Environmental Science and Pollution Research

, Volume 21, Issue 18, pp 10815–10822 | Cite as

Biochemical and standard toxic effects of acetaminophen on the macrophyte species Lemna minor and Lemna gibba

  • Bruno Nunes
  • Glória Pinto
  • Liliana Martins
  • Fernando Gonçalves
  • Sara C. Antunes
Research Article


Acetaminophen is globally one of the most prescribed drugs due to its antipyretic and analgesic properties. However, it is highly toxic when the dosage surpasses the detoxification capability of an exposed organism, with involvement of an already described oxidative stress pathway. To address the issue of the ecotoxicity of acetaminophen, we performed acute exposures of two aquatic plant species, Lemna gibba and Lemna minor, to this compound. The selected biomarkers were number of fronds, biomass, chlorophyll content, lipid peroxidation (TBARS assay), and proline content. Our results showed marked differences between the two species. Acetaminophen caused a significant decrease in the number of fronds (EC50 = 446.6 mg/L), and the establishment of a dose-dependent peroxidative damage in L. minor, but not in L. gibba. No effects were reported in both species for the indicative parameters chlorophyll content and total biomass. However, the proline content in L. gibba was substantially reduced. The overall conclusions point to the occurrence of an oxidative stress scenario more prominent for L. minor. However, the mechanisms that allowed L. gibba to cope with acetaminophen exposure were distinct from those reported for L. minor, with the likely involvement of proline as antioxidant.


Aquatic plants Pharmaceutical drugs Paracetamol Oxidative stress Biomarkers 



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. Glória Pinto was hired under the programme Ciência 2008 (FCT, Portugal) and Bruno Nunes under the programme Investigador FCT co-funded by the Human Potential Operational Programme (National Strategic Reference Framework 2007–2013) and European Social Fund (EU).


  1. Aarti PD, Tanaka R, Tanaka A (2006) Effects of oxidative stress on chlorophyll biosynthesis in cucumber (Cucumis sativus) cotyledons. Physiol Plant 128:186–197CrossRefGoogle Scholar
  2. Antunes SC, Freitas R, Figueira E, Gonçalves F, Nunes B (2013) Biochemical effects of acetaminophen in aquatic species: edible clams Venerupis decussata and Venerupis philippinarum. Environ Sci Pollut Res 20(8):6658–6666CrossRefGoogle Scholar
  3. Bates LS, Waldren RP, Teari T (1973) Rapid determination of free proline for water stress studies. Plant Soil 39:205–207CrossRefGoogle Scholar
  4. Benotti MJ, Trenholm RA, Vanderford BJ, Holady JC, Stanford BD, Snyder SA (2009) Pharmaceuticals and endocrine disrupting compounds in U.S. drinking water. Environ Sci Technol 43:597–603CrossRefGoogle Scholar
  5. Bohnert HJ, Jensen RG (1996) Strategies for engineering water-stress tolerance in plants. Trends Biotechnol 14(3):89–97CrossRefGoogle Scholar
  6. Boleda MA, Galceran MA, Ventura F (2011) Behavior of pharmaceuticals and drugs of abuse in a drinking water treatment plant (DWTP) using combined conventional and ultrafiltration and reverse osmosis (UF/RO) treatments. Environ Pollut 159(6):1584–1591CrossRefGoogle Scholar
  7. Boxall AB, Rudd M, Brooks BW, Caldwell D, Choi K, Hickmann S, Innes E, Ostapyk K, Staveley JP, Verslycke T (2012) Pharmaceuticals and personal care products in the environment: what are the big questions. Environ Health Perspect 120:1221–1229CrossRefGoogle Scholar
  8. Brain RA, Johnson DJ, Richards SM, Hanson ML, Sanderson H, Lam MW, Young C, Mabury SA, Sibley PK, Solomon KR (2004) Microcosm evaluation of the effects of an eight pharmaceutical mixture to the aquatic macrophytes Lemna gibba and Myriophyllum sibiricum. Aquat Toxicol 70:23–40CrossRefGoogle Scholar
  9. Brain RA, Hanson ML, Solomon KR, Brooks BW (2008) Aquatic plants exposed to pharmaceuticals: effects and risks. Rev Environ Contam Toxicol 192:67–115Google Scholar
  10. Brandão FP, Pereira JL, Gonçalves F, Nunes B (2011) The impact of paracetamol on selected biomarkers of the mollusk species Corbicula fluminea. Environ Toxicol 1–10Google Scholar
  11. Brind AM (2007) Drugs that damage the liver. Medicine 35(1):26–30CrossRefGoogle Scholar
  12. Buege JA, Aust SD (1978) Microsomal lipid peroxidation. Methods Enzymol 52:302–310CrossRefGoogle Scholar
  13. Bull RJ, Crook J, Whittaker M, Cotruvo JA (2011) Therapeutic dose as the point of departure in assessing potential health hazards from drugs in drinking water and recycled municipal wastewater. Regul Toxicol Pharmacol 60(1):1–19CrossRefGoogle Scholar
  14. Corcoran J, Winter MJ, Tyler CR (2010) Pharmaceuticals in the aquatic environment: a critical review of the evidence for health effects in fish. Crit Rev Toxicol 40(4):287–304CrossRefGoogle Scholar
  15. Crane M, Watts C, Boucard T (2006) Chronic aquatic environmental risks from exposure to human pharmaceuticals. Sci Total Environ 367:23–41CrossRefGoogle 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. Davy M, Petrie R, Smrchek J, Kuchnicki T, Francois D (2001) Proposal to update non-target plant toxicity testing under NAFTA. USEPA, Washington, DCGoogle Scholar
  18. Dogan M, Saygideger SD, Ugur Colak U (2009) Effect of lead toxicity on aquatic macrophyte Elodea canadensis Michx. Bull Environ Contam Toxicol 83:249–254CrossRefGoogle Scholar
  19. Eaton AD, Clesceri LS, Greenberg AE (1995) Standard methods for the examination of water and wastewater, 19th edn. American Public Health Association, American Water Works Association, and Water Environment Federation, Washington, DCGoogle Scholar
  20. Elkahoui E, Hernández JA, Abdelly C, Ghrir R, Limama F (2005) Effects of salt on lipid peroxidation and antioxidant enzyme activities of Catharanthus roseus suspension cells. Plant Sci 168:607–613CrossRefGoogle Scholar
  21. Fent K, Weston A, Caminada D (2006) Ecotoxicology of human pharmaceuticals. Aquat Toxicol 76:122–159CrossRefGoogle Scholar
  22. Ferrari B, Paxéus N, Lo Giudice R, Pollio A, Garric J (2003) Ecotoxicological impact of pharmaceuticals found in treated wastewaters: study of carbamazepine, clofibric acid, and diclofenac. Ecotoxicol Environ Saf 55:359–370CrossRefGoogle Scholar
  23. Gapper C, Dolan L (2006) Control of plant development by reactive oxygen species. Plant Physiol 141:341–345CrossRefGoogle Scholar
  24. García SO, Pinto GP, Encina PG, Mata RI (2013) Consumption and occurrence of pharmaceutical and personal care products in the aquatic environment in Spain. Sci Total Environ 444:451–465CrossRefGoogle Scholar
  25. Hinson JA, Reid AB, McCullough SS, James LP (2004) Acetaminophen-induced hepatotoxicity: role of metabolic activation, reactive oxygen/nitrogen species, and mitochondrial permeability transition. Drug Metab Rev 36Google Scholar
  26. Huang L, Lu D, Zhang P, Diao J, Zhou Z (2012) Enantioselective toxic effects of hexaconazole enantiomers against Scenedesmus obliquus. Chirality 24(8):610–614CrossRefGoogle Scholar
  27. Huber C, Bartha B, Harpaintner R, Schröder P (2009) Metabolism of acetaminophen (paracetamol) in plants—two independent pathways result in the formation of a glutathione and a glucose conjugate. Environ Sci Pollut Res 16:206–213CrossRefGoogle Scholar
  28. Huber C, Bartha B, Schröder P (2012) Metabolism of diclofenac in plants—hydroxylation is followed by glucose conjugation. J Hazard Mater 243:250–256CrossRefGoogle Scholar
  29. Jaeschke H, Bajt ML (2006) Intracellular signaling mechanisms of acetaminophen-induced liver cell death. Toxicol Sci 89(1):31–41CrossRefGoogle Scholar
  30. Jaeschke H, Knight TR, Bajt ML (2003) The role of oxidant stress and reactive nitrogen species in acetaminophen hepatotoxicity. Toxicol Lett 144:279–288CrossRefGoogle Scholar
  31. Jaleel CA, Gopi R, Sankar B, Manivannan P, Kishorekumar A, Sridharan R, Panneerselvam R (2007) Studies on germination, seedling vigour, lipid peroxidation and proline metabolism in Catharanthus roseus seedlings under salt stress. S Afr J Bot 73:190–195CrossRefGoogle Scholar
  32. Jones OAH, Voulvoulis N, Lester JN (2002) Aquatic environmental assessment of the top 25 English prescription pharmaceuticals. Water Res 36:5013–5022CrossRefGoogle Scholar
  33. Klaassen CD (2001) Casarett & Doull’s toxicology: the basic science of poisons, 6th edn. Mc Graw Hill, New YorkGoogle Scholar
  34. Kolpin DW, Furlong ET, Meyer MT, Thurman EM, Zaugg SD, Barber LB, Buxton HT (2002) Pharmaceuticals, hormones, and other organic wastewater contaminants in US streams, 1999–2000: a national reconnaissance. Environ Sci Technol 36(6):1202–1211CrossRefGoogle Scholar
  35. Lewis MA, Wang W (1999) In: Gerhardt A (ed) Biomonitoring using aquatic vegetation. Environmental Science Forum 9. Trans Tech Publications, Switzerland, pp 243–274Google Scholar
  36. Li W, Shi Y, Gao L, Liu J, Cai Y (2012) Occurrence of antibiotics in water, sediments, aquatic plants, and animals from Baiyangdian Lake in North China. Chemosphere 89(11):1307–1315CrossRefGoogle Scholar
  37. Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol 148:350–382CrossRefGoogle Scholar
  38. Lourenção BC, Medeiros RA, Filho RCR, Mazo LH, Filho OF (2009) Simultaneous voltammetric determination of paracetamol and caffeine in pharmaceutical formulations using a boron-doped diamond electrode. Talanta 78:748–752CrossRefGoogle Scholar
  39. Lushchak VI (2011) Adaptive response to oxidative stress: bacteria, fungi, plants and animals. Comp Biochem Physiol C 153:175–190Google Scholar
  40. Matamoros V, Nguyen LX, Arias CA, Salvadó V, Brix H (2012) Evaluation of aquatic plants for removing polar microcontaminants: a microcosm experiment. Chemosphere 88(10):1257–1264CrossRefGoogle Scholar
  41. Mattioli R, Costantino P, Trovato M (2009) Proline accumulation in plants: not only stress. Plant Signal Behav 4:1016–1018CrossRefGoogle Scholar
  42. Metcalfe CD, Koenig BG, Bennie DT, Servos M, Ternes TA, Hirsch R (2003a) Occurrence of neutral and acidic drugs in the effluents of Canadian sewage treatment plants. Environ Toxicol Chem 22:2872–2880CrossRefGoogle Scholar
  43. Metcalfe CD, Miao X-S, Koenig BG, Struger J (2003b) Distribution of acidic and neutral drugs in surface waters near sewage treatment plants in the lower Great Lakes, Canada. Environ Toxicol Chem 22:2881–2889CrossRefGoogle Scholar
  44. Micheu S, Crailsheim K, Leonhard B (2000) Importance of proline and other amino acids during honeybee flight (Apis mellifera carnica POLLMANN). Amino Acids 18:157–175CrossRefGoogle Scholar
  45. Misra N, Gupta AK (2005) Effect of salt stress on proline metabolism in two high yielding genotypes of greengram. Plant Sci 169:331–339CrossRefGoogle Scholar
  46. Nunes B, Carvalho F, Guilhermino L (2004) Acute and chronic effects of clofibrate and clofibric acid on the enzymes acetylcholinesterase, lactate dehydrogenase and catalase of the mosquitofish, Gambusia holbrooki. Chemosphere 57:1581–1589CrossRefGoogle Scholar
  47. Nunes B, Carvalho F, Guilhermino L (2005) Acute toxicity of widely used pharmaceuticals in aquatic species: Gambusia holbrooki, Artemia parthenogenetica and Tetraselmis chuii. Ecotoxicol Environ Saf 61:413–419CrossRefGoogle Scholar
  48. OECD (2006) Lemna sp. growth inhibition test. OECD–Guideline for Testing of Chemicals, Paris, p 221Google Scholar
  49. Patel M, Tang BK, Kalow W (1993) Variability of acetaminophen metabolism in Caucasians and Orientals. Pharmacogenetics 2(1):38–45CrossRefGoogle Scholar
  50. Pérez S, Barceló D (2007) Application of advanced MS techniques to analysis and identification of human and microbial metabolites of pharmaceuticals in the aquatic environment. TrAC Trends Anal Chem 26(6):494–514CrossRefGoogle Scholar
  51. Prescott LF (1980) Kinetics and metabolism of paracetamol and phenacetin. Br J Clin Pharmacol 10:291S–298SCrossRefGoogle Scholar
  52. Rejmánková E (1975) Comparison of Lemna gibba and Lemna minor from the production ecological viewpoint. Aquat Bot 1:423–427CrossRefGoogle Scholar
  53. Roberts P, Thomas K (2006) The occurrence of selected pharmaceuticals in wastewater effluent and surface waters of the lower Tyne catchment. Sci Total Environ 356:143–153CrossRefGoogle Scholar
  54. Rodrigues S, Antunes SC, Brandão FP, Castro BB, Gonçalves F, Nunes B (2012) Effects of anticholinesterase drugs on biomarkers and behavior of pumpkinseed, Lepomis gibbosus (Linnaeus, 1758). J Environ Monit 14(6):1638–1644CrossRefGoogle Scholar
  55. Sanderson H, Johnson DJ, Reitsma T, Brain RA, Wilson CJ, Solomon KR (2004) Ranking and prioritization of environmental risks of pharmaceuticals in surface waters. Regul Toxicol Pharmacol 39:158–183CrossRefGoogle Scholar
  56. Solé M, Shaw JP, Frickers PE, Readman JW, Hutchinson TH (2010) Effects on feeding rate and biomarker responses of marine mussels experimentally exposed to propranolol and acetaminophen. Anal Bioanal Chem 396:649–656CrossRefGoogle Scholar
  57. Stewart PM, Scribailo RW, Simon TP (1999) In: Gerhardt A (ed) The use of aquatic macrophytes in monitoring and in assessment of biological integrity. Environmental Science Forum 9. Trans Tech Publications, Switzerland, pp 275–302Google Scholar
  58. Szabados L, Savouré A (2009) Proline: a multifunctional amino acid. Trends Plant Sci 15(2):13650–1385Google Scholar
  59. Ternes TA (1998) Occurrence of drugs in German sewage treatment plants and rivers. Water Res 32:3245–3260CrossRefGoogle Scholar
  60. Thounaojam TC, Panda P, Mazumdar P, Kumar D, Sharma GD, Sahoo L, Panda SK (2012) Excess copper induced oxidative stress and response of antioxidants in rice. Plant Physiol Biochem 53:33–39CrossRefGoogle Scholar
  61. Xu JJ, Hendriks BS, Zhao J, Graaf D (2008) Multiple effects of acetaminophen and p38 inhibitors: towards pathway toxicology. FEBS Lett 582:1276–1282CrossRefGoogle Scholar
  62. Yan S, Zhou Q (2011) Toxic effects of Hydrilla verticillata exposed to toluene, ethylbenzene and xylene and safety assessment for protecting aquatic macrophytes. Chemosphere 85:1088–1094CrossRefGoogle Scholar
  63. Yang L, Yu LE, Ray MB (2008) Degradation of paracetamol in aqueous solutions by TiO2 photocatalysis. Water Res 42:3480–3488CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Bruno Nunes
    • 1
    • 2
  • Glória Pinto
    • 1
    • 2
  • Liliana Martins
    • 3
  • Fernando Gonçalves
    • 1
    • 2
  • Sara C. Antunes
    • 1
    • 4
  1. 1.Centro de Estudos do Ambiente e do Mar (CESAM)Universidade de AveiroAveiroPortugal
  2. 2.Departamento de Biologia da Universidade de AveiroCampus Universitário de SantiagoAveiroPortugal
  3. 3.Faculdade de Ciências da Saúde da Universidade Fernando Pessoa (FCS-UFP)PortoPortugal
  4. 4.Departamento de Biologia da Faculdade de Ciências da Universidade do PortoPortoPortugal

Personalised recommendations