, Volume 23, Issue 3, pp 449–458 | Cite as

Cholinesterase activity on Echinogammarus meridionalis (Pinkster) and Atyaephyra desmarestii (Millet): characterisation and in vivo effects of copper and zinc

  • C. Quintaneiro
  • M. Monteiro
  • A. M. V. M. Soares
  • J. Ranville
  • A. J. A. Nogueira


Metals are released into freshwater ecosystems from natural and anthropogenic sources, compromising their structural and functional equilibrium. As early warning tools, cholinesterases (ChEs) are usually used to assess the effects of organophosphate and carbamate pesticides, but are also known to be inhibited by metals. The objectives of this work were to characterise the activity of ChE present in the amphipod Echinogammarus meridionalis and the shrimp Atyaephyra desmarestii and to evaluate the in vivo effects of the metals copper and zinc in their ChE activity. To achieve this, firstly the activity of ChE forms were characterised using different in vitro assays with substrates and selective inhibitors. Then, the in vivo effects of 48 h exposures to increasing concentrations of copper and zinc on ChE activity were determined. The ChE form present in both species was acetylcholinesterase (AChE) since both revealed preference for the acetylthiocholine iodide substrate, total inhibition with eserine, the inhibitor of ChEs, and with 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide, the specific inhibitor of AChE, and presented insensitivity to iso-OMPA, a specific inhibitor of butyrylcholinesterase. The activity of ChEs was inhibited by zinc exposures in the amphipod species, but was not affected by copper. Exposure to copper and zinc did not affect ChEs activity in the shrimp at the concentrations tested. This work is a relevant contribution as foundation for the use of AChE in freshwater crustaceans in further studies including biomonitoring campaigns in different contamination scenarios.


Acetylcholinesterase Amphipod Crustacean Essential metals Neurotoxicity Shrimp 



The work of C. Quintaneiro was funded by FCT-Portuguese Foundation for Science and Technology through a doctoral and pos-doctoral fellowships (SFRH/BD/28705/2006 and SFRH/BPD/89951/2012) and M. Monteiro by a pos-doctoral fellowship (SFRH/BPD/45911/2008). We are very extremely grateful to Sr. Eduardo from Quinta do Alcaide who provided the access to Lena River sampling site.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Arufe MI, Arellano JM, Garcia L, Albendin G, Sarasquete C (2007) Cholinesterase activity in gilthead seabream (Sparus aurata) larvae: characterization and sensitivity to the organophosphate azinphosmethyl. Aquat Toxicol 84(3):328–336CrossRefGoogle Scholar
  2. Bonacci S, Corsi I, Focardi S (2009) Cholinesterases in the Antarctic scallop Adamussium colbecki: characterization and sensitivity to pollutants. Ecotoxicol Environ Saf 72(5):1481–1488CrossRefGoogle Scholar
  3. 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
  4. Brown RJ, Galloway TS, Lowe D, Browne MA, Dissanayake A, Jones MB, Depledge MH (2004) Differential sensitivity of three marine invertebrates to copper assessed using multiple biomarkers. Aquat Toxicol 66(3):267–278CrossRefGoogle Scholar
  5. Chuiko GM (2000) Comparative study of acetylcholinesterase and butyrylcholinesterase in brain and serum of several freshwater fish: specific activities and in vitro inhibition by DDVP, an organophosphorus pesticide. Comp Biochem Physiol C 127(3):233–242Google Scholar
  6. Cunha I, Garcia LM, Guilhermino L (2005) Sea-urchin (Paracentrotus lividus) glutathione S-transferases and cholinesterase activities as biomarkers of environmental contamination. J Environ Monit 7(4):288–294CrossRefGoogle Scholar
  7. Cunha I, Mangas-Ramirez E, Guilhermino L (2007) Effects of copper and cadmium on cholinesterase and glutathione S-transferase activities of two marine gastropods (Monodonta lineata and Nucella lapillus). Comp Biochem Physiol Toxicol Pharmacol 145(4):648–657CrossRefGoogle Scholar
  8. Diamantino TC, Almeida E, Soares AM, Guilhermino L (2003) Characterization of cholinesterases from Daphnia magna Straus and their inhibition by zinc. Bull Environ Contam Toxicol 71(2):219–225CrossRefGoogle Scholar
  9. Ellman GL, Courtney KD, Andres V, Featherstone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7(2):88–95CrossRefGoogle Scholar
  10. Elumalai M, Antunes C, Guilhermino L (2002) Effects of single metals and their mixtures on selected enzymes of Carcinus maenas. Water Air Soil Pollut 141(1–4):273–280CrossRefGoogle Scholar
  11. Eto M (1974) Organophosphorus pesticides; organic and biological chemistry. CRC Press, OhioGoogle Scholar
  12. Ferreira NGC, Rosário F, Domingues I, Calhôa CF, Soares AMVM, Loureiro S (2010) Acetylcholinesterase characterization in the terrestrial isopod Porcellionides pruinosus. In: Hamamura N, Suzuki S, Mendo S, Barroso CM, Iwata H, Tanabe S (eds) Interdisciplinary Studies on Environmental Chemistry—Biological Responses to Contaminants. TERRAPUB, pp. 227–236Google Scholar
  13. Forget J, Bocquene G (1999) Partial purification and enzymatic characterization of acetylcholinesterase from the intertidal marine copepod Tigriopus brevicornis. Comp Biochem Physiol Part B 123:345–350CrossRefGoogle Scholar
  14. Frasco MF, Fournier D, Carvalho F, Guilhermino L (2005) Do metals inhibit acetylcholinesterase (AChE)? Implementation of assay conditions for the use of AChE activity as a biomarker of metal toxicity. Biomarkers 10(5):360–375CrossRefGoogle Scholar
  15. Frasco MF, Fournier D, Carvalho F, Guilhermino L (2006) Cholinesterase from the common prawn (Palaemon serratus) eyes: catalytic properties and sensitivity to organophosphate and carbamate compounds. Aquat Toxicol 77(4):412–421CrossRefGoogle Scholar
  16. Frasco MF, Colletier JP, Weik M, Carvalho F, Guilhermino L, Stojan J, Fournier D (2007) Mechanisms of cholinesterase inhibition by inorganic mercury. Fed Eur Biochem Soc J 274(7):1849–1861Google Scholar
  17. Fulton MH, Key PB (2001) Acetylcholinesterase inhibition in estuarine fish and invertebrates as an indicator of organophosphorus insecticide exposure and effects. Environ Toxicol Chem 20(1):37–45CrossRefGoogle Scholar
  18. Gagnaire B, Geffard O, Xuereb B, Margoum C, Garric J (2008) Cholinesterase activities as potential biomarkers: characterization in two freshwater snails, Potamopyrgus antipodarum (Mollusca, Hydrobiidae, Smith 1889) and Valvata piscinalis (Mollusca, Valvatidae, Müller 1774). Chemosphere 71(3):553–560CrossRefGoogle Scholar
  19. Garcia LM, Castro B, Ribeiro R, Guilhermino L (2000) Characterization of cholinesterase from guppy (Poecilia reticulata) muscle and its in vitro inhibition by environmental contaminants. Biomarkers 5(4):274–284CrossRefGoogle Scholar
  20. Garric J, Gagnaire B, Geffard O, Xuereb B, Margoum C (2008) Cholinesterase activities as potential biomarkers: characterization in two freshwater snails, Potamopyrgus antipodarum (Mollusca, Hydrobiidae, Smith 1889) and Valvata piscinalis (Mollusca, Valvatidae, Muller 1774). Chemosphere 71(3):553–560CrossRefGoogle Scholar
  21. Guilhermino L, Lopes MC, Carvalho AP, Soares AMVM (1996) Inhibition of acetylcholinesterase activity as effect criterion in acute tests with juvenile Daphnia magna. Chemosphere 32(4):727–738CrossRefGoogle Scholar
  22. Guilhermino L, Elumalai M, Antunes C (2007) Enzymatic biomarkers in the crab Carcinus maenas from the Minho River estuary (NW Portugal) exposed to zinc and mercury. Chemosphere 66(7):1249–1255CrossRefGoogle Scholar
  23. Hamza-Chaffai A, Romeo M, Gnassia-Barelli M, El Abed A (1998) Effect of copper and lindane on some biomarkers measured in the clam Ruditapes decussatus. Bull Environ Contam Toxicol 61(3):397–404CrossRefGoogle Scholar
  24. Ibrahim H, Kheir R, Helmi S, Lewis J, Crane M (1998) Effects of organophosphorus, carbamate, pyrethroid and organochlorine pesticides, and a heavy metal on survival and cholinesterase activity of Chironomus riparius Meigen. Bull Environ Contam Toxicol 60(3):448–455CrossRefGoogle Scholar
  25. Key PB, Fulton MH (2002) Characterization of cholinesterase activity in tissues of the grass shrimp (Palaemonetes pugio). Pestic Biochem Physiol 72(3):186–192CrossRefGoogle Scholar
  26. Key PB, Fulton MH, Harman-Fetcho JA, McConnell LL (2003) Acetylcholinesterase activity in grass shrimp and aqueous pesticide levels from South Florida drainage canals. Arch Environ Contam Toxicol 45(3):371–377Google Scholar
  27. Lehtonen KK, Leinio S (2003) Effects of exposure to copper and malathion on metallothionein levels and acetylcholinesterase activity of the mussel Mytilus edulis and the clam Macoma balthica from the northern Baltic sea. Bull Environ Contam Toxicol 71(3):489–496CrossRefGoogle Scholar
  28. Lionetto MG, Caricato R, Giordano ME, Pascariello MF, Marinosci L, Schettino T (2003) Integrated use of biomarkers (acetylcholinesterase and antioxidant enzymes activities) in Mytilus galloprovincialis and Mullus barbatus in an Italian coastal marine area. Mar Pollut Bull 46(3):324–330CrossRefGoogle Scholar
  29. Locatello L, Matozzo V, Marin MG (2009) Biomarker responses in the crab Carcinus aestuarii to assess environmental pollution in the Lagoon of Venice (Italy). Ecotoxicology 18(7):869–877CrossRefGoogle Scholar
  30. Macedo-Sousa JA, Pestana JLT, Gerhardt A, Nogueira AJA, Soares AMVM (2007) Behavioural and feeding responses of Echinogammarus meridionalis (Crustacea, Amphipoda) to acid mine drainage. Chemosphere 67(8):1663–1670CrossRefGoogle Scholar
  31. Macedo-Sousa JA, Gerhardt A, Brett CMA, Nogueira AJA, Soares AMVM (2008) Behavioural responses of indigenous benthic invertebrates (Echinogammarus meridionalis, Hydropsyche pellucidula and Choroterpes picteti) to a pulse of acid mine drainage: a laboratorial study. Environ Pollut 156(3):966–973CrossRefGoogle Scholar
  32. Mack A, Robitzki A (2000) The key role of butyrylcholinesterase during neurogenesis and neural disorders: an antisense-5’ butyrylcholinesterase-DNA study. Prog Neurobiol 60(6):607–628CrossRefGoogle Scholar
  33. Masson P, Lockridge O (2010) Butyrylcholinesterase for protection from organophosphorus poisons: catalytic complexities and hysteretic behavior. Arch Biochem Biophys 494(2):107–120CrossRefGoogle Scholar
  34. Massoulie J (2002) The origin of the molecular diversity and functional anchoring of cholinesterases. Neurosignals 11(3):130–143CrossRefGoogle Scholar
  35. McLoughlin N, Yin DQ, Maltby L, Wood RM, Yu HX (2000) Evaluation of sensitivity and specificity of two crustacean biochemical biomarkers. Environ Toxicol Chem 19(8):2085–2092CrossRefGoogle Scholar
  36. Minic J, Chatonnet A, Krejci E, Molgó J (2003) Butyrylcholinesterase and acetylcholinesterase activity and quantal transmitter release at normal and acetylcholinesterase knockout mouse neuromuscular junctions. Br J Pharmacol 138(1):177–187CrossRefGoogle Scholar
  37. Monserrat JM, Bianchini A (1998) Some kinetic and toxicological characteristics of thoracic ganglia cholinesterase of Chasmagnathus granulata (Decapoda, Grapsidae). Comp Biochem Physiol C 120(2):193–199Google Scholar
  38. Monserrat JM, Bianchini A (2001) Anticholinesterase effect of eserine (physostigmine) in fish and crustacean species. Braz Arch Biol Technol 44:63–68CrossRefGoogle Scholar
  39. Monteiro M, Quintaneiro C, Morgado F, Soares AMVM, Guilhermino L (2005) Characterization of the cholinesterases present in head tissues of the estuarine fish Pomatoschistus microps: application to biomonitoring. Ecotoxicol Environ Saf 62(3):341–347CrossRefGoogle Scholar
  40. OECD (1992) Test no. 203: fish acute toxicity test. OECD, ParisCrossRefGoogle Scholar
  41. Payne JF, Mathieu A, Melvin W, Fancey LL (1996) Acetylcholinesterase, an old biomarker with a new future? Field trials in association with two urban rivers and a paper mill in Newfoundland. Mar Pollut Bull 32(2):225–231CrossRefGoogle Scholar
  42. Peakal DB (ed) (1992) Animal biomarkers as pollution indicators. Chapman & Hall, LondonGoogle Scholar
  43. Pestana JLT, Re A, Nogueira AJA, Soares AMVM (2007) Effects of cadmium and zinc on the feeding behaviour of two freshwater crustaceans: Atyaephyra desmarestii (Decapoda) and Echinogammarus meridionalis (Amphipoda). Chemosphere 68(8):1556–1562CrossRefGoogle Scholar
  44. Quintaneiro C, Monteiro M, Pastorinho R, Soares AMVM, Nogueira AJA, Morgado F, Guilhermino L (2006) Environmental pollution and natural populations: a biomarkers case study from the Iberian Atlantic coast. Mar Pollut Bull 52(11):1406–1413CrossRefGoogle Scholar
  45. Quintaneiro C, Ranville J, Nogueira AJA (2013a) Effects of essential metals in two freshwater detritivores species: biochemical approach. Ecotoxicology (submitted)Google Scholar
  46. Quintaneiro C, Ranville J, Nogueira AJA (2013b) Physiological effects of essential metals on two detritivores: Atyaephyra desmarestii (Millet) and Echinogammarus meridionalis (Pinkster). Arch Environ Contam Toxicol (submitted)Google Scholar
  47. Rodrigues SR, Caldeira C, Castro BB, Gonçalves F, Nunes B, Antunes SC (2011) Cholinesterase (ChE) inhibition in pumpkinseed (Lepomis gibbosus) as environmental biomarker: ChE characterization and potential neurotoxic effects of xenobiotics. Pestic Biochem Phys 99(2):181–188CrossRefGoogle Scholar
  48. Romani R, Antognelli C, Baldracchini F, De Santis A, Isani G, Giovannini E, Rosi G (2003) Increased acetylcholinesterase activities in specimens of Sparus auratus exposed to sublethal copper concentrations. Chem Biol Interact 145(3):321–329CrossRefGoogle Scholar
  49. Sanchez-Hernandez JC (2006) Ecotoxicological perspectives of B-esterases in the assessment of pesticide contamination. In: Plattenberg RH (ed) Environmental pollution: new research. Nova Science, New York, pp 1–45Google Scholar
  50. Suresh A, Sivaramakrishna B, Victoriamma PC, Radhakrishnaiah K (1992) Comparative study on the inhibition of acetylcholinesterase activity in the freshwater fish Cyprinus carpio by mercury and zinc. Biochem Int 26(2):367–375Google Scholar
  51. Tu HT, Silvestre F, Scippo ML, Thome JP, 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
  52. Varo I, Navarro JC, Amat F, Guilhermino L (2002) Characterisation of cholinesterases and evaluation of the inhibitory potential of chlorpyrifos and dichlorvos to Artemia salina and Artemia parthenogenetica. Chemosphere 48(6):563–569CrossRefGoogle Scholar
  53. Vieira LR, Gravato C, Soares AM, Morgado F, Guilhermino L (2009) Acute effects of copper and mercury on the estuarine fish Pomatoschistus microps: linking biomarkers to behaviour. Chemosphere 76(10):1416–1427CrossRefGoogle Scholar
  54. Wilkinson GN (1961) Statistical estimations in enzyme kinetics. Biochem J 80:324–332Google Scholar
  55. Xuereb B, Noury P, Felten V, Garric J, Geffard O (2007) Cholinesterase activity in Gammarus pulex (Crustacea Amphipoda): characterization and effects of chlorpyrifos. Toxicology 236(3):178–189CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • C. Quintaneiro
    • 1
  • M. Monteiro
    • 1
  • A. M. V. M. Soares
    • 1
  • J. Ranville
    • 2
  • A. J. A. Nogueira
    • 1
  1. 1.CESAM and Departamento de BiologiaUniversidade de AveiroAveiroPortugal
  2. 2.Department of Chemistry and GeochemistryColorado School of MinesGoldenUSA

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