Effect of Individual and Combined Treatments of Pesticide, Fertilizer, and Salt on Growth and Corticosterone Levels of Larval Southern Leopard Frogs (Lithobates sphenocephala)

  • Rose Adelizzi
  • Julia Portmann
  • Robin Van MeterEmail author


Human activities have introduced a variety of chemicals, including pesticides, fertilizers, and salt, into the environment, which may have deleterious effects on the organisms inhabiting these areas. Amphibians are especially susceptible to absorption of chemical pollutants. To determine the possible combined effects of these chemicals on amphibian development and stress levels, Southern leopard frog (Lithobates sphenocephala) larvae were exposed to one of eight individual or combined treatments of atrazine, ammonium nitrate fertilizer, and sodium chloride salt. Stress levels, indicated by release of the stress hormone corticosterone, were measured premetamorphosis at week 8 of development. Water hormone samples were processed to analyze corticosterone levels. Changes in tadpole growth were determined by surface area measurements taken from biweekly photographs. The combined chemical treatment of atrazine, salt, and fertilizer had a significant interactive effect by increasing stress levels before metamorphosis (p = 0.003). After a month of larval development, tadpoles exposed to ammonium nitrate had larger surface area (p = 0.035). Tadpoles exposed to atrazine had a lower growth rate throughout larval development (p = 0.025) and the lowest number of individuals reaching metamorphosis at 33%. However, the frogs in the atrazine treatment that did successfully metamorphose did so in fewer days (p = 0.002). Because amphibians are exposed to multiple chemicals simultaneously in the environment, assessing the effects of a combination of contaminants is necessary to improve application strategies and ecosystem health.



Many thanks to Dr. Leslie Sherman for her editorial assistance with this manuscript. Help with statistical analyses was provided by Dr. George Spilich from the Psychology Department. The ELx808 Ultra Microplate Reader at 405 nm (Biotek Instruments Inc., Winooski, VT, USA) was provided by Psychology Department at Washington College.

Authors’ Contribution

All authors contributed to experimental design, data collection, data analysis, and writing the paper equally.


This research was funded by the Biology & Environmental Science and Studies Departments at Washington College, John S. Toll Fellows Program, and Douglas Cater Society of Junior Fellows.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical Approval

Southern leopard frog eggs were collected under the Maryland State Scientific Collecting Permit #55848 and euthanized according to IACUC protocol #SP17-003.

Data Availability

Research data pertaining to the present study are located at


  1. Adams AJ, Pessier AP, Briggs CJ (2017) Rapid extirpation of a North American frog coincides with an increase in fungal pathogen prevalence: historical analysis and implications for reintroduction. Ecol Evol. Google Scholar
  2. Allran JW, Karasov WH (2000) Effects of atrazine and nitrate on northern leopard frog (Rana pipiens) larvae exposed in the laboratory from posthatch through metamorphosis. Environ Toxicol Chem 19:2850–2855. Google Scholar
  3. Boone MD, Bridges CM, Fairchild JF, Little EE (2005) Multiple sublethal chemicals affect tadpoles of the green frog Rana clamitans. Environ Toxicol Chem 24(5):1267–1272Google Scholar
  4. Brand A, Snodgrass J, Gallagher M, Casey R, Van Meter R (2010) Lethal and sublethal effects of embryonic and larval exposure of Hyla versicolor to stormwater pond sediments. Arch Environ Contam Toxicol 58(2):325–331. Google Scholar
  5. Burgett AA, Wright CD, Smith GR, Fortune DR, Johnson SL (2007) The impact of ammonium nitrate on wood frog (Rana sylvatica) tadpoles: effects on survivorship and behavior. Herpetol Conserv Biol 2(1):29–34Google Scholar
  6. Burraco P, Gomez-Mestre I (2016) Physiological stress responses in amphibian larvae to multiple stressors reveal marked anthropogenic effects even below lethal levels. Physiol Biochem Zool 89(6):462–472Google Scholar
  7. Chambers DL (2011) Increased conductivity affects corticosterone levels and prey consumption in larval amphibians. J Herpetol 45(2):219–223Google Scholar
  8. Charbonnier JF, Pearlmutter J, Vonesh JR, Gabor CR, Forsburg ZR, Grayson KL (2018) Cross-life stage effects of aquatic larval density and terrestrial moisture on growth and corticosterone in the spotted salamander. Divers 10(3):1–16. Google Scholar
  9. Chinathamby K, Reina RD, Bailey PCE, Lees BK (2006) Effects of salinity on the survival, growth and development of the brown tree frog (Litoria ewingii). Aust J Zool 54(2):97–105Google Scholar
  10. Correll DL, Jordan TE, Weller DE (1992) Cross media inputs to eastern U.S. watersheds and their significance to estuarine water quality. Water Sci Technol 26(12):2675–2683Google Scholar
  11. Dananay KL, Krynak KL, Jrynak TJ, Benard MF (2015) Legacy of road salt: apparent positive larval effects counteracted by negative postmetamorphic effects in wood frogs. Environ Toxicol Chem 34(10):2417–2424. Google Scholar
  12. Davis AK, Connell LL, Grosse A, Maerz JC (2008) A fast, non-invasive method of measuring growth in tadpoles using image analysis. Herpetol Rev 39(1):56–58Google Scholar
  13. DeNoyelles F, Kettle WD, Sinn DE (1982) The response of plankton communities in experimental ponds to atrazine, the most heavily used pesticide in the United States. Ecology 63:1285–1293Google Scholar
  14. Gabor CR, Bosch J, Fries JN, Davis DR (2013) A non-invasive water borne hormone assay for amphibians. Amphibia-Reptilia 34:151–162Google Scholar
  15. Gabor CR, Fisher MC, Bosch J (2015) Elevated corticosterone levels and changes in amphibian behavior are associated with Batrachochytrium dendrobatidis (Bd) infection and Bd lineage. PLoS ONE 10(4):1–13. Google Scholar
  16. Gabor CR, Davis DR, Kim DS, Zabierek KC, Bendik NF (2018) Urbanization is associated with elevated corticosterone in Jollyville Plateau salamanders. Ecol Indic 85:229–235. Google Scholar
  17. Gallant N, Teather K (2001) The differences in size, pigmentation, and fluctuating asymmetry in stressed and nonstressed northern leopard frogs (Rana pipiens). Ecoscience 8(4):430–436Google Scholar
  18. Glinski DA, Purucker ST, Van Meter RJ, Black MC, Henderson WM (2018) Analysis of pesticides in surface water, stemflow, and throughfall in an agricultural area in South Georgia, USA. Chemosphere 209:496–507Google Scholar
  19. Hall EM, Brady SP, Mattheus NM, Earley RL, Diamond M, Crespi EJ (2017) Physiological consequences of exposure to salinized roadside ponds on wood frog larvae and adults. Biol Conserv 209:98–106. Google Scholar
  20. Harman-Fetcho JA, McConnell LL, Rice CP, Baker JE (2000) Wet deposition and air–water gas exchange of currently used pesticides to a subestuary of the Chesapeake Bay. Environ Sci Technol 34(8):1462–1468. Google Scholar
  21. Hauer FR, Lamberti GA (eds) (2006) Methods in stream ecology, 2nd edn. Academic, San DiegoGoogle Scholar
  22. Howe GE, Gillis R, Mowbray RC (1998) Effect of chemical synergy and larval stage on the toxicity of atrazine and alachlor to amphibian larvae. Environ Toxicol Chem 17(3):519–525Google Scholar
  23. Karraker NE, Gibbs JP, Vonesh JR (2008) Impacts of road deicing salt on the demography of vernal pool-breeding amphibians. Ecol Appl 18(3):724–734. Google Scholar
  24. Kaushal SS, Groffman PM, Likens GE, Belt KT, Stack WP, Kelly VR, Band LE, Fisher GT (2005) Increased salinization of fresh water in the northeastern United States. PNAS USA 102(38):13517–13520Google Scholar
  25. Kaushal SS, Likens GE, Pace ML, Utz RM, Haq S, Gorman J, Grese M (2018) Freshwater salinization syndrome on a continental scale. PNAS. Google Scholar
  26. Kelly VR, Lovett GM, Weathers KC, Findlay SE, Strayer DL, Burns DJ, Likens GE (2008) Long-term sodium chloride retention in a rural watershed: legacy effects of road salt on stream water concentration. Environ Sci Technol 42(2):410–415. Google Scholar
  27. Kuang Z, McConnell LL, Torrents A, Meritt D, Tobash S (2003) Atmospheric deposition of pesticides to an agricultural watershed of the Chesapeake Bay. J Environ Qual 32:1611–1622Google Scholar
  28. Kulkarni SS, Buchholz DR (2014) Corticosteroid signaling in frog metamorphosis. Gen Comp Endocrinol 203:225–231Google Scholar
  29. Lowrance R, Leonard RA, Asmussen LE, Todd RL (1985) Nutrient budgets for agricultural watersheds in the southeastern coastal plain. Ecology 66(1):287–296Google Scholar
  30. Mann RM, Hyne RV, Choung CB, Wilson SP (2009) Amphibians and agricultural chemicals: review of the risks in a complex environment. Environ Pollut 157(11):2903–2907Google Scholar
  31. McDiarmid RW, Altig R (1999) Tadpoles: the biology of anuran larvae. Nature 1–44Google Scholar
  32. McMahon TA, Halstead NT, Johnson S, Raffel TR, Roman JM, Crumrine PW, Boughton RK, Martin LB, Rohr JR (2011) The fungicide chlorothalonil is nonlinearly associated with corticosterone levels, immunity, and mortality in amphibians. Environ Health Perspect 119(8):1098–1103Google Scholar
  33. McMahon TA, Boughton RK, Martin LB, Rohr J (2017) Exposure to the herbicide atrazine nonlinearly affects tadpole corticosterone levels. J Herpetol 51(2):270–273Google Scholar
  34. Ortiz ME, Marco A, Saiz N, Lizana M (2004) Impact of ammonium nitrate on growth and survival of six european amphibians. Arch Environ Contam Toxicol 47(2):234–239. Google Scholar
  35. Ortiz-Santaliestra ME, Marco A, Jose M, Ndez F, Lizana M (2006) Influence of developmental stage on sensitivity to ammonium nitrate of aquatic stages of amphibians. Environ Toxicol Chem 25(1):105–111. Google Scholar
  36. Pounds JA, Bustamante MR, Coloma LA, Consuegra JA, Fogden MPL, Puschendorf R, Ron SR, Sanchez-Azofeifa GA, Still CJ, Young BE (2006) Widespread amphibian extinctions from epidemic disease driven by global warming. Nature 439(7073):161–167. Google Scholar
  37. Relyea R, Schoeppner NM, Hoverman JT (2005) Pesticides and amphibians: the importance of community context. Ecol Appl 15(4):1125–1134Google Scholar
  38. Rouse JD, Bishop CA, Struger J (1999) Nitrogen pollution: an assessment of its threat to amphibian survival. Environ Health Perspect 107(10):799–803. Google Scholar
  39. Royer TV, David MD, Gentry LE (2006) Timing of riverine export of nitrate and phosphorus from agricultural watersheds in Illinois: implications for reducing nutrient loading to the Mississippi River. Environ Sci Technol 40(13):4126–4131. Google Scholar
  40. Santymire RM, Manjerovic MB, Sacerdote-Velat A (2018) A novel method for the measurement of glucocorticoids in dermal secretions of amphibians. Conserv Physiol 6(1):1–12Google Scholar
  41. Sanzo D, Hecnar H (2006) Effects of road de-icing salt (NaCl) on larval wood frogs (Rana sylvatica). Environ Pollut 140(2):247–256. Google Scholar
  42. Sapolsky RM, Romero LM, Munck AU (2000) How do glucocorticoids influence stress response? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr Rev 1(1):55–89Google Scholar
  43. Smalling KL, Orlando JL, Calhoun D, Battaglin WA, Kuivila KM (2012) Occurrence of pesticides in water and sediment collected from amphibian habitats located throughout the United States, 2009–10. U.S. Geol Surv Data Ser 707:1–36Google Scholar
  44. Solomon KR, Baker DB, Richards RP, Dixon KR, Klaine SJ, La Point TW, Kendall RJ, Wisskopf CP, Giddings JM, Giesy JP, Hall LW Jr, Williams WM (1996) Ecological risk assessment of atrazine in North American surface waters. Environ Toxicol Chem 15(1):31–76. Google Scholar
  45. Stoltz K, Carlson R, Wilcoxen TE (2015) Effects of corticosterone on development and immunocompetence in Western Chorus Frogs (Pseudacris triseriata) and Southern Leopard Frogs (Lithobates sphenocephalus). BIOS 86(2):91–98Google Scholar
  46. Storrs SI, Kiesecker JM (2004) Survivorship patterns of larval amphibians exposed to low concentrations of atrazine. Environ Health Perspect 112(10):1054–1057Google Scholar
  47. 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(5702):1783–1786Google Scholar
  48. Sullivan KB, Spence KM (2003) Effects of sublethal concentrations of atrazine and nitrate on metamorphosis of the African clawed frog. Environ Toxicol Chem 22(3):627–635.;2 Google Scholar
  49. Van Meter RJ, Swan CM, Leips J, Snodgrass JW (2011) Road salt induces novel food web structure and interactions. Wetlands 31(5):843–851Google Scholar
  50. Wood L, Welch AM (2015) Assessment of interactive effects of elevated salinity and three pesticides on life history and behavior of southern toad (Anaxyrus terrestris) tadpoles. Environ Toxicol Chem 34(3):667–676Google Scholar
  51. Zaya RM, Amini Z, Whitaker AS, Kohler SL, Ide CF (2011) Atrazine exposure affects growth, body condition and liver health in Xenopus laevis tadpoles. Aquat Toxicol 104(3–4):243–253Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Rose Adelizzi
    • 1
  • Julia Portmann
    • 1
  • Robin Van Meter
    • 1
    Email author
  1. 1.Departments of Biology and Environmental Science & StudiesWashington CollegeChestertownUSA

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