Acute Effects of Tetracycline Exposure in the Freshwater Fish Gambusia holbrooki: Antioxidant Effects, Neurotoxicity and Histological Alterations

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


A large body of evidence was compiled in the recent decades showing a noteworthy increase in the detection of pharmaceutical drugs in aquatic ecosystems. Due to its ubiquitous presence, chemical nature, and practical purpose, this type of contaminant can exert toxic effects in nontarget organisms. Exposure to pharmaceutical drugs can result in adaptive alterations, such as changes in tissues, or in key homeostatic mechanisms, such as antioxidant mechanisms, biochemical/physiological pathways, and cellular damage. These alterations can be monitored to determine the impact of these compounds on exposed aquatic organisms. Among pharmaceutical drugs in the environment, antibiotics are particularly important because they include a variety of substances widely used in medical and veterinary practice, livestock production, and aquaculture. This wide use constitutes a decisive factor contributing for their frequent detection in the aquatic environment. Tetracyclines are the individual antibiotic subclass with the second highest frequency of detection in environmental matrices. The characterization of the potential ecotoxicological effects of tetracycline is a much-required task; to attain this objective, the present study assessed the acute toxic effects of tetracycline in the freshwater fish species Gambusia holbrooki by the determination of histological changes in the gills and liver, changes in antioxidant defense [glutathione S-transferase (GST), catalase (CAT), and lipoperoxidative damage] as well as potential neurotoxicity (acetylcholinesterase activity). The obtained results suggest the existence of a cause-and-effect relationship between the exposure to tetracycline and histological alterations (more specifically in gills) and enzymatic activity (particularly the enzyme CAT in liver and GST in gills) indicating that this compound can exert a pro-oxidative activity.


Tetracycline Tacrine Gill Tissue Pharmaceutical Drug Histological Alteration 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This research was supported by the European Regional Development Fund through the COMPETE—Operational Competitiveness Program and by national funds through the Foundation for Science and Technology under the projects PEst-C/MAR/LA0015/2013 and PTDC/AMB/70431/2006.

Supplementary material

244_2014_101_MOESM1_ESM.docx (12 kb)
Supplementary material 1 (DOCX 12 kb)
244_2014_101_MOESM2_ESM.png (647 kb)
Supplementary material 2 (PNG 647 kb)


  1. Aebi H (1984) Catalase in vitro. Methods Enzymol 6:105–121Google Scholar
  2. Alazemi BM, Lewis JW, Andrews EB (1996) Gill damage in the fresh water fish Gnathonemus petersii (family: Mormyridae) exposed to selected pollutants: an ultrastructural study. Environ Technol 17(3):225–238CrossRefGoogle Scholar
  3. Amacher DE, Martin BA (1997) Tetracycline-induced steatosis in primary canine hepatocyte cultures. Fundam App Toxicol 40(2):256–263CrossRefGoogle Scholar
  4. Asha KK, Sankar TV, Nair PGV (2007) Effect of tetracycline on pancreas and liver function of adult male albino rats. J Pharmacy Pharmacol 59:1241–1248CrossRefGoogle Scholar
  5. Barceló D (2003) Emerging pollutants in water analysis. Trends Anal Chem 22(10):15–16CrossRefGoogle Scholar
  6. 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):25–34CrossRefGoogle Scholar
  7. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  8. Brunelli E, Mauceri A, Maisano M, Bernabo I, Giannetto A, De Domenico E et al (2011) Ultrastructural and immunohistochemical investigation on the gills of the teleost, Thalassoma pavo L., exposed to cadmium. Acta Histochem 113:201–213CrossRefGoogle Scholar
  9. Bruno DW (1989) An investigation into oxytetracycline residues in Atlantic salmon, Salmo salar L. J Fish Dis 12(2):77–86CrossRefGoogle Scholar
  10. Buege JA, Aust SD (1978) Microsomal lipid peroxidation. Methods Enzymol 52:302–310CrossRefGoogle Scholar
  11. Cabello FC (2006) Heavy use of prophylactic antibiotics in aquaculture: a growing problem for human and animal health and for the environment. Environ Microbiol 8(7):1137–1144CrossRefGoogle Scholar
  12. Cabral A, Marques C (1999) Life history, population dynamics and production of eastern mosquito fish, Gambusia holbrooki (Pisces, Poeciliidae) in rice fields of the lower Mondego River Valley, Western Portugal. Acta Oecol 20(6):607–620CrossRefGoogle Scholar
  13. Camargo MMP, Martinez CBR (2007) Histopathology of gills, kidney and liver of a neotropical fish caged in an urban stream. Neotrop Ichthyol 5(3):327–336CrossRefGoogle Scholar
  14. Castiglioni S, Bagnati R, Calamari D, Fanelli R, Zuccato E (2005) A multiresidue analytical method using solid-phase extraction and high-pressure chromatography tandem mass spectrometry to measure pharmaceuticals of different therapeutic classes in urban waste waters. J Chromatogr A 1092(2):206–215CrossRefGoogle Scholar
  15. Cengiz E, Unlu E (2006) Sublethal effects of commercial deltamethrin on the structure of the gill, liver and gut tissues of mosquitofish, Gambusia affinis: a microscopic study. Environ Toxicol Pharmacol 21:246–253CrossRefGoogle Scholar
  16. Cerqueira MA, Vieira FN, Ferreira RV, Silva JF (2005) The water quality of the Cértima River Basin (Central Portugal). Environ Monit Assess 111:297–306CrossRefGoogle Scholar
  17. Chambers HF (2001) Cloramphenicol, tetracyclines, macrolides, clindamycin and streptogramins. In: Katzung BG (ed) Basic and clinical pharmacology, 8th edn. McGraw-Hill, New York, pp 774–783Google Scholar
  18. Chen WR, Huang CH (2009) Transformation of tetracyclines mediated by Mn(II) and Cu(II) ions in the presence of oxygen. Environ Sci Technol 43(2):401–407CrossRefGoogle Scholar
  19. Cleuvers M (2003) Aquatic ecotoxicity of pharmaceuticals including the assessment of combination effects. Toxicol Lett 142(3):185–194CrossRefGoogle Scholar
  20. Daughton CG, Ternes TA (1999) Pharmaceuticals and personal care products in the environment: agents of subtle changes? Environ Health Perspect 107(6):907–938CrossRefGoogle Scholar
  21. De Domenico E, Mauceri A, Giordano G, Maisano M, Gioffrè G, Natalotto A et al (2011) Effects of “in vivo” exposure to toxic sediments on juveniles of sea bass (Dicentrarchus labrax). Aquat Toxicol 105:688–697CrossRefGoogle Scholar
  22. De Domenico E, Mauceri A, Giordano D, Maisano M, Giannetto A, Parrino V et al (2013) Biological responses of juvenile European sea bass (Dicentrarchus labrax) exposed to contaminated sediments. Ecotoxicol Environ Saf 97:114–123CrossRefGoogle Scholar
  23. Deblonde T, Cossu-Leguilla C, Hartemann P (2011) Emerging pollutants in wastewater: a review of the literature. Int J Hyg Environ Health 214:442–448CrossRefGoogle Scholar
  24. Díaz-Cruz MS, Alda MJL, Barceló D (2003) Environmental behavior and analysis of veterinary and human drugs in soils, sediments and sludge. Trends Anal Chem 22(6):340–351CrossRefGoogle Scholar
  25. Dong L, Gao J, Xie X, Zhou Q (2012) DNA damage and biochemical toxicity of antibiotics in soil on the earthworm Eisenia fetida. Chemosphere 89(1):44–51CrossRefGoogle Scholar
  26. Ellman GL, Courtney KD, Andres V, Featherstone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7:88–95CrossRefGoogle Scholar
  27. 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
  28. Ezemonye LI, Ikpesu TO (2011) Evaluation of sub-lethal effects of endosulfan on cortisol secretion, glutathione S-transferase and acetylcholinesterase activities in Clarias gariepinus. Food Chem Toxicol 49(9):1898–1903CrossRefGoogle Scholar
  29. Fatta-Kassinos D, Meric S, Nikolaou A (2011) Pharmaceutical residues in environmental waters and wastewater: current state of knowledge and future research. Anal Bioanal Chem 399:251–275CrossRefGoogle Scholar
  30. Fernandes MN, Mazon AF (2003) Environmental pollution and fish gill morphology. In: Val AL, Kapoor BG (eds) Fish adaptations. Science, Enfield, pp 203–231Google Scholar
  31. 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
  32. Gravato C, Guimarães L, Santos J, Faria M, Alves A, Guilhermino L (2010) Comparative study about the effects of pollution on glass and yellow ells (Anguilla anguilla) from the estuaries of Minho, Lima and Douro Rivers (NM Portugal). Ecotoxicol Environ Saf 73(4):524–533CrossRefGoogle Scholar
  33. Gu C, Karthikeyan KG (2005) Interaction of tetracycline with aluminum and iron hydrous oxides. Environ Sci Technol 39(8):2660–2667CrossRefGoogle Scholar
  34. 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
  35. Halling-Sorensen B, Nielson BN, Lanzky PF, Ingerslev F, Lutzhoft HCH, Jorgensen SE (1998) Occurrence, fate and effects of pharmaceutical substances in the environment—a review. Chemosphere 36(2):357–393CrossRefGoogle Scholar
  36. Halling-Sorensen B, Sengelov G, Tjornelund J (2002) Toxicity of teracyclines and tetracycline degradation products to environmentally relevant bacteria, including selected tetracycline-resistant bacteria. Arch Environ Contam Toxicol 44:7–16CrossRefGoogle Scholar
  37. 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. Gulf Res Rep 7(4):297–309Google Scholar
  38. Hirsch R, Ternes R, Haberer K, Kratz KL (1999) Occurrence of antibiotics in the aquatic environment. Sci Total Environ 225(1–2):109–118CrossRefGoogle Scholar
  39. Hughes GM, Perry SF (1976) Morphometric study of trout gills: a light-microscope method suitable for the evaluation of pollutant action. J Exp Biol 64:447–460Google Scholar
  40. Jia A, Xiao Y, Hu J, Asami M, Kunikane S (2009) Simultaneous determination of tetracyclines and their degradation products in environmental waters by liquid chromatography–electrospray tandem mass spectrometry. J Chromatogr A 1216:4655–4662CrossRefGoogle Scholar
  41. Jones OAH, Voulvoulis N, Lester JN (2002) Aquatic environmental assessment of the top 25 English prescription pharmaceuticals. Water Res 36(20):5013–5022CrossRefGoogle Scholar
  42. Kang JJ, Fang H (1997) Polycyclic aromatic hydrocarbons inhibit the activity of acetylcholinesterase purified from electric eel. Biochem Biophys Res Commun 238(2):367–369CrossRefGoogle Scholar
  43. Kraus RL, Pasieczny R, Lariosa-Willingham K, Turner MS, Jiang A, Trauger JW (2005) Antioxidant properties of minocycline: neuroprotection in an oxidative stress assay and direct radical scavenging activity. J Neurochem 94(3):818–827CrossRefGoogle Scholar
  44. Kummerer K (2009) Antibiotics in the aquatic environment—a review—part I. Chemosphere 75:417–434CrossRefGoogle Scholar
  45. Machado ALS, Brandão AAH, da Silva CMOM, da Rocha RF (2003) Influence of tetracycline in the hepatic and renal development of rat’s offspring. Braz Ach Biol Technol 46(1):47–51Google Scholar
  46. Martinez CBR, Nagae MY, Zaia CTBV, Zaia DAM (2004) Morphological and physiological acute effects of lead in the neotropical fish Prochilodus lineatus. Braz J Biol 64(4):797–807CrossRefGoogle Scholar
  47. Michael I, Rizzo L, McArdell CS, Manaia CM, Merlin C, Schwartz T et al (2013) Urban wastewater treatment plants as hotspots for the release of antibiotics in the environment: a review. Water Res 47(3):957–995CrossRefGoogle Scholar
  48. Nero V, Farwell A, Lee LEJ, Van Meer T, MacKinnon MD, Dixon DG (2006) The effects of salinity on naphthenic acid toxicity to yellow perch: gill and liver histopathology. Ecotoxicol Environ Saf 65(2):252–264CrossRefGoogle Scholar
  49. Nico L, Fuller P (2013) Gambusia holbrooki. USGS Nonindigenous Aquatic Species Database, Gainesville, FL. Accessed: May 4, 2013
  50. Nunes B (2011) The use of cholinesterases in ecotoxicology. Rev Environ Contam Toxicol 212:29–59Google Scholar
  51. 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. Ecotoxicol Environ Saf 72(2):341–354CrossRefGoogle Scholar
  52. 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
  53. 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. AJB 5:2480–2487Google Scholar
  54. Organisation for Economic Co-operation and Development (1992) OECD guidelines for the testing of chemicals, Section 2-fish, early-life stage toxicity test (guideline no. 210). OECD Publishing. doi: 10.1787/9789264070103-en
  55. Ozmen M, Ayas Z, Gungurdu A, Ekmekci GF, Yerli S (2008) Ecotoxicological assessment of water pollution in Sariyar Dam Lake, Turkey. Ecotoxicol Environ Safe 70(1):167–173CrossRefGoogle Scholar
  56. Pari L, Gnanasoundari M (2006) Influence of naringenin on oxytetracyclie mediated oxidative damage in rat liver. Basic Clin Pharmacol Toxicol 98(5):456–461CrossRefGoogle Scholar
  57. Péry AR, Gust M, Vollat B, Mons R, Ramil M, Fink G et al (2008) Fluoxetine effects assessment on the life cycle of aquatic invertebrates. Chemosphere 73(3):300–304CrossRefGoogle Scholar
  58. 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
  59. Ramos AS, Antunes SC, Gonçalves F, Nunes B (2014) The gooseneck barnacle (Pollicipes pollicipes) as a candidate sentinel species for coastal contamination. Arch Environ Toxicol 66(317–326):1Google Scholar
  60. Ribeiro SHM (2012) Estimativa dos benefícios da melhoria na qualidade da água no Cértima Master thesis [in Portugese]. University of Aveiro, PortugalGoogle Scholar
  61. Said AA, Matsuki N, Kasuya Y (1995) Effects of aminoglycoside antibiotics on cholinergic autonomic nervous transmission. Pharmacol Toxicol 76(2):128–132CrossRefGoogle Scholar
  62. Sarmah AK, Meyer MT, Boxall AB (2006) A global perspective on the use, sales, exposure pathways, occurrence, fate and effects of veterinary antibiotics (VAs) in the environment. Chemosphere 65(5):725–759CrossRefGoogle Scholar
  63. Sayed AEDH, Mekkawy IA, Mahmoud UM (2012) Histopathological alterations in some body organs of adult Clarias gariepinus (Burchell, 1822) exposed to 4-nonylphenol, zoology. In: Garcia MD (ed) zoology. InTech, West Palm Beach, pp 163–184Google Scholar
  64. Shabana MB, Ibrahim HM, Khadre SEM, Elemam MG (2012) Influence of rifampicin and tetracycline administration on some biochemical and histological parameters in albino rats. J Basic App Zool 65(5):299–308CrossRefGoogle Scholar
  65. Snavely SR, Hodges GR (1984) The neurotoxicity of antibacterial agents. Ann Intern Med 101(1):92–104CrossRefGoogle Scholar
  66. Soory M (2008) A role for non-antimicrobial actions of tetracyclines in combating oxidative stress in periodontal and metabolic diseases: a literature review. Open Dent J 2:5–12CrossRefGoogle Scholar
  67. Spilovska K, Korabecny J, Kral J, Horova A, Musilek K, Soukup O et al (2013) 7-Methoxytacrine-adamantylamine heterodimers as cholinesterase inhibitors in Alzheimer’s disease treatment—synthesis, biological evaluation and molecular modeling studies. Molecules 18:2397–2418CrossRefGoogle Scholar
  68. Stentiford GD, Longshaw M, Lyons BP, Jones G, Green M, Feist SW (2003) Histopathological biomarkers in estuarine fish species for the assessment of biological effects of contaminants. Mar Environ Res 55(2):137–159CrossRefGoogle Scholar
  69. Stevens DE (1992) Gill morphometry of the red drum, Sciaenops ocellatus. Fish Physiol Biochem 10(2):169–176CrossRefGoogle Scholar
  70. Sturve J, Almroth BC, Forlin L (2008) Oxidative stress in rainbow trout (Oncorhynchus mykiss) exposed to sewage treatment plant effluent. Ecotoxicol Environ Safe 70(3):446–452CrossRefGoogle Scholar
  71. Temmink JHM, Bowmieister PJ, Jong P, van der Berg JHJ (1983) An ultra-structural study of chromate-induced hyperplasia in the gill of rainbow trout, Salmo gairdneri. Aquat Toxicol 4(2):165–179CrossRefGoogle Scholar
  72. Thomas RJ (1994) Neurotoxicity of antibacterial therapy. South Med J 87(9):869–874CrossRefGoogle Scholar
  73. Tu HT, Silvestre F, Scippo M-L, Thome J-P, Phuong NT, Kestemont P (2009) Acetylcholinesterase activity as a biomarker of exposure to antibiotics and pesticides in the black tiger shrimp (Penaeus monodon). Ecotoxicol Environ Saf 72(5):1463–1470CrossRefGoogle Scholar
  74. van den Booggard AE, Stobberingh EE (1999) Antibiotic usage in animals: impact on bacterial resistance and public health. Drugs 58(4):589–607CrossRefGoogle Scholar
  75. Van Dyk JC, Pieterse GM, Van Vuren JHJ (2007) Histological changes in the liver of Oreochromis mossambicus (Cichlidae) after exposure to cadmium and zinc. Ecotoxicol Environ Safe 66:432–440CrossRefGoogle Scholar
  76. Van Heerden D, Vosloo A, Nikinmaa M (2004) Effects of short-term, copper exposure on gill structure metallothionein and hypoxia-inducible factor-1a (HIF-1a) levels in rainbow trout (Oncorhynchus mykiss). Aquat Toxicol 69:271–280CrossRefGoogle Scholar
  77. Verlicchi P, Galleti A, Petrovic M, Barceló D (2010) Hospital effluents as a source of emerging pollutants: an overview of micropollutants and sustainable treatment options. J Hydrol 389(3–4):416–428CrossRefGoogle Scholar
  78. Wang QQ, Yates SR (2008) Laboratory study of oxytetracycline degradation kinetics in animal manure and soil. J Agric Food Chem 56(5):1683–1688CrossRefGoogle Scholar
  79. Wilson JM, Bunte RM, Anthony J, 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
  80. Wood CM, Soivio A (1991) Environmental effects on gill function: an introduction. Physiol Zool 64:1–3Google Scholar
  81. 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
  82. Yonar ME (2012) The effect of lycopene on oxytetracycline-induced oxidative stress and immunosuppression in rainbow trout (Oncorhynchus mykiss, W.). Fish Shellfish Immunol 32(6):994–1001CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • B. Nunes
    • 1
    • 5
  • S. C. Antunes
    • 4
    • 5
  • R. Gomes
    • 3
  • J. C. Campos
    • 3
  • M. R. Braga
    • 3
  • A. S. Ramos
    • 4
  • A. T. Correia
    • 2
    • 3
  1. 1.Departamento de BiologiaUniversidade de Aveiro, Campus Universitário de SantiagoAveiroPortugal
  2. 2.Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR/CIMAR)PortoPortugal
  3. 3.Faculdade de Ciências da SaúdeUniversidade Fernando Pessoa (UFP)PortoPortugal
  4. 4.Faculdade de CiênciasUniversidade do Porto (FCUP)PortoPortugal
  5. 5.Centro de Estudos do Ambiente e do MarUniversidade de Aveiro, Campus Universitário de SantiagoAveiroPortugal

Personalised recommendations