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Evaluation in situ of genotoxicity and stress in South American common toad Rhinella arenarum in environments related to fluorite mine

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Abstract

Little attention has been paid to the impact of wastewater generated by mining activities on fluoride. In this study, we evaluated the hematology responses of common South American toad Rhinella arenarum inhabiting natural and artificial environments associated with a fluorite mine from central Argentina. We analyzed three sampling stations associated with the fluorite mine: (I) Los Cerros Negros stream (CN), which runs on granitic rock with a high fluorite content; (II) Los Vallecitos stream (LV), which runs on metamorphic rock with low fluorite content; and (III) artificial decantation ponds (DP) containing sediments produced by fluorite flotation process. We calculated frequencies of micronuclei, erythrocyte nuclear abnormalities, mitosis, and immature erythrocytes. In addition, we performed a differential leukocyte count and determined neutrophils/lymphocyte ratio as a stress response estimator. We found high micronucleus (MN) and erythrocyte nuclear abnormality (ENA) frequencies in DP and CN but low frequencies in LV. The neutrophil/lymphocyte ratio was different among sites, with a significant increase in individuals from DP. Values registered in DP could be caused by exposure to mixture of compounds registered in dams that hold wastewater, while high values registered in CN stream might be due to natural concentrations of fluoride. Our results suggest that blood is an effective and non-destructive sensitive indicator for monitoring genotoxic agents in freshwater ecosystems.

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References

  • Addy K, Green L, Herron E (2004) pH and alkalinity. University of Rhode Island, Kingston

    Google Scholar 

  • Antunes SC, Castro BB, Nunes B, Pereira R, Gonçalves F (2008) In situ bioassay with Eisenia andrei to assess soil toxicity in an abandoned uranium mine. Ecotoxicol Environ Saf 71:620–631

    Article  CAS  Google Scholar 

  • APHA-AWWA (1999) Standard methods for the examination of water and wastewater. 20th edition. Lenore, Clescerl, Greenberg y Eaton (eds.) American Public Health Assoc., American Water Works Assoc., Water Environment Federation pp 1325

  • Attademo AM, Cabagna Zenklusen M, Lajmanovich RC, Peltzer PM, Junges C, Bassó A (2011) B-esterase activities and blood cell morphology in the frog Leptodactylus chaquensis (Amphibia: Leptodactylidae) on rice agroecosystems from Santa Fe Province (Argentina). Ecotoxicology 20:274–282

    Article  CAS  Google Scholar 

  • Attademo AM, Peltzer PM, Lajmanovich RC, Cabagna Zenklusen MC, Junges CM, Basso A (2013) Biological endpoints, enzyme activities, and blood cell parameters in two anuran tadpole species in rice agroecosystems of mid-eastern Argentina. Environ Monit Ass 186:635–649

    Article  Google Scholar 

  • Ayllon F, Garcia Vazquez E (2000) Induction of micronuclei and other nuclear abnormalities in European minnow Phoxinus phoxinus and mollie Poecilia latipinna: an assessment of the fish micronucleus test. Mutat Res-Gen Tox En 467(2):177–186

    Article  CAS  Google Scholar 

  • Babini MS, Bionda CL, Salas NE, Martino AL (2015) Health status of tadpoles and metamorphs of Rhinella arenarum (Anura, Bufonidae) that inhabit agroecosystems and its implications for land use. Ecotoxicol Environ Saf 118:118–125

    Article  CAS  Google Scholar 

  • Barbier O, Arreola Mendoza L, Del Razo LM (2010) Molecular mechanisms of fluoride toxicity. Chem Biol Interact 188:319–333

    Article  CAS  Google Scholar 

  • Barni S, Boncompagni E, Grosso A, Bertone V, Freitas I, Fasola M, Fenoglio C (2007) Evaluation of Rana snkesculenta blood cell response to chemical stressors in the environment during the larval and adult phases. Aquat Toxicol 81:45–54

    Article  CAS  Google Scholar 

  • Beutler E (1985) Chemical toxicity of the erythrocyte. In: Irons RD (ed) Toxicology of the blood and bone marrow. Raven press, New York, p 182

    Google Scholar 

  • Bionda CL, Lajmanovich RC, Salas NE, Martino AL, di Tada IE (2011) Reproductive ecology of the common South American toad Rhinella arenarum (Anura: Bufonidae): reproductive effort, clutch size, fecundity, and mate selection. J Herpetol 45:261–264

    Article  Google Scholar 

  • Bosch B, Mañas F, Gorla N, Aiassa D (2011) Micronucleus test in post metamorphic Odontophrynus cordobae and Rhinella arenarum (Amphibia: Anura) for environmental monitoring. J Toxicol Environ Health Sci 3(6):154–163

    Google Scholar 

  • Boyd CE (1982) Water quality management for pond fish culture. Elsevier Scientific Publication Company, Netherland, p 318

    Google Scholar 

  • Cairns J Jr, Heath AG, Parker BC (1975) The effects of temperature upon the toxicity of chemicals to aquatic organisms. Hydrobiologia 47:135–171

    Article  CAS  Google Scholar 

  • Camargo JA (2003) Fluoride toxicity to aquatic organisms: a review. Chemosphere 50:251–264

    Article  Google Scholar 

  • Canadian Council of Ministers of Environment (CCME) (2002) Canadian water quality guidelines for protection of aquatic life: inorganic fluorides. pp 1–4

  • Cañedo Argüelles M, Kefford BJ, Piscart C, Prat N, Schäfer RB, Schulz CJ (2013) Salinisation of rivers: an urgent ecological issue. Environ Pollut 173:157–167

    Article  Google Scholar 

  • Cantú MP, Degiovanni SB (1984) Geomorfología de la región centro sur de la provincia de Córdoba. In IX Congreso Geológico Argentino, Asociación Geológica Argentina. Actas 4:76–92

    Google Scholar 

  • Carrasco KR, Tilbury KL, Mayers MS (1990) Assessment of the piscine micronuclei test as in situ biological indicator of chemical contaminants effects. Can J Fish Aquat Sci 47:2123–2136

    Article  CAS  Google Scholar 

  • Castro BB, Guilhermino L, Ribeiro R (2003) In situ bioassay chambers and procedures for assessment of sediment toxicity with Chironomus riparius. Environ Pollut 125(3):325–335

    Article  CAS  Google Scholar 

  • Collins SJ, Russell RW (2009) Toxicity of road salt to Nova Scotia amphibians. Environ Pollut 157(1):320–324

    Article  CAS  Google Scholar 

  • Coniglio J (2006). Evolución petrológica y metalogenética del batolito Cerro Áspero en relación con el ciclo geoquímica endógeno del Flúor, Sierras de Comechingones, Córdoba, Argentina. Tesis doctoral UNRC pp 163 in Spanich

  • Coniglio J, D’Eramo, F, Pinotti L, Demartis M, Petrelli H (2006) Magmatismo devónico de las sierras de córdoba: fuente posible de flúor de las mineralizaciones mesozoicas el ejemplo del batolito cerro áspero. VIII Congreso de Mineralogía y Metalogenia. Actas 1 pp 227–232 in Spanich

  • Crott JW, Mashiyama ST, Ames BC, Fenech M (2001) The effect of folic acid deficiency and MTHFR C677T polymorphism on chromosome damage in human lymphocytes in vitro. Cancer Epidemiol Biomarkers Prev 10:1089–1096

    CAS  Google Scholar 

  • da Silva Corrêa SA, de Souza Abessa DM, Gomes do Santos L, Bezerra da Silva E, Seriani R (2016) Differential blood counting in fish as a non-destructive biomarker of water contamination exposure. Toxicol Environ Chemistry:1–10

  • da Silva Souza T, Fontanetti CS (2006) Micronucleus test and observation of nuclear alterations in erythrocytes of Nile tilapia exposed to waters affected by refinery effluent. Toxicol Environ Mut 605:87–93

    Article  Google Scholar 

  • Dacie JV, Lewis SM (1984) Practical hematology. Churchill Livingstone, New York

    Google Scholar 

  • Davis AK (2009) Metamorphosis-related changes in leukocyte profiles of larval bullfrogs (Rana catesbeiana). Comp Clin Pathol 18(2):181–186

    Article  Google Scholar 

  • Davis AK, Maney DL, Maerz JC (2008) The use of leukocyte profiles to measure stress in vertebrates: a review for ecologists. Funct Ecol 22:760–772

    Article  Google Scholar 

  • Di Rienzo JA, Casanoves F, Balzarini MG, Gonzalez L, Tablada M, Robledo CW (2012). Grupo InfoStat Professional, FCA. Universidad Nacional de Córdoba, Argentina http://www.infostat.com.ar S in Spanich

  • Djomo JE, Ferrier V, Békaert C (2000) Amphibian micronucleus test in vivo (Jaylet test) to evaluate the genotoxicity of petrochemical waste waters. Bull Environ Contam Toxicol 65:168–174

    Article  CAS  Google Scholar 

  • Duellman WE, Trueb L (1994) Biology of amphibians. John Hopkins University Press, Baltimore

    Google Scholar 

  • Eggen RI, Behra R, Burkhardt Holm P, Escher BI, Schweigert N (2004) Peer reviewed: challenges in ecotoxicology. Environ Sci Technol 38(3):58A–64A

    Article  CAS  Google Scholar 

  • Ergene S, Çavaş T, Çelik A, Köleli N, Kaya F, Karahan A (2007) Monitoring of nuclear abnormalities in peripheral erythrocytes of three fish species from the Goksu Delta (Turkey): genotoxic damage in relation to water pollution. Ecotoxicology 16:385–391

    Article  CAS  Google Scholar 

  • Fenech M (2000) The in vitro micronucleus technique. Mutat Res 455:81–95

    Article  CAS  Google Scholar 

  • Fenech M, Kirsch Volders M, Natarajan AT, Surralles J, Crott JW, Parry J, Norppa H, Eastmond DA, Tucker JD, Thomas P (2011) Molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells. Mutagenesis 26:125–132

    Article  CAS  Google Scholar 

  • García G, Fontúrbel F (2003). Propuestas para un desarrollo sostenible. Lago Titikaka por estrategas K. Ed. Publicaciones Integrales. La Paz in Spanich

  • Gauthier L, Tardy E, Mouchet F, Marty J (2004) Biomonitoring of the genotoxic potential (micronucleus assay) and detoxifying activity (EROD induction) in the River Dadou (France), using the amphibian Xenopus laevis. Sci Total Environ 323(1):47–61

    Article  CAS  Google Scholar 

  • Gonzalo C, Camargo JA (2012) Fluoride bioaccumulation in the signal crayfish Pacifastacus leniusculus (Dana) as suitable bioindicator of fluoride pollution in freshwater ecosystems. Ecol Indic 20:244–251

    Article  CAS  Google Scholar 

  • Guilherme S, Válega M, Pereira ME, Santos MA, Pacheco M (2008) Erythrocytic nuclear abnormalities in wild and caged fish (Liza aurata) along an environmental mercury contamination gradient. Ecotoxicol Environ Saf 70(3):411–421

    Article  CAS  Google Scholar 

  • Heyer WR, Donnelly MA, Mcdiarmid RW, Hayek LC, Foster MS (1994) Measuring and monitoring biological diversity: standard methods for amphibians. (Eds.). Washington, Smithsonian Institution Press, pp 364

  • Hoffman DJ, Rattner BA, Burton Jr GA, Cairns Jr J (eds) (2010) Handbook of ecotoxicology. CRC Press, London, New York, Washington

  • 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):519–525

    Article  CAS  Google Scholar 

  • Karraker NE, Ruthig GR (2009) Effect of road deicing salt on the susceptibility of amphibian embryos to infection by water molds. Environ Res 109(1):40–45

    Article  CAS  Google Scholar 

  • Lajmanovich RC, Cabagna Zenklusen MC, Attademo AM, Junges CM, Peltzer PM, Bassó A, Lorenzatti E (2014) Induction of micronuclei and nuclear abnormalities in tadpoles of the common toad (Rhinella arenarum) treated with the herbicides Liberty® and glufosinate-ammonium. Toxicol Environ Mutagen 769:7–12

    Article  CAS  Google Scholar 

  • Lanctôt C, Bennett W, Wilson S, Fabbro L, Leusch FDL, Melvin SD (2016) Behaviour, development and metal accumulation in striped marsh frog tadpoles (Limnodynastes peronii) exposed to coal mine wastewater. Aquat Toxicol 173:218–227

    Article  Google Scholar 

  • Lavoie I, Lavoie M, Fortin C (2012) A mine of information: benthic algal communities as biomonitors of metal contamination from abandoned tailings. Sci Total Environ 425:231–241

    Article  CAS  Google Scholar 

  • Law 24051 (1992) Ley Nacional de Residuos Peligrosos (Regime for hazardous wastes). Secretaría de Ambiente y Desarrollo Sustentable. Argentina. On line http://www2.medioambiente.gov.ar/mlegal/residuos/ley24051.htm. http://www2.medioambiente.gov.ar/mlegal/residuos/dec831/dec831_anxIV.htm Access 19 Feb 2016 in Spanich

  • Livingstone DR (1993) Biotechnology and pollution monitoring: use of molecular biomarkers in the aquatic environment. J Chem Technol Biot 57:195–211

    Article  CAS  Google Scholar 

  • Machado da Rocha C (2011) The micronucleus test in erythrocytes of amphibian larvae as tool for xenobiotic exposure risk assessment: a brief review and an example using Lithobates catesbeianus exposed to copper sulphate. J Sci Res 8(1):23–29

    Google Scholar 

  • Marques SM, Antunes SC, Pissarra H, Pereira ML, Gonçalves F, Pereira R (2009) Histopathological changes and erythrocytic nuclear abnormalities in Iberian green frogs (Rana perezi Seoane) from a uranium mine pond. Aquat Toxicol 91:187–195

    Article  CAS  Google Scholar 

  • Martino AL, Sinsch U (2002) Speciation by polyploidy in Odontophrynus americanus. J Zool 257:67–81

    Article  Google Scholar 

  • Nöller HG (1959) Eine einfache Technik der Blutentnahme beim Frosch. Pflüg Arch Physiol 269:98–100

    Article  Google Scholar 

  • Pollo FE, Salas NE, Mancini MA, Martino AL (2012) Estudio comparativo de la frecuencia de micronúcleos y anormalidades nucleares en eritrocitos de tres especies ícticas. Acta Toxicol Arg 20:62–67 in Spanish

    Google Scholar 

  • Pollo FE, Bionda CL, Salinas ZA, Salas NE, Martino AL (2015) Common toad Rhinella arenarum (Hensel, 1867) and its importance in assessing environmental health: test of micronuclei and nuclear abnormalities in erythrocytes. Environ Monit Assess 187(9):1–9

    Article  CAS  Google Scholar 

  • Pollo FE, Grenat PR, Otero MA, Salas NE, Martino AL (2016) Assessment in situ of genotoxicity in tadpoles and adults of frog Hypsiboas cordobae (Barrio 1965) inhabiting aquatic ecosystems associated to fluorite mine. Ecotoxicol Environ Saf 133:466–474

    Article  CAS  Google Scholar 

  • Prieto Z, Incio JL, 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). Rev Peru Med Exp Salud Publica 25:51–58

    Google Scholar 

  • Romanova EB, Egorikhina MN (2006) Changes in hematological parameters of Rana frogs in a transformed urban environment. Russ J Ecol 37(3):188–192

    Article  Google Scholar 

  • Sanabria EA, Quiroga LB, Acosta JC (2007) Sitios de oviposición y esfuerzo reproductivo en Chaunus arenarum (Anura: Bufonidae) en el desierto del Monte, Argentina. Rev Esp Herpet 21:49–53 in Spanich

    Google Scholar 

  • Sanzo D, Hecnar SJ (2006) Effects of road de-icing salt (NaCl) on larval wood frogs (Rana sylvatica). Environ Pollut 140(2):247–256

    Article  CAS  Google Scholar 

  • Schmid W (1975) The micronucleus test. Mutat Res 31:9–15

    Article  CAS  Google Scholar 

  • Seriani R, Abessa DM, Moreira LB, Cabrera JP, Sanches JQ, Silva CL, Amorim FA, Rivero D, Silva FL, Fitorra L, Carvalho Oliveira R, Macchione M, Ranzani Paiva M (2015) In vitro mucus transportability, cytogenotoxicity, and hematological changes as non-destructive physiological biomarkers in fish chronically exposed to metals. Ecotoxicol Environ Saf 112:162–168

    Article  CAS  Google Scholar 

  • Serrano García L, Montero Montoya R (2001) Micronuclei and chromatid buds are the result of related genotoxic events. Environ Mol Mutagen 38:38–45

    Article  Google Scholar 

  • Shimizu N, Itoh N, Utiyama H, Wahl GM (1998) Selective entrapment of extrachromosomally amplified DNA by nuclear budding and micronucleation during S phase. J Cell Biol 140:1307–1320

    Article  CAS  Google Scholar 

  • Strunjak Perovic I, Popovic NT, Coz Rakovac R, Jadan M (2009) Nuclear abnormalities of marine fish erythrocytes. J Fish Biol 74:2239–2249

    Article  CAS  Google 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 306:1783–1786

    Article  CAS  Google Scholar 

  • Tolbert PE, Shy CM, Allen JW (1992) Micronuclei and other anomalies in buccal smears: methods development. Mutat Res 271:69–77

    Article  CAS  Google Scholar 

  • Udroiu I (2006) The micronucleus test in piscine erythrocytes. Aquat Toxicol 79:201–204

    Article  CAS  Google Scholar 

  • Valenzuela AE, Silva VM, Klempau AE (2006) Qualitative and quantitative effects of constant light photoperiod on rainbow trout (Oncorhynchus mykiss) peripheral blood erythrocytes. Aquaculture 251:596–602

    Article  Google Scholar 

  • Venturino A, Rosenbaum E, Caballero De Castro A, Anguiano OL, Gauna L, Fonovich De Schroeder T, Pechen De D'Angelo AM (2003) Biomarkers of effect in toads and frogs. Biomarkers 8(3–4):167–186

    Article  CAS  Google Scholar 

  • Vera Candioti JV, Natale GS, Soloneski S, Ronco AE, Larramendy ML (2010) Sublethal and lethal effects on Rhinella arenarum (Anura, Bufonidae) tadpoles exerted by the pirimicarb-containing technical formulation insecticide Aficida®. Chemosphere 78:249–255

    Article  Google Scholar 

  • Young B, Stuart S, Chanson J, Cox N, Boucher T (2004) Joyas que están desapareciendo: El estado de los anfibios en el nuevo mundo. Nature Serve, Arlington, p 53 in Spanish

    Google Scholar 

  • Zocche JJ, Damiani AP, Hainzenreder G, Mendonça RÁ, Peres PB, dos Santos CEI, Debastiani R, Ferraz Dias J, de Andrade VM (2013) Assessment of heavy metal content and DNA damage in Hypsiboas faber (anuran amphibian) in coal open-casting mine. Environ Toxicol Pharmacol 36(1):194–201

    Article  CAS  Google Scholar 

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Acknowledgements

The authors gratefully acknowledge the Fluorite Córdoba SA Company for allowing access to Los Cerros Negros mine and providing logistic support.

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Correspondence to Favio E. Pollo.

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This research was primarily supported by a grant from the Secretary of Research and Technology of National University of Río Cuarto (PPI 18/C416) and National Agency for Scientific and Technological Promotion FONCYT (BID-PICT 0932-2012; BID-PICT 2533-2014). The first, second, third, and four authors thank CONICET - Argentina (Argentinean National Research Council for Science and Technology) for fellowships granted. The investigation was conducted according to the state law “Protection and Conservation of Wild Fauna” (Argentina National Law No. 22.421). Our study was authorized by Cordoba Environmental Agency (A.C.A.S.E.), Environmental Secretary of Córdoba Government.

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Pollo, F.E., Grenat, P.R., Salinas, Z.A. et al. Evaluation in situ of genotoxicity and stress in South American common toad Rhinella arenarum in environments related to fluorite mine. Environ Sci Pollut Res 24, 18179–18187 (2017). https://doi.org/10.1007/s11356-017-9479-2

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