Final Report: Models and Mechanisms: Understanding Multiple Stressor Effects on an Amphibian Population

EPA Grant Number: R829086
Title: Models and Mechanisms: Understanding Multiple Stressor Effects on an Amphibian Population
Investigators: Palmer, Brent D. , Elskus, Adria , Sih, Andy , Shepherd, Brian , Crowley, Philip
Institution: University of Kentucky
EPA Project Officer: Packard, Benjamin H
Project Period: August 1, 2001 through July 31, 2004 (Extended to November 25, 2005)
Project Amount: $522,832
RFA: Wildlife Risk Assessment (2001) RFA Text |  Recipients Lists
Research Category: Environmental Justice , Biology/Life Sciences , Ecological Indicators/Assessment/Restoration , Aquatic Ecosystems


This research aimed to further our understanding of the effects of multiple environmental stressors, both natural and anthropogenic, on a wildlife population of the streamside salamander, Ambystoma barbouri. The objectives of this research project were to: (1) build, implement, and evaluate a spatially explicit, individual-based population model; (2) conduct experiments to measure the effects of multiple stressors on parameters that enter into the model (e.g. survival, fecundity, growth, extinction rates); and (3) measure physiological variables to investigate potential mechanisms underlying effects of multiple stressors on the parameters that enter into the model. A. barbouri was selected because of increasing concern over the apparent global decline of amphibians and because amphibian life history characteristics make them particularly vulnerable to multiple stressors. To produce a realistic, situation-specific model, the combinatorial effects of multiple stressors likely to be encountered by this population were used to model population demography (growth, survival, fecundity, extinction rates). These stressors included habitat ephemerality (desiccation), predator-prey interactions (chemoreception and locomotion), and anthropogenic pollutants (atrazine).

Summary/Accomplishments (Outputs/Outcomes):

Agricultural contaminants may be contributing to worldwide amphibian declines, but little is known about which agrichemicals pose the greatest threat to particular species. One reason for this is that tests of multiple contaminants under ecologically relevant conditions rarely are conducted concurrently. We examined the effects of 37-day exposure to the agrichemicals atrazine (4, 40, and 400 μg/L), carbaryl (0.5, 5, and 50 μg/L), endosulfan (0.1, 1, and 10 μg/L for 31 days and 0.1, 10, and 100 μg/L for the last 6 days), and octylphenol (5, 50, and 500 μg/L) and to a solvent control on streamside salamanders (A. barbouri) in the presence and absence of food. We found that none of the agrichemicals significantly affected embryo survival, but hatching was delayed by the highest concentration of octylphenol. In contrast to embryos, larval survival was reduced by the highest concentrations of carbaryl, endosulfan, and octylphenol. Growth rates were lower in the highest concentrations of endosulfan and octylphenol than in all other treatments, and the highest concentration of endosulfan caused respiratory distress. Significantly more carbaryl, endosulfan, and octylphenol tanks had larvae with limb deformities than did control tanks. Refuge use was independent of chemical exposure, but 10 μg/L of endosulfan and 500 μg/L of octylphenol decreased larval activity. Systematically tapping tanks caused a greater activity increase in larvae exposed to 400 μg/L of atrazine and 10 μg/L of endosulfan relative to solvent controls, suggesting underlying nervous system malfunction. Hunger stimulated a decrease in refuge use and an increase in activity, but this response was least pronounced in larvae exposed to the highest concentration of any of the four agrichemicals, possibly because these larvae were the most lethargic.

Under our ecologically relevant test conditions, octylphenol seemed to have the greatest detrimental effects on A. barbouri. Chronic exposure to octylphenol induced the greatest mortality, delay in hatching, growth reduction, and lethargy. Endosulfan also had deleterious effects, including increased mortality, reduced growth rates, respiratory distress, limb deformities, and altered behavior. Carbaryl caused significant larval mortality at the highest concentration and produced the greatest percent of malformed larvae but did not significantly affect behavior relative to controls. Although atrazine did not induce significant mortality, it did seem to affect motor function. More studies are needed that concurrently examine the effect of multiple stressors and multiple agrichemicals on amphibians so we can better identify and mitigate the effects of the agrichemicals that pose the greatest threat.

In addition, amphibian populations can be affected adversely by multiple biotic and abiotic stressors that together can contribute to their local and global decline. In other experiments, we focused on the combined effects of food limitation, drying conditions, and exposure to possibly the most abundant and widely used herbicide in the world, atrazine. We used a factorial design to evaluate the effects of exposure to four ecologically relevant doses of atrazine (approximate measured doses: 0, 4, 40, and 400 μg/L), two food levels (limited and unlimited food), and two hydroperiods (presence or absence of a dry down) on the survival, life history, and behavior of the streamside salamander, A. barbouri, from the embryo stage through metamorphosis. In general, food and atrazine levels did not interact statistically, and atrazine affected dependent variables in a standard, dose-dependent manner. Exposure to 400 μg/L of atrazine decreased embryo survival and increased time to hatching. Drying conditions and food limitation decreased larval survival, whereas 400 μg/L of atrazine only reduced larval survival in one of the two years tested, suggesting that the lethality of atrazine may be condition dependent. Sublethal effects included elevated activity and reduced shelter use associated with increasing concentrations of atrazine and food limitation. The larval period was lengthened by food limitation and shortened by 400 μg/L of atrazine. Drying conditions accelerated metamorphosis for larvae exposed to 0 and 4 μg/L of atrazine but did not affect timing of metamorphosis for larvae exposed to 40 or 400 μg/L of atrazine. Food limitation, drying conditions, and 400 μg/L of atrazine reduced size at metamorphosis without affecting body condition (relationship between mass and length), even though feeding rates did not differ significantly among atrazine concentrations at any time during development. This suggests that high atrazine levels may have increased larval energy expenditures. Because smaller size at metamorphosis can lower terrestrial survival and lifetime reproduction, resource limitations, drying conditions, and environmentally realistic concentrations of atrazine have the potential to contribute to amphibian declines in impacted systems.

We further demonstrated that detrimental effects are persistent long after exposure. We examined the effects of premetamorphic exposure (mean exposure 64 days) to ecologically relevant concentrations of the globally common herbicide atrazine (0, 4, 40, and 400 μg/L) on the behavior and water retention of individual and groups of postmetamorphic salamanders. Salamanders exposed to 40 and 400 μg/L of atrazine exhibited greater activity, fewer water-conserving behaviors, and accelerated water loss at 4 and 8 months after exposure compared to controls. The effects were independent of the presence of conspecifics, and no recovery from atrazine exposure was detected. These results indicate that the effects of contaminants and adverse climatic conditions or even climate change may interact to harm postmetamorphic amphibians. In addition, these data show that the two stressors can be temporally separate events and still interact. These results emphasize the both latent and cumulative effects of temporal stressors play roles in amphibian survival.

Our chronic study of atrazine exposure during development in salamanders provided the opportunity to investigate the long-term consequences after exposure. For instance, we were interested to see if survival after exposure was influenced or if toxic effects appeared following exposure, a concept termed “carry-over effects.” In addition, contaminant-induced mortality can be compensated for by reduced competition later, a phenomenon known as “density-mediated compensation.” Using the exposure regime above (atrazine at 0, 4, 40, and 400μg/L during development), we later quantified the survival during the following 14 months after exposure. We demonstrated that atrazine-induced mortality during exposure was ameliorated by density-dependent survival after exposure, but complete density-mediated compensation was precluded by significant carryover effects of atrazine. Consequently, all treatment groups had significantly lower survival than controls 14 months after exposure. The greatest change in survival occurred at the lowest exposure concentrations. These nonlinear, long-term, postexposure effects of atrazine have similarities to effects of early developmental exposure to other endocrine disruptors. Together with evidence from other laboratories of low levels of atrazine impairing amphibian gonadal development, these results raise concerns about the role of atrazine in amphibian declines and highlight the importance of considering persistent, postexposure effects when evaluating the impact of xenobiotics on environmental health.

These data demonstrate that developmental exposure to environmentally relevant concentrations of commonly used agrichemicals has significant physiological, behavioral, and life history impacts on salamanders. Many of these effects are not tested traditionally. These data indicate that additional testing procedures are necessary to understand effects of agrichemicals on amphibians and to protect wildlife populations from adverse effects.

Our computer model, implemented in MATLAB 7, can be run on current high-performance microcomputers. Our intended use of the model focuses on understanding interactions among three key stressors (predation, desiccation, environmental chemicals) on the growth, survival, fecundity, and extinction rates of a stream salamander population. We are confident that the model, once fully refined and validate, will prove adaptable to similar scenarios in other aquatic systems and that predictions based on stressor interactions will generate both testable hypotheses and management approaches applicable to a wide range of environmental scenarios.


We have shown that atrazine exposure to a larval streamside salamander (A. barbouri) has numerous deleterious consequences, including behavioral and physiological effects and mortality. Some of these effects may occur more than a year after exposure occurs. The A. barbouri previously exposed to concentrations of atrazine as low as 4 ppb had significantly lower survival 421 days after exposure, a result that was apparent only when considering the accumulation of both exposure and carryover effects. Atrazine at 4 ppb is only 1 ppb greater than the maximum allowable level in U.S. drinking water and a concentration to which amphibians may be chronically exposed. In the process of measuring the effects of atrazine on A. barbouri survival, we developed a laboratory framework for quantifying exposure, carryover, density-mediated, and net effects of stressors on survival that may serve as a more practical alternative to the rigorous demands and complexities of population growth rate analyses.

We demonstrated that developmental exposure in A. barbouri was associated with hyperactivity and increased desiccation risk 8 months after exposure, with no detectable recovery from atrazine exposure. These data suggest that exposure to atrazine early in development may have permanent effects on these salamanders. This conclusion is supported by our finding that the effect of atrazine during exposure did not differ from the magnitude of its carryover effect, once again suggesting that there was no recovery from atrazine exposure.

Various studies in other laboratories have reported nonlinear relationships between atrazine concentration and amphibian responses, and many of these relationships have been nonmonotonic, with the largest change in response occurring at low exposure levels. Endocrine disruptors commonly induce nonmonotonic dose-response curves, and thus the endocrine-disrupting potential of atrazine has been suggested as the cause of the documented nontraditional nonmonotonic dose responses. The relationship between atrazine concentration and survival in this study was nonlinear (logarithmic) but monotonic. The responses to atrazine recorded in A. barbouri have many consistencies with endocrine disruption. The largest adverse change in survival occurred at low exposure concentrations. Further, exposure to endocrine-disrupting compounds during critical developmental stages often induces irreversible effects, not unlike the long-term, postexposure effects observed here. Virtually every response to atrazine we have quantified in this species using these concentrations has had the greatest response change at low concentrations, suggesting that A. barbouri may be sensitive to marginal atrazine inputs into aquatic systems.

The deleterious, long-term effects of larval atrazine suggest that contaminant exposure after metamorphosis may not be necessary for contaminants to harm postmetamorphic amphibians, a life stage that often disproportionately affects population dynamics. This is important because exposure to substantial concentrations of contaminants probably is more likely before metamorphosis, because most amphibian embryos and larvae are strictly aquatic and cannot readily escape water bodies where many contaminants accumulate and concentrate.

This series of studies raises concerns about the role of this widespread, persistent, and mobile herbicide in the international decline of amphibians. Certainly, more research on the effects of atrazine on amphibians is necessary. In addition to concerns for amphibians, the persistent effects of atrazine should be of general concern because some of the most catastrophic effects of contaminants on wildlife and human populations have been associated with lasting, postexposure effects, such as the enduring effects of organochlorine insecticides (e.g., DDT) and the delayed neurotoxicity of organophosphorus pesticides and various metals. Despite these historical cases, chronic studies focusing on carryover effects of contaminants remain surprisingly rare. This is especially disconcerting when one considers that short-term effects typically are inferior to long-term effects for explaining population level changes and are more likely to give the erroneous impression that certain xenobiotics are innocuous. Although it can be challenging to quantify long-term effects of stressors across temporal scales, it likely will be necessary to understand fully the impacts of xenobiotics on environmental health.

Journal Articles on this Report : 5 Displayed | Download in RIS Format

Other project views: All 25 publications 5 publications in selected types All 5 journal articles
Type Citation Project Document Sources
Journal Article Rohr JR, Elskus AA, Shepherd BS, Crowley PH, McCarthy TM, Niedzwiecki JH, Sager T, Sih A, Palmer BD. Lethal and sublethal effects of atrazine, carbaryl, endosulfan, and octylphenol on the streamside salamander (Ambystoma barbouri). Environmental Toxicology and Chemistry 2003;22(10):2385-2392. R829086 (2002)
R829086 (2003)
R829086 (Final)
  • Abstract: SETAC Journals Abstract
  • Journal Article Rohr JR, Elskus AA, Shepherd BS, Crowley PH, McCarthy TM, Niedzwiecki JH, Sager T, Sih A, Palmer BD. Multiple stressors and salamanders:effects of an herbicide, food limitation, and hydroperiod. Ecological Applications 2004;14(4):1028-1040. R829086 (2003)
    R829086 (Final)
  • Full-text: University of South Florida PDF
  • Abstract: ESA Journals Abstract
  • Journal Article Rohr JR, Crumrine PW. Effects of an herbicide and an insecticide on pond community structure and processes. Ecological Applications 2005;15(4):1135-1147. R829086 (Final)
  • Full-text: University of South Florida PDF
  • Abstract: ESA Journals Abstract
  • Journal Article Rohr JR, Palmer BD. Aquatic herbicide exposure increases salamander desiccation risk eight months later in a terrestrial environment. Environmental Toxicology and Chemistry 2005;24(5):1253-1258. R829086 (Final)
  • Abstract from PubMed
  • Journal Article Rohr JR, Sager T, Sesterhenn TM, Palmer BD. Exposure, postexposure, and density-mediated effects of atrazine on amphibians: breaking down net effects into their parts. Environmental Health Perspectives 2006;114(1):46-50. R829086 (Final)
  • Abstract from PubMed
  • Full-text: EHP HTML
  • Other: EHP PDF
  • Supplemental Keywords:

    Atrazine, octylphenol, carbaryl, endosulfan, salamander, larval, metamorphosis, development, behavior, water, exposure, risk assessment, effects, ecological effects, vulnerability, sensitive populations, dose-response, animal, organism, population, stressor, cumulative effects, chemicals, toxics, terrestrial, aquatic, habitat, life-history, conservation, environmental chemistry, biology, endocrinology, ecology, modeling, agriculture,, RFA, Health, Scientific Discipline, Air, Ecosystem Protection/Environmental Exposure & Risk, Aquatic Ecosystems & Estuarine Research, exploratory research environmental biology, wildlife, Ecosystem/Assessment/Indicators, climate change, Air Pollution Effects, Aquatic Ecosystem, Susceptibility/Sensitive Population/Genetic Susceptibility, Monitoring/Modeling, Ecological Effects - Environmental Exposure & Risk, Environmental Monitoring, genetic susceptability, Ecological Risk Assessment, Atmosphere, predicting risk, sensitive populations, anthropogenic stress, ecological exposure, demographic data, stressors, watersheds, endocrine disruption , amphibians, dose-response, endocrine disrupting chemical, exposure, amphibian, endocrine disruptors, modeling, multiple stressors, Wildlife Risk Assessment, animal models, salamander eggs, ecosystem stress, watershed assessment, ecological response, toxics, environmental hazard exposures, modeling ecosystems, amphibian population

    Progress and Final Reports:

    Original Abstract
  • 2002 Progress Report
  • 2003 Progress Report
  • 2004
  • 2005