Zebrafish embryo neonicotinoid developmental neurotoxicity in the FET test and behavioral assays

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Rebecca von Hellfeld
Viktoriia Ovcharova
Samantha Bevan
Maria-Agapi Lazaridi
Caroline Bauch
Paul Walker
Susanne Hougaard Bennekou
Anna Forsby
Thomas Braunbeck


The need for reliable, sensitive (developmental) neurotoxicity testing of chemicals has steadily increased. Given the limited capacities for routine testing according to accepted regulatory guidelines, there is potential risk to human health and the environment. Most toxicity studies are based on mammalian test systems, which have been questioned for low sensitivity, limited relevance for humans, and animal welfare considerations. This increased the need for alternative models, one of which is the zebrafish (Danio rerio) embryo. This study assessed selected neonicotinoids at sub-lethal concentrations for their effects on embryonic development and behavior. The fish embryo acute toxicity test (OECD TG 236) determined the lowest observable effective concentrations, which were used as the highest test concentrations in subsequent behavioral assays. In the FET test, no severe compound-induced sublethal effects were seen at < 100 µM. In the coiling assay, exposure to ≥ 1.25 µM nicotine (positive control) affected both the burst duration and burst count per minute, whereas ≥ 50 µM thiacloprid affected the mean burst duration. Exposure to ≥ 50 µM acetamiprid and imidacloprid induced significant alterations in both mean burst duration and burst count per minute. In the swimming assay, 100 µM acetamiprid induced alterations in the frequency and extent of movements, whilst nicotine exposure only induced non-significant changes. All behavioral changes could be correlated to findings in mammalian studies. Given the quest for alternative test methods of (developmental) neurotoxicity, zebrafish embryo behavior testing could be integrated into a future tiered testing scheme.

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von Hellfeld, R., Ovcharova, V., Bevan, S., Lazaridi, M.-A., Bauch, C., Walker, P., Hougaard Bennekou, S., Forsby, A. and Braunbeck, T. (2022) “Zebrafish embryo neonicotinoid developmental neurotoxicity in the FET test and behavioral assays”, ALTEX - Alternatives to animal experimentation, 39(3), pp. 367–387. doi: 10.14573/altex.2111021.

Ali, S., Champagne, D. L. and Richardson, M. K. (2012). Behavioral profiling of zebrafish embryos exposed to a panel of 60 water-soluble compounds. Behav Brain Res 228, 272-283. doi:10.1016/j.bbr.2011.11.020

Aschner, M., Ceccatelli, D., Daneshian, M. et al. (2017). Reference compounds for alternative test methods to indicate developmental neurotoxicity (DNT) potential of chemicals: Example lists and criteria for their selection and use. ALTEX 34, 49-73. doi:10.14573/altex.1604201

Auteri, D., Arena, M., Barmaz, S. et al. (2017). Neonicotinoids and bees: The case of the European regulatory risk assessment. Sci Total Environ 579, 966-971. doi:10.1016/j.scitotenv.2016.10.158

Babeľová, J., Šefčíková, Z., Čikoš, Š. et al. (2017). Exposure to neonicotinoid insecticides induces embryotoxicity in mice and rabbits. Toxicology 392, 71-80. doi:10.1016/j.tox.2017.10.011

Bailey, J., Thew, M. and Balls, M. (2014). An analysis of the use of animal models in predicting human toxicology and drug safety. Altern Lab Anim 42, 181-199. doi:10.1177/026119291404200306

Bambino, K. and Chu, J. (2017). Zebrafish in toxicology and environmental health. Curr Top Dev Biol 124, 331-367. doi:10.1016/bs.ctdb.2016.10.007

Basnet, R., Zizioli, D., Taweedet, S. et al. (2019). Zebrafish larvae as a behavioral model in neuropharmacology. Biomedicines 7, 23. doi:10.3390/biomedicines7010023

Bass, S. L. S. and Gerlai, R. (2008). Zebrafish (Danio rerio) responds differentially to stimulus fish: The effects of sympatric and allopatric predators and harmless fish. Behav Brain Res 186, 107-117. doi:10.1016/j.bbr.2007.07.037

Bayne, K. A. L., Beaver, B. V., Mench, J. A. et al. (2015). Chapter 38 – Laboratory animal behavior. In J. G. Fox, L. C. Anderson, G. M. Otto et al. (eds), Laboratory Animal Medicine (1617-1651). 3rd edition. Academic Press. doi:10.1016/B978-0-12-409527-4.00038-9

Bencan, Z. and Levin, E. D. (2008). The role of α7 and α4β2 nicotinic receptors in the nicotine-induced anxiolytic effect in zebrafish. Physiol Behav 95, 408-412. doi:10.1016/j.physbeh.2008.07.009

Braunbeck, T., Kais, B., Lammer, E. et al. (2015). The fish embryo test (FET): Origin, applications, and future. Environ Sci Pollut Res Int 22, 16247-16261. doi:10.1007/s11356-014-3814-7

Burke, A. P., Niibori, Y., Terayama, H. et al. (2018). Mammalian susceptibility to a neonicotinoid insecticide after fetal and early postnatal exposure. Sci Rep 8, 16639. doi:10.1038/s41598-018-35129-5

Chang, L. W. (1998). Introduction. In W. Slikker and L. W. Chang (eds.), Handbook of Developmental Neurotoxicology (1-2). Academic Press. doi:10.1016/B978-012648860-9.50003-0

Clark, M. and Steger-Hartmann, T. (2018). A big data approach to the concordance of the toxicity of pharmaceuticals in animals and humans. Regul Toxicol Pharmacol 96, 94-105. doi:10.1016/j.yrtph.2018.04.018

Crosby, E. B., Bailey, J. M., Oliveri, A. N. et al. (2015). Neurobehavioral impairments caused by developmental imidacloprid exposure in zebrafish. Neurotoxicol Teratol 49, 81-90. doi:10.1016/j.ntt.2015.04.006

Cunha-Oliveira, T., Rego, A. C. and Oliveira, C. R. (2008). Cellular and molecular mechanisms involved in the neurotoxicity of opioid and psychostimulant drugs. Brain Res Rev 58, 192-208. doi:10.1016/j.brainresrev.2008.03.002

de Koning, C., Beekhuijzen, M., Tobor-Kapłon, M. et al. (2015). Visualizing compound distribution during zebrafish embryo development: The effects of lipophilicity and DMSO. Birth Defects Res Part B Dev Reprod Toxicol 104, 253-272. doi:10.1002/bdrb.21166

Delp, J., Gutbier, S., Klima, S. et al. (2018). A high-throughput approach to identify specific neurotoxicants/developmental toxicants in human neuronal cell function assays. ALTEX 35, 235-253. doi:10.14573/altex.1712182

Drapeau, P., Saint-Amant, L., Buss, R. R. et al. (2002). Development of the locomotor network in zebrafish. Prog Neurobiol 68, 85-111. doi:10.1016/S0301-0082(02)00075-8

Dubovický, M., Kovačovský, P., Ujházy, E. et al. (2008). Evaluation of developmental neurotoxicity: Some important issues focused on neurobehavioral development. Interdiscip Toxicol 1, 206-210. doi:10.2478/v10102-010-0042-y

EC (2011). Directive 98/8/EC concerning the placing of biocidal products on the market. Imidacloprid Product-type 18 (Insecticides, Acaricides and Product to control other Arthropods). https://echa.europa.eu/documents/10162/225b9c58-e24c-6491-cc8d-7d85564f3912

EC (2019). Regulation (EU) No 528/2012 concerning the making available on the market and use of biocidal products. Dinotefuran Product-type 18 (Insecticides, acaricides and to control other arthropods). https://echa.europa.eu/documents/10162/6f1a5175-f1eb-4e6b-4b49-8cea13efcf5a

EFSA – European Food Safety Authority (2013). Scientific opinion on the developmental neurotoxicity potential of acetamiprid and imidacloprid. EFSA J 11, 3471. doi:10.2903/j.efsa.2013.3471

Engel, A. G., Lambert, E. H., Mulder, D. M. et al. (1982). A newly recognized congenital myasthenic syndrome attributed to a prolonged open time of the acetylcholine-induced ion channel. Ann Neurol 11, 553-569. doi:10.1002/ana.410110603

Engel, A. G., Ohno, K., Shen, X.-M. et al. (2003). Congenital myasthenic syndromes: Multiple molecular targets at the neuromuscular junction. Ann N Y Acad Sci 998, 138-160. doi:10.1196/annals.1254.016

EU – European Union (2010). Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. OJ L 276, 33-79. https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32010L0063&qid=1648544293278&from=EN

FAO – Food and Agriculture Organization (2016). FAO Specifications and Evaluations for Agricultural Pesticides. Clothianidin. https://www.fao.org/3/ca7726en/ca7726en.pdf

Faria, M., Prats, E., Novoa-Luna, K. A. et al. (2019). Development of a vibrational startle response assay for screening environmental pollutants and drugs impairing predator avoidance. Sci Total Environ 650, 87-96. doi:10.1016/j.scitotenv.2018.08.421

Faria, M., Wu, X., Luja-Mondragón, M. et al. (2020). Screening anti-predator behaviour in fish larvae exposed to environmental pollutants. Sci Total Environ 714, 136759. doi:10.1016/j.scitotenv.2020.136759

Feng, Y., Caiping, M., Li, C. et al. (2010). Fetal and offspring arrhythmia following exposure to nicotine during pregnancy. J Appl Toxicol 30, 53-58. doi:10.1002/jat.1471

Franks, M. E., Macpherson, G. R. and Figg, W. D. (2004). Thalidomide. Lancet 363, 1802-1811. doi:10.1016/S0140-6736(04)16308-3

Freeman, G. B., Sherman, K. A. and Gibson, G. E. (1987). Locomotor activity as a predictor of times and dosages for studies of nicotine’s neurochemical actions. Pharmacol Biochem Behav 26, 305-312. doi:10.1016/0091-3057(87)90123-7

Fritsche, E. (2017). OECD/EFSA workshop on developmental neurotoxicity (DNT): The use of non-animal test methods for regulatory purposes. ALTEX 34, 311-315. doi:10.14573/altex.1701171

Fung, Y. K. and Lau, Y. S. (1989). Effects of prenatal nicotine exposure on rat striatal dopaminergic and nicotinic systems. Pharmacol Biochem Behav 33, 1-6. doi:10.1016/0091-3057(89)90419-x

Galloway, T. S., Dogra, Y., Garrett, N. et al. (2017). Ecotoxicological assessment of nanoparticle-containing acrylic copolymer dispersions in fairy shrimp and zebrafish embryos. Environ Sci Nano 4, 1981-1997. doi:10.1039/C7EN00385D

Gao, L., Li, S., Zhang, J. et al. (2016). Excess imidacloprid exposure causes the heart tube malformation of chick embryos. J Agric Food Chem 64, 9078-9088. doi:10.1021/acs.jafc.6b03381

Gervais, J. A., Luukinen, B., Buhl, K. et al. (2012). Imidacloprid technical fact sheet. Natl Pestic Inf Cent. http://npic.orst.edu/factsheets/archive/imidacloprid.html

Giniatullin, R., Nistri, A. and Yakel, J. L. (2005). Desensitization of nicotinic ACh receptors: Shaping cholinergic signaling. Trends Neurosci 28, 371-378. doi:10.1016/j.tins.2005.04.009

Gomez, C. M., Maselli, R. A., Groshong, J. et al. (2002). Active calcium accumulation underlies severe weakness in a panel of mice with slow-channel syndrome. J Neurosci 22, 6447-6457. doi:10.1523/JNEUROSCI.22-15-06447.2002

Health & Chemicals (2002). Minamata Disease the History and Measures. http://www.env.go.jp/en/chemi/hs/minamata2002/

Hellpointner, E. (1990). Determination of the quantum yield and assessment of the environmental half-life of the direct photodegradation of imidacloprid in water. Report No. PF3422.

Henn, K. and Braunbeck, T. (2011). Dechorionation as a tool to improve the fish embryo toxicity test (FET) with the zebrafish (Danio rerio). Comp Biochem Physiol C Toxicol Pharmacol 153, 91-98. doi:10.1016/j.cbpc.2010.09.003

Hirano, T., Yanai, S., Takada, T. et al. (2018). NOAEL-dose of a neonicotinoid pesticide, clothianidin, acutely induce anxiety-related behavior with human-audible vocalizations in male mice in a novel environment. Toxicol Lett 282, 57-63. doi:10.1016/j.toxlet.2017.10.010

Hoelting, L., Klima, S., Karreman, C. et al. (2016). Stem cell-cerived immature human dorsal root ganglia neurons to identify peripheral neurotoxicants. Stem Cells Transl Med 5, 476-487. doi:10.5966/sctm.2015-0108

Kagawa, N. and Nagao, T. (2018). Neurodevelopmental toxicity in the mouse neocortex following prenatal exposure to acetamiprid. J Appl Toxicol 38, 1521-1528. doi:10.1002/jat.3692

Kalueff, A. V., Gebhardt, M., Stewart, A. M. et al. (2013). Towards a comprehensive catalog of zebrafish behavior 1.0 and beyond. Zebrafish 10, 70-86. doi:10.1089/zeb.2012.0861

Kara, M., Yumrutas, O., Demir, C. F. et al. (2015). Insecticide imidacloprid influences cognitive functions and alters learning performance and related gene expression in a rat model. Int J Exp Pathol 96, 332-337. doi:10.1111/iep.12139

Kimmel, C. B., Patterson, J. and Kimmel, R. O. (1974). The development and behavioral characteristics of the startle response in the zebra fish. Dev Psychobiol 7, 47-60. doi:10.1002/dev.420070109

Kimmel, C., Ballard, W., Kimmel, S. et al. (1995). Stages of embryonic development of the zebrafish. Am J Anat 203, 253-310. doi:10.1002/aja.1002030302

Kitamura, S., Miyata, C., Tomita, M. et al. (2020). A central nervous system disease of unknown cause that occurred in the minamata region: Results of an epidemiological study. J Epidemiol 30, 3-11. doi:10.2188/jea.JE20190173

Klüver, N., König, M., Ortmann, J. et al. (2015). Fish embryo toxicity test: Identification of compounds with weak toxicity and analysis of behavioral effects to improve prediction of acute toxicity for neurotoxic compounds. Environ Sci Technol 49, 7002-7011. doi:10.1021/acs.est.5b01910

Kokel, D., Bryan, J., Laggner, C. et al. (2010). Rapid behavior-based identification of neuroactive small molecules in the zebrafish. Nat Chem Biol 6, 231-237. doi:10.1038/nchembio.307

Kozikowski, B. A., Burt, T. M., Tirey, D. A. et al. (2003). The effect of freeze/thaw cycles on the stability of compounds in DMSO. J Biomol Screen 8, 210-215. doi:10.1177/1087057103252618

Krohn, J. and Hellpointner, E. (2002). Environmental fate of imidacloprid. Pflanzenschutz Nachrichten Bayer 55, 1-25. http://cues.cfans.umn.edu/old/pollinators/pdf-pesticides/2006CAImidclprdfate.pdf

Lammer, E., Carr, G. J., Wendler, K. et al. (2009). Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential alternative for the fish acute toxicity test? Comp Biochem Physiol C Toxicol Pharmacol 149, 196-209. doi:10.1016/j.cbpc.2008.11.006

Leonard, J. P. and Salpeter, M. M. (1979). Agonist-induced myopathy at the neuromuscular junction is mediated by calcium. J Cell Biol 82, 811-819. doi:10.1083/jcb.82.3.811

Letcher, R. J., Bustnes, J. O., Dietz, R. et al. (2010). Exposure and effects assessment of persistent organohalogen contaminants in arctic wildlife and fish. Sci Total Environ 408, 2995-3043. doi:10.1016/j.scitotenv.2009.10.038

Levin, E. D. and Cerutti, D. T. (2009). Behavioral neuroscience of zebrafish. In J. J. Buccafusco (ed.), Methods of Behavior Analysis in Neuroscience (Chapter 15). 2nd edition. CRC Press/Taylor & Francis.

Liu, X., Zhang, Q., Li, S. et al. (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. doi:10.1016/j.chemosphere.2018.01.176

Loser, D., Hinojosa, M. G., Blum, J. et al. (2021). Functional alterations by a subgroup of neonicotinoid pesticides in human dopaminergic neurons. Arch Toxicol 95, 2081-2107. doi:10.1007/s00204-021-03031-1

Ma, X., Li, H., Xiong, J. et al. (2019). Developmental toxicity of a neonicotinoid insecticide, acetamiprid to zebrafish embryos. J Agric Food Chem 67, 2429-2436. doi:10.1021/acs.jafc.8b05373

Masjosthusmann, S., Blum, J., Bartmann, K. et al. (2020). Establishment of an a priori protocol for the implementation and interpretation of an in-vitro testing battery for the assessment of developmental neurotoxicity. EFSA Support Publ 17, 1938E. doi:10.2903/sp.efsa.2020.EN-1938

Matturri, L., Ottaviani, G., Ramos, S. G. et al. (2000). Sudden infant death syndrome (SIDS): A study of cardiac conduction system. Cardiovasc Pathol 9, 137-145. doi:10.1016/S1054-8807(00)00035-1

Millot, F., Decors, A., Mastain, O. et al. (2017). Field evidence of bird poisonings by imidacloprid-treated seeds: A review of incidents reported by the French SAGIR network from 1995 to 2014. Environ Sci Pollut Res 24, 5469-5485. doi:10.1007/s11356-016-8272-y

Monticello, T. M., Jones, T. W., Dambach, D. M. et al. (2017). Current nonclinical testing paradigm enables safe entry to first-in-human clinical trials: The IQ consortium nonclinical to clinical translational database. Toxicol Appl Pharmacol 334, 100-109. doi:10.1016/j.taap.2017.09.006

Nishimura, H. and Nakai, K. (1958). Developmental anomalies in offspring of pregnant mice treated with nicotine. Science 127, 877-878. doi:10.1126/science.127.3303.877

OECD (2007). Test No. 426: Developmental Neurotoxicity Study. OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publishing, Paris. doi:10.1787/9789264067394-en

OECD (2013). Test No. 236: Fish Embryo Acute Toxicity (FET) Test. OECD Guidelines for the Testing of Chemicals, Section 2. OECD Publishing, Paris. doi:10.1787/9789264203709-en

OECD (2018). Test No. 443: Extended One-Generation Reproductive Toxicity Study. OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publishing, Paris. doi:10.1787/9789264185371-en

Ogungbemi, A. O., Teixido, E., Massei, R. et al. (2020). Optimization of the spontaneous tail coiling test for fast assessment of neurotoxic effects in the zebrafish embryo using an automated workflow in KNIME®. Neurotoxicol Teratol 81, 106918. doi:10.1016/j.ntt.2020.106918

Ohno, S., Ikenaka, Y., Onaru, K. et al. (2020). Quantitative elucidation of maternal-to-fetal transfer of neonicotinoid pesticide clothianidin and its metabolites in mice. Toxicol Lett 322, 32-38. doi:10.1016/j.toxlet.2020.01.003

Osterauer, R. and Köhler, H. R. H.-R. (2008). Temperature-dependent effects of the pesticides thiacloprid and diazinon on the embryonic development of zebrafish (Danio rerio). Aquat Toxicol 86, 485-494. doi:10.1016/j.aquatox.2007.12.013

Özdemir, H. H., Kara, M., Yumrutas, O. et al. (2014). Determination of the effects on learning and memory performance and related gene expressions of clothianidin in rat models. Cogn Neurodyn 8, 411-416. doi:10.1007/s11571-014-9293-1

Paine, M. (2017). Therapeutic disasters that hastened safety testing of new drugs. Clin Pharmacol Ther 101, 430-434. doi:10.1002/cpt.613

Palpant, N. J., Hofsteen, P., Pabon, L. et al. (2015). Cardiac development in zebrafish and human embryonic stem cells is inhibited by exposure to tobacco cigarettes and e-cigarettes. PLoS One 10, e0126259. doi:10.1371/journal.pone.0126259

Paparella, M., Bennekou, S. H. and Bal-Price, A. (2020). An analysis of the limitations and uncertainties of in vivo developmental neurotoxicity testing and assessment to identify the potential for alternative approaches. Reprod Toxicol 96, 327-336. doi:10.1016/j.reprotox.2020.08.002

Papke, R. L., Ono, F., Stokes, C. et al. (2012). The nicotinic acetylcholine receptors of zebrafish and an evaluation of pharmacological tools used for their study. Biochem Pharmacol 84, 352-365. doi:10.1016/j.bcp.2012.04.022

Parker, B. and Connaughton, V. P. (2007). Effects of nicotine on growth and development in larval zebrafish. Zebrafish 4, 59-68. doi:10.1089/zeb.2006.9994

Pisa, L., Goulson, D., Yang, E.-C. et al. (2021). An update of the worldwide integrated assessment (WIA) on systemic insecticides. Part 2: Impacts on organisms and ecosystems. Environ Sci Pollut Res 28, 11749-11797. doi:10.1007/s11356-017-0341-3

Raftery, T. D. and Volz, D. C. (2015). Abamectin induces rapid and reversible hypoactivity within early zebrafish embryos. Neurotoxicol Teratol 49, 10-18. doi:10.1016/j.ntt.2015.02.006

Richendrfer, H., Creton, R. and Colwill, R. M. (2014). The embryonic zebrafish as a model system to study effects of environmental toxicants on behavior. In C. Lessman and E. A. Carver (eds.), Zebrafish (245-265). Nova Science Publishers, Inc. https://www.novapublishers.com/wp-content/uploads/2019/05/978-1-63117-558-9_ch12.pdf

Rihel, J., Prober, D. A., Arvanites, A. et al. (2011). Zebrafish behavioural profiling links drugs to biological targets and rest/wake regulation. Nat Chem Biol 327, 348-351. doi:10.1126/science.1183090.Zebrafish

Russell, W. M. S. and Burch, R. L. (1959). The Principles of Humane Experimental Techniques. London, UK: Methuen.

Saint-Amant, L. and Drapeau, P. (1998). Time course of the development of motor behaviors in the zebrafish embryo. J Neurobiol 37, 622-632. doi:10.1002/(SICI)1097-4695(199812)37:4<622::AID-NEU10>3.0.CO;2-S

Sano, K., Isobe, T., Yang, J. et al. (2016). In utero and lactational exposure to acetamiprid induces abnormalities in socio-sexual and anxiety-related behaviors of male mice. Front Neurosci 10, 228-240. doi:10.3389/fnins.2016.00228

Selderslaghs, I. W. T., Hooyberghs, J., De Coen, W. et al. (2010). Locomotor activity in zebrafish embryos: A new method to assess developmental neurotoxicity. Neurotoxicol Teratol 32, 460-471. doi:10.1016/j.ntt.2010.03.002

Selderslaghs, I. W. T., Hooyberghs, J., Blust, R. et al. (2013). Assessment of the developmental neurotoxicity of compounds by measuring locomotor activity in zebrafish embryos and larvae. Neurotoxicol Teratol 37, 44-56. doi:10.1016/j.ntt.2013.01.003

Sheets, L. P. (1994). An acute oral neurotoxicity screening study with technical grade imidacloprid (NTN 33893) in rats. Unpublished report from Miles Inc. report No. MOB7221, dated 16 February 1994, GLP, supplement report No. MOB7221, dated 7 June 1994. Submitted to WHO by Bayer AG, Mannheim, Germany.

Sobanska, M., Scholz, S., Nyman, A.-M. M. et al. (2018). Applicability of the fish embryo acute toxicity (FET) test (OECD 236) in the regulatory context of registration, evaluation, authorisation, and restriction of chemicals (REACH). Environ Toxicol Chem 37, 657-670. doi:10.1002/etc.4055

Sparks, T. C. and Nauen, R. (2015). IRAC: Mode of action classification and insecticide resistance management. Pestic Biochem Physiol 121, 122-128. doi:10.1016/j.pestbp.2014.11.014

Strähle, U., Scholz, S., Geisler, R. et al. (2012). Zebrafish embryos as an alternative to animal experiments – A commentary on the definition of the onset of protected life stages in animal welfare regulations. Reprod Toxicol 33, 128-132. doi:10.1016/j.reprotox.2011.06.121

Svoboda, K. R., Vijayaraghavan, S. and Tanguay, R. L. (2002). Nicotinic receptors mediate changes in spinal motoneuron development and axonal pathfinding in embryonic zebrafish exposed to nicotine. J Neurosci 22, 10731-10741. http://www.ncbi.nlm.nih.gov/pubmed/12486166

Takada, T., Yoneda, N., Hirano, T. et al. (2018). Verification of the causal relationship between subchronic exposures to dinotefuran and depression-related phenotype in juvenile mice. J Vet Med Sci 80, 720-724. doi:10.1292/jvms.18-0022

Tanaka, T. (2012). Reproductive and neurobehavioral effects of clothianidin administered to mice in the diet. Birth Defects Res Part B Dev Reprod Toxicol 95, 151-159. doi:10.1002/bdrb.20349

Terayama, H., Endo, H., Tsukamoto, H. et al. (2016). Acetamiprid accumulates in different amounts in murine brain regions. Int J Environ Res Public Health 13, 937. doi:10.3390/ijerph13100937

Tian, X., Hong, X., Yan, S. et al. (2020). Neonicotinoids caused oxidative stress and DNA damage in juvenile Chinese rare minnows (Gobiocypris rarus). Ecotoxicol Environ Saf 197, 110566. doi:10.1016/j.ecoenv.2020.110566

Tomizawa, M. and Casida, J. E. (2005). Neonocotinoid insecticide toxicology: Mechanisms of selective action. Annu Rev Pharmacol Toxicol 45, 247-268. doi:10.1146/annurev.pharmtox.45.120403.095930

Vargesson, N. (2015). Thalidomide-induced teratogenesis: History and mechanisms. Birth Defects Res Part C Embryo Today Rev 105, 140-156. doi:10.1002/bdrc.21096

Vargesson, N. (2019). The teratogenic effects of thalidomide on limbs. J Hand Surg Eurl 44, 88-95. doi:10.1177/1753193418805249

Vignet, C., Cappello, T., Fu, Q. et al. (2019). Imidacloprid induces adverse effects on fish early life stages that are more severe in Japanese medaka (Oryzias latipes) than in zebrafish (Danio rerio). Chemosphere 225, 470-478. doi:10.1016/j.chemosphere.2019.03.002

Vliet, S. M., Ho, T. C. and Volz, D. C. (2017). Behavioral screening of the LOPAC1280 library in zebrafish embryos. Toxicol Appl Pharmacol 329, 241-248. doi:10.1016/j.taap.2017.06.011

von Hellfeld, R., Brotzmann, K., Baumann, L. et al. (2020). Adverse effects in the fish embryo acute toxicity (FET) test: A catalogue of unspecific morphological changes versus more specific effects in zebrafish (Danio rerio) embryos. Environ Sci Eur 32, 122. doi:10.1186/s12302-020-00398-3

Vorhees, C. V., Williams, M. T., Hawkey, A. B. et al. (2021). Translating neurobehavioral toxicity across species from zebrafish to rats to humans: Implications for risk assessment. Front Toxicol 3, 629229. doi:10.3389/ftox.2021.629229

Wang, Y., Yang, G., Dai, D. et al. (2017). Individual and mixture effects of five agricultural pesticides on zebrafish (Danio rerio) larvae. Environ Sci Pollut Res 24, 4528-4536. doi:10.1007/s11356-016-8205-9

Wang, Y., Li, X., Yang, G. et al. (2020). Changes of enzyme activity and gene expression in embryonic zebrafish co-exposed to beta-cypermethrin and thiacloprid. Environ Pollut 256, 113437. doi:10.1016/j.envpol.2019.113437

Welsh, L., Tanguay, R. L. and Svoboda, K. R. (2009). Uncoupling nicotine mediated motoneuron axonal pathfinding errors and muscle degeneration in zebrafish. Toxicol Appl Pharmacol 237, 29-40. doi:10.1016/j.taap.2008.06.025

Wilt, F. H. and Hake, S. (2003). Principles of Developmental Biology. W.W. Norton & Company.

Wu, S., Li, X., Liu, X. et al. (2018). Joint toxic effects of triazophos and imidacloprid on zebrafish (Danio rerio). Environ Pollut 235, 470-481. doi:10.1016/j.envpol.2017.12.120

Yoneda, N., Takada, T., Hirano, T. et al. (2018). Peripubertal exposure to the neonicotinoid pesticide dinotefuran affects dopaminergic neurons and causes hyperactivity in male mice. J Vet Med Sci 80, 634-637. doi:10.1292/jvms.18-0014

Yoo, M. H., Rah, Y. C., Park, S. et al. (2018). Impact of nicotine exposure on hair cell toxicity and embryotoxicity during zebrafish development. Clin Exp Otorhinolaryngol 11, 109-117. doi:10.21053/ceo.2017.00857

Yoshida, H. (1989). Hydrolysis of NTN 33893. Report No. NR1276. Japan.

Zhang, H. and Zhao, L. (2017). Influence of sublethal doses of acetamiprid and halosulfuron-methyl on metabolites of zebrafish (Brachydanio rerio). Aquat Toxicol 191, 85-94. doi:10.1016/j.aquatox.2017.08.002

Zindler, F., Beedgen, F., Brandt, D. et al. (2019a). Analysis of tail coiling activity of zebrafish (Danio rerio) embryos allows for the differentiation of neurotoxicants with different modes of action. Ecotoxicol Environ Saf 186, 109754. doi:10.1016/j.ecoenv.2019.109754

Zindler, F., Beedgen, F. and Braunbeck, T. (2019b). Time-course of coiling activity in zebrafish (Danio rerio) embryos exposed to ethanol as an endpoint for developmental neurotoxicity (DNT) – Hidden potential and underestimated challenges. Chemosphere 235, 12-20. doi:10.1016/j.chemosphere.2019.06.154

Zindler, F., Stoll, S., Baumann, L. et al. (2020). Do environmentally relevant concentrations of fluoxetine and citalopram impair stress-related behavior in zebrafish (Danio rerio) embryos? Chemosphere 261, 127753. doi:10.1016/j.chemosphere.2020.127753

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