Water quality assessment and amphibian sensitivity in a protected area of the Atlantic Forest Biome
Resumo
The Atlantic Forest biome harbors exceptionally high biodiversity and endemism. Turvo State Park protects the largest remnant of Semideciduous Seasonal Forest in Rio Grande do Sul. Despite this biological richness, stressors such as pesticide contamination threaten species survival and development. Amphibians, due to their ecological and physiological traits, are particularly sensitive to environmental disturbances. In this study, we assessed the sensitivity of Leptodactylus luctator tadpoles in lentic environments located inside the park and within its buffer zone. Ten lentic habitats were evaluated (five in each area). Water samples from the 10 sites were analyzed for pesticide residues, and a spawn of L. luctator was collected from a lake inside the park. The spawn was reared in the laboratory until tadpoles reached Gosner stage 25, after which they were transferred to the study sites. After 14 days, tadpoles were measured, weighed, and examined for the presence of micronuclei (MN) and erythrocyte nuclear abnormalities (ENA). Water analyses revealed clomazone and chlorpyrifos inside the park, while atrazine, azoxystrobin, and imidacloprid were detected in the buffer zone. MN and ENAs—including binucleate, anucleate, notched, and lobed nuclei, nuclear buds, karyolysis, apoptosis, and micronuclei—were observed in tadpoles from both areas. These findings highlight the importance of biomonitoring in protected areas, particularly given the anthropogenic pressures exerted on their borders.
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Referências
Agostini, M. G., Roesler, I., Bonetto, C., Ronco, A. E., & Bilenca, D. (2020). Pesticides in the real world: The consequences of GMO-based intensive agriculture on native amphibians. Biological Conservation, 241, 108355. https://doi.org/10.1016/j.biocon.2019.108355
Agrolink. (2022). Bula Azoxistrobina. Amistar 500 WG. https://www.agrolink.com.br/agrolinkfito/produto/amistar-500-wg_7485.html
Arcaute, C. R., Pérez-Iglesias, J. M., Nikoloff, N., Natale, G. S., Soloneski, S., & Larramendy, M. L. (2014). Genotoxicity evaluation of the insecticide imidacloprid circulating blood cells of Montevideo tree frog Hypsiboas pulchellus tadpoles (Anura, Hylidae) by comet and micronucleus bioassays. Ecological Indicators, 45, 632–639. https://doi.org/10.1016/j.ecolind.2014.05.034
Ascoli-Morrete, T., Bandeira, N. M. G., Signor, E., Gazola, H. A., Homrich, I. S., Biondo, R., Rossato-Grando, L. G., & Zanella, N. (2022). Bioaccumulation of pesticides and genotoxicity in anurans from southern Brazil. Environmental Science and Pollution Research, 29(30), 45549–45559. https://doi.org/10.1007/s11356-022-19042-z
Atkins, J. B., Reisz, R. R., & Maddin, H. C. (2019). Braincase simplification and the origin of lissamphibians. PLoS ONE, 14(3), Article e0213694. https://doi.org/10.1371/journal.pone.0213694
Attademo, A. M., Peltzer, P. M., Lajmanovich, R. C., Cabagna-Zenklusen, M. C., Junges, C. M., & Basso, A. (2014). Biological endpoints, enzyme activities, and blood cell parameters in two anuran tadpole species in rice agroecosystems of mid-eastern Argentina. Environmental Monitoring and Assessment, 186, 635–649. https://doi.org/10.1007/s10661-013-3404-z
Bahl, M. F., Costa, C. S., Demetrio, P. M., Mac Loughlin, T. M., Arruti, M. E., Brodeur, J. M. C., & Natale, G. S. (2024). Integration of a battery of biomarkers to evaluate the health status of field-collected frogs of Leptodactylus luctator living in ecosystems with different anthropogenic disturbances. Science of The Total Environment, 933, 173174. https://doi.org/10.1016/j.scitotenv.2024.173174
Barni, S., Boncompagni, E., Grosso, A., Bertone, V., Freitas, I., Fasola, M., & Fenoglio, C. (2007). Evaluation of Rana esculenta blood cell response to chemical stressors in the environment during the larval and adult phases. Aquatic Toxicology, 81(1), 45–54. https://doi.org/10.1016/j.aquatox.2006.10.012
Borges, R. E., Santos, L. R. D., Benvindo-Souza, M., Modesto, R. S., Assis, R. A., & De Oliveira, C. (2019). Genotoxic evaluation in tadpoles associated with agriculture in the central Cerrado, Brazil. Archives of Environmental Contamination and Toxicology, 77(1), 22–28. https://doi.org/10.1007/s00244-019-00623-y
Braga, L. G. T., & Lima, S. L. (2001). Influência da temperatura ambiente no desempenho da rã-touro, Rana catesbeiana (Shaw, 1802) na fase de recria. Revista Brasileira de Zootecnia, 30(6), 1659–1663. https://doi.org/10.1590/S1516-35982001000700001
Brasil. (2012). Lei nº 12.651, de 25 de maio de 2012. Dispõe sobre a proteção da vegetação nativa e dá outras providências. Diário Oficial da União. http://www.planalto.gov.br/ccivil_03/_ato2011-2014/2012/lei/l12651.htm
Brito, W. J. B., & Costa, R. D. L. (2022). Niche partitioning between species of the Leptodactylidae family: A brief account of siblings. Research, Society and Development, 11(3), Article e37111326826. https://doi.org/10.33448/rsd-v11i3.26826
Carrasco, K. R., Tilbury, K. L., & Myers, M. S. (1990). Assessment of the piscine micronucleus test as an in situ biological indicator of chemical contaminant effects. Canadian Journal of Fisheries and Aquatic Sciences, 47(11), 2123–2136. https://doi.org/10.1139/f90-237
Çavas, T., & Ergene-Gözükara, S. (2005). Induction of micronuclei and nuclear abnormalities in Oreochromis niloticus following exposure to petroleum refinery and chromium processing plant effluents. Aquatic Toxicology, 74(3), 264–271. https://doi.org/10.1016/j.aquatox.2005.06.001
Cushman, S. A. (2006). Effects of habitat loss and fragmentation on amphibians: A review and prospectus. Biological Conservation, 128(2), 231–240. https://doi.org/10.1016/j.biocon.2005.09.031
De Oliveira-Filho, A. T., Eisenlohr, P. V., & Prado, J. (2015). The Brazilian Atlantic Forest: New findings, challenges and prospects in a shrinking hotspot. Biodiversity and Conservation, 24, 2129–2133. https://doi.org/10.1007/s10531-015-0995-4
Di Muccio, A., Fidente, P., Barbini, D. A., Dommarco, R., Seccia, S., & Morrica, P. (2006). Application of solid-phase extraction and liquid chromatography-mass spectrometry to the determination of neonicotinoid pesticide residues in fruit and vegetables. Journal of Chromatography A, 1108(1), 1–6. https://doi.org/10.1016/j.chroma.2005.12.111
Donato, F. F., Martins, M. L., Munaretto, J. S., Prestes, O. D., Adaime, M. B., & Zanella, R. (2015). Development of a multiresidue method for pesticide analysis in drinking water by solid phase extraction and determination by gas and liquid chromatography with triple quadrupole tandem mass spectrometry. Journal of the Brazilian Chemical Society, 26(10), 2077–2087. https://doi.org/10.5935/0103-5053.20150192
Dyck, A., Robinson, S. A., Young, S. D., Renaud, J. B., Sabourin, L., Lapen, D. R., & Pick, F. R. (2021). The effects of ditch management in agroecosystems on embryonic and tadpole survival, growth, and development of northern leopard frogs (Lithobates pipiens). Archives of Environmental Contamination and Toxicology, 81(1), 107–122. https://doi.org/10.1007/s00244-021-00836-0
Edge, C. B., Thompson, D. G., Hao, C., & Houlahan, J. E. (2014). The response of amphibian larvae to exposure to a glyphosate-based herbicide (Roundup WeatherMax) and nutrient enrichment in an ecosystem experiment. Ecotoxicology and Environmental Safety, 109, 124–132. https://doi.org/10.1016/j.ecoenv.2014.07.040
Environmental Protection Agency. (2003). Imidacloprid: Pesticide tolerances. US EPA.
Fenech, M., Chang, W. P., Kirsch-Volders, M., Holland, N., Bonassi, S., & Zeiger, E. (2003). HUMN project: Detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 534(1-2), 65–75. https://doi.org/10.1016/S1383-5718(02)00249-8
Fenech, M., Kirsch-Volders, M., Natarajan, A. T., Surralles, J., Crott, J. W., Parry, J., Norppa, H., Eastmond, D. A., Tucker, J. D., & Thomas, P. (2011). Molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells. Mutagenesis, 26(1), 125–132. https://doi.org/10.1093/mutage/geq052
Frost, D. R. (2024). Amphibian species of the world: An online reference (Version 6.2) [Base de dados]. American Museum of Natural History. https://amphibiansoftheworld.amnh.org/index.php. https://doi.org/10.5531/db.vz.0001
Gauthier, L., Van Der Gaag, M. A., L’Haridon, J., Ferrier, V., & Fernandez, M. (1993). In vivo detection of waste water and industrial effluent genotoxicity: Use of the Newt Micronucleus Test (Jaylet Test). Science of the Total Environment, 138(1-3), 249–269. https://doi.org/10.1016/0048-9697(93)90419-7
Getelina, M. A., Schwantes, J. B., Graichen, D. A. S., & Schuch, A. P. (2022). Influence of anthropogenic pressure on the genetic diversity and chromosomal instability of an endangered forest-specialist anuran. Hydrobiologia, 849, 2463–2475. https://doi.org/10.1007/s10750-022-04840-w
Glomski, C. A., Tamburlin, J., Hard, R., & Chainani, M. (1997). The phylogenetic odyssey of the erythrocyte. IV. The amphibians. Histology and Histopathology, 12(1), 147–170. https://pubmed.ncbi.nlm.nih.gov/9046052/
Gonçalves, M. W., De Campos, C. B. M., Godoy, F. R., Gambale, P. G., Nunes, H. F., Nomura, F., Bastos, R. P., Da Cruz, A. D., & De Melo E Silva, D. (2019). Assessing genotoxicity and mutagenicity of three common amphibian species inhabiting agroecosystem environment. Archives of Environmental Contamination and Toxicology, 77, 409–420. https://doi.org/10.1007/s00244-019-00647-4
Gosner, K. L. (1960). A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica, 16(3), 183–190.
Grott, S. C., Israel, N., Lima, D., Bitschinski, D., Abel, G., Alves, T. C., Silva, E. B., Albuquerque, C. A. C., Mattos, J. J., Bainy, A. C. D., & Almeida, E. A. (2022). Influence of temperature on growth, development and thyroid metabolism of American bullfrog tadpoles (Lithobates catesbeianus) exposed to the herbicide tebuthiuron. Environmental Toxicology and Pharmacology, 94, Article 103910. https://doi.org/10.1016/j.etap.2022.103910
Harabawy, A. S. A., & Mosleh, Y. Y. I. (2014). The role of vitamins A, C, E and selenium as antioxidants against genotoxicity and cytotoxicity of cadmium, copper, lead and zinc on erythrocytes of Nile tilapia, Oreochromis niloticus. Ecotoxicology and Environmental Safety, 104, 28–35. https://doi.org/10.1016/j.ecoenv.2014.02.015
Hartmann, M. T., Hartmann, P. A., & Müller, C. (2023). Pesticide effects on tadpole's survival. In E. A. Almeida & J. S. Freitas (Eds.), Toxicology of amphibian tadpoles (pp. 1–20). CRC Press.
Herek, J. S., Vargas, L., Trindade, S. A. R., Rutkoski, C. F., Macagnan, N., Hartmann, P. A., & Hartmann, M. T. (2021). Genotoxic effects of glyphosate on Physalaemus tadpoles. Environmental Toxicology and Pharmacology, 81, Article 103516. https://doi.org/10.1016/j.etap.2020.103516
Hudson, W. H. (1892). The naturalist in La Plata. Chapman and Hall.
Instituto Brasileiro de Geografia e Estatística. (2012). Manual técnico da vegetação brasileira (2. ed.). IBGE.
Iop, S., Caldart, V. M., Dos Santos, T. G., & Cechin, S. Z. (2011). Anurans of Turvo State Park: Testing the validity of Seasonal Forest as a new biome in Brazil. Journal of Natural History, 45(39-40), 2443–2461. https://doi.org/10.1080/00222933.2011.596951
Iop, S., Caldart, V. M., Dos Santos, T. G., & Cechin, S. Z. (2012). What is the role of heterogeneity and spatial autocorrelation of ponds in the organization of frog communities in Southern Brazil? Zoological Studies, 51(7), 1094–1104.
IUCN SSC Amphibian Specialist Group. (2023). Leptodactylus luctator. The IUCN Red List of Threatened Species 2023. https://dx.doi.org/10.2305/IUCN.UK.2023-1.RLTS.T194295016A194423836.en
Kiechle, F. L., & Zhang, X. (2002). Apoptosis: Biochemical aspects and clinical applications. Clinica Chimica Acta, 326(1-2), 27–45. https://doi.org/10.1016/S0009-8981(02)00297-8
Koehler, A. M., & Shew, H. D. (2018). Field efficacy and baseline sensitivities of Septoria steviae to fungicides used for the management of Septoria leaf spot of stevia. Crop Protection, 109, 95–101. https://doi.org/10.1016/j.cropro.2018.03.006
Lajmanovich, R. C., Cabagna, M., Peltzer, P. M., Stringhini, G. A., & Attademo, A. M. (2005). Micronucleus induction in erythrocytes of the Hyla pulchella tadpoles (Amphibia: Hylidae) exposed to insecticide endosulfan. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 587(1-2), 67–72. https://doi.org/10.1016/j.mrgentox.2005.08.001
Larsen, E. H., & Ramløv, H. (2013). Role of cutaneous surface fluid in frog osmoregulation. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 165(3), 365–370. https://doi.org/10.1016/j.cbpa.2013.04.005
Lima, R. A. F., Oliveira, A. A., Pitta, G. R., De Gasper, A. L., Vibrans, A. C., Chave, J., Ter Steege, H., & Prado, P. I. (2020). The erosion of biodiversity and biomass in the Atlantic Forest biodiversity hotspot. Nature Communications, 11, Article 6347. https://doi.org/10.1038/s41467-020-20217-w
Maciel, T. A., & Juncá, F. A. (2009). Effects of temperature and volume of water on the growth and development of tadpoles of Pleurodema diplolister and Rhinella granulosa (Amphibia: Anura). Zoologia (Curitiba), 26(3), 413–418. https://doi.org/10.1590/S1984-46702009000300005
Macagnan, N., Rutkoski, C. F., Folador, A., Skovronski, V. J., Müller, C., Hartmann, P. A., & Hartmann, M. T. (2023). Mortality and toxicity of a commercial formulation of cypermethrin in Physalaemus gracilis tadpoles. Scientific Reports, 13, Article 18002. https://doi.org/10.1038/s41598-023-45090-7
Magalhães, F. M., Lyra, M. L., Carvalho, T. R., Baldo, D., Brusquetti, F., Burella, P., Colli, G. R., Gehara, M. C., Giaretta, A. A., Haddad, C. F. B., Langone, J. A., López, J. A., Napoli, M. F., Santana, D. J., De Sá, R. O., & Garda, A. A. (2020). Taxonomic review of South American Butter frogs: Phylogeny, geographic patterns, and species delimitation in the Leptodactylus latrans species group (Anura: Leptodactylidae). Herpetological Monographs, 34(1), 131–177. https://doi.org/10.1655/0733-1347-34.1.131
Mikolić, A., & Karačonji, I. B. (2018). Imidacloprid as reproductive toxicant and endocrine disruptor: Investigations in laboratory animals. Archives of Industrial Hygiene and Toxicology, 69(2), 103–108. https://doi.org/10.2478/aiht-2018-69-3144
Myers, N., Mittermeier, R. A., Mittermeier, C. G., Fonseca, G. A. B., & Kent, J. (2000). Biodiversity hotspots for conservation priorities. Nature, 403, 853–858. https://doi.org/10.1038/35002501
Norppa, H., & Falck, G. C. M. (2003). What do human micronuclei contain? Mutagenesis, 18(3), 221–233. https://doi.org/10.1093/mutage/18.3.221
Nüsse, M., Miller, B. M., Viaggi, S., & Grawé, J. (1996). Analysis of the DNA content distribution of micronuclei using flow sorting and fluorescent in situ hybridization with a centromeric DNA probe. Mutagenesis, 11(4), 405–413. https://doi.org/10.1093/mutage/11.4.405
Obiakor, M. O., Okonkwo, J. C., Nnabude, P. C., & Ezeonyejiaku, C. D. (2012). Eco-genotoxicology: Micronucleus assay in fish erythrocytes as in situ aquatic pollution biomarker: A review. Journal of Animal Science Advances, 2(1), 123–133.
Pavan, F. A., Samojeden, C. G., Rutkoski, C. F., Folador, A., Fré, S. P., Müller, C., Hartmann, P. A., & Hartmann, M. T. (2021). Morphological, behavioral and genotoxic effects of glyphosate and 2,4-D mixture in tadpoles of two native species of South American amphibians. Environmental Toxicology and Pharmacology, 85, Article 103637. https://doi.org/10.1016/j.etap.2021.103637
Pérez-Iglesias, J. M., Soloneski, S., Nikoloff, N., Natale, G. S., & Larramendy, M. L. (2015). Toxic and genotoxic effects of the imazethapyr-based herbicide formulation Pivot H® on Montevideo tree frog Boana pulchellus tadpoles (Anura, Hylidae). Ecotoxicology and Environmental Safety, 119, 15–24. https://doi.org/10.1016/j.ecoenv.2015.04.045
Pérez-Lucas, G., Vela, N., El Aatik, A., & Navarro, S. (2019). Environmental risk of groundwater pollution by pesticide leaching through the soil profile. In M. Larramendy & S. Soloneski (Eds.), Pesticides: Use and misuse and their impact in the environment (pp. 1–27). IntechOpen. https://doi.org/10.5772/intechopen.82418
Pereira, G., Riero, M., Lajmanovich, R. C., & Maneiro, R. (2024). Acute toxicity and genotoxicity of the S-metolachlor-based herbicide Dual Gold® on Leptodactylus luctator (Hudson, 1892) tadpoles (Anura: Leptodactylidae). Limnetica, 43(2), 221–234. https://www.limnetica.net/pt/acute-toxicity-and-genotoxicity-s-metolachlor-based-herbicide-dual-gold%C2%AE-leptodactylus-luctator
Prieto, Z., León-Incio, J., Quijano-Jara, C., Fernández, R., Polo-Benites, E., Vallejo-Rodríguez, R., & Villegas-Sanchez, L. (2008). Efecto genotóxico del dicromato de potasio en eritrocitos de sangre periférica de Oreochromis niloticus (Tilapia). Revista Peruana de Medicina Experimental y Salud Pública, 25(1), 51–58. http://www.scielo.org.pe/scielo.php?script=sci_arttext&pid=S1726-46342008000100008
Riaz, U., Rafi, F., Naveed, M., Mehdi, S. M., Murtaza, G., Niazi, A. G., & Mehmood, H. (2021). Pesticide pollution in an aquatic environment. In Freshwater Pollution and Aquatic Ecosystems (pp. 131-163). Apple Academic Press.
Rocha, M. C., Santos, M. B., Zanella, R., Prestes, O. D., Gonçalves, A. S., & Schuch, A. P. (2020). Preserved riparian forest protects endangered forest-specialists amphibian species against the genotoxic impact of sunlight and agrochemicals. Biological Conservation, 249. https://doi.org/10.1016/j.biocon.2020.108746
Saccol, S. S. A., Ucha, J. L. C. D., Madalozzo, B., Cechin, S. Z., & Santos, T. G. (2022). Influence of land use on the diversity of pond-breeding anurans in South Brazilian grasslands. Biodiversity and Conservation, 31, 21–37. https://doi.org/10.1007/s10531-021-02317-1
Samojeden, C. G., Pavan, F. A., Rutkoski, C. F., Folador, A., Fré, S. P., Müller, C., Hartmann, P. A., & Hartmann, M. T. (2022). Toxicity and genotoxicity of imidacloprid in the tadpoles of Leptodactylus luctator and Physalaemus cuvieri (Anura: Leptodactylidae). Scientific Reports, 12, Article 11926. https://doi.org/10.1038/s41598-022-16039-z
Santos, G. dos, Rutkoski, C. F., Folador, A., Skovronski, V. J., Müller, C., Pompermaier, A., Hartmann, P. A., & Hartmann, M. T. (2024). 2,4-D-based herbicide underdoses cause mortality, malformations, and nuclear abnormalities in Physalaemus cuvieri tadpoles. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 277, Article 109840. https://doi.org/10.1016/j.cbpc.2024.109840
Scaramella, N., Mausbach, J., Laurila, A., & Räsänen, K. (2022). Short-term responses of Rana arvalis tadpoles to pH and predator stress: Adaptive divergence in behavioural and physiological plasticity? Journal of Comparative Physiology B, 192, 669–682. https://doi.org/10.1007/s00360-022-01449-2
Schvezov, N., Caffetti, J. D., Silva, C. J., Boeris, J. M., Baldo, D., & Lajmanovich, R. C. (2023). Impact of soil from monoculture pine plantations on two anuran species from the Atlantic Forest: Odontophrynus reigi and Leptodactylus luctator. Science of The Total Environment, 869. https://doi.org/10.1016/j.scitotenv.2023.161769
Schuch, A. P., Santos, M. B., Lipinski, V. M., Peres, L. V., Santos, C. P., Cechin, S. Z., Schuch, N. J., Pinheiro, D. K., & Loreto, E. L. S. (2015). Identification of influential events concerning the Antarctic ozone hole over southern Brazil and the biological effects induced by UVB and UVA radiation in an endemic treefrog species. Ecotoxicology and Environmental Safety, 118, 190–198. https://doi.org/10.1016/j.ecoenv.2015.04.029
Secretaria do Meio Ambiente. (2005). Plano de manejo do Parque Estadual do Turvo - RS. Divisão de Unidades de Conservação do Estado do Rio Grande do Sul. https://www.sema.rs.gov.br/upload/arquivos/201610/24172430-plano-manejo-peturvo.pdf
Sigurdsen, T., & Green, D. M. (2011). The origin of modern amphibians: A re-evaluation. Zoological Journal of the Linnean Society, 162(2), 457–469. https://doi.org/10.1111/j.1096-3642.2010.00683.x
Silva, F. L., Prado, I. S., Fraga, R. E., Rocha, M. A., Juncá, F. A., & Da Silva, M. B. (2021). Swimming ability in tadpoles of Physalaemus cf. cuvieri, Scinax x-signatus and Leptodactylus latrans (Amphibia: Anura) exposed to the insecticide chlorpyrifos. Ecotoxicology and Environmental Contamination, 16(1), 13-18. https://doi.org/10.5132/eec.2021.01.02
Sparling, D. W., Bickham, J., Cowman, D., Fellers, G. M., Lacher, T., Matson, C. W., & McConnell, L. (2015). In situ effects of pesticides on amphibians in the Sierra Nevada. Ecotoxicology, 24(2), 262–278. https://doi.org/10.1007/s10646-014-1375-7
Wells, K. D. (2007). The ecology and behavior of amphibians. University of Chicago Press.
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