Low doses of imidacloprid induce neurotoxic effects in adult marsh frogs: GFAP, NfL, and angiostatin as biomarkers

  • S. V. Yermolenko Oles Honchar Dnipro National University
  • V. S. Nedzvetsky Dnipro State Agrarian and Economic University
  • V. Y. Gasso Oles Honchar Dnipro National University
  • V. A. Spirina Dalhousie University
  • V. B. Petrushevskyi Oles Honchar Dnipro National University
  • V. V. Kyrychenko Oles Honchar Dnipro National University
Keywords: neonicotinoids; insecticides; neurotoxicity; amphibians; brain proteins


Imidacloprid is one of the most widely used insecticides in the world. The neurotoxicity of imidacloprid in adult amphibians has not been studied thoroughly. We investigated the expression of glial fibrillary acidic protein (GFAP), neurofilament light chain (NfL) and angiostatin in the amphibian brain to identify valid biomarkers of low dose imidacloprid exposure. For the experiment, 30 individuals of the marsh frog Pelophylax ridibundus were selected. The amphibians were divided into five groups. The duration of the experiment was 7 and 21 days. The exposure concentrations were 10 and 100 µg/L. The results of the study revealed a decrease in the expression of GFAP after 7 days in the exposure groups of 10 and 100 μg/L. An increase in the level of NfL was observed in the group exposed to 10 μg/L after 21 days of the experiment. The angiostatin level was increased after 7 days at 10 µg/L and after 21 days at 100 µg/L. The data obtained indicate that low concentrations of imidacloprid can cause neurotoxic effects in the brain of P. ridibundus. Such effects can have a significant impact on amphibian populations. According to the results of the study of the expression level of GFAP, NfL and angiostatin, it can be stated that imidacloprid has a neurotoxic effect on adult marsh frogs. The studied indicators can be promising biomarkers of environmental pollution by neonicotinoids.


Abou-Donia, M. B., Goldstein, L. B., Bullman, S., Tu, T., Khan, W. A., Dechkovskaia, A. M., & Abdel-Rahman, A. A. (2008). Imidacloprid induces neurobehavioral deficits and increases expression of glial fibrillary acidic protein in the motor cortex and hippocampus in offspring rats following in utero exposure. Journal of Toxicology and Environmental Health, Part A, 71(2), 119–130.

Anadón, A., Ares, I., Martínez, M., Martínez-Larrañaga, M.-R., & Martínez, M.-A. (2019). Neurotoxicity of neonicotinoids. Advances in Neurotoxicology, 4, 167–207.

Bonmatin, J. M., Giorio, C., Girolami, V., Goulson, D., Kreutzweiser, D. P., Krupke, C., Liess, M., Long, E., Marzaro, M., Mitchell, E. A., Noome, D. A., Simon-Delso, N., & Tapparo, A. (2015). Environmental fate and exposure: Neonicotinoids and fipronil. Environmental Science and Pollution Research International, 22(1), 35–67.

Campbell, K. S., Keller, P. G., Heinzel, L. M., Golovko, S. A., Seeger, D. R., Golovko, M. Y., & Kerby, J. L. (2022). Detection of imidacloprid and metabolites in northern leopard frog (Rana pipiens) brains. The Science of the Total Environment, 813, 152424.

Crist, E., Mora, C., & Engelman, R. (2017). The interaction of human population, food production, and biodiversity protection. Science, 356(6335), 260–264.

De Arcaute, C. R., Pérez-Iglesias, J. M., Nikoloff, N., Natale, G. S., Soloneski, S., & Larramendy, M. L. (2014). Genotoxicity evaluation of the insecticide imidacloprid on circulating blood cells of Montevideo tree frog Hypsiboas pulchellus tadpoles (Anura, Hylidae) by comet and micronucleus bioassays. Ecological Indicators, 45, 632–639.

Eliasson, C., Sahlgren, C., Berthold, C. H., Stakeberg, J., Celis, J. E., Betsholtz, C., Eriksson, J. E., & Pekny, M. (1999). Intermediate filament protein partnership in astrocytes. The Journal of Biological Chemistry, 274(34), 23996–24006.

Eser, N., Cicek, M., Yoldas, A., Demir, M., & Deresoy, F. A. (2022). Caffeic acid phenethyl ester ameliorates imidacloprid-induced acute toxicity in the rat cerebral cortex. Environmental Toxicology and Pharmacology, 96, 103980.

Flaskos, J. (2014). The neuronal cytoskeleton as a potential target in the developmental neurotoxicity of organophosphorothionate insecticides. Basic and Clinical Pharmacology and Toxicology, 115(2), 201–208.

Gaetani, L., Blennow, K., Calabresi, P., Di Filippo, M., Parnetti, L., & Zetterberg, H. (2019). Neurofilament light chain as a biomarker in neurological disorders. Journal of Neurology, Neurosurgery and Psychiatry, 90(8), 870–881.

Gasso, V. Y., Hahut, A. N., Yermolenko, S. V., Hasso, I. A., Agca, C. A., Nedzvetsky, V. S., & Sukharenko, E. V. (2020). Local industrial pollution induces astrocyte cytoskeleton rearrangement in the dice snake brain: GFAP as a biomarker. Biosystems Diversity, 28(3), 250–256.

Gasso, V., Nedzvetsky, V., Novitskyi, R., & Yermolenko, S. (2021). Λ-cyhalothrin causes oxidative stress accompanied by reduced glutathione alteration and modulation of regulatory protein p53 expression in the fish brain. Ecology and Noospherology, 32(2), 71–76 (in Ukranian).

Gibbons, D., Morrissey, C., & Mineau, P. (2015). A review of the direct and indirect effects of neonicotinoids and fipronil on vertebrate wildlife. Environmental Science and Pollution Research, 22(1), 103–118.

Guzyk, M. M., Tykhomyrov, A. A., Nedzvetsky, V. S., Prischepa, I. V., Grinenko, T. V., Yanitska, L. V., & Kuchmerovska, T. M. (2016). Poly (ADP-ribose) polymerase-1 (PARP-1) inhibitors reduce reactive gliosis and improve angiostatin levels in retina of diabetic rats. Neurochemical Research, 41(10), 2526–2537.

Hall, Z. J., & Tropepe, V. (2020). Using teleost fish to discern developmental signatures of evolutionary adaptation from phenotypic plasticity in brain structure. Frontiers in Neuroanatomy, 14, 10.

Harmanşa, Y. K., & Erbaş, O. (2022). Diagnostic and therapeutic biomarkers for neurodegeneration. Journal of Experimental and Basic Medical Sciences, 3(1), 47–53.

Hnasko, T. S., & Hnasko, R. M. (2015). The western blot. Methods in Molecular Biology, 1318, 87–96.

Huslystyi, A., Nedzvetsky, V., Yermolenko, S., Gasso, V., Petrushevskyi, V., & Sukharenko, E. (2021). Low doses of imidacloprid induce oxidative stress and neural cell disruption in earthworm Eisenia fetida. International Letters of Natural Sciences, 84, 1–11.

Kozak, V. M., Brygadyrenko, V. V., & Romanenko, E. R. (2020). Influence of herbicides, insecticides and fungicides on food consumption and body weight of Rossiulus kessleri (Diplopoda, Julidae). Biosystems Diversity, 28(3), 272–280.

Liang, Y. Z., Zeng, Z. L., Hua, L. L., Li, J. F., Wang, Y. L., & Bi, X. Z. (2016). Expression and significance of angiostatin, vascular endothelial growth factor and matrix metalloproteinase-9 in brain tissue of diabetic rats with ischemia reperfusion. Asian Pacific Journal of Tropical Medicine, 9(6), 587–591.

Liem, R. K. (1993). Molecular biology of neuronal intermediate filaments. Current Opinion in Cell Biology, 5(1), 12–16.

Liu, C. J., Men, W. J., Liu, Y. J., & Zhang, H. (2002). The pollution of pesticides in soils and its bioremediation. System Sciences and Comprehensive Studies in Agriculture, 18(4), 295–297.

Loser, D., Grillberger, K., Hinojosa, M. G., Blum, J., Haufe, Y., Danker, T., Johansson, Y., Möller, C., Nicke, A., Bennekou, S. H., Gardner, I., Bauch, C., Walker, P., Forsby, A., Ecker, G. F., Kraushaar, U., & Leist, M. (2021). Acute effects of the imidacloprid metabolite desnitro-imidacloprid on human nACh receptors relevant for neuronal signaling. Archives of Toxicology, 95(12), 3695–3716.

Lu, C. H., Macdonald-Wallis, C., Gray, E., Pearce, N., Petzold, A., Norgren, N., Giovannoni, G., Fratta, P., Sidle, K., Fish, M., Orrell, R., Howard, R., Talbot, K., Greensmith, L., Kuhle, J., Turner, M. R., & Malaspina, A. (2015). Neurofilament light chain: A prognostic biomarker in amyotrophic lateral sclerosis. Neurology, 84(22), 2247–2257.

Malkiewicz, K., Koteras, M., Folkesson, R., Brzezinski, J., Winblad, B., Szutowski, M., & Benedikz, E. (2006). Cypermethrin alters glial fibrillary acidic protein levels in the rat brain. Environmental Toxicology and Pharmacology, 21(1), 51–55.

Manzo, L., Castoldi, A. F., Coccini, T., & Prockop, L. D. (2001). Assessing effects of neurotoxic pollutants by biochemical markers. Environmental research, 85(1), 31–36.

Martynov, V. O., Titov, O. G., Kolombar, T. M., & Brygadyrenko, V. V. (2019). Influence of essential oils of plants on the migration activity of Tribolium confusum (Coleoptera, Tenebrionidae). Biosystems Diversity, 27(2), 177–185.

Meter, R. J., Glinski, D. A., Henderson, W. M., & Purucker, S. T. (2016). Soil organic matter content effects on dermal pesticide bioconcentration in American toads (Bufo americanus). Environmental Toxicology and Chemistry, 11(35), 2734–2741.

Müller, Y. M., Kobus, K., Schatz, J. C., Ammar, D., & Nazari, E. M. (2012). Prenatal lead acetate exposure induces apoptosis and changes GFAP expression during spinal cord development. Ecotoxicology and Environmental Safety, 75(1), 223–229.

Nedzvetsky, V., Gasso, V., Novitskiy, R., & Yermolenko, S. (2020). Influence of the insecticide λ-cyhalothrin on oxidative stress and expression of replicative protein A in the brain of fish. Agrology, 3(4), 214–218 (in Ukranian).

Nkontcheu, D. B. K., Tchamadeu, N. N., Ngealekeleoh, F., & Nchase, S. (2017). Ecotoxicological effects of imidacloprid and lambda-cyhalothrin (insecticide) on tadpoles of the African common toad, Amietophrynus regularis (Reuss, 1833) (Amphibia: Bufonidae). Emerging Science Journal, 1(2), 49–53.

Pandey, A., Jauhari, A., Singh, T., Singh, P., Singh, N., Srivastava, A. K., Khan, F., Pant, A. B., Parmar, D., & Yadav, S. (2015). Transactivation of P53 by cypermethrin induced miR-200 and apoptosis in neuronal cells. Toxicology Research, 4(6), 1578–1586.

Pérez-Iglesias, J. M., de Arcaute, C. R., Nikoloff, N., Dury, L., Soloneski, S., Natale, G. S., & Larramendy, M. L. (2014). The genotoxic effects of the imidacloprid-based insecticide formulation glacoxan imida on Montevideo tree frog Hypsiboas pulchellus tadpoles (Anura, Hylidae). Ecotoxicology and Environmental Safety, 104, 120–126.

Popp, J., Pető, K., & Nagy, J. (2013). Pesticide productivity and food security. A review. Agronomy for Sustainable Development, 33(1), 243–255.

Saunders, D. E., DiCerbo, J. A., Williams, J. R., & Hannigan, J. H. (1997). Alcohol reduces neurofilament protein levels in primary cultured hippocampal neurons. Alcohol, 14(5), 519–526.

SERA (2005). Imidacloprid–human health and ecological risk assessment–final report. Report from Syracuse Environmental Research Associates to USDA, Forest Service.

Shiyntum, H. N., Dovban, O. O., Kovalchuk, Y. P., Yaroshenko, T. Y., & Ushakova, G. A. (2017). Corvitin restores metallothionein and glial fibrillary acidic protein levels in rat brain affected by pituitrin-izadrin. Ukrainian Biochemical Journal, 89(3), 36–45.

Sidiropoulou, E., Sachana, M., Flaskos, J., Harris, W., Hargreaves, A. J., & Woldehiwet, Z. (2009). Diazinon oxon interferes with differentiation of rat C6 glioma cells. Toxicology in Vitro, 23(8), 1548–1552.

Sindi, R. A., Harris, W., Arnott, G., Flaskos, J., Mills, C. L., & Hargreaves, A. J. (2016). Chlorpyrifos-and chlorpyrifos oxon-induced neurite retraction in pre-differentiated N2a cells is associated with transient hyperphosphorylation of neurofilament heavy chain and ERK 1/2. Toxicology and Applied Pharmacology, 308, 20–31.

Sweeney, M. R., Thompson, C. M., & Popescu, V. D. (2021). Sublethal, behavioral, and developmental effects of the neonicotinoid pesticide imidacloprid on larval wood frogs (Rana sylvatica). Environmental Toxicology and Chemistry, 40(7), 1838–1847.

Tiwari, M. (2012). Apoptosis, angiogenesis and cancer therapies. Journal of Cancer Therapeutics and Research, 1(1), 3.

Topal, A., Alak, G., Ozkaraca, M., Yeltekin, A. C., Comaklı, S., Acıl, G., Kokturk, M., & Atamanalp, M. (2017). Neurotoxic responses in brain tissues of rainbow trout exposed to imidacloprid pesticide: Assessment of 8-hydroxy-2-deoxyguanosine activity, oxidative stress and acetylcholinesterase activity. Chemosphere, 175, 186–191.

Tykhomyrov, А. A., Pavlova, A. S., & Nedzvetsky, V. S. (2016). Glial fibrillary acidic protein (GFAP): On the 45th anniversary of its discovery. Neurophysiology, 48(1), 54–71.

Udeigwe, T. K., Teboh, J. M., Eze, P. N., Stietiya, M. H., Kumar, V., Hendrix, J., Mascagni Jr., H. J., Ying, T., & Kandakji, T. (2015). Implications of leading crop production practices on environmental quality and human health. Journal of Environmental Management, 151, 267–279.

Valenzuela, R., Costa-Besada, M. A., Iglesias-Gonzalez, J., Perez-Costas, E., Villar-Cheda, B., Garrido-Gil, P., Melendez-Ferro, M., Soto-Otero, R., Lanciego, J. L., Henrion, D., Franco, R., & Labandeira-Garcia, J. L. (2016). Mitochondrial angiotensin receptors in dopaminergic neurons. Role in cell protection and aging-related vulnerability to neurodegeneration. Cell Death and Disease, 7(10), e2427.

Van Meter, R. J., Glinski, D. A., Hong, T., Cyterski, M., Henderson, W. M., & Purucker, S. T. (2014). Estimating terrestrial amphibian pesticide body burden through dermal exposure. Environmental Pollution, 193, 262–268.

Yesipova, N., Marenkov, O., Sharamok, T., Nesterenko, O., & Kurchenko, V. (2022). Development of the regulation of hydrobiological monitoring in circulation cooling system of the Zaporizhzhia Nuclear Power Plant. Eastern-European Journal of Enterprise Technologies, 116, 6–17.

Zhang, W. (2018). Global pesticide use: Profile, trend, cost/benefit and more. Proceedings of the International Academy of Ecology and Environmental Sciences, 8(1), 1–27.

Zikankuba, V. L., Mwanyika, G., Ntwenya, J. E., & James, A. (2019). Pesticide regulations and their malpractice implications on food and environment safety. Cogent Food and Agriculture, 5(1), 1601544.

How to Cite
Yermolenko, S. V., Nedzvetsky, V. S., Gasso, V. Y., Spirina, V. A., Petrushevskyi, V. B., & Kyrychenko, V. V. (2022). Low doses of imidacloprid induce neurotoxic effects in adult marsh frogs: GFAP, NfL, and angiostatin as biomarkers . Regulatory Mechanisms in Biosystems, 13(4), 426-430. https://doi.org/10.15421/022256