (Gunderson et al., 2011; Veldhoen and Helbing, 2001), and northern or southern leopard frogs (Lithobates pipiens or Lithobates sphenocephalus) (Hogan et al., 2008; Langlois et al., 2010; Storrs and Semlitsch, 2008; Tsai et al., 2005), are more commonly "
[Show abstract][Hide abstract] ABSTRACT: Populations of amphibians are in decline in certain locations around the world, and the possible contribution of environmental contaminants, including estrogenic compounds, to these declines is of potential concern. In the current study, responses of the wood frog (Lithobates sylvaticus) to exposure to 17α-ethynylestradiol (EE2), the synthetic estrogen used in oral contraceptives, during the larval period were characterized. Exposure of L. sylvaticus to 1.08, 9.55, or 80.9μg EE2/L had no effects on survival, growth, or metamorphic endpoints monitored in the current study. However, there were significant effects of exposure to EE2 on phenotypic sex ratios. In general, lesser proportions of L. sylvaticus developed as phenotypic males and greater proportions developed as phenotypic females or with mixed sex phenotypes at all concentrations of EE2 tested. Utilizing the data collected in the current study, the EC(50) for complete feminization of L. sylvaticus was determined to be 7.7μg EE2/L, and the EC(50) for partial feminization was determined to be 2.3μg EE2/L. In addition, after chronic exposure, abundances of transcripts of vitellogenin A2, high density lipoprotein binding protein, and 7-dehydrocholesterol reductase were 1.8-280-fold greater in livers from L. sylvaticus exposed to EE2 compared to controls. Overall, there were significant effects of exposure to all concentrations of EE2 tested, the least of which was within about 2-fold of estrogen equivalent concentrations previously measured in the environment.
"However, this was not consistent with previous laboratory studies and may have been as a consequence of the low temperatures experienced during the study, which is known to affect sex ratios in amphibians (Hayes, 2005). Anuran gonads are most susceptible to malformation during the period of sexual differentiation and the time in which this takes place has been demonstrated to differ between species (Ogielska and Bartmanska, 1999; Storrs and Semlitsch, 2008). Since the majority of amphibian toxicology literature is based on northern hemisphere species, the direct threats to Australian amphibians inhabiting agricultural surface waters are uncertain. "
[Show abstract][Hide abstract] ABSTRACT: Exposures of Limnodynastes tasmaniensis tadpoles to atrazine (0.1, 1, 3 and 30microgL(-1)), metolachlor (0.1, 1 and 10microgL(-1)) and thiobencarb (90, 180 and 360microgL(-1)) from Gosner stage 28 to 42 under controlled laboratory conditions gave no significant effects on tadpole growth, development and sex ratios. A binary mixture of atrazine and thiobencarb as well as a ternary mixture of all three herbicides also had no significant effects on the developing larvae to show no evidence of interactive toxicity. Abnormal gonad morphology was observed on two occasions; both from 0.1microgL(-1) atrazine treatments with one tadpole observed with testicular ovarian follicles. The low frequencies of abnormal gonadal morphology and testicular ovarian follicles did not indicate a concentration associated response to herbicide exposure. No significantly unbalanced sex ratios were observed to suggest any evidence of chemically induced feminisation. These observations suggest that environmentally relevant concentrations of atrazine, metolachlor and thiobencarb do not present a significant threat to the normal development of L. tasmaniensis larvae in surface waters of irrigated agricultural areas.
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