Competition and pesticide exposure affect development of invasive (Rhinella marina) and native (Fejervarya vittigera) rice paddy amphibian larvae

Abstract

Increased pesticide use in rice agricultural ecosystems may alter competitive interactions between invasive and native amphibian species. We conducted an experiment with two rice paddy amphibians found in Luzon, Philippines, the invasive cane toad (Rhinella marina) and the endemic Luzon wart frog (Fejervarya vittigera), to determine whether exposure to a common herbicide, butachlor, drives competitive interactions in favor of the invasive amphibian. Our results revealed that competition had a strong effect on the development of both species, but in opposing directions; Luzon wart frog tadpoles were smaller and developed slower than when raised alone, whereas cane toad tadpoles were larger and developed faster. Contrary to our predictions, development and survival of endemic wart frog tadpoles was not affected by butachlor, whereas invasive cane toad tadpoles were affected across several endpoints including gene expression, body size, and survival. We also observed an interaction between pesticide exposure and competition for the cane toad, where survival declined but body size and expression of thyroid sensitive genes increased. Taken together, our findings indicate that the success of the cane toad larvae in rice fields may be best explained by increased rates of development and larger body sizes of tadpoles in response to competition with native Luzon wart frog tadpoles rather than lower sensitivity to a common pesticide. Our results for the cane toad also provide evidence that butachlor can disrupt thyroid hormone mediated development in amphibians, and further demonstrate that important species interactions such as competition can be affected by pesticide exposure in aquatic ecosystems.

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References

  1. Abigail MEA, Samuel SM, Ramalingam C (2015) Addressing the environmental impacts of butachlor and the available remediation strategies: a systematic review. Int J Environ Sci Technol 12:4025–4036. https://doi.org/10.1007/s13762-015-0866-2

    Article  Google Scholar 

  2. Attademo AM, Peltzer PM, Lajmanovich RC (2005) Amphibians occurring in soybean and implications for biological control in Argentina. Agric Ecosyst Environ 106:389–394. https://doi.org/10.1016/j.agee.2004.08.012

    Article  Google Scholar 

  3. Bambaradeniya C, Edirisinghe JP, De Silva DN, Ranawana KB, Wijekoon S (2004) Biodiversity associated with an irrigated rice agro-ecosystem in Sri Lanka. Biodivers Conserv 12:1715–1753

    Article  Google Scholar 

  4. Bridges CM, Semlitsch RD (2000) Variation in pesticide tolerance of tadpoles among and within species of Ranidae and patterns of amphibian decline. Conserv Biol 14:1490–1499

    Article  Google Scholar 

  5. Bustin SA, Beaulieu J-F, Huggett J et al. (2010) MIQE précis: practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments. BMC Mol Biol 11:74. https://doi.org/10.1186/1471-2199-11-74

    Article  Google Scholar 

  6. Cabrera-Guzmán E, Crossland MR, Brown GP, Shine R (2013a) Larger body size at metamorphosis enhances survival, growth and performance of young cane toads (Rhinella marina). PLoS ONE. 8:7. https://doi.org/10.1371/journal.pone.0070121

  7. Cabrera-Guzmán E, Crossland MR, Shine R (2013b) Competing tadpoles: Australian native frogs affect invasive cane toads (Rhinella marina) in natural waterbodies. Austral Ecol 38:896–904. https://doi.org/10.1111/aec.12029

    Article  Google Scholar 

  8. Cabrera-Guzmán E, Crossland MR, Shine R (2012) Predation on the eggs and larvae of invasive cane toads (Rhinella marina) by native aquatic invertebrates in tropical Australia. Biol Conserv 153:1–9. https://doi.org/10.1016/j.biocon.2012.04.012

    Article  Google Scholar 

  9. Campero M, Slos S, Ollevier F, Stoks R. (2007) Sublethal pesticide concentrations and predation jointly shape life history: behavioral and physiological mechanisms. Ecol Appl 17:2111–2122

    Article  Google Scholar 

  10. Chang J, Liu S, Zhou S et al (2011) Effects of butachlor on reproduction and hormone levels in adult zebrafish (Danio rerio). Exp Toxicol Pathol 65:205–209. https://doi.org/10.1016/j.etp.2011.08.007

    Article  Google Scholar 

  11. Chapman PM (2002) Integrating toxicology and ecology: putting the ‘eco’ into ecotoxicology. Mar Pollut Bull 44:7–15. https://doi.org/10.1016/S0025-326X(01)00253-3

    CAS  Article  Google Scholar 

  12. Choksi NY, Jahnke GD, St. Hilaire C, Shelby M (2003) Role of thyroid hormones in human and laboratory animal reproductive health. Birth Defects Res Part B–Dev Reprod Toxicol 68:479–491. https://doi.org/10.1002/bdrb.10045

    CAS  Article  Google Scholar 

  13. Cohen JE, Schoenly K, Heong KL, Justo H, Arida G, Barrion AT, Litsinger JA (2014) A food web approach to evaluating the effect of insecticide spraying on insect pest population dynamics in a Philippine irrigated rice ecosystem. J Appl Ecol 31:747–763

    Article  Google Scholar 

  14. Cunha-Filho GA, Resck IS, Cavalcanti BC, Pessoa CÓ, Moraes MO, Ferreira JRO, Rodrigues FAR, dos Santos ML (2010) Cytotoxic profile of natural and some modified bufadienolides from toad Rhinella schneideri parotoid gland secretion. Toxicon 56:339–348. https://doi.org/10.1016/j.toxicon.2010.03.021

    CAS  Article  Google Scholar 

  15. Denver RJ (1997) Environmental stress as a developmental cue: corticotropin-releasing hormone is a proximate mediator of adaptive phenotypic plasticity in amphibian metamorphosis. Horm Behav 31:169–179. https://doi.org/10.1006/hbeh.1997.1383

    CAS  Article  Google Scholar 

  16. Denver RJ (2009) Stress hormones mediate environment-genotype interactions during amphibian development. Gen Comp Endocrinol 164:20–31. https://doi.org/10.1016/j.ygcen.2009.04.016

    CAS  Article  Google Scholar 

  17. Diesmos AC (2008) Ecology and diversity of herpetofaunal communities in fragmented lowland rainforests in the Philippines. PhD Thesis, National University of Singapore.

  18. Diesmos AC, Diesmos ML, Brown RM (2006) Status and distribution of alien invasive frogs in the Philippines. J Environ Sci Manag 9:41–53

    Google Scholar 

  19. Diesmos AC, Watters JL, Huron NA, Davis DR, Alcala AC, Crombie RI, Afuang LE, Gee-Das G, Sison RV, Sanguila MB, Penrod ML, Labonte MJ, Davey CS, Leone EA, Diesmos ML, Sy EY, Welton LJ, Brown RM, Cameron DS (2015) Amphibians of the Philippines, Part I: checklist of the Species. Proc Calif Acad Sci 62:457–539

    Google Scholar 

  20. Edirisinghe JP, Bambaradeniya CNB (2006) Rice fields: an ecosystem rich in biodiversity. J Natl Sci Found Sri Lanka 34:57–59. https://doi.org/10.4038/jnsfsr.v34i2.2084

    Article  Google Scholar 

  21. Fort DJ, Degitz S, Tietge J, Touart LW (2007) The hypothalamic-pituitary-thyroid (HPT) axis in frogs and its role in frog development and reproduction. Crit Rev Toxicol 37:117–161. https://doi.org/10.1080/10408440601123545

    CAS  Article  Google Scholar 

  22. García-Muñoz E, Guerrero F, Parra G (2010) Intraspecific and interspecific tolerance to copper sulphate in five Iberian amphibian species at two developmental stages. Arch Environ Contam Toxicol 59:312–321. https://doi.org/10.1007/s00244-010-9473-x

    Article  Google Scholar 

  23. Geng BR, Yao D, Xue QQ (2005) Acute toxicity of the pesticide dichlorvos and the herbicide butachlor to tadpoles of four anuran species. Bull Environ Contam Toxicol 75:343–349. https://doi.org/10.1007/s00128-005-0759-z

    CAS  Article  Google Scholar 

  24. Gosner KL (1960) A simplified table for staging anuran embryos and larvae with notes on identification. Herpetol Leag 16:183–190

    Google Scholar 

  25. GRiSP (Global Rice Science Partnership) (2013) Rice Almanac, 4th edn. International Rice Research Institute, Los Baños

    Google Scholar 

  26. Hayes RA, Crossland MR, Hagman M, Hagman M, Capon RJ, Shine R (2009) Ontogenetic variation in the chemical defenses of cane toads (Bufo marinus): toxin profiles and effects on predators. J Chem Ecol 35:391–399. https://doi.org/10.1007/s10886-009-9608-6

    CAS  Article  Google Scholar 

  27. Hocking DJ, Babbitt KJ (2014) Amphibian contributions to ecosystem services. Herpetol Conserv Biol 9:1–17

    Google Scholar 

  28. Holzer KA, Bayers RP, Nguyen TT, Lawler SP (2017) Habitat value of cities and rice paddies for amphibians in rapidly urbanizing Vietnam. J Urban Ecol 3:1–12. https://doi.org/10.1093/jue/juw007

    Article  Google Scholar 

  29. Horgan FG, Ramal AF, Bernal CC et al (2016) Applying ecological engineering for sustainable and resilient rice production systems. Procedia Food Sci. 6:7–15. https://doi.org/10.1016/j.profoo.2016.02.002

    Article  Google Scholar 

  30. Howard BD (2015) Catching evidence with frogs through a focused ethnographic research with a Filipino rice farming community. MSc Thesis. Northern Arizona University

  31. Hua J, Jones DK, Mattes BM, Cothran RD, Relyea RA, Hoverman JT (2015b) The contribution of phenotypic plasticity to the evolution of insecticide tolerance in amphibian populations. Evol Appl 8:586–596. https://doi.org/10.1111/eva.12267

    CAS  Article  Google Scholar 

  32. Hua J, Jones DK, Mattes BM, Cothran RD, Relyea RA, Hoverman JT (2015a) Evolved pesticide tolerance in amphibians: predicting mechanisms based on pesticide novelty and mode of action. Environ Pollut 206:56–63. https://doi.org/10.1016/j.envpol.2015.06.030

    CAS  Article  Google Scholar 

  33. Huarong GUO, Licheng YIN, Shicui Z, Wenrong F (2010) The toxic mechanism of high lethality of herbicide butachlor in marine flatfish flounder, Paralichthys olivaceus. Ocean Coast Sea Res 9:257–264. https://doi.org/10.1007/s11802-010-1693-1

    Google Scholar 

  34. Johansson F, Lederer B, Lind MI (2010) Trait performance correlations across life stages under environmental stress conditions in the common frog Rana temporaria. PLoS One 5:e11680. https://doi.org/10.1371/journal.pone.0011680

    Article  Google Scholar 

  35. Janssens L, Stoks R (2013) Exposure to a widespread non-pathogenic bacterium magnifies sublethal pesticide effects in the damselfly Enallagma cyathigerum: from the suborganismal level to fitness-related traits. Environ Pollut 177:143–149

  36. Jones DK, Hammond JI, Relyea RA (2011) Competitive stress can make the herbicide Roundup® more deadly to larval amphibians. Environ Toxicol Chem 30:446–454. https://doi.org/10.1002/etc.384

    CAS  Article  Google Scholar 

  37. Kanamori A, Brown DD (1992) The regulation of thyroid hormone receptor beta genes by thyroid hormone in Xenopus laevis. J Biol Chem 267:739–745

    CAS  Google Scholar 

  38. Khatiwada JR, Ghimire S, Khatiwada PS, Paudel B, Bischof R, Jiang J, Haugaasen T (2016) Frogs as potential biological control agents in the rice fields of Chitwan, Nepal. Agric Ecosyst Environ 230:307–314. https://doi.org/10.1016/j.agee.2016.06.025

    Article  Google Scholar 

  39. Knoedler JR, Denver RJ (2014) Kruppel-like factors are effectors of nuclear receptor signaling. Gen Comp Endocrinol 203:49–59. https://doi.org/10.1016/j.ygcen.2014.03.003

    CAS  Article  Google Scholar 

  40. Köhler HR, Triebskorn R (2013) Wildlife ecotoxicology of pesticides: can we track effects to the population level and beyond? Science 80:759–765. https://doi.org/10.1126/science.1237591

    Article  Google Scholar 

  41. Kulkarni SS, Buchholz DR, (2012) Beyond synergy: corticosterone and thyroid hormone have numerous interaction effects on gene regulation in Xenopus tropicalis tadpoles. Endocrinology 153:5309–5324. https://doi.org/10.1210/en.2012-1432

  42. Kuwagata T, Hamasaki T, Watanabe T (2008) Modeling water temperature in a rice paddy for agro-environmental research. Agric For Meteorol 148:1754–1766. https://doi.org/10.1016/j.agrformet.2008.06.011

  43. Lande R, Shannon S (1996) The role of genetic variation in adaptation and population persistence in a changing environment. Evolution 50:434–437. doi: 134.114.101.46

    Article  Google Scholar 

  44. Lee DY, Yasuda M, Yamamoto T, Yoshida T, Kuroiwa Y (1997) Bufalin inhibis endothelial cell proliferation and angiogenesis in vitro. Life Sci 60:127–134

    CAS  Article  Google Scholar 

  45. Leney JL, Drouillard KG, Haffner D (2006) Metamorphosis increases biotransformation of polychlorinated biphenyls: a comparative study of polychlorinated bipheny metabolism in green frogs (Rana clamitans) and leopard frogs (Rana pipiens) at various life stages. Environ Toxicol Chem 25:2971–2980. https://doi.org/10.1897/05-561r1.1

    CAS  Article  Google Scholar 

  46. Li S, Li M, Wang Q, Gui W, Zhu G (2016) Exposure to butachlor causes thyroid endocrine disruption and promotion of metamorphosis in Xenopus laevis. Chemosphere 152:158–165. https://doi.org/10.1016/j.chemosphere.2016.02.098

    CAS  Article  Google Scholar 

  47. Liu W-YY, Wang C-YY, Wang T-SS, Fellers GM, Lai BC, Kam YC (2011) Impacts of the herbicide butachlor on the larvae of a paddy field breeding frog (Fejervarya limnocharis) in subtropical Taiwan. Ecotoxicology 20:377–384. https://doi.org/10.1007/s10646-1010

    CAS  Article  Google Scholar 

  48. Loman J (2001) Intraspecific competition in tadpoles of Rana arvalis: does it matter in nature? A field experiment. Popul Ecol 43:253–263. https://doi.org/10.1007/s10144-001-8189-1

    Article  Google Scholar 

  49. Mamun MIR, Park JH, Choi J-H, Kim HK, Choi WJ, Han SS, Hwang K, Jang NI, Assayed ME, El-Dib MA, Shin HC, Abd EA, Shim JH (2009) Development and validation of a multiresidue method for determination of 82 pesticides in water using GC. J Sep Sci 32:559–574. https://doi.org/10.1002/jssc.200800606

    CAS  Article  Google Scholar 

  50. McKinlay R, Plant Ja, Bell JNB, Voulvoulis N (2008) Endocrine disrupting pesticides: implications for risk assessment. Environ Int 34:168–183. https://doi.org/10.1016/j.envint.2007.07.013

    CAS  Article  Google Scholar 

  51. Mcleod DS, Siler CD, Diesmos AC et al (2011) Amphibians and reptiles of Luzon island, v: the herpetofauna of Angat dam watershed, Bulacan province, Luzon island, philippines. Asian Herpetol Res 2:177–198. https://doi.org/10.3724/SP.J.1245.2011.00177

    Google Scholar 

  52. Mills NE, Semlitsch RD (2004) Competition and predation mediate the indirect effects of an insecticide on southern leopard frogs. Ecol Appl. 14:1041–1054. https://doi.org/10.1890/02-5134

    Article  Google Scholar 

  53. Molloy K, Henderson W (2006) Science of cane toad invasion and control. Proceedings of the Invasive Animals CRC/ CSIRO/Qld NRM&W cane toad workshop. In: Animals. Invasive Animals Cooperative Research Centre, Canberra

  54. Opitz R, Lutz I, Nguyen N-H et al (2006) Analysis of thyroid hormone receptor betaA mRNA expression in Xenopus laevis tadpoles as a means to detect agonism and antagonism of thyroid hormone action. Toxicol Appl Pharmacol. 212:1–13. https://doi.org/10.1016/j.taap.2005.06.014

    CAS  Article  Google Scholar 

  55. Parsons KC, Mineau P, Renfrew RB (2010) Effects of esticide use in rice fields on birds. Waterbirds. 33:193–218. https://doi.org/10.1675/063.033.s115

    Article  Google Scholar 

  56. Pestana JLT, Loureiro S, Baird DJ, Soares AMVM (2009) Fear and loathing in the ben- thos: responses of aquatic insect larvae to the pesticide imidacloprid in the presence of chemical signals of predation risk. Aquat Toxicol 93:138–149. https://doi.org/10.1016/j.aquatox.2009.04.008

    CAS  Article  Google Scholar 

  57. Pizzatto L, Shine R (2008) The behavioral ecology of cannibalism in cane toads (Bufo marinus). Behav Ecol Sociobiol 63:123–133. https://doi.org/10.1007/s00265-008-0642-0

    Article  Google Scholar 

  58. Prentis PJ, Wilson JRU, Dormontt EE et al (2008) Adaptive evolution in invasive species. Trends Plant Sci 13:288–294. https://doi.org/10.1016/j.tplants.2008.03.004

    CAS  Article  Google Scholar 

  59. Punhali PL, PAR (1995) Impact oPlease check collab name “PAR” in ref. Punhali 1995f pesticides on farmer health and the rice environment. Kluwer Academic Publishers, Norwell, MA

    Google Scholar 

  60. Ramirez NEC (2014) The Influence of the frequency of rice crops per year on anuran abundance and diversity in Candaba Swamp, central Luzon, Philippines. MSc Thesis, University of the Philippines Diliman

  61. Rasmussen JJ, Nørum U, Jerris MR et al (2013) Pesticide impacts on predator-prey interactions across two levels of organisation. Aquat Toxicol 140–141:340–345. https://doi.org/10.1016/j.aquatox.2013.06.019

    Article  Google Scholar 

  62. Relyea RA (2002) Competitor-induced plasticity in tadpoles: consequences, cues, and connections to predator-induced plasticity. Ecol Monogr 72:523–540. https://doi.org/10.1890/0012-9615(2002)072[0523:CIPITC]2.0.CO;2

    Article  Google Scholar 

  63. Relyea RA, Hoverman JT (2008) Interactive effects of predators and a pesticide on aquatic communities. Oikos 117:1647–1658. https://doi.org/10.1111/j.1600-0706.2008.16933.x

    Article  Google Scholar 

  64. Rollins LA, Richardson MF, Shine R (2015) A genetic perspective on rapid evolution in cane toads (Rhinella marina). Mol Ecol 24:2264–2276. https://doi.org/10.1111/mec.13184

    Article  Google Scholar 

  65. Rowley J, Brown R, Bain R et al (2010) Impending conservation crisis for Southeast Asian amphibians. Biol Lett 6:336–338. https://doi.org/10.1098/rsbl.2009.0793

    Article  Google Scholar 

  66. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101–1108. https://doi.org/10.1038/nprot.2008.73

    CAS  Article  Google Scholar 

  67. Shi Y-B, Matsuura K, Fujimoto K, Wen W, Fu L (2012) Thyroid hormone receptor actions on transcription in amphibia: the roles of histone modification and chromatin disruption. Cell Biosci 2:42. https://doi.org/10.1186/2045-3701-2-42

    CAS  Article  Google Scholar 

  68. Shine BR (2010) The ecological impact of invasive cane toads (Bufo Marinus) in Australia. Q Rev Biol 85:253–291. https://doi.org/10.1086/655116

    Article  Google Scholar 

  69. Snelder DJ, Masipiquen MD, De Snoo GR (2008) Risk assessment of pesticide usage by smallholder farmers in the Cagayan Valley (Philippines). Crop Prot 27:747–762. https://doi.org/10.1016/j.cropro.2007.10.011

  70. Sodhi NS, Koh LP, Brook BW, Ng PKL (2004) Southeast Asian biodiversity: an impending disaster. Trends Ecol Evol 19:654–660. https://doi.org/10.1016/j.tree.2004.09.006

    Article  Google Scholar 

  71. Therneau T (2016) A package for survival analysis in S. (R package version 2.36–12, 2012)

  72. Toan PV, Sebesvari Z, Bläsing M, Rosendahl I, Renaud FG (2013) Pesticide management and their residues in sediments and surface and drinking water in the Mekong Delta, Vietnam. Sci Total Environ 452–453:28–39. https://doi.org/10.1016/j.scitotenv.2013.02.026

    Article  Google Scholar 

  73. Veldhoen N, Propper CR, Helbing CC (2014) Enabling comparative gene expression studies of thyroid hormone action through the development of a flexible real-time quantitative PCR assay for use across multiple anuran indicator and sentinel species. Aquat Toxicol 148:162–173. https://doi.org/10.1016/j.aquatox.2014.01.008

    CAS  Article  Google Scholar 

  74. Wake DB, Vredenburg VT (2008) Colloquium paper: are we in the midst of the sixth mass extinction? A view from the world of amphibians. Proc Natl Acad Sci USA 105:Suppl:11466–Suppl:11473. https://doi.org/10.1073/pnas.0801921105

    Article  Google Scholar 

  75. Williamson I (1999) Competition between the larvae of the introduced cane toad Bufo marinus (Anura: Bufonidae) and native anurans from the Darling Downs area of southern Queensland. Aust J Ecol 24:636–643. https://doi.org/10.1046/j.1442-9993.1999.00993.x

    Article  Google Scholar 

  76. Wolff SE, Veldhoen N, Helbing CC, Ramirez CA, Malpas JM, Propper CR (2015) Estrogenic environmental contaminants alter the mRNA abundance profiles of genes involved in gonadal differentiation of the American bullfrog. Sci Total Environ 521–522:380–387. https://doi.org/10.1016/j.scitotenv.2015.02.033

    Article  Google Scholar 

  77. Yap SMS, Demayo CG (2015) Farmers’ knowledge and understanding of pesticide use and field spraying practices: a case study of rice farmers in the municipality of Molave, Zamboanga Del Sur, Philippines. Adv Environ Biol 9:134–142. https://doi.org/10.3390/bs5030384

    Google Scholar 

  78. Zhu L, Li W, Zha J et al (2014) Butachlor causes disruption of HPG and HPT axes in adult female rare minnow (Gobiocypris rarus). Chem Biol Interact 221:119–126. https://doi.org/10.1016/j.cbi.2014.07.016

    CAS  Article  Google Scholar 

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Acknowledgements

We thank Frank von Hippel and Rachel Rubin for their editorial support. We are grateful to Linsey Benally, Alexander McCain, Renee Lorica, and Calsey Richardson for their assistance in field and laboratory data collection. We thank the anonymous reviewers who helped to improve the manuscript. Funding was provided by the Merriam Powell Center for Environmental Research’s Integrative Graduate Education, Research, and Traineeship Program (IGERT) Fellowship, the Achievement Rewards for College Scientists (ARCS) Foundation, the Closing Rice Yield Gaps in Asia with a Reduced Environmental Footprint (CORIGAP) funded by the Swiss Agency for Development and Cooperation, the National Institute on Minority Health and Health Disparities of the NIH, Award Number T37MD008626 to CRP, and the National Cancer Institute of the NIH award for the Partnership of Native American Cancer Prevention U54CA143925 to Northern Arizona University.

Author contributions

M.S.G., G.S., and C.R.P. conceived and designed the study. M.S.G. executed the study, analyzed the data, and wrote the manuscript. G.S. and C.R.P. contributed significant editorial guidance on the manuscript.

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Correspondence to Molly E. Shuman-Goodier.

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The authors declare that they have no competing interests associated with this study, and that all applicable institutional and/or national guidelines for the care and use of animals were followed and approved by Northern Arizona’s Institutional Animal Care and Use Committee (IACUC).

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Shuman-Goodier, M.E., Singleton, G.R. & Propper, C.R. Competition and pesticide exposure affect development of invasive (Rhinella marina) and native (Fejervarya vittigera) rice paddy amphibian larvae. Ecotoxicology 26, 1293–1304 (2017). https://doi.org/10.1007/s10646-017-1854-8

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Keywords

  • Butachlor
  • Cane Toad
  • Endocrine Disruption
  • Luzon Wart Frog
  • Southeast Asia
  • Thyroid