, Volume 24, Issue 9, pp 1896–1905 | Cite as

Ecotoxicological effect of zinc pyrithione in the freshwater fish Gambusia holbrooki

  • B. Nunes
  • M. R. Braga
  • J. C. Campos
  • R. Gomes
  • A. S. Ramos
  • S. C. Antunes
  • A. T. Correia


Currently diverse biocidal agents can be used for distinct applications, such as personal hygiene, disinfection, antiparasitic activity, and antifouling effects. Zinc pyrithione is an organometallic biocide, with bactericidal, algicidal and fungicidal activities. It has been recently incorporated in antifouling formulas, such as paints, which prevent the establishment of a biofilm on surfaces exposed to the aquatic environment. It has also been used in cosmetics, such as anti-dandruff shampoos and soaps. Previously reported data has shown the presence of this substance in the aquatic compartment, a factor contributing to the potential exertion of toxic effects, and there is also evidence that photodegradation products of zinc pyrithione were involved in neurotoxic effects, namely by inhibiting cholinesterases in fish species. Additional evidence points to the involvement of zinc pyrithione in alterations of metal homeostasis and oxidative stress, in both aquatic organisms and human cell models. The present work assesses the potential ecotoxicity elicited by zinc pyrithione in the freshwater fish Gambusia holbrooki after an acute (96 h) exposure. The oxidative stress was assessed by the quantification of the activities of specific enzymes from the antioxidant defense system, such as catalase, and glutathione-S-transferases; and the extent of peroxidative damage was quantified by measuring the thiobarbituric acid reactive substances levels. Neurotoxicity was assessed through measurement of acetylcholinesterase activity; and a standardized method for the description and assessment of histological changes in liver and gills of was also used. Zinc pyrithione caused non-specific and reversible tissue alterations, both in liver and gills of exposed organisms. However, histopathological indices were not significantly different from the control group. In terms of oxidative stress biomarkers, none of the tested biomarkers indicated the occurrence of pro-oxidative effects, suggesting that the oxidative pathway is not the major toxicological outcome of exposure to zinc pyrithione.


Antifouling Organometallics Biomarkers Histological damage Mosquitofish Oxidative stress Neurotoxicity 



This work was supported by European Funds through COMPETE and by National Funds through the Portuguese Science Foundation (FCT) within project PEst-C/MAR/LA0017/2013. Bruno Nunes was hired under the programme Investigador FCT, co-funded by the Human Potential Operational Programme (National Strategic Reference Framework 2007–2013) and European Social Fund (EU). We would like to thank the highly valuable contribution of Dr. Jonathan Wilson for the revision of the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Aebi H (1984) Catalase in vitro. Methods Enzymol 6:105–121Google Scholar
  2. Ahmad I, Pacheco M, Santos M (2006) Anguilla anguilla L. oxidative stress biomarkers: an in situ study of freshwater wetland ecosystem (Pateira de Fermentelos, Portugal). Chemosphere 65(6):952–962CrossRefGoogle Scholar
  3. Alazemi BM, Lewis JW, Andrews EB (1996) Gill damage in the freshwater fish Gnathonemus petersii (Family: Mormyridae) exposed to selected pollutants: an ultrastructural study. Environ Technol 17(3):225–238CrossRefGoogle Scholar
  4. Álvarez-Muñoz D, Gómez-Pana A, Blasco J, Sarasquete C, González-Mazo E (2009) Oxidative stress and histopathology damage related to the metabolism of dodecylbenzene sulfonate in Senegalese sole. Chemosphere 74(9):1216–1223CrossRefGoogle Scholar
  5. Bao VWW, Leung KMY, Kwok KWH, Zhang AQ, Lui GCS (2008) Synergistic toxic effects of zinc pyrithione and copper to three marine species: implications on setting appropriate water quality criteria. Mar Pollut Bull 57(6–12):616–623CrossRefGoogle Scholar
  6. Bellas J, Granmo A, Beiras R (2005) Embryotoxicity of the antifouling biocide zinc pyrithione to sea urchin (Paracentrotus lividus) and mussel (Mytilus edulis). Mar Pollut Bull 50(11):1382–1385CrossRefGoogle Scholar
  7. Bernet D, Schmidt H, Meier W, Burkhardt-Holm P, Wahli T (1999) Histopathology in fish: proposal for a protocol to access aquatic pollution. J Fish Dis 22(1):25–34CrossRefGoogle Scholar
  8. Borg DA, Trombetta LD (2010) Toxicity and bioaccumulation of the booster biocide copper pyrithione, copper 2-pyridinethiol-1-oxide, in gill tissues of Salvelinus fontinalis (brook trout). Toxicol Ind Health 26(3):139–150CrossRefGoogle Scholar
  9. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1–2):248–254CrossRefGoogle Scholar
  10. Brandão F, Correia AT, Gonçalves F, Bruno Nunes B (2013) Effects of anthropogenic metallic contamination on cholinesterases of Gambusia holbrooki. Mar Poll Bull 76:72–76CrossRefGoogle Scholar
  11. Buege JA, Aust SD (1978) Microsomal lipid peroxidation. Method Enzymol 52:302–310CrossRefGoogle Scholar
  12. Cabral A, Marques C (1999) Life history, population dynamics and production of eastern mosquitofish, Gambusia holbrooki (Pisces, Poeciliidae) in rice fields of the lower Mondego River Valley, Western Portugal. Acta Oecol 20(6):607–620CrossRefGoogle Scholar
  13. Camargo M, Martinez C (2007) Histopathology of gills, kidney and liver of a neotropical fish caged in an urban stream. Neotrop Icthyol 5(3):327–336CrossRefGoogle Scholar
  14. Cengiz E, Unlü E (2003) Histopathological of gills in mosquitofish, Gambusia affinis after long-term exposure to sublethal concentrations of malathion. J Environ Sci Health B 38(5):581–589CrossRefGoogle Scholar
  15. Costa PM, Diniz MS, Caeiro S, Lobo J, Martins M, Ferreira AM, Caetano M, Vale C, DelValls TA, Costa MH (2009) Histological biomarkers in liver and gills of juvenile Solea senegalensis exposed to contaminated estuarine sediments: a weighted indices approach. Aquat Toxicol 92(3):202–212CrossRefGoogle Scholar
  16. Daughton CG, Ternes TA (1999) Pharmaceuticals and personal care products in the environment: agents of subtle change? Environ Health Perspect 107(Suppl 6):907–938CrossRefGoogle Scholar
  17. Ellman G, Courtney KD, Vjr Andres, Featherstone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7(2):88–90CrossRefGoogle Scholar
  18. Evans DH, Piermarini PM, Choe KP (2005) The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste. Physiol Rev 85:97–177CrossRefGoogle Scholar
  19. Fernandes MN, Mazon AF (2003) Environmental pollution and fish gill morphology. In: Val AL, Kapoor BG (eds) Fish adaptations. Science Publishers, Enfield, pp 203–231Google Scholar
  20. Fernandes C, Fontaínhas-Fernandes A, Rocha E, Salgado MA (2008) Monitoring pollution in Esmoriz-Paramos lagoon, Portugal: liver histological and biochemical effects in Liza saliens. Environ Monit Assess 145(1–3):315–322CrossRefGoogle Scholar
  21. Ferreira G, Simas T, Nobre A, Silva C, Shiffereggen K, Lencart-Silva J (2003). Identification of sensitive areas and vulnerable zones in transitional and coastal Portuguese systems: spplication of the United States National Estuarine Eutrophication Assessment to the Minho, Lima, Douro, Ria de Aveiro, Mondego, Tagus, Sado, Mira, Ria Formosa and Guadiana systems. INAG/IMARGoogle Scholar
  22. Gonçalves A, Padrão J, Gonçalves F, Nunes B (2010) In vivo acute effects of several pharmaceutical drugs (diazepam, clofibrate, clofibric acid) and detergents (sodium dodecylsulphate and benzalkonium chloride) on cholinesterases from Gambusia holbrooki. Fresen Environ Bull 4:1–12Google Scholar
  23. Guardiola FA, Cuesta A, Mesequer A, Esteban MA (2012) Risks of using antifouling biocides in aquaculture. Int J Mol Sci 13(2):1541–1560CrossRefGoogle Scholar
  24. Habig WH, Pabst MJ, Jakoby WB (1974) Glutathione-S-transferases: the first enzymatic step in mercapturic acid formation. J Biol Chem 249(22):7130–7139Google Scholar
  25. Hawkins WE, Overstreet RM, Provancha MJ (1984) Effects of space shuttle exhaust plumes on gills of some estuarine fishes: a light and electron microscopic study (vol 513), Faculty Publications from the Harold W, Manter Laboratory of ParasitologyGoogle Scholar
  26. Jagoe CH, Faivre A, Newman MC (1996) Morphological and morphometric changes in the gills of mosquitofish (Gambusia holbrooki) after exposure to mercury (II). Aquat Toxicol 34(2):163–183CrossRefGoogle Scholar
  27. Kobayashi N, Okamura H (2002) Effects of new antifouling compounds on the development of sea urchin. Mar Pollut Bull 44(8):748–751CrossRefGoogle Scholar
  28. Lamore SD, Wondrak GT (2011) Zinc pyrithione impairs zinc homeostasis and upregulates stress response gene expression in reconstructed human epidermis. Biometals 24(5):875–890CrossRefGoogle Scholar
  29. Lang T, Wosniok W, Baršienė J, Broeg K, Kopecka J, Parkkonen J (2006) Liver histopathology in Baltic flounder (Platichtys flesus) as indicator of biological effects of contaminants. Mar Pollut Bull 53(8–9):488–496CrossRefGoogle Scholar
  30. Mackie DS, van den Berg CMG, Readman JW (2004) Determination of pyrithione in natural waters by cathodic stripping voltammetry. Anal Chim Acta 511:47–53CrossRefGoogle Scholar
  31. Madsen T, Gustavsson K, SamsØe-Petersen L. Simonsen F, Jakobsen J, Foverskov S, Larsen MM (2000) Ecotoxicological assessments of antifouling biocides and nonbiocidal paints. Environmental Project no. 531. Danish Environmental Protection AgencyGoogle Scholar
  32. Maraldo K, Dahllöf I (2004) Indirect estimation of degradation time for zinc pyrithione and copper pyrithione in seawater. Mar Pollut Bull 48(9–10):894–901CrossRefGoogle Scholar
  33. Marcheselli M, Azzoni P, Mauri M (2011) Novel antifouling agent-zinc pyrithione: stress induction and genotoxicity to the marine mussel Mytilus galloprovincialis. Aquat Toxicol 102(1–2):39–47CrossRefGoogle Scholar
  34. Mazon AF, Monteiro EA, Pinheiro GH, Fernandes MN (2002) Hematological and physiological changes induced by short-term exposure to copper in the freshwater fish, Prochilodus scrofa. Braz J Biol 62(4A):621–631CrossRefGoogle Scholar
  35. Mochida K, Ito K, Harino H, Kakuno A, Fuji K (2006) Acute toxicity of pyrithione antifouling biocides and joint toxicity with copper to red sea bream (Pagrus major) and toy shrimp (Heptacarpus futilirostris). Environ Toxicol Chem 25(11):3058–3064CrossRefGoogle Scholar
  36. Mochida K, Ito K, Harino H, Tanaka H, Onduka T, Kakuno A, Fujii K (2009) Inhibition of acetylcholinesterase by metabolites of copper pyrithione (CuPT) and its possible involvement in vertebral deformity of a CuPT-exposed marine teleostean fish. Comp Biochem Phys C 149(4):624–630Google Scholar
  37. Neihof RA, Bailey CA, Patouillet C, Hannan PJ (1979) Photodegradation of mercaptopyridine-N-oxide biocides. Arch Environ Contam Toxicol 8(3):355–368CrossRefGoogle Scholar
  38. Nico L, Fuller P (2013) Gambusia holbrooki. USGS Nonindigenous Aquatic Species Database, Gainesville, Fl. Accessed 05 Apr 2013
  39. Nunes B, Carvalho F, Guilhimermino L (2003) Characterization of total head cholinesterases of Gambusia holbrooki (mosquitofish), and the assessment of effects induced by two environmental contaminants. J Vet Pharmacol Ther 26(1):260–261Google Scholar
  40. Nunes B, Gaio AR, Carvalho F, Guilhermino L (2008) Behaviour and biomarkers of oxidative stress in Gambusia holbrooki after acute exposure to widely used pharmaceuticals and a detergent. Ecotox Environ Safe 71(2):341–354CrossRefGoogle Scholar
  41. Nunes B, Barbosa AR, Antunes SC, Gonçalves F (2014) Combination effects of anticholinesterasics in acetylcholinesterase of a fish species: effects of a metallic compound, an organophosphate pesticide, and a pharmaceutical drug. Environ Sci Pollut Res 21:6258–6262CrossRefGoogle Scholar
  42. Nunes B, Antunes SC, Gomes R, Campos JC, Braga MR, Ramos AS, Correia AT (2015) Acute effects of tetracycline exposure in the freshwater fish Gambusia holbrooki: antioxidant effects, neurotoxicity and histological alterations. Arch Environ Contam Toxicol 68(2):371–381CrossRefGoogle Scholar
  43. OECD (1992) OECD Guideline for the testing of chemicals. Test 203: Fish, Acute Toxicity TestGoogle Scholar
  44. Olsson PE, Larsson A, Haux C (1996) Influence of seasonal changes in water temperature on cadmium inducibility of hepatic and renal metallothionein in rainbow trout. Mar Environ Res 42:41–44CrossRefGoogle Scholar
  45. Olurin KB, Olojo EAA, Mbaka GO, Akindele AT (2006) Histopathological responses of the gill and liver tissues of Clarias gariepinus fingerlings to the herbicide, glyphosate. Afr J Biotechnol 5(24):2480–2487Google Scholar
  46. Onduka T, Mochida K, Harino H, Ito K, Kakuno A, Fujii K (2010) Toxicity of metal pyrithione photodegradation products to marine organisms with indirect evidence for their presence in seawater. Arch Environ Contam Toxicol 58(4):991–997CrossRefGoogle Scholar
  47. Oyama TM, Saito M, Yonezama T, Okano Y, Oyama Y (2012) Nanomolar concentrations of zinc pyrithione increase cell susceptibility to oxidative stress induced by hydrogen peroxide in rat thymocytes. Chemosphere 87(11):1316–1322CrossRefGoogle Scholar
  48. Poleksic V, Mitrovic-Tutundzic V (1994) Fish gills as a monitor of sublethal and chronic effects of pollution. In: Müller R, Lloyd R (eds) Sublethal and chronic effects of pollutants on freshwater fish. Fishing New Books, OxfordGoogle Scholar
  49. Reeder NL, Kaplan J, Xu J, Youngguist RS, Wallace J, Hu P, Juhlin KD, Schwartz JR, Grant RA, Fieno A, Nerneth S, Reichling T, Tiesman JP, Mills T, Steinke M, Wang SL, Saunders CW (2011) Zinc pyrithione inhibits yeast growth through copper influx and inactivation of iron-sulfur proteins. Antimicrob Agents Chem 55(12):5753–5760CrossRefGoogle Scholar
  50. Richmonds C, Dutta H (1989) Histopathological changes induced by malathion in the gills of bluegill Lepomis macrochirus. B Environ Contam Toxicol 43:123–130CrossRefGoogle Scholar
  51. Rudolf E, Cervinka M (2011) Stress responses of human dermal fibroblasts exposed to zinc pyrithione. Toxicol Lett 204(2–3):164–173CrossRefGoogle Scholar
  52. Sakkas VA, Shibata K, Yamaguchi Y, Sugasawa S, Albanis T (2007) Aqueous phototransformation of zinc pyrithione: degradation kinetics and byproduct identification by liquid chromatography–atmospheric pressure chemical ionisation mass spectrometry. J Chromatogr A 1144(2):175–182CrossRefGoogle Scholar
  53. Schwartz JR, Shah R, Krigbaum H, Sacha J, Vogt A, Blume-Peytavi U (2011) New insights on dandruff/seborrhoeic dermatitis: the role of the scalp follicular infundibulum in effective treatment strategies. Br J Dermatol 165(Suppl 2):18–23CrossRefGoogle Scholar
  54. Sismeiro-Vivas J, Abrantes N, Pereira JL, Castro BB, Gonçalves F (2007) Short-term effects of Quirlan® (Chlorfenvinphos) on the behavior and acetylcholinesterase activity of Gambusia holbrooki. Environ Toxicol 22:194–202CrossRefGoogle Scholar
  55. Takashima F, Hibiya T (1995) An atlas of fish histology: normal and pathological features, 2nd edn. Kodensha Ltd., TokyoGoogle Scholar
  56. Turley PA, Fenn RJ, Ritter JC, Callow ME (2005) Pyrithiones as antifoulants: environmental fate and loss of toxicity. Biofouling 21(1):31–40CrossRefGoogle Scholar
  57. Wilson JM, Bunte RM, Carty AJ (2009) Evaluation of rapid cooling and tricaine methanesulfonate (MS222) as methods of euthanasia in zebrafish (Danio rerio). J Am Assoc Lab Anim Sci 48(6):785–789Google Scholar
  58. Wood CM, Soivio A (1991) Environmental effects on gill function: an introduction. Physiol Zool 64(1):1–3Google Scholar
  59. Xuereb B, Lefèvre E, Garric J, Geffard O (2009) Acetylcholinesterase activity in Gammarus fossarum (Crustacea Amphipoda): linking AChE inhibition and behavioural alteration. Aquat Toxicol 94(2):114–122CrossRefGoogle Scholar
  60. Yasokawa D, Murata S, Iwahashi Y, Kitagawa E, Kishi K, Okumura Y, Iwahashi H (2010) DNA microarray analysis suggests that zinc pyrithione causes iron starvation to the yeast Saccharomyces cerevisiae. J Biosci Bioeng 109(5):479–486CrossRefGoogle Scholar
  61. Yasser AG, Naser MD (2011) Impact of pollutants on fish collected from different parts of Shatt Al-Arab River: a histopathological study. Environ Monit Assess 181(1–4):175–182CrossRefGoogle Scholar
  62. Yebra DM, Kiil S, Dam-Johansen K (2004) Antifouling technology-past, present and future steps towards efficient and environmentally friendly antifouling coatings. Prog Org Coat 50(2):75–104CrossRefGoogle Scholar
  63. Zha J, Wang Z, Wang N, Ingersoll C (2007) Histological alternation of vitellogenin induction in adult rare minnow (Gobiocypris rarus) after exposure to ethynylestradiol and nonylphenol. Chemosphere 66(3):488–495CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • B. Nunes
    • 1
    • 2
  • M. R. Braga
    • 3
  • J. C. Campos
    • 3
  • R. Gomes
    • 3
  • A. S. Ramos
    • 4
  • S. C. Antunes
    • 1
    • 4
  • A. T. Correia
    • 3
    • 5
  1. 1.Centro de Estudos do Ambiente e do Mar (CESAM), Campus Universitário de SantiagoUniversidade de AveiroAveiroPortugal
  2. 2.Departamento de Biologia, Centro de Estudos do Ambiente e do Mar (CESAM), Campus Universitário de SantiagoUniversidade de AveiroAveiroPortugal
  3. 3.Faculdade de Ciências da Saúde da Universidade Fernando Pessoa (FCS-UFP)PortoPortugal
  4. 4.Departamento de BiologiaFaculdade de Ciências da Universidade do Porto (FCUP)PortoPortugal
  5. 5.Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR - CIMAR)PortoPortugal

Personalised recommendations