Bruce D. Pauli

Toxicity of glyphosate‐based pesticides to four North American frog species

Glyphosate-based herbicides are among the most widely used pesticides in the world. We compared the acute toxicity of the glyphosate end-use formulation Roundup Original to four North American amphibian species (Rana clamitans, R. pipiens, R. sylvatica, and Bufo americanus) and the toxicity of glyphosate technical, the polyethoxylated tallowamine surfactant (POEA) commonly used in glyphosate-based herbicides, and five newer glyphosate formulations to R. clamitans. For R. clamitans, acute toxicity values in order of decreasing toxicity were POEA > Roundup Original > Roundup Transorb > Glyfos AU; no significant acute toxicity was observed with glyphosate technical material or the glyphosate formulations Roundup Biactive, Touchdown, or Glyfos BIO. Comparisons between the four amphibian species showed that the toxicity of Roundup Original varied with species and developmental stage. Rana pipiens tadpoles chronically exposed to environmentally relevant concentrations of POEA or glyphosate formulations containing POEA showed decreased snout-vent length at metamorphosis and increased time to metamorphosis, tail damage, and gonadal abnormalities. These effects may be caused, in some part, by disruption of hormone signaling, because thyroid hormone receptor beta mRNA transcript levels were elevated by exposure to formulations containing glyphosate and POEA. Taken together, the data suggest that surfactant composition must be considered in the evaluation of toxicity of glyphosate-based herbicides.

Gonadal differentiation in frogs exposed to estrogenic and antiestrogenic compounds

Exposure of amphibians to endocrine disrupting compounds (EDCs) may alter differentiation of gonads, especially when exposures begin during early life stages. Gonadal differentiation was observed in leopard frogs (Rana pipiens) and wood frogs (Rana sylvatica) exposed as tadpoles to estrogenic (estradiol, ethinylestradiol, nonylphenol) and antiestrogenic compounds (an aromatase inhibitor, flavone, and an antiestrogen, ICI 182780). Exposure to all compounds at micrograms/L concentrations altered gonadal differentiation in some animals by inducing either complete feminization or an intersex condition, and altered testicular tubule morphology, increased germ cell maturation (vitellogenesis), and oocyte atresia. Comparisons between the two species indicate that R. pipiens are more susceptible to sex reversal and development of intersex gonads. However, R. sylvatica also showed alterations to testicular morphology, germ cell maturation, and ooctye atresia. These laboratory results indicate that amphibians could be susceptible to altered gonadal differentiation and development when exposed to estrogenic and antiestrogenic compounds in aquatic environments, such as those impacted by agricultural, industrial, and municipal runoff.

Effects of chytrid fungus and a glyphosate-based herbicide on survival and growth of wood frogs (Lithobates sylvaticus)

Anthropogenic-derived stressors in the environment, such as contaminants, are increasingly considered important cofactors that may decrease the immune response of amphibians to pathogens. Few studies, however, have integrated amphibian disease and contaminants to test this multiple-stressor hypothesis for amphibian declines. We examined whether exposure to sublethal concentrations of a glyphosate-based herbicide and two strains of the pathogenic chytrid fungus, Batrachochrytrium dendrobatidis (Bd) could: (1) sublethally affect wood frogs (Lithobates sylvaticus) by altering the time to and size at metamorphosis, and (2) directly affect survivability of wood frogs after metamorphosis. Neither Bd strain nor herbicide exposure alone significantly altered growth or time to metamorphosis. The two Bd strains did not differ in their pathogenicity, and both caused mortality in post-metamorphic wood frogs. There was no evidence of an interaction between treatments, indicating a lack of herbicide-induced susceptibility to Bd. However, the trends in our data suggest that exposure of wood frogs to a high concentration of glyphosate-based herbicide may reduce Bd-caused mortality compared to animals exposed to Bd alone. These results exemplify the complexities inherent when populations are coping with multiple stressors. In this case, the perceived stressor, glyphosate-based herbicide, appeared to affect the pathogen more than the hosts immune system, relieving the host from disease-caused effects. This suggests caution when invoking multiple stressors as a cause for increased disease susceptibility and indicates that the effects of multiple stressors on disease outcome depend on the interrelationships of stressors to both the pathogen and the host.

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.

Exposure of juvenile green frogs (Lithobates clamitans) in littoral enclosures to a glyphosate-based herbicide.

The majority of studies on the toxicity of glyphosate-based herbicides to amphibians have focused on larval life stages exposed in aqueous media. However, adult and juvenile amphibians may also be exposed directly or indirectly to herbicides. The potential for such exposures is of particular interest in the littoral zone surrounding wetlands as this is preferred habitat for many amphibian species. Moreover, it may be argued that potential herbicide effects on juvenile or adult amphibians could have comparatively greater influence on overall recruitment, reproductive potential and thus stability of local populations than effects on larvae. In this experiment, juvenile green frogs (Lithobates clamitans) were exposed to two concentrations (2.16 and 4.27 kg a.e./ha) of a glyphosate-based herbicide formulation (VisionMax®), which were based on typical application scenarios in Canadian forestry. The experimental design employed frogs inhabiting in situ enclosures established at the edge of small naturalized wetlands that were split in half using an impermeable plastic barrier. When analyzed using nominal target application rates, exposure to the glyphosate-based herbicide had no significant effect on survival, body condition, liver somatic index or the observed rate of Batrachochytrium dendrobatidis infection. However, there were marginal trends in both ANOVA analysis and post-hoc regressions regarding B. dendrobatidis infection rates and liver somatic index in relation to measured exposure estimates. Results from this study highlight the importance of field research and the need to include multiple endpoints when examining potential effects of a contaminant on non-target organisms.

Environmental dependency of amphibian–ranavirus genotypic interactions: evolutionary perspectives on infectious diseases

The context‐dependent investigations of host–pathogen genotypic interactions, where environmental factors are explicitly incorporated, allow the assessment of both coevolutionary history and contemporary ecological influences. Such a functional explanatory framework is particularly valuable for describing mortality trends and identifying drivers of disease risk more accurately. Using two common North American frog species (Lithobates pipiens and Lithobates sylvaticus) and three strains of frog virus 3 (FV3) at different temperatures, we conducted a laboratory experiment to investigate the influence of host species/genotype, ranavirus strains, temperature, and their interactions, in determining mortality and infection patterns. Our results revealed variability in host susceptibility and strain infectivity along with significant host–strain interactions, indicating that the outcome of an infection is dependent on the specific combination of host and virus genotypes. Moreover, we observed a strong influence of temperature on infection and mortality probabilities, revealing the potential for genotype–genotype–environment interactions to be responsible for unexpected mortality in this system. Our study thus suggests that amphibian hosts and ranavirus strains genetic characteristics should be considered in order to understand infection outcomes and that the investigation of coevolutionary mechanisms within a context‐dependent framework provides a tool for the comprehensive understanding of disease dynamics.

Effects of herbicides and the chytrid fungus Batrachochytrium dendrobatidis on the health of post-metamorphic northern leopard frogs (Lithobates pipiens)

Effects of exposure to contaminants such as pesticides along with exposure to pathogens have been listed as two major contributors to the global crisis of declining amphibian populations. These two factors have also been linked in explanations of the causes of these population declines. We conducted a combined exposure experiment to test the hypothesis that exposure to two agricultural herbicides would increase the susceptibility of post-metamorphic northern leopard frogs (Lithobates pipiens) to the amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd). We assessed the independent and interactive effects of these exposures on the health and survival of the frogs. Wild-caught frogs underwent a 21-day exposure to a nominal concentration of either 2.1 μg/L atrazine (Aatrex(®) Liquid 480) or 100 μg a.e./L glyphosate (Roundup(®) Original), followed by Bd, and then were observed until 94 days post-initial exposure to the herbicides. Actual levels of atrazine were between 4.28 ± 0.04 μg/L and 1.70 ± 0.26 μg/L while glyphosate degraded from 100 μg a.e./L to approximately 7 μg a.e./L within 6 days of initial exposure to the herbicides. Compared to controls, the glyphosate formulation reduced the snout-vent length of frogs during the pesticide exposure (at Day 21), and the atrazine formulation reduced gain in mass up to Day 94. No treatment affected survival, splenosomatic or hepatosomatic indices, the densities and sizes of hepatic and splenic melanomacrophage aggregates, the density and size of hepatic granulomas, proportions of circulating leucocytes, the ratio of neutrophils to lymphocytes, or the ratio of leucocytes to erythrocytes. Histological assessment of samples collected at Day 94 revealed no evidence of Bd infection in any Bd-exposed frogs, while real-time PCR detected only one case of light infection in a single atrazine- and Bd-exposed frog. Frogs exposed to Bd shed their skin significantly more frequently than Bd-unexposed frogs, which may have helped them resist or clear infection, and could explain why no interaction between the herbicides and Bd was detected. The results suggest that these frogs were resistant to Bd infection and that pre-exposure to the herbicides did not alter this resistance. The effects seen on the growth following herbicide exposure is a concern, as reduced growth can lower the reproductive success and survival of the amphibians.

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.

Effects of Nonylphenol on Rates of Tail Resorption and Metamorphosis in Rana Catesbeiana Tadpoles

Nonylphenol (NP) is a persistent, lipophilic, and toxic chemical that can be endocrine disrupting (estrogenic) at sublethal concentrations. Since amphibian metamorphosis is a hormone-driven process and a delicate balance of hormone levels is required for successful metamorphosis, exposure of larval amphibians to NP might disrupt metamorphic processes. This study tested whether NP exposure influenced rate of metamorphic progression and tail resorption in bullfrog (Rana catesbeiana) tadpoles. Premetamorphic bullfrog tadpoles were exposed for 7 d to one of 3 nominal concentrations of NP (234 μg/L, 468 μg/L, or 936 μg/L) with or without the addition of exogenous 3,3′,5-triiodothyronine (T3). In the absence of exogenous T3, NP significantly increased the rate of tail growth (as measured by tail length) at 936 μg/L. There was no significant effect of NP alone on tail width, limb development, or the process of cranial transformation. When T3 was added to the treatments, increasing NP concentrations were associated with a significant decrease in the rate of cranial transformation, and at the highest dose, the rate of tail resorption was significantly lower than in the controls. Overall, NP had an inhibitory effect on the rate of bullfrog tadpole metamorphic progression and tail resorption. The authors thank the Georgia Basin Ecosystem Initiative (GBEI), Toxic Substance Research Initiative (Health Canada and Environment Canada), Pacific Environmental Science Centre, and Dr. Ken Hall.

Validation of an amphibian sperm inhibition toxicological test method using zinc

Analysis of sperm has been investigated as a possible method to examine the toxicity of environmental contaminants. The amphibian sperm inhibition toxicological test (ASITT) method examines the effects of contaminants on Xenopus laevis (African clawed frog) sperm motility and path trajectories. As part of a preliminary validation of the method, the effects of increasing divalent metal ion, zinc (Zn2+), on X. laevis sperm motility were examined. We hypothesized that Zn2+ concentration would have significant inhibitory effects on percent sperm motility, velocities, and trajectories. The Zn2+ was added to a control solution in concentrations from 0 to 1,417 microg/L. Sperm cells were videotaped at 30 frames per second under x 400 microscope, and percent motility was recorded and paths were mapped by marking the change in position of the sperm head over a period of 1 s. Sperm motility was categorized as progressive, hyperactivated, idle, or nonmotile, and velocities and trajectories were calculated on the basis of x,y coordinates. Increasing Zn2+ concentrations caused a significant exponential decay in percent total motility and progressive motility. Straight-line velocity increased with increasing Zn2+ concentrations. Overall, results suggest that Zn2+ may be interfering with cellular processes, such as cellular respiration, flagellar bending, or ion exchange, thereby inhibiting sperm motility.