, Volume 6, Issue 1, pp 91–98 | Cite as

Combined Effects of Atrazine and Chlorpyrifos on Susceptibility of the Tiger Salamander to Ambystoma tigrinum Virus

Original Contribution


Several hypotheses have been examined as potential causes of global amphibian declines, including emerging infectious diseases and environmental contaminants. Although these factors are typically studied separately, animals are generally exposed to both stressors simultaneously. We examined the effects of the herbicide atrazine and the insecticide chlorpyrifos on the susceptibility of tiger salamander larvae, Ambystoma tigrinum, to a viral pathogen, Ambystoma tigrinum virus (ATV). Environmentally relevant concentrations of atrazine (0, 20, 200 μg/L) and chlorpyrifos (0, 2, 20, 200 μg/L) were used along with ATV in a fully factorial experimental design whereby individually housed, 4-week-old larvae were exposed for 2 weeks. Atrazine alone was not lethal to larvae, and chlorpyrifos alone was lethal only at the highest concentration. When combined with ATV, chlorpyrifos increased susceptibility to viral infection and resulted in increased larval mortality. A significant interactive effect between atrazine and ATV was detected. Atrazine treatments slightly decreased survival in virus-exposed treatments, yet slightly increased survival in the virus-free treatments. These findings corroborate earlier research on the impacts of atrazine, in particular, on disease susceptibility, but exhibit greater effects (i.e., reduced survival) when younger larvae were examined. This study is the first of its kind to demonstrate decreases in amphibian survival with the combination of pesticide and a viral disease. Further examination of these multiple stressors can provide key insights into potential significance of environmental cofactors, such as pesticides, in disease dynamics.


pesticide disease multiple stressors amphibian salamander virus 



The authors thank J. Baumsteiger, J. Eastman, J. Stewart, K. Benyo, and S. Spear for their help in setting up and taking down the experiment. J. Roth and J. Collins provided the salamander larvae used in this experiment. Additional support was provided by A. Hart, D. Fisher, and B. Han. Funding for this project was provided by a grant from the National Science Foundation. The authors also thank the anonymous reviewers for comments to improve this manuscript.


  1. Barron MG, Woodburn KB (1995) Ecotoxicology of chlorpyrifos. Reviews of Environmental Contamination and Toxicology 144:1–93Google Scholar
  2. Belden JB, Lydy MJ (2000) Impact of atrazine on organophoshate insecticide Toxicity. Environmental Toxicology and Chemistry 19:2266–2274CrossRefGoogle Scholar
  3. Berger L, Speare R, Daszak P, Green DE, Cunningham AA Goggin CL, Slocombe R, Ragan MA, Hyatt AD, McDonald KR, Hines HB, Lips KR, Marantelli G, Parkes H (1998) Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America. Proceedings of the National Academy of Sciences USA 95:9031–9036CrossRefGoogle Scholar
  4. Blaustein AR, Kiesecker JM (2002) Complexity in conservation: lessons from the global decline of amphibian populations. Ecology Letters 5:597–608CrossRefGoogle Scholar
  5. Boone MD, James SM (2003) Interactions of an insecticide, herbicide and natural stressors in amphibian community mesocosms. Ecological Applications 13:829–841CrossRefGoogle Scholar
  6. Brunner JL, Schock DM, Davidson EW, Collins JP (2004) Intraspecific reservoirs: complex life history and the persistence of a lethal ranavirus. Ecology 85:560–566CrossRefGoogle Scholar
  7. Brunner JL, Richards K, Collins JP (2005) Dose and host characteristics influence virulence of ranavirus infections. Oecologia 44: 399-406CrossRefGoogle Scholar
  8. Carey C (2000) Infectious disease and worldwide declines of amphibian populations, with comments on emerging diseases in coral reef organisms and in humans. Environmental Health Perspectives 108:143–150CrossRefGoogle Scholar
  9. Carey C (1993) Hypothesis concerning the causes of the disappearance of boreal toads from the mountains of Colorado. Conservation Biology 7:355–362CrossRefGoogle Scholar
  10. Charlemagne J (1979) Thymus independent anti-horse erythrocyte antibody response and suppressor T cells in the Mexican axolotl (Amphibia, Urodela, Ambystoma mexicanum). Immunology 36:643–648Google Scholar
  11. Chinchar VG (2002) Ranaviruses (family Iridoviridae): emerging cold-blooded killers. Archives of Virology 147:447–470CrossRefGoogle Scholar
  12. Christin MS, Gendron AD, Brousseau P, Me’Nard L, Marcogliese DJ, Cyr D, Ruby S, Fournier M (2003) Effects of agricultural pesticides on the immune system of Rana pipiens and on its resistance to parasitic infection. Environmental Toxicology and Chemistry 22:1127–1133CrossRefGoogle Scholar
  13. Collins JP, Storfer AS (2003) Global amphibian declines: sorting the hypotheses. Diversity and Distributions 9:89–98CrossRefGoogle Scholar
  14. Collins JP, Halliday T (2005) Forecasting changes in amphibian biodiversity: aiming at a moving target. Philosophical Transactions of the British Royal Society: B 360:309–314CrossRefGoogle Scholar
  15. Colombo A, Federica O, Bonfanti P (2005) Exposure to the organophosphorus pesticide chlorpyrifos inhibits acetylcholinesterase activity and affects muscular integrity in Xenopus laevis larvae. Chemosphere 61:1665–1671CrossRefGoogle Scholar
  16. Daszak P, Berger L, Cunningham AA, Hyatt AD, Green DE, Speare R (1999) Emerging infectious diseases and amphibian population declines. Emerging Infectious Disease 5:735748CrossRefGoogle Scholar
  17. Daszak P, Cunningham AA, Hyatt AD (2000) Emerging infectious diseases of wildlife- threats to biodiversity and human health. Science 287:443–449CrossRefGoogle Scholar
  18. Daszak P, Cunningham AA, Hyatt AD (2003) Infectious disease and amphibian population declines. Diversity and Distributions 9:141–150CrossRefGoogle Scholar
  19. De Castro F, Bolker B (2005) Mechanisms of disease-induced extinction. Ecology Letters 8:117–126CrossRefGoogle Scholar
  20. Diana SG, Resetarits WJ, Schaeffer DJ, Beckmen KB, Beasley VR (2000) Effects of atrazine on amphibian growth and survival in artificial aquatic communities. Environmental Toxicology and Chemistry 19:2961–2967CrossRefGoogle Scholar
  21. Forson D, Storfer AS (2006a) Atrazine increases ranavirus susceptibility in the tiger salamander, Ambystoma tigrinum. Ecological Applications 16:2325–2332CrossRefGoogle Scholar
  22. Forson D, Storfer AS (2006b) Effects of atrazine and iridovirus infection on survival and life history characteristics in long-toed salamanders, Ambystoma macrodactylum. Environmental Toxicology and Chemistry 25:168–173CrossRefGoogle Scholar
  23. Hayes TB, Collins A, Lee M, Mendoza M, Noriega N, Stuart AA, Vonk A (2002) Hermaphroditic, demasculinized frogs after exposure to the herbicide atrazine at low ecologically relevant doses. Proceedings of the National Academy of Sciences USA 99:5476–5480CrossRefGoogle Scholar
  24. Hayes TB, Haston K, Tsui M, Hoang A, Haeffele C, Vonk A (2003) Atrazine-induced hermaphroditism at 0.1 ppb in American leopard frogs (Rana pipiens): laboratory and field evidence. Environmental Health Perspectives 111:568–575Google Scholar
  25. Hurlbert SH, Mulla MS, Keith JO, Westlake WE, Düsch ME (1970) Biological effects and persistence of Dursban in freshwater ponds. Journal of Economic Entomology 1:43–52Google Scholar
  26. Jancovich JK, Davidson EW, Parameswaran N, Mao J, Chinchar VG, Collins JP, Jacobs BL, Storfer AS (2005) Evidence for emergence of an amphibian iridoviral disease because of human-enhanced spread. Molecular Ecology 14:213–224CrossRefGoogle Scholar
  27. Jancovich JK, Davidson EW, Morado JF, Jacobs BL, Collins JP (1997) Isolation of a lethal virus from the endangered tiger salamander Ambystoma tigrinum stebbinsi. Diseases of Aquatic Organisms 31:161–167CrossRefGoogle Scholar
  28. Johnson TJ, Chase JM, Dosch KL, Hartson RB, Gross JA, Larson DJ, Sutherland DR, Carpenter SR (2007) Aquatic eutrophication promotes pathogenic infection in amphibians. Proceedings of the National Academy of Sciences USA 104:15781–15786CrossRefGoogle Scholar
  29. Kiely T, Donaldson D, Grude A (2004) Pesticide industry sales and usage: 2000 and 2001 market estimates. EPA(7503C)-733-R-04-001. Final/Technical Report. U.S. Environmental Protection Agency, Washington, DCGoogle Scholar
  30. Kiesecker JM (2002) Synergism between trematode infection and pesticide exposure: a link to amphibian limb deformities in nature? Proceedings of the National Academy of Sciences USA 99:9900–9904CrossRefGoogle Scholar
  31. Larson DL, McDonald S, Fivizzani AJ, Newton WE, Hamilton SJ (1998) Effects of the herbicide atrazine on Ambystoma tigrinum metamorphosis: Duration, larval growth, and hormonal response. Physiological Zoology 71:671 – 679Google Scholar
  32. Manzanti L, Rice C, Bialek K, Sparling D, Stevenson C, Johnson WE, Kangas P, Rheinstein J (2003) Aqueous-phase disappearance of atrazine, metolachlor, and chlorpyrifos in laboratory aquaria and outdoor macrocosms. Archives of Environmental Contamination and Toxicology 44:67–76CrossRefGoogle Scholar
  33. McConnell LL, LeNoir JS, Datta S, Seiber JN (1998) Wet deposition of current-use pesticides in the Sierra Nevada mountain range, California, USA. Environmental Toxicology and Chemistry 17:1908–1916CrossRefGoogle Scholar
  34. Hamish McCallum H, Dobson A (1995) Detecting disease and parasite threats to endangered species and ecosystems. Trends in Ecology and Evolution 10:190–194CrossRefGoogle Scholar
  35. Relyea R, Hoverman J (2006) Assessing the ecology in ecotoxicology: a review and synthesis in freshwater systems. Ecology Letters 9:1157–1171CrossRefGoogle Scholar
  36. Rohr JR, Schotthoefer AM, Raffel TR, Carrick HJ, Halstead N, Hoverman JT, Johnson CM, Johnson LB, Lieske C, Piwoni MD, Schoff PK, Beasley VR (2008) Agrochemicals increase trematode infections in a declining amphibian species. Nature doi: 10.1038/nature07281
  37. Rohr JR, Sager T, Sesterhenn TM, Palmer BD (2006) Exposure, postexposure, and density-mediated effects of atrazine on amphibians: Breaking down net effects into their parts. Environmental Health Perspectives 114:46–50CrossRefGoogle Scholar
  38. Solomon KR, Baker DB, Richards RP, Dixon KR, Klaine SJ, La Point TW, Kendall RJ, Weisskopf CP, Giddings JM, Giesy JP, Hall LW Jr, Williams WM (1996) Ecological risk assessment of atrazine in North American surface waters. Environmental Toxicology and Chemistry 15:31–76CrossRefGoogle Scholar
  39. Storfer A, Alfaro ME, Ridenhour BJ, Jancovich JK, Mech SG, Parris MJ, Collins JP (2007) Phylogenetic concordance analysis shows an emerging pathogen is novel and endemic. Ecology Letters 10:1075–1083CrossRefGoogle Scholar
  40. Storrs SI, Kiesecker JM (2004) Survivorship patterns of larval amphibians exposed to low concentrations of atrazine. Environmental Health Perspectives 112:1054–1057Google Scholar
  41. Stuart SN, Chanson JS, Cox NA, Young BE, Rodrigues ASL, Fischman DL, Waller RW (2004) Status and trends of amphibian declines and extinctions worldwide. Science 306:1783–1786CrossRefGoogle Scholar
  42. van den Brink PJ, van Wijngaarden RPA, Lucassen WGH, Brock TCM., Leeuwangh P (1996) Effects of the insecticide Dursban 4E (active ingredient chlorpyrifos) in outdoor experimental ditches: II. Invertebrate community responses and recovery. Environmental Toxicology and Chemistry 15:1133–1142CrossRefGoogle Scholar
  43. Wacksman MN, Maul JD, Lydy MJ (2006) Impact of atrazine on chlorpyrifos toxicity in four aquatic vertebrates. Archives of Environmental Contamination and Toxicology 51:681689CrossRefGoogle Scholar
  44. Widder PD, Bidwell JR (2006) Cholinesterase activity and behavior in chlorpyrifos-exposed Rana sphenocephala tadpoles. Environmental Toxicology and Chemistry 25:2446–2454CrossRefGoogle Scholar

Copyright information

© International Association for Ecology and Health 2009

Authors and Affiliations

  1. 1.Biology DepartmentUniversity of South DakotaVermillionUSA
  2. 2.Biological SciencesWashington State UniversityPullmanUSA

Personalised recommendations