Toxicokinetics and bioaccumulation of polycyclic aromatic compounds in wood frog tadpoles (Lithobates sylvaticus) exposed to Athabasca oil sands sediment.

We performed accumulation-elimination experiments of polycyclic aromatic compounds (PACs) in wood frog tadpoles (Lithobates sylvaticus) using river sediment from Canada's Athabasca oil sands region. The PACs in wood frog tadpoles were ∼2x higher on average when the animals were in direct contact with PAC-contaminated sediment than when they were separated from the sediment with a screen and exposed only to aqueous PACs. These results suggest that sediment exposure/ingestion contributes as much to PAC accumulation in tadpoles as exposure via aqueous pathways. Alkyl-substituted PAC concentrations in exposed tadpoles exceeded those of the unsubstituted (parent) PACs by about 10 × . Bioaccumulation factors ranged between 0.01 and 4.93, with parent PACs having higher bioaccumulation factors than alkylated PACs. Wood frog tadpoles efficiently eliminated and metabolized most parent and alkyl-substituted PACs, though some compounds (e.g., C4-naphthalenes) had higher bioaccumulation potential and may serve as effective markers of exposure. Here we present a comprehensive analysis of the toxicokinetics and bioaccumulation of PACs (52 analytes) in amphibian larvae, and highlight the importance of sediment exposure when considering the bioaccumulation and potential biological impact of PACs in benthic and epibenthic organisms.

Effects of glyphosate-based herbicides on survival, development, growth and sex ratios of wood frog (Lithobates sylvaticus) tadpoles. II: agriculturally relevant exposures to Roundup WeatherMax® and Vision® under laboratory conditions.

Glyphosate-based herbicides are currently the most commonly used herbicides in the world. They have been shown to affect survival, growth, development and sexual differentiation of tadpoles under chronic laboratory exposures but this has not been investigated under more environmentally realistic conditions. The purpose of this study is (1) to determine if an agriculturally relevant exposure to Roundup WeatherMax®, a relatively new and understudied formulation, influences the development of wood frog tadpoles (Lithobates sylvaticus) through effects on the mRNA levels of genes involved in the control of metamorphosis; (2) to compare results to the well-studied Vision® formulation (containing the isopropylamine salt of glyphosate [IPA] and polyethoxylated tallowamine [POEA] surfactant) and to determine which ingredient(s) in the formulations are responsible for potential effects on development; and (3) to compare results to recent field studies that used a similar experimental design. In the present laboratory study, wood frog tadpoles were exposed to an agriculturally relevant application (i.e., two pulses) of Roundup WeatherMax® and Vision® herbicides as well as the active ingredient (IPA) and the POEA surfactant of Vision®. Survival, development, growth, sex ratios and mRNA levels of genes involved in tadpole metamorphosis were measured. Results show that Roundup WeatherMax® (2.89 mg acid equivalent (a.e.)/L) caused 100% mortality after the first pulse. Tadpoles treated with a lower concentration of Roundup WeatherMax® (0.21 mg a.e./L) as well as Vision® (2.89 mg a.e./L), IPA and POEA had an increased condition factor (based on length and weight measures in the tadpoles) relative to controls at Gosner stage (Gs) 36/38. At Gs42, tadpoles treated with IPA and POEA had a decreased condition factor. Also at Gs42, the effect on condition factor was dependent on the sex of tadpoles and significant treatment effects were only detected in males. In most cases, treatment reduced the normal mRNA increase of key genes controlling development in tadpoles between Gs37 and Gs42, such as genes encoding thyroid hormone receptor beta in brain, glucocorticoid receptor in tail and deiodinase enzyme in brain and tail. We conclude that glyphosate-based herbicides have the potential to alter mRNA profiles during metamorphosis. However, studies in natural systems have yet to replicate these negative effects, which highlight the need for more ecologically relevant studies for risk assessment.

Effects of glyphosate-based herbicides on survival, development, growth and sex ratios of wood frogs (Lithobates sylvaticus) tadpoles. I: chronic laboratory exposures to VisionMax®.

The purpose of this study was to determine if chronic exposure to the glyphosate-based herbicide VisionMax(®) affects the survival, development, growth, sex ratios and expression of specific genes involved in metamorphosis of wood frog tadpoles (Lithobates sylvaticus). We hypothesized that exposure to this herbicide will affect developmental rates by disrupting hormone pathways, sex ratios and/or gonadal morphology. Tadpoles were chronically exposed in the laboratory from Gosner developmental stage 25 to 42 to four different concentrations of VisionMax(®) (ranging from 0.021 to 2.9 mg acid equivalents/L). Chronic exposures to VisionMax(®) had direct effects on the metamorphosis of L. sylvaticus tadpoles by decreasing development rates, however, there was a decrease in survival only in the group exposed to the highest dose of VisionMax(®) (2.9 mg a.e./L; from approximately 96% in the control group to 77% in the treatment group). There was a decrease in the number of tadpoles reaching metamorphic climax, from 78% in the control group to 42% in the VisionMax(®) (2.9 mg a.e./L) group, and a 7-day delay to reach metamorphic climax in the same treatment group. No effects of exposure on sex ratios or gonadal morphology were detected in tadpoles exposed to any of the concentrations of VisionMax(®) tested. Gene expression analyses in brain and tail tissues demonstrated that exposure to VisionMax(®) alters the expression of key genes involved in development. Results showed significant interaction (two-way ANOVA, P<0.05) between developmental Gosner stage and treatment in brain corticotropin-releasing factor, deiodinase type II (dio2) and glucocorticotiroid receptor (grII) and tail dio2 and grII. This demonstrates that mRNA levels may be differently affected by treatment depending on the developmental stage at which they are assessed. At the same time there was a clear dose-response effect for VisionMax(®) to increase thyroid hormone receptor ß in tadpole brain (F(2,69)=3.475, P=0.037) and tail (F(2,69)=27.569, P<0.001), regardless of developmental stage. Interestingly, delays in development (or survival) were only observed in the group exposed to 2.9 mg a.e./L of VisionMax(®), suggesting that tadpoles need to be exposed to a "threshold" concentration of glyphosate-based herbicide to exhibit phenotypic observable effects. We suggest that the upregulation of genes that trigger metamorphosis following VisionMax(®) herbicide exposure might result from a compensatory response for the delays in development observed. Further studies are needed to determine if disruption of expression of these key genes leads to long-term effects when metamorphs reach adult stages.

The importance of social dimension and maturation stage for the probabilistic maturation reaction norm in Poecilia reticulata.

Maturation is an important event in an organism's life history, with important implications on dynamics of both wild and captive populations. The probabilistic maturation reaction norm (PMRN) has emerged as an important method to describe variation in maturation in wild fish. Because most PMRNs are based on age and size only, it is important to understand limitations of these variables in explaining maturation. We experimentally assessed (i) the sensitivity of age- and size-based PMRNs to unaccounted sources of plasticity, (ii) the role of social environment on maturation and (iii) the significance of estimating PMRNs early and late in the maturation process (initiation and completion of maturation, respectively). We reared male guppies (Poecilia reticulata) under laboratory conditions, subjected to two food levels and three different social cues. We found that growth and social environment affected the maturation in a way that could not be accounted for by their effect on age and size. PMRNs estimated for the initiation stage were less plastic (growth differences and social cues influenced the PMRN shape only little) than those for completion. The initiation of maturation is probably closer to the maturation 'decision' and allows determining factors influencing maturation decision most accurately.

The aromatase inhibitor fadrozole and the 5-reductase inhibitor finasteride affect gonadal differentiation and gene expression in the frog Silurana tropicalis.

Aromatase (cyp19) and the 5alpha- and 5beta-reductases (srd5alpha and srd5beta) are important enzymes for vertebrate sexual development. We investigated the effects of inhibition of cyp19 by fadrozole (FAD), and srd5alpha and srd5beta by finasteride (FIN) during anuran larval development. Chronic exposures of Silurana (Xenopus) tropicalis from Nieuwkoop-Faber stage 12 until stage 60 were performed using either 2 microM FAD or 25 microM FIN. Histological analysis of exposed metamorphic frogs revealed that both treatments induced intersex individuals (presence of testicular oocytes). FAD treatment resulted in 55% male, 30% female and 15% intersex, while FIN treatment produced 27% male, 53% female and 20% intersex. Real-time RT-PCR analysis of hepatic sex steroid- and thyroid hormone-related gene expression demonstrated that FAD-induced intersex animals had higher srd5alpha1, srd5alpha2 and eralpha mRNA levels than control and FAD males. In contrast, FIN-induced intersex had low srd5alpha1, srd5alpha2, srd5beta and dio3 and high dio2 mRNA levels. FIN-treated males exhibited high trbeta, dio2 and a lower dio3 mRNA levels. We conclude that chemically induced intersex animals display different gene expression profiles than non-exposed animals and that, although morphologically similar, intersex animals produced by different chemicals have different endocrine pathophysiologies.

Frog virus 3-like infections in aquatic amphibian communities.

Frog virus 3 (FV3) and FV3-like viruses, are members of the genus Ranavirus (family Iridoviridae), and they have been associated with infectious diseases that may be contributing to amphibian population declines. We examined the mode of transmission of an FV3-like virus, and potential hosts and reservoirs of the virus in a local amphibian community. Using the polymerase chain reaction to detect infected animals, we found an FV3-like virus in south-central Ontario, Canada, amphibian communities, where it infects sympatric amphibian species, including ranid and hylid tadpoles (Rana sylvatica, Hyla versicolor, and Pseudacris spp.), larval salamanders (Ambystoma spp.), and adult eastern-spotted newts (Notophthalmus viridescens). The high prevalence of FV3-like infections in caudate larvae suggests that salamanders are likely to be both hosts and reservoirs. In laboratory FV3 challenges of R. sylvatica, the rate of infection was dependent on the amount of virus to which the animals were exposed. In addition, although vertical transmission was suspected, horizontal transmission through exposure to infected pond water is the most likely route of infection in tadpoles. Based on our observations, a simple model of FV3/FV3-like virus transmission postulates that, in aquatic amphibian communities, transmission of the virus occurs between anuran and urodele species, with ambystomatid salamanders the most likely reservoir for the ranavirus in our study.

Impacts of agriculture on the parasite communities of northern leopard frogs (Rana pipiens) in southern Quebec, Canada.

Given that numerous amphibians are suffering population declines, it is becoming increasingly important to examine the relationship between disease and environmental disturbance. Indeed, while many studies relate anthropogenic activity to changes in the parasitism of snails and fishes, little is known of the impact on the parasites of amphibians, particularly from agriculture. For 2 years, the parasite communities of metamorphic northern leopard frogs from 7 agricultural wetlands were compared with those from 2 reference wetlands to study differences in parasite community diversity and abundance of various species under pristine conditions and 3 categories of disturbance: only agricultural landscape, only pesticides, and agricultural landscape with pesticides. Agricultural (and urban) area was negatively related to species richness, and associated with the near absence of adult parasites and species that infect birds or mammals. We suggest that agriculture and urbanization may hinder parasite transmission to frogs by limiting access of other vertebrate hosts of their parasites to wetlands. The only parasite found at all localities was an unidentified echinostome infecting the kidneys. This parasite dominated communities in localities surrounded by the most agricultural land, suggesting generalist parasites may persist in disrupted habitats. Community composition was associated with dissolved organic carbon and conductivity, but few links were found with pesticides. Pollution effects may be masked by a strong impact of land use on parasite transmission.

Effects of chlorinated solvents on four species of North American amphibians.

Tetrachloroethylene (PCE), a dry cleaning and degreasing solvent, can enter groundwater through accidental leaks or spills, and concentrations as high as 75 mg/L have been reported in Canadian aquifers. Amphibians in wetlands receiving contaminated groundwater may be exposed to PCE and its degradation products, but little information is available on the impacts of these compounds on indigenous amphibian species. Acute (96-h static renewal) exposures to PCE and its major degradation products, trichloroethylene (TCE) and cisand trans-dichloroethylene, were conducted on embryos of four North American amphibian species: wood frogs (Rana sylvatica), green frogs (R. clamitans), American toads (Bufo americanus), and spotted salamanders (Ambystoma maculatum). Subsequently, chronic exposures to PCE and TCE were conducted with the larvae of American toads. Both PCE and TCE were teratogenic to amphibian embryos; median effective concentrations (EC50s) for developmental deformities produced by PCE and TCE exposure for wood frogs and green frogs were 12 and 40 mg/L, respectively. Embryonic survivorship, however, was not compromised at these concentrations. American toads were less sensitive; the EC50 for developmental abnormalities was not attained at the highest test concentrations, 45 and 85 mg/L PCE and TCE, respectively. These results are pertinent in assessing the impact of groundwater pollution on an aquifer-fed wetland.

Influence of the menstrual cycle on airway function in asthmatic and normal subjects.

Investigations of premenstrual asthma (PMA) have been based on studies of asthmatics already aware of a deterioration of asthma premenstrually. Little is known, therefore, about relationships between the menstrual cycle and airway function in asthmatics who do not complain of PMA or in normal subjects. We investigated airway function in both of these groups for three or four consecutive menstrual cycles. Daily records of asthma symptoms and peak expiratory flow rates were maintained by 11 asthmatics and 29 normal control subjects. Standard spirometry and serum estradiol and progesterone levels were measured during the follicular, midluteal, and late luteal phases of the menstrual cycle. Airway reactivity to methacholine was tested during the follicular and luteal phases. The normal group showed no significant changes in symptoms, peak flow rates, spirometric parameters, or airway reactivity. Although the asthmatic group also demonstrated no significant changes in spirometry and airway reactivity, asthma symptoms (shortness-of-breath, cough, wheeze, and chest tightness) deteriorated significantly (p less than 0.001) from the follicular to the luteal phase, as did the morning peak flows of the asthmatics (p = 0.045). Airway function and reactivity were not related to hormone levels in either group. This study indicates that asthmatics not previously aware of PMA will record a premenstrual worsening of asthma symptoms and peak expiratory flow rates. These changes are not related to a deterioration in spirometry and airway reactivity or to the absolute levels of circulating progesterone and estradiol.