Effect of Individual and Combined Treatments of Pesticide, Fertilizer, and Salt on Growth and Corticosterone Levels of Larval Southern Leopard Frogs (Lithobates sphenocephala)
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.
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.
Southern leopard frog eggs were collected under the Maryland State Scientific Collecting Permit #55848 and euthanized according to IACUC protocol #SP17-003.
Research data pertaining to the present study are located at https://github.com/rvanmeter2/AECT-mixed-agrochemicals.git.
- 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
- 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
- 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
- Chambers DL (2011) Increased conductivity affects corticosterone levels and prey consumption in larval amphibians. J Herpetol 45(2):219–223Google Scholar
- 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
- 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
- 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
- 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
- Gabor CR, Bosch J, Fries JN, Davis DR (2013) A non-invasive water borne hormone assay for amphibians. Amphibia-Reptilia 34:151–162Google Scholar
- 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
- 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
- Hauer FR, Lamberti GA (eds) (2006) Methods in stream ecology, 2nd edn. Academic, San DiegoGoogle Scholar
- 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
- 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
- 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
- Kulkarni SS, Buchholz DR (2014) Corticosteroid signaling in frog metamorphosis. Gen Comp Endocrinol 203:225–231Google Scholar
- 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
- 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
- McDiarmid RW, Altig R (1999) Tadpoles: the biology of anuran larvae. Nature 1–44Google Scholar
- 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
- 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
- Relyea R, Schoeppner NM, Hoverman JT (2005) Pesticides and amphibians: the importance of community context. Ecol Appl 15(4):1125–1134Google Scholar
- 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
- 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
- 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
- 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. https://doi.org/10.1002/etc.5620150105 Google Scholar
- 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
- Storrs SI, Kiesecker JM (2004) Survivorship patterns of larval amphibians exposed to low concentrations of atrazine. Environ Health Perspect 112(10):1054–1057Google Scholar
- 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
- 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. https://doi.org/10.1897/1551-5028(2003)022%3c0627:EOSCOA%3e2.0.CO;2 Google Scholar
- 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
- 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
- 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