Assessment of the genotoxic effect of thiamethoxam in Cyprinus carpio by the micronucleus and Comet assays

Assessment of the genotoxic effect of thiamethoxam in Cyprinus carpio by the micronucleus and Comet assays

Ayşegül Ergenler, Funda Turan

ORCID IDs: A.E. 0000-0001-9186-3909; F.T. 0000-0002-0257-6009

Faculty of Marine Science and Technology, Iskenderun Technical University, 31200, Iskenderun, Hatay, TÜRKİYE


Pesticides are compounds formed by natural or various chemical processes that kill harmful organisms, remove pests from their environment, reduce population density or completely eliminate them. Monitoring for toxic effects and screening for different insecticides is vital and crucial for reducing adverse effects on aquatic organisms and public health. Therefore, in this study, we aimed to determine genotoxic effect of thiamethoxamine in a model fish species, Cyprinus carpio, using the micronucleus test and Comet assay. Common carp (average weight of 1.56±0.31g) were exposed to control and three different concentrations of thiamethoxamine (1.0, 1.5 and 2.0 mg L-1) based on previously detected aquatic environmental concentrations for ten days. At the end of study, the Damage frequency (%), Arbitrary unit and Genetic damage index (%) were evaluated in gill and liver cells of carp by Comet assay. Micronucleus frequencies and erythrocyte abnormalities were also determined in erythrocytes cells of carp by micronucleus test. The highest micronucleus frequency and erythrocyte abnormalities were significantly observed in 2.0 mg L-1 group (p<0.001) and, the highest damage frequencies (%) as 74.000±1.732 and 68.000±1.732 were significantly observed in 2.0 mg L-1 group in gill and liver cells, respectively (p<0.001). Our results revealed significant increases in the frequencies of micronuclei and DNA strand breaks in C. carpio, following exposure to thiamethoxamine, thus demonstrated the genotoxic potential of this pesticide on fish.

Keywords: DNA damage, thiamethoxam, micronucleus test, Comet assay, pesticide

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Albaseer, S.S. (2019) Factors controlling the fate of pyrethroids residues during post-harvest processing of raw agricultural crops: An overview. Food Chemistry 295: 58-63.

Ayllon, F., Garcia-Vazquez, E. (2000) Induction of micronuclei and other nuclear abnormalities in European minnow Phoxinus phoxinus and mollie Poecilia latipinna: an assessment of the fish micronucleus test. Mutation Research 467: 177-186.

Barganska, Z., Slebioda, M., Namiesnik, J. (2013) Pesticide residues levels in honey from apiaries located of Northern Poland. Food Control 31: 196-201.

Bogoni, J.A., Armiliato, N., Araldi-Favassa, C.T., Techio, V.H. (2014) Genotoxicity in Astyanax bimaculatus (Twospot Astyanax) exposed to the waters of Engano River (Brazil) as determined by micronucleus tests in erythrocytes. Archives of Environmental Contamination Toxicology 66(3): 441-449.

Bonmatin, J.M., Noome, D.A., Moreno, H., Mitchell, E.A., Glauser, G., Soumana, O.S., Sánchez-Bayo, F. (2019) A survey and risk assessment of neonicotinoids in water, soil and sediments of Belize. Environmental Pollution 249: 949-958.

Brenerman, B.M., Illuzzi, J.L., Wilson III, D.M. (2014) Base excision repair capacity in informing healthspan. Carcinogenesis 35(12): 2643-2652.

Carrasco, K.R., Tilbury, K.L., Myers, M.S. (1990) Assessment of the Piscine Micronucleus Test as an in situ biological indicator of chemical contaminant effects. Canadian Journal of Fisheries and Aquatic Sciences 47(11): 2123-2136.

Cavalcante, D.G.S. M., Martinez, C.B.R., Sofia, S.H. (2008) Genotoxic effects of Roundup on the fish Prochilodus lineatus. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 655(1-2): 41-46.

Cavallaro, M.C., Morrissey, C.A., Headley, J.V., Peru, K.M., Liber, K. (2017) Comparative chronic toxicity of imidacloprid, clothianidin, and thiamethoxam to Chironomus dilutus and estimation of toxic equivalency factors. Environmental Toxicology and Chemistry 36(2): 372-382.

Cavas, T., Ergene-Gozukara S. (2005) Micronucleus test in fish cells: A bioassay for in situ monitoring of genotoxic pollution in the marine environment. Environmental and Molecular Mutagenesis 46: 64-70.

Cavas, T., Könen, S. (2007) Detection of cytogenetic and DNA damage in peripheral erythrocytes of goldfish (Carassius auratus) exposed to a glyphosate formulation using the micronucleus test and the comet assay. Mutagenesis 22(4): 263-268.

Cavusoglu, K., Yalcin, E., Turkmen, Z., Yapar, K., Sagir, S. (2012) Physiological, anatomical, biochemical, and cytogenetic effects of thiamethoxam treatment on Allium cepa (Amaryllidaceae) L. Environmental Toxicology 27: 635-643.

Chen, J., Chen, X., Zhao, J., Liu, S., Chi, Z. (2020) Instrument-free and visual detection of organophosphorus pesticide using a smartphone by coupling aggregation-induced emission nanoparticle and two-dimension MnO2 nanoflake. Biosensors and Bioelectronics 170: 112668.

Collins, A.R. (2004) The comet assay for DNA damage and repair. Molecular Biotechnology 26(3): 249-261.

Da Silva Souza, T., Fontanetti, C.S. (2006) Micronucleus test and observation of nuclear alterations in erythrocytes of Nile tilapia exposed to waters affected by refinery effluent. Mutation Research 605: 87-93.

Ding, T.T., Zhang, Y.H., Zhu, Y., Du, S.L., Zhang, J., Cao, Y., Wang, Y.Z., Wang, G.T., He, L.S. (2019) Deriving water quality criteria for China for the organophosphorus pesticides dichlorvos and malathion. Environmental Science and Pollution Research 26(33): 34622-34632.

EFSA (2013) EFSA press release: EFSA identifies risks to bees from neonicotinoids. European Food Safety Authority website. Available at: (accessed 11 Sep 2014).

Ernst, F., Alonso, B., Colazzo, M., Pareja, L., Cesio, V., Pereira, A., Márquez, A., Errico, E., Segura, A.M., Heinzen, H., Pérez-Parada, A. (2018) Occurrence of pesticide residues in fish from south American rainfed agroecosystems. Science of the Total Environment 631: 169-179.

European Commission (2013) Europa press release—Bees & pesticides: Commission to proceed with plan to better protect bees. Available at: 379_ en.htm (accessed 11 Sep 2014).

Finnegan, M.C., Baxter, L.R., Maul, J.D., Hanson, M.L., Hoekstra, P.F. (2017) Comprehensive characterization of the acute and chronic toxicity of the neonicotinoid insecticide thiamethoxam to a suite of aquatic primary producers, invertebrates, and fish. Environmental Toxicology and Chemistry 36(10): 2838-2848.

Georgieva, E., Stoyanova, S., Velcheva, I., Yancheva, V. (2014) Histopathological alterations in common carp (Cyprinus carpio L.) gills caused by thiamethoxam. Brazilian Archives of Biology and Technology 57: 991-996.

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.

Guillen, J., Bielza, P. (2013) Thiamethoxam acts as a target-site synergist of spinosad in resistant strains of Frankliniella occidentalis. Pest Management Science 69: 188-194.

Jones, M.M., Robertson, J.L., Weinzierl, R.A. (2012) Toxicity of thiamethoxam and mixtures of chlorantraniliprole plus acetamiprid, esfenvalerate, or thiamethoxam to neonates of oriental fruit moth (Lepidoptera: Tortricidae). Journal of Economic Entomology 105: 1426-1431.

Karmakar, R., Kulshrestha, G. (2009) Persistence, metabolism and safety evaluation of thiamethoxam in tomato crop. Pest Management Science 65: 931-937.

Liu, X., Zhang, Q., Li, S., Mi, P., Chen, D., Zhao, X., Feng, X. (2018) Developmental toxicity and neurotoxicity of synthetic organic insecticides in zebrafish (Danio rerio): A comparative study of deltamethrin, acephate, and thiamethoxam. Chemosphere 199: 16-25.

Martins da Costa, E., Azarias Guimarães, A., Pereira Vicentin, R., de Almeida Ribeiro, P.R., Ribas Leao, A.C., Balsanelli, E., Lebbe, L., Aerts, M., Willems, A., de Souza Moreira, F.M. (2017) Bradyrhizobium brasilense sp. nov., a symbiotic nitrogen-fixing bacterium. Archives of Microbiology 199(8): 1211-1221.

Mitkovska, V.I., Dimitrov, H.A., Chassovnikarova, T.G. (2017) In vivo genotoxicity and cytotoxicity assessment of allowable concentrations of nickel and lead: comet assay and nuclear abnormalities in acridine orange stained erythrocytes of common carp (Cyprinus carpio L.). Acta Zoologia Bulgarica 8: 47-56.

Morrissey, C.A., Mineau, P., Devries, J.H., Sanchez-Bayo, F., Liess, M., Cavallaro, M.C., Liber, K. (2015) Neonicotinoid contamination of global surface waters and associated risk to aquatic invertebrates: A review. Environment International 74: 291-303.

Nordborg, M., Arvidsson, R., Finnveden, G., Cederberg, C., Sörme, L., Palm, V., Stamyr, k., Molander, S. (2017) Updated indicators of Swedish national human toxicity and ecotoxicity footprints using USEtox 2.01. Environmental Impact Assessment Review 62: 110-114.

Norusis, M.J. (1993) Advanced Statistics. SPSS for Windows, Release 6.0.

Nowell, L.H., Norman, J.E., Moran, P.W., Martin, J.D., Stone, W.W. (2014) Pesticide toxicity index a tool for assessing potential toxicity of pesticide mixtures to freshwater aquatic organisms. Science of the Total Environment 476: 144-157.

Pena, A., Rodriguez-Liebana, J.A., Mingorance, M.D. (2011) Persistence of two neonicotinoid insecticides in wastewater, and in aqueous solutions of surfactants and dissolved organic matter. Chemosphere 84: 464-470.

Prusty, A.K., Meena, D.K., Mohapatra, S., Panikkar, P., Das, P., Gupta, S.K., Behera, B.K. (2015) Synthetic pyrethroids (Type II) and freshwater fish culture: Perils and mitigations. International Aquatic Research 7(3): 163-191.

Rejczak, T., Tuzimski, T. (2015) A review of recent developments and trends in the QuEChERS sample preparation approach. Open Chemistry 13: 980-1010.

Riaz, M.A., Chandor-Proust, A., Dauphin-Villemant, C., Poupardin, R., Jones, C. M., Strode, C., Régent-Kloeckner, M., David, J.P., Reynaud, S. (2013) Molecular mechanisms associated with increased tolerance to the neonicotinoid insecticide imidacloprid in the dengue vector Aedes aegyptiAquatic Toxicology 126: 326-337.

Saraiva, A.S., Sarmento, R.A., Rodrigues, A.C.M, Campos, D., Fedorova, G., Žlábek, V., Gravato, C., Pestana, J.L.T., Soares, A.M.V.M. (2017) Assessment of thiamethoxam toxicity to Chironomus ripariusEcotoxicology and Environmental Safety 137: 240-246.

Silva, V., Mol, H.G., Zomer, P., Tienstra, M., Ritsema, C.J., Geissen, V. (2019) Pesticide residues in European agricultural soils – a hidden reality unfolded. Science of the Total Environment 653: 1532-1545.

Singh, N.P., Danner, D.B., Tice, R.R., Brant, L., Schneider, E.L. (1990) DNA damage and rpair with age in individual human lymphocytesMutation Research/DNAging 237(3-4): 123-130.

Souders II, C.L., Rushin, A., Sanchez, C.L., Toth, D., Adamovsky, O., Martyniuk, C.J. (2021) Mitochondrial and transcriptome responses following exposure to the insecticide fipronil on rat dopaminergic neural cells. Neurotoxicology 85: 173-185.

Stevens, M.M., Helliwell, S., Hughes, P.A. (2005) Toxicity of Bacillus thuringiensis var. israelensis formulations, spinosad, and selected synthetic insecticides to Chironomus tepperi larvae. Journal of the American Mosquito Control Association 21(4): 446-450.

Temiz, Ö., Kargın, D., Çoğun, H.Y. (2021) In vivo effects on stress protein, genotoxicity, and oxidative toxicity parameters in Oreochromis niloticus tissues exposed to Thiamethoxam. Water Air and Soil Pollution 232(6): 1-17.

Thany, S.H. (2010) Neonicotinoid insecticides: historical evolution and resistance mechanisms. Advances Experimental Medicine and Biology 683: 75-83.

Tian, X., Yang, W., Wang, D., Zhao, Y., Yao, R., Ma, L., Ge, C., Li, X., Huang, Z., He, L., Jiao, W., Lin, A. (2018) Chronic brain toxicity response of juvenile Chinese rare minnows (Gobiocypris rarus) to the neonicotinoid insecticides imidacloprid and nitenpyram. Chemosphere 210: 1006-1012.

Topal, A., Alak, G., Ozkaraca, M., Yeltekin, A. C., Comaklı, S., Acıl, G. (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.

Turan, F., Ergenler, A. (2019) Assessment of DNA damage by Comet assay in Trachinotus ovatus cells from Mersin Bay in the Northeastern Mediterranean. NESciences 4(3): 25-31.

Turan, F., Ergenler, A. (2021a) The effect of transport process on the micronucleus frequency in rrythrocytes of the common carp Cyprinus carpio L. In: EJONS 12th International Conference on Mathematics, Engineering, Natural & Medical Sciences 9-11 Jully 2021, Paris France, Conference Full Text Book, (eds., Erkmen, O., Akhmetov, N.), EJONS Publications, pp.124-128.

Turan, F., Ergenler, A. (2021b) The genotoxic effect of noise pollution on carp (Cyprinus carpio) by micronucleus test. In: Cukurova 6th International Scientific Researches Conference 5 – 6 March 2021, Adana, Conference Full Text Book, (eds., Kardaslar, A., Kidiryuz, M.), ISPEC Publications, pp.247-250.

Turan, F., Ergenler, A. (2021c) DNA damage in hybrid tilapia (Oreochromis niloticus x O. aureus) exposed to short-transport process. NESciences 6(3): 190-196.

Turan, F., Ergenler, A. (2022) Investigation of the genotoxic effect of acetamiprid in Cyprinus carpio using the micronucleus analysis and the Comet assay. Turkish Journal of Maritime and Marine Sciences (in press). doi: 10.52998/trjmms.1037906

Turan, F., Eken, M., Ozyilmaz, G., Karan, S., Uluca, H. (2020a) Heavy metal bioaccumulation, oxidative stress and genotoxicity in African catfish Clarias gariepinus from Orontes river. Ecotoxicology 29(9): 1522-1537.

Turan, F., Karan, S., Ergenler, A. (2020b) Effect of heavy metals on toxicogenetic damage of European eels Anguilla anguilla. Environmental Science and Pollution Research 27(30): 38047-38055.

Uğurlu, P., Ünlü, E., Satar, E.I. (2015) The toxicological effects of thiamethoxam on Gammarus kischineffensis (Schellenberg 1937)(Crustacea: Amphipoda). Environmental Toxicology and Pharmacology 39(2): 720-726.

Van den Brink, P.J., Van Smeden, J.M., Bekele, R.S., Dierick, W., De Gelder, D.M., Noteboom, M., Roessink, I. (2016) Acute and chronic toxicity of neonicotinoids to nymphs of a mayfly species and some notes on seasonal differences. Environmental Toxicology and Chemistry 35: 128-133.

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. Current Opinion in Environmental Sustainability 5(3-4):293-305.

Wan, Y., Hussain, S., Merchant, A., Xu, B., Xie, W., Wang, S., Zhang, Y., Zhou, X., Wu, Q. (2020) Tomato spotted wilt orthotospovirus influences the reproduction of its insect vector, western flower thrips, Frankliniella occidentalis, to facilitate transmission. Pest Management Science 76(7): 2406-2414.

Whiteside, M., Mineau, P., Morrison, C., Knopper, L.D. (2008) Comparison of a score‐based approach with risk‐based ranking of in‐use agricultural pesticides in Canada to aquatic receptors. Integrated Environmental Assessment and Management 4(2): 215-236.

Yan, S.H., Wang, J.H., Zhu, L.S., Chen, A.M., Wang, J. (2016) Thiamethoxam induces oxidative stress and antioxidant response in zebrafish (Danio rerio) livers. Environmental Toxicology 31(12): 2006-2015.

Yanar, M., Genç, E. (2004) Anaesthetic effects of quinaldine sulphate together with the use of Diazepam on Oreochromis niloticus L. 1758 (Cichlidae) at different temperatures. Turkish Journal of Veterinary and Animal Sciences 28: 1001-1005.

Yang, H., Liu, H.J., Hu, Z.B., Liang J., Pang, H., Yi, B. (2014) Consideration on degradation kinetics and mechanism of thiamethoxam by reactive oxidative species (ROSs) during photocatalytic process. Chemical Engineering Journal 245: 24-33.

Zhao, X.P., Wu, C.X., Wang, Y.H., Cang, T., Chen, L.P., Yu, R.X., Wang, Q. (2012) Assessment of toxicity risk of insecticides used in rice ecosystem on Trichogramma japonicum, an egg parasitoid of rice lepidopterans. Journal of Economic Entomology 105: 92-101.