Evaluation of the cytotoxic effects of chlorpyrifos ethyl in fish cell lines

Pınar Arslan Yüce

doi:10.3153/AR26006

EN

Evaluation of the cytotoxic effects of chlorpyrifos ethyl in fish cell lines

Abstract

Chlorpyrifos-ethyl (CPE) is a widely used organophosphate pesticide that poses a significant risk to aquatic ecosystems. The present study aimed to investigate the cytotoxicity of CPE on two fish cell lines: rainbow trout gonad (RTG-2) and common carp epithelioma (EPC). The cells were exposed to a range of CPE concentrations (0.488 - 250 ppm) for 24 h. Cytotoxicity was evaluated using two complementary colourimetric assays, MTT and Neutral Red (NR), which measure mitochondrial activity and lysosomal membrane integrity, respectively. The results demonstrated that CPE induced a significant, concentration-dependent decrease in both assays in both cell lines. The calculated half-inhibitory concentration (IC50) values for the RTG-2 and EPC cells showed variations, indicating differences in sensitivity between the cell types and the metabolic endpoints assessed. The cell morphologies were changed when exposed to CPE concentrations in both cell lines. The in vitro cytotoxicity data were consistent with the acute toxicity ranges (LC50) reported in the literature for in vivo fish species, validating the use of these piscine cell lines as reliable and ethical tools for the preliminary ecotoxicological screening of pesticides. This study confirms the high cytotoxic potential of CPE and underscores the utility of fish cell lines in environmental risk assessment.

Keywords

Supporting Institution

Çankırı Karatekin Universtiy Scientific Research Projects Presidency

Project Number

FF210621B12

Ethical Statement

Not applicable. The study involved only in vitro experiments using established fish cell lines and did not include experiments on live animals.

Thanks

Special thanks to Prof. Dr. A. Cağlan Gunal for providing chlorpyrifos-ethyl, to Prof. Dr. Gülçin Akça for using the cell culture laboratory access and to Prof. Dr. Begüm Yurdakök for using the SpectraMax microplate reader.

References

Abdul Majeed, S., Nambi, K.S.N., Taju, G., Sundar Raj, N., Madan, N., & Sahul Hameed, A.S. (2013). Establishment and characterisation of a permanent cell line from the gill tissue of Labeo rohita (Hamilton) and its application in gene expression and toxicology. Cell Biology and Toxicology, 29(1), 59–73. https://doi.org/10.1007/s10565-012-9237-7
Abdul Majeed, S., Nambi, K.S.N., Taju, G., Babu, V.S., Farook, M.A., & Hameed, A.S. (2014). Development and characterization of a new gill cell line from air breathing fish Channa striatus (Bloch, 1793) and its application in toxicology: direct comparison to the acute fish toxicity. Chemosphere, 96, 89-98. https://doi.org/10.1016/j.chemosphere.2013.07.045
Almeida, M., Martins, M.A., Soares, A.M., Cuesta, A., & Oliveira, M. (2019). Polystyrene nanoplastics alter the cytotoxicity of human pharmaceuticals on marine fish cell lines. Environmental Toxicology and Pharmacology, 69, 57–65. https://doi.org/10.1016/j.etap.2019.03.019
Anonymous (2025). Türk Gıda Kodeksi Pestisitlerin Maksimum Kalıntı Limitleri Yönetmeliği. https://mevzuat.gov.tr/mevzuat?MevzuatNo=38965&MevzuatTur=7&MevzuatTertip=5
Arslan, P. & Ozeren, S.C. (2021). A case study of 38 micro‐organic pollutants contamination in Kirmir Stream, Turkey. Environmental Quality Management, 31(4), 283-290. https://doi.org/10.1002/tqem.21811
Arslan, P. & Ozeren, S.C. (2022). Physiological and histopathological alterations in Capoeta baliki and Squalius pursakensis after caused by some environmental pollutants. Environmental Monitoring and Assessment, 194(3), 1-13. https://doi.org/10.1007/s10661-022-09830-y
Belek, N., Erkmen, B., Dinçel, A.S., & Günal, A.Ç. (2022). Does persistent organic pollutant PFOS (perfluorooctane sulfonate) negative impacts on the aquatic invertebrate organism, Astacus leptodactylus [Eschscholtz, 1823]. Ecotoxicology, 31, 1217–1230. https://doi.org/10.1007/s10646-022-02579-7
Brodeur, J.C., Damonte, M.J., Rojas, D.E., Cristos, D., Vargas, C., Poliserpi, M.B., & Andriulo, A.E. (2022). Concentration of current-use pesticides in frogs from the Pampa region and correlation of a mixture toxicity index with biological effects. Environmental Research, 204, 112354. https://doi.org/10.1016/j.envres.2021.112354

Davila, J. C., Rodriguez, R. J., Melchert, R. B., & Acosta Jr, D. (1998). Predictive value of in vitro model systems in toxicology. Annual Review of Pharmacology and Toxicology, 38(1), 63–96. https://doi.org/10.1146/annurev.pharmtox.38.1.63
Erkekoğlu, P. & Baydar, T. (2017). Current In Vitro Cytotoxicity Tests. Hacettepe University Journal of the Faculty of Pharmacy, 41(1), 45-63.
Erkmen, B., Günal, A.Ç., Polat, H., Erdoğan, K., Civelek, H., & Erkoç, F. (2022). Sublethal effects of acrylamide on thyroid hormones, complete blood count and micronucleus frequency of vertebrate model organism (Cyprinus carpio). Turkish Journal of Biochemistry, 47(6), 811-818. https://doi.org/10.1515/tjb-2022-0025
EU 2020. Commission Implementing Regulation (EU) 2020/18 of 10 January 2020 concerning the non-renewal of the approval of the active substance chlorpyrifos, in accordance with Regulation (EC) No 1107/2009 of the European Parliament and of the Council concerning the placing of plant protection products on the market and amending the Annex to Commission Implementing Regulation (EU) No 540/2011. http://data.europa.eu/eli/reg_impl/2020/18/oj
Fotakis, G. & Timbrell, J.A. (2006). In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicology Letters, 160(2),171–7. https://doi.org/10.1016/j.toxlet.2005.07.001
Ministry of Food, Agriculture and Livestock (2016). General Directorate of Food and Control, Department of Plant Protection Products, decision regarding Chlorpyrifos-ethyl. https://bku.tarim.gov.tr/Duyuru/ DuyuruDetay/1.
Handy, R.D., Clark, N.J., Boyle, D., Vassallo, J., Green, C., Nasser, F., Botha, T.L., Wepener, V., van den Brick, F., & Svendsen, C. (2022). The bioaccumulation testing strategy for nanomaterials: correlations with particle properties and a meta-analysis of in vitro fish alternatives to in vivo fish tests. Environmental Science: Nano, https://doi.org/10.1039/D1EN00694K
John, E.M. & Shaike, J.M. (2015). Chlorpyrifos: pollution and remediation. Environmental Chemistry Letters, 13, 269–291. https://doi.org/10.1007/s10311-015-0513-7
Kumar, R., Sankhla, M. S., Kumar, R., & Sonone, S.S. (2021). Impact of pesticide toxicity in aquatic environment. Biointerface Research in Applied Chemistry, 11(3), 10131–10140. https://doi.org/10.33263/BRIAC113.1013110140
Miller, T.H., Ng, K.T., Bury, S.T., Bury, S.E., Bury, N.R., & Barron, L.P. (2019). Biomonitoring of pesticides, pharmaceuticals and illicit drugs in a freshwater invertebrate to estimate toxic or effect pressure. Environment International, 129, 595–606. https://doi.org/10.1016/j.envint.2019.04.038
Mojiri, A., Zhou, J.L., Robinson, B., Ohashi, A., Ozaki, N., Kindaichi, T., Farrajji, H., & Vakili, M. (2020). Pesticides in aquatic environments and their removal by adsorption methods. Chemosphere, 253, 126646. https://doi.org/10.1016/j.chemosphere.2020.126646
Muir, D., Savinova, T., Savinov, V., Alexeeva, L., Potelov, V., & Svetochev, V. (2003). Bioaccumulation of PCBs and chlorinated pesticides in seals, fishes and invertebrates from the White Sea, Russia. Science of the Total Environment, 306(1-3), 111–131. https://doi.org/10.1016/S0048-9697(02)00488-6
Nasiri, M., Ahmadzadeh, H., & Amiri, A. (2020). Sample preparation and extraction methods for pesticides in aquatic environments: A review. TrAC Trends in Analytical Chemistry, 123, 115772. https://doi.org/10.1016/j.trac.2019.115772
OECD (2019). Test No. 203: Fish, Acute Toxicity Test. OECD Publishing. https://www.oecd.org/en/publications/test-no-203-fish-acute-toxicity-test_9789264069961-en.html
Oruc, E. (2012). Oxidative stress responses and recovery patterns in the liver of Oreochromis niloticus exposed to chlorpyrifos-ethyl. Bulletin of Environmental Contamination and Toxicology, 88(5), 678–684. https://doi.org/10.1007/s00128-012-0548-4
Otieno, P.O., Schramm, K.W., Pfister, G., Lalah, J.O., Ojwach, S., & Virani, M. (2012). Spatial Distribution and Temporal Trend in Concentration of Carbofuran, Diazinon and Chlorpyrifos Ethyl Residues in Sediment and Water in Lake Naivasha, Kenya. Bulletin of Environmental Contamination and Toxicology, 88, 526–532. https://doi.org/10.1007/s00128-012-0529-7
Otieno, P.O., Owuor, P.O., Lalah, J.O., Pfister, G., & Schramm, K.W. (2013). Comparative evaluation of ELISA kit and HPLC DAD for the determination of chlorpyrifos ethyl residues in water and sediments. Talanta, 117, 250–257. https://doi.org/10.1016/j.talanta.2013.09.014
Pandit, G.G., Rao, A.M., Jha, S.K., Krishnamoorthy, T.M., Kale, S.P., Raghu, K., & Murthy, N.B.K. (2001). Monitoring of organochlorine pesticide residues in the Indian marine environment. Chemosphere, 44(2), 301–305. https://doi.org/10.1016/S0045-6535(00)00179-X
Pehlivan, A., Atmaca, E., & Aksoy, A. (2018). Ekotoksikoloji Alanında Balık Hücre Hatlarının Kullanımı. Etlik Veteriner Mikrobiyoloji Dergisi, 29(2), 175-180. https://doi.org/10.35864/evmd.513576
Phipps, G.L. & Holcombe, G.W. (1985). A method for aquatic multiple species toxicant testing: acute toxicity of 10 chemicals to 5 vertebrates and 2 invertebrates. Environmental Pollution A, 38(2), 141–157. https://doi.org/10.1016/0143-1471(85)90073-X
Ruiz-Arias, M.A., Medina-Díaz, I.M., Bernal-Hernández, Y.Y. et al. (2023). The situation of chlorpyrifos in Mexico: a case study in environmental samples and aquatic organisms. Environmental Geochemistry and Health, 45, 6323–6351. https://doi.org/10.1007/s10653-023-01618-4
Ruiz-Palacios, M., Esteban, M. Á., & Cuesta, A. (2020). Establishment of a brain cell line (SaB-1) from gilthead seabream and its application to fish virology. Fish & Shellfish Immunology, 106, 161-166. https://doi.org/10.1016/j.fsi.2020.07.065
Salihu, S.O. & Iyya, Z. (2022). Assessment of Physicochemical parameters and Organochlorine pesticide residues in selected vegetable farmlands soil in Zamfara State, Nigeria. Science Progress and Research (SPR), 2(2). https://doi.org/10.52152/spr/2022.171
Schirmer, K. (2006). Proposal to improve vertebrate cell cultures to establish them as substitutes for the regulatory testing of chemicals and effluents using fish. Toxicology, 224, 163–183. https://doi.org/10.1016/j.tox.2006.04.042
Surinlert, P., Petmunee, L., Jiraarpassorn, C., Salika, T., Watthanard, M., Bunnuang, K., ... & Tipbunjong, C. (2025). Chlorpyrifos abolished C2C12 myoblast cell proliferation and differentiation via mitochondrial stress. Toxicology Reports, 102041. https://doi.org/10.1016/j.toxrep.2025.102041
Taju, G., Majeed, S.A., Nambi, K.S.N., Farook, M.A., Vimal, S., & Hameed, A.S. (2014). In vitro cytotoxic, genotoxic and oxidative stress of cypermethrin on five fish cell lines. Pesticide Biochemistry and Physiology, 113, 15–24. https://doi.org/10.1016/j.pestbp.2014.06.006
Tokur, O. & Aksoy, A. (2017). In vitro sitotoksisite testleri. Harran Üniversitesi Veteriner Fakültesi Dergisi, 6(1), 112-118. https://doi.org/10.31196/huvfd.325794
Tomlin, C.D.S. (2009). The pesticide manual: a world compendium. British Crop Production Council, Alton.
Top, Z.N., Tiryaki, O., Polat. B. (2023). Monitoring and environmental risk assessment of agricultural fungicide and insecticides in water, sediment from Kumkale Plain, Çanakkale-Turkey. Journal of Environmental Science and Health, Part B. 58(4), 304-315. https://doi.org/10.1080/03601234.2023.2187598
UNEP Stockholm Convention on Persistent Organic Pollutants (2024). Risk management evaluation for chlorpyrifos. Report of the Persistent Organic Pollutants Review Committee on the work of its twentieth meeting. UNEP/POPS/POPRC.20/10/Add.1
US EPA (2020). Chlorpyrifos Proposed Interim Registration Review Decision Case Number 0100. https://www.epa.gov/ingredients-used-pesticide-products/proposed-interim-decision-registration-review-chlorpyrifos
Varga, E., Prause, H. C., Riepl, M. et al. (2024). Cytotoxicity of Prymnesium parvum extracts and prymnesin analogs on epithelial fish gill cells RTgill-W1 and the human colon cell line HCEC-1CT. Archieves of Toxicology, 98, 999–1014. https://doi.org/10.1007/s00204-023-03663-5
Wang, S., Zhang, X., Gui, B., Xu, X., Su, L., Zhao, Y. H., & Martyniuk, C. J. (2022). Comparison of modes of action between fish, cell and mitochondrial toxicity based on toxicity correlation, excess toxicity and QSAR for class-based compounds. Toxicology, 470, 153155. https://doi.org/10.1016/j.tox.2022.153155
Wu, Y. J., Kang, K., Yang, W., Wu, Q., Chen, R., & Zhou, X. (2025). Combined Toxicity of Heavy Metal Cadmium and Insecticide Chlorpyrifos to Drosophila S2 Cells. Bulletin of Environmental Contamination and Toxicology, 115(2), 24. https://doi.org/10.1007/s00128-025-04097-8
Xing, H., Wang, X., Sun, G., Gao, X., Xu, S., & Wang, X. (2012). Effects of atrazine and chlorpyrifos on activity and transcription of glutathione S-transferase in common carp (Cyprinus carpio L.). Environmental Toxicology and Pharmacology, 33(2), 233–244. https://doi.org/10.1016/j.etap.2011.12.014
Xu, Y., Zhong, Z., Zhang, Z., Feng, Y., Zhao, L., Jiang, Y., & Wang, Y. (2022). Establishment and characterisation of the gonadal cell lines derived from large yellow croaker (Larimichthys crocea) for gene expression studies. Aquaculture, 546, 737300. https://doi.org/10.1016/j.aquaculture.2021.737300
Yurdakok-Dikmen, B., Alpay, M., Kismali, G., Filazi, A., Kuzukiran, O., & Sireli, U.T. (2015). In vitro effects of phthalate mixtures on colorectal adenocarcinoma cell lines. Journal of Environmental Pathology, Toxicology and Oncology, 34(2). https://doi.org/10.1615/JEnvironPatholToxicolOncol.2015013256

Details

Primary Language

English

Subjects

Aquatic Toxicology , Fish Biology

Journal Section

Research Article

Authors

Pınar Arslan Yüce ^*
0000-0001-5910-2835
Türkiye

Early Pub Date

January 15, 2026

Publication Date

January 15, 2026

Submission Date

November 24, 2025

Acceptance Date

January 12, 2026

Published in Issue

Year 2026 Volume: 9 Number: 1

DOI

https://doi.org/10.3153/AR26006

IZ

https://izlik.org/JA53GX67NU

Cite

RIS / Bibtex

APA

Arslan Yüce, P. (2026). Evaluation of the cytotoxic effects of chlorpyrifos ethyl in fish cell lines. Aquatic Research, 9(1), 54-62. https://doi.org/10.3153/AR26006

Öz

Evaluation of the cytotoxic effects of chlorpyrifos ethyl in fish cell lines

Abstract

Keywords

Supporting Institution

Project Number

Ethical Statement

Thanks

References

Details

Primary Language

Subjects

Journal Section

Authors

Early Pub Date

Publication Date

Submission Date

Acceptance Date

Published in Issue

DOI

IZ

Cite