Inhibitory Effects of Four Neonicotinoid Active Ingredients on Acetylcholine Esterase Activity


There is a great concern about the decline of pollinators, and neonicotinoids emerging bee disorders are assumed to play a significant role. Since changes in learning ability has been observed in honey bees exposed to some acetylcholine esterase (AChE) inhibitors, we therefore, tested in vitro the effect of four neonicotinoids on purified eel AChE. AChE activity was inhibited in a concentration-dependent manner, and calculated IC50 values for thiamethoxam (IC50 = 414 μM) and clothianidin (IC50 = 160 μM) were found to be much higher compared to acetamiprid (IC50 = 75.2 μM) and thiacloprid (IC50 = 87.8 μM). The Lineweaver–Burk reciprocal plots for acetamiprid shows unchanged Vmax and increased Km values with inhibitor concentrations, while analysis of Michaelis–Menten plots shows predominantly competitive mechanism. The inhibition constant value (Ki = 24.3 μM) indicates strong binding of the acetamiprid complex to AChE. Finally, the four tested neonicotinoids are not a uniform group regarding their blocking ability. Our results suggest a previously not established, direct AChE blocking mechanism of neonicotinoids tested, thus the neuronal AChE enzyme is likely among the direct targets of the neonicotinoid insecticides. We conclude, that these AChE inhibitory effects may also contribute to toxic effects on the whole exposed animal.





acetylcholine esterase




acetylthiocholine iodide


Colony Collapse Disorder




5,5′-dithiobis(2-nitrobenzoic acid)


nicotinic acetylcholine receptors






  1. 1.

    Anhalt, J. C., Moorman, T. B., Koskinen, W. C. (2008) Degradation and sorption of imidacloprid in dissimilar surface and subsurface soils. J. Environ. Sci. Health. 43, 207–213.

    CAS  Article  Google Scholar 

  2. 2.

    Badiou, A., Meled, M., Belzunces, L. P. (2008) Honeybee Apis mellifera acetylcholinesterase - a biomarker to detect deltamethrin exposure. Ecotoxicol. Environ. Saf. 69, 246–253.

    CAS  Article  Google Scholar 

  3. 3.

    Blacquiere, T., Smagghe, G., van Gestel, C. A., Mommaerts, V. (2012) Neonicotinoids in bees: a review on concentrations, side-effects and risk assessment. Ecotoxicolog. 21, 973–992.

    CAS  Article  Google Scholar 

  4. 4.

    Boily, M., Sarrasin, B., Deblois, C., Aras, P., Chagnon, M. (2013) Acetylcholinesterase in honey bees (Apis mellifera) exposed to neonicotinoids, atrazine and glyphosate: laboratory and field experiments. Environ. Sci. Pollut. Res. Int. 20, 5603–5614.

    CAS  Article  Google Scholar 

  5. 5.

    Cortes, A., Cascante, M., Cardenas, M. L., Cornish-Bowden, A. (2001) Relationships between inhibition constants, inhibitor concentrations for 50% inhibition and types of inhibition: new ways of analysing data. Biochem. J. 357, 263–268.

    CAS  Article  Google Scholar 

  6. 6.

    Deglise, P., Grunewald, B., Gauthier, M. (2002) The insecticide imidacloprid is a partial agonist of the nicotinic receptor of honeybee Kenyon cells. Neurosci. Lett. 321, 13–16.

    CAS  Article  Google Scholar 

  7. 7.

    DeLorenzo, M. E., Thompson, B., Cooper, E., Moore, J., Fulton, M. H. (2012) A long-term monitoring study of chlorophyll, microbial contaminants, and pesticides in a coastal residential stormwater pond and its adjacent tidal creek. Environ. Monit. Assess. 184, 343–359.

    CAS  Article  Google Scholar 

  8. 8.

    El Hassani, A. K., Dacher, M., Gary, V., Lambin, M., Gauthier, M., Armengaud, C. (2008) Effects of sublethal doses of acetamiprid and thiamethoxam on the behavior of the honeybee (Apis mellifera). Arch. Environ. Contam. Toxicol. 54, 653–661.

    CAS  Article  Google Scholar 

  9. 9.

    Ellman, G. L., Courtney, K. D., Andres, V., Jr., Feather-Stone, R. M. (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95.

    CAS  Article  Google Scholar 

  10. 10.

    Elston, C., Thompson, H. M., Walters, K. F. A. (2013) Sub-lethal effects of thiamethoxam, a neonicotinoid pesticide, and propiconazole, a DMI fungicide, on colony initiation in bumblebee (Bombus terrestris) micro-colonies. Apidologi. 44, 563–574.

    CAS  Article  Google Scholar 

  11. 11.

    Girolami, V., Mazzon, L., Squartini, A., Mori, N., Marzaro, M., Di Bernardo, A., Greatti, M., Giorio, C., Tapparo, A. (2009) Translocation of neonicotinoid insecticides from coated seeds to seedling guttation drops: a novel way of intoxication for bees. J. Econ. Entomol. 102, 1808–1815.

    CAS  Article  Google Scholar 

  12. 12.

    Goulson, D. (2013) REVIEW: An overview of the environmental risks posed by neonicotinoid insecticides. J. Appl. Ecol. 50, 977–987.

    Article  Google Scholar 

  13. 13.

    Iwasa, T., Motoyama, N., Ambrose, J. T., Roe, R. M. (2004) Mechanism for the differential toxicity of neonicotinoid insecticides in the honey bee, Apis mellifera. Crop Protectio. 23, 371–378.

    CAS  Article  Google Scholar 

  14. 14.

    Jeschke, P., Nauen, R. (2008) Neonicotinoids-from zero to hero in insecticide chemistry. Pest Manag. Sci. 64, 1084–1098.

    CAS  Article  Google Scholar 

  15. 15.

    Jeschke, P., Nauen, R., Schindler, M., Elbert, A. (2011) Overview of the status and global strategy for neonicotinoids. J. Agric. Food Chem. 59, 2897–2908.

    CAS  Article  Google Scholar 

  16. 16.

    Krupke, C. H., Hunt, G. J., Eitzer, B. D., Andino, G., Given, K. (2012) Multiple Routes of Pesticide Exposure for Honey Bees Living Near Agricultural Fields. PLoS One 7.

    Google Scholar 

  17. 17.

    Lamers, M., Anyusheva, M., La, N., Nguyen, V. V., Streck, T. (2011) Pesticide Pollution in Surfaceand Groundwater by Paddy Rice Cultivation: A Case Study from Northern Vietnam (vol 39, pg 356, 2011). Clean-Soil Air Wate. 39, 508–5008.

    CAS  Article  Google Scholar 

  18. 18.

    Matsuda, K., Buckingham, S. D., Kleier, D., Rauh, J. J., Grauso, M., Sattelle, D. B. (2001) Neonicotinoids: insecticides acting on insect nicotinic acetylcholine receptors. Trends Pharmacol. Sci. 22, 573–580.

    CAS  Article  Google Scholar 

  19. 19.

    Morakchi, S., Maiza, A., Farine, P., Aribi, N., Soltani, N. (2005) Effects of a neonicotinoid insecticide (acetamiprid) on acetylcholinesterase activity and cuticular hydrocarbons profil in German cockroaches. Commun. Agric. Appl. Biol. Sci. 70, 843–848.

    CAS  PubMed  Google Scholar 

  20. 20.

    Palmer, M. J., Moffat, C., Saranzewa, N., Harvey, J., Wright, G. A., Connolly, C. N. (2013) Cholinergic pesticides cause mushroom body neuronal inactivation in honeybees. Nat. Commun. 4.

    Google Scholar 

  21. 21.

    Pettis, J. S., Lichtenberg, E. M., Andree, M., Stitzinger, J., Rose, R., Vanengelsdorp, D. (2013) Crop Pollination Exposes Honey Bees to Pesticides Which Alters Their Susceptibility to the Gut Pathogen Nosema ceranae. PLoS One 8.

    Google Scholar 

  22. 22.

    Reetz, J. E., Zuhlke, S., Spiteller, M., Wallner, K. (2011) Neonicotinoid insecticides translocated in guttated droplets of seed-treated maize and wheat: a threat to honeybees? Apidologi. 42, 596–606.

    CAS  Article  Google Scholar 

  23. 23.

    Starner, K., Goh, K. S. (2012) Detections of the neonicotinoid insecticide imidacloprid in surface waters of three agricultural regions of California, USA, 2010–2011. Bull. Environ. Contam. Toxicol. 88, 316–321.

    CAS  Article  Google Scholar 

  24. 24.

    Tapparo, A., Giorio, C., Marzaro, M., Marton, D., Solda, L., Girolami, V. (2011) Rapid analysis of neonicotinoid insecticides in guttation drops of corn seedlings obtained from coated seeds. J. Environ. Monit. 13, 1564–1568.

    CAS  Article  Google Scholar 

  25. 25.

    Tomizawa, M., Casida, J. E. (2003) Selective toxicity of neonicotinoids attributable to specificity of insect and mammalian nicotinic receptors. Annu. Rev. Entomol. 48, 339–364.

    CAS  Article  Google Scholar 

  26. 26.

    Tomizawa, M., Casida, J. E. (2005) Neonicotinoid insecticide toxicology: Mechanisms of selective action. Annu. Rev. Pharmacol. Toxicol. 45, 247–267.

    CAS  Article  Google Scholar 

  27. 27.

    van der Sluijs, J. P., Simon-Delso, N., Goulson, D., Maxim, L., Bonmatin, J. M., Belzunces, L. P. (2013) Neonicotinoids, bee disorders and the sustainability of pollinator services. Curr. Opin. Env. Sust. 5, 293–305.

    Article  Google Scholar 

  28. 28.

    van Engelsdorp, D., Evans, J. D., Saegerman, C., Mullin, C., Haubruge, E., Nguyen, B. K., Frazier, M., Frazier, J., Cox-Foster, D., Chen, Y. P., Underwood, R., Tarpy, D. R., Pettis, J. S. (2009) Colony Collapse Disorder: A Descriptive Study. PLoS One 4.

    Google Scholar 

  29. 29.

    Williamson, S. M., Wright, G. A. (2013) Exposure to multiple cholinergic pesticides impairs olfactory learning and memory in honeybees. J. Exp. Biol. 216, 1799–1807.

    CAS  Article  Google Scholar 

  30. 30.

    Wu, J. Y., Smart, M. D., Anelli, C. M., Sheppard, W. S. (2012) Honey bees (Apis mellifera) reared in brood combs containing high levels of pesticide residues exhibit increased susceptibility to Nosema (Microsporidia) infection. J. Invert. Pathol. 109, 326–329.

    CAS  Article  Google Scholar 

Download references

Author information



Corresponding author

Correspondence to Ágnes Vehovszky.

Rights and permissions

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Győri, J., Farkas, A., Stolyar, O. et al. Inhibitory Effects of Four Neonicotinoid Active Ingredients on Acetylcholine Esterase Activity. BIOLOGIA FUTURA 68, 345–357 (2017).

Download citation


  • Neonicotinoids
  • acetamiprid
  • clothianidin
  • thiamethoxam
  • in vitro acetylcholinesterase assay