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Genotoxicity of spent pot liner as determined with the zebrafish (Danio rerio) experimental model


Spent pot liner (SPL) is a solid waste generated during the primary smelting of aluminum, and its toxicity is attributed to the presence of fluoride, cyanide, and aluminum salts, which can be leached into aquatic ecosystems. Since the effects of this waste on aquatic life forms have not yet been investigated, the objective of our study was to evaluate the toxicity of simulated leachates of SPL on zebrafish (Danio rerio). Animals were exposed to 0 (control), 0.32, 0.64, or 0.95 g L−1 of SPL for 24, 72, and 96 h, and genotoxicity was accessed through micronucleus and comet assays. All of the tested treatments induced DNA fragmentation, and the observed frequency of micronuclei and damaged nucleoids generally increased with increasing SPL concentration. The highest frequency of micronuclei (3.3 per 3000 erythrocytes) was detected after 96 h of exposure with 0.95 g L−1 SPL. In the comet assay, nucleoids classified with highest level of damage in relation to the control were observed principally after 24 and 96 h of exposure. The data obtained in this study confirm the genotoxicaction and mutagenic potential of SPL and indicate that open-air deposits of the waste material could represent a health risk to humans and ecosystems alike.

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  • AL-SABTI K, METCALFE CD (1995) Fish micronuclei for assessing genotoxicity in water. Mutation Res Genetic Toxicology 343(2):121–135.

    Article  CAS  Google Scholar 

  • ANDRADE LF, CAMPOS JMS, DAVIDE LC (2008) Cytogenetic alterations induced by SPL (spent pot liner) in meristematic cells of plant bioassays. Ecotoxicol Environ Saf 71(3):706–710.

    Article  CAS  Google Scholar 

  • ANDRADE LF, DAVIDE LC, GEDRAITE LS (2010) The effect of cyanide compounds, fluorides, aluminum, and inorganic oxides present in spent pot liner on germination and root tip cells of Lactuca sativa. Ecotoxicol Environ Saf 73(4):626–631.

    Article  CAS  Google Scholar 

  • ANDRADE-VIEIRA LF, GEDRAITE LS, CAMPOS JMS, DAVIDE LC (2011) Spent Pot Liner (SPL) induced DNA damage and nuclear alterations in root tip cells of Allium cepa as a consequence of programmed cell death. Ecotoxicol Environ Saf 74(4):882–888.

    Article  CAS  Google Scholar 

  • ANDRADE-VIEIRA, L.F., DE CAMPOS, J.M.S., AND DAVIDE, L.C., (2012). Effects of Spent Pot Liner on mitotic activity and nuclear DNA content in meristematic cells of Allium cepa.J Environ Manag,107, 140–146, DOI:

  • BERNET D, SCHMIDT H, MEIER W, BURKHARDT-HOLM P, WAHLI T (1999) Histopathology in fish: proposal for a protocol to assess aquatic pollution. J Fish Dis 22(1):25–34.

    Article  Google Scholar 

  • CARRASCO KR, TILBURY KL, MAYERS MS (1990) Assessment of the piscine micronuclei test in situ biological indicator of chemical contaminants effects. Canad J Fish Aquatic Science 47(11):2123–2136.

    Article  CAS  Google Scholar 

  • CHANDRA S, CHAUHAN LKS, MURTHY RC, SAXENA PN, PANDE PN, GUPTA SK (2005) Comparative biomonitoring of leachates from hazardous solid waste of two industries using Allium test. Sci Total Environ 347(1-3):46–52.

    Article  CAS  Google Scholar 

  • COLLINS AR (2004) The comet assay for DNA damage and repair. Principles, applications, and limitation. Rev Mol Biotechnol 26(3):249–261.

    Article  CAS  Google Scholar 

  • COLLINS AR, DOBSON VL, DUSINSKÁ M, KENNEDY G, STĔTINA R (1997) The comet assay: what can it really tell us? Mutat Res 375(2):183–193.

    Article  CAS  Google Scholar 

  • CONOLLY RB, LUTZ WK (2004) Nonmonotonic dose-response relationships: mechanistic basis, kinetic modeling, and implications for risk assessment. Toxicological Sciences 77(1):151–157

    Article  CAS  Google Scholar 

  • DAVID WM, MITCHELL DL, WALTER RB (2004) DNA repair in hybrid fish of the genus Xiphophorus. Review Comp Biochem Physiol 138:301–309

    Google Scholar 

  • DOS REIS GB, ANDRADE-VIEIRA LF, DE CAMPOS MORAES I, CÉSAR PHS, MARCUSSI S, DAVIDE LC (2017) Reliability of plant root comet assay in comparison with human leukocyte comet assay for assessment environmental genotoxic agents. Ecotoxicol Environ Saf 142:110–116.

    Article  Google Scholar 

  • FAßBENDER C, BRAUNBECK T (2013) Assessment of genotoxicity in gonads, liver and gills of zebrafish (Danio rerio) by use of the comet assay and micronucleus test after in vivo exposure to methyl methanesulfonate. Bull Environ Contam Toxicol 91(1):89–95.

    Article  Google Scholar 

  • Fenech, M. et al. (2000). The HUman MicroNucleous Project—an international collaborative study on the use of micronucleous technique for measuring DNA damage in humans. Mutation Res, Amsterdam 428 (½), 271-283

  • FRACÁCIO R, VERANI NF, ESPÍNDOLA ELG, ROCHA O, RIGOLIN-SÁ O, ANDRADE CA (2003) Alterations on growth and gill morphology of Danio rerio (Pisces, Ciprinidae) exposed to the toxic sediments. Braz Arch Biol Technol 46(4):685–695.

    Article  Google Scholar 

  • FREITAS AS, CUNHA IMF, ANDRADE-VIEIRA LF, TECHIO VH (2016) Effect of SPL (Spent Pot Liner) and its main components on root growth, mitotic activity and phosphorylation of Histone H3 in Lactuca sativa L. Ecotoxicol Environ Saf 124:426–434.

    Article  CAS  Google Scholar 

  • JIA X, ZHANG H, LIU X (2011) Low levels of cadmium exposure induce DNA damage and oxidative stress in the liver of Oujiang colored common carp Cyprinus carpio var. color. Fish Physiol Biochem 37(1):97–103.

    Article  CAS  Google Scholar 

  • KARI G, RODECK U, DICKER AP (2007) Zebrafish: an emerging model system for human disease and drug disco: very. Clinical Pharmacology Therapeutics, Saint Louis 82(1):70–80.

    Article  CAS  Google Scholar 

  • KARLSSON-NORRGREN, L.; RUNN, P.; HAUX, C. AND FORLIN, L., (1985). Cadmiun-induced changes in gill morphology of zebrafish, Brachydanio rerio (Hamilton-Buchanan), and rainbow trout, Salmo gairdneri Richardson.J Fish Biol,27, 81–95, 1, DOI:

  • LICHTENFELS AJFC, LORENZI-FILHO G, GUIMARAES ET, MACCHIONE M, SALDIVA PHN (1996) Effects of water pollution on the gill apparatus of fish. J Comp Pathol 115(1):47–60.

    Article  CAS  Google Scholar 

  • LIESCHKE JG, CURRIE PD (2007) Animal models of human disease: zebrafish swim into view. Nature Revi-Genetics, London 8(5):353–367.

    Article  CAS  Google Scholar 

  • LISBONA DF, STEEL KM (2008) Recovery of fluoride values from spent pot-lining: precipitation of an aluminium hydroxyfluoride hydrate product. Sep Purif Technol 61(2):182–192.

    Article  CAS  Google Scholar 

  • LISBONA DF, SOMERFIELD C, STEEL KM (2012) Leaching of spent pot-lining with aluminum anodizing wastewaters: fluoride extraction and thermodynamic modeling of aqueous speciation. Ind Eng Chem Res 51(25):8366–8377.

    Article  CAS  Google Scholar 

  • MARCUSSI S, SANTOS PR, MENALDO DL, SILVEIRA LB, SANTOS-FILHO NA, MAZZI MV, DA SILVA SL, STÁBELI RG, ANTUNES LM, SOARES AM (2011) Evaluation of the genotoxicity of Crotalus durissusterrificus snake venom and its isolated toxins on human lymphocytes. Mutat Res 724(1–2):59–63.

    Article  CAS  Google Scholar 

  • MARCUSSI S, STÁBELI RG, SANTOS-FILHO NA, MENALDO DL, SILVA PEREIRA LL, ZULIANI JP, CALDERON LA, DA SILVA SL, ANTUNES LM, SOARES AM (2013) Genotoxic effect of Bothrops snake venoms and isolated toxins on human lymphocyte DNA. Toxicon 65:9–14.

    Article  CAS  Google Scholar 

  • Narwal, SS., Catalán, CAN., Sampietro, DA., Vattuone, MA., Politycka, B., (2008). Plant bioassays, Studium Press, Houston pp1–344

  • NTUK, U., WHITE, E., TAIT, S., STEEL, K., (2013). Prediction of solubilities of aluminium hydroxyfluoride hydrate recovered from spent pot lining. In: Proceedings of Chemeca 2013: challenging tomorrow. Chemeca 2013: Challenging tomorrow, Brisbane, QLD, Australia, (1–6). 29

  • OLIVEIRA RC, FANTA E, TURCATTI NM, CARDOSO, R.J. AND CARVALHO, C.S. (1996) Lethal effects of inorganic mercury on cells and tissues of Trichomycterus brasiliensis (Pisces; Siluroidei). Biocell 20(3):171–178

  • PALMIERI MJ, LUBER J, ANDRADE-VIEIRA LF, DAVIDE LC (2014) Cytotoxic and phytotoxic effects of the main chemical components of spent pot-liner: a comparative approach. Mutat Res Genet Toxicol Environ Mutagen 763:30–35.

    Article  CAS  Google Scholar 

  • PALMIERI MJ, ANDRADE-VIEIRA LF, TRENTO MVC, ELEUTÉRIO MWF, LUBER J, DAVIDE LC, MARCUSSI S (2016) Cytogenotoxic effects of Spent Pot Liner (SPL) and its main components on human leukocytes and meristematic cells of Allium cepa. Water Air Soil Pollut 227(5):156–166.

    Article  Google Scholar 

  • PARAÍBA LC, BOEIRA CR, JONSSON CM, CARRASCO JM (2006) Fator de bioconcentração de poluentes orgânicos de lodos em frutos de laranjeiras. Revista Ecotoxicologia e meio ambiente 16:125–134

    Google Scholar 

  • R DEVELOPMENT CORE TEAM,2014. R: a language and environment for statistical computing. R Foundation for Statistical Computing

  • RIXIAN L, HUASHENG H, XINHONG W, Kejian W, CHUNGUANG W (2005) The genotoxic effects of benzo[a]pyrene and methamidophos on black porgy evaluated by comet assay. Chin J Oceanol Limnol 23(4):455–460.

    Article  Google Scholar 

  • VILCHES, M., (2009). Análise genotóxica do Rio Cadeia/RS através do ensaio cometa e teste de micronúcleo e anormalidades nucleares utilizando peixes como bioindicadores. Dissertação (Mestrado em Qualidade Ambiental). Centro Universitário Feevale, Novo Hamburgo, Brasil

  • WONG CKC, YEUNG HY, WOO PS, WONG MH (2001) Specific expression of cytochrome P4501A1 gene in gills, intestines and liver tilapia exposed to coastal sediments. Aquat Toxicol 54(1-2):69–80.

    Article  CAS  Google Scholar 

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We thank Pedro Henrique Cesar for support in the formatting of Fig. 4.


This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the Coordenação de Aperfeiçoamento de Nível Superior (CAPES), and the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG).

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Correspondence to Luis D. Solis-Murgas.

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The handling of animals and the experiments conducted with them were carried out in strict accordance with institutional (Animal Experimentation Ethics Committee of the Federal University of Lavras (UFLA), Lavras, MG, Brazil) and national (National Council for Control of Animal Experimentation (CONCEA)) guidelines for the care and use of laboratory animals.

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The authors declare that they have no conflict of interest.

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Responsible editor: Markus Hecker

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Castro, T.F.D., Paiva, I.M., Carvalho, A.F.S. et al. Genotoxicity of spent pot liner as determined with the zebrafish (Danio rerio) experimental model. Environ Sci Pollut Res 25, 11527–11535 (2018).

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