The Zebrafish as an Alternative Animal Model for Ecotoxicological Research and Testing

Braga, Ana Paula Andrade; Souza, Leticia Rosa de; Lima, Maria Gabriela Franco; Cruz, Jaqueline Matos; Souza, Ana Cristina Zullo de; Costa, Mariana Santos; Castro, Vera Lucia Scherholz Salgado de; Marin-Morales, Maria Aparecida

doi:10.1590/1678-4324-2024220968

Ana Paula Andrade Braga

Universidade Estadual Paulista, Departamento de Biologia Geral e Microbiologia, Instituto de Biociências, Rio Claro, São Paulo, Brasil

https://orcid.org/0000-0003-4493-0245

Leticia Rosa de Souza

Universidade Estadual Paulista, Departamento de Biologia Geral e Microbiologia, Instituto de Biociências, Rio Claro, São Paulo, Brasil

https://orcid.org/0000-0002-4069-9341

Maria Gabriela Franco Lima

Universidade Estadual Paulista, Departamento de Biologia Geral e Microbiologia, Instituto de Biociências, Rio Claro, São Paulo, Brasil

https://orcid.org/0000-0001-6794-9833

Jaqueline Matos Cruz

Universidade Estadual Paulista, Departamento de Biologia Geral e Microbiologia, Instituto de Biociências, Rio Claro, São Paulo, Brasil

https://orcid.org/0000-0002-3349-4858

Ana Cristina Zullo de Souza

Universidade Estadual Paulista, Departamento de Biologia Geral e Microbiologia, Instituto de Biociências, Rio Claro, São Paulo, Brasil

https://orcid.org/0000-0002-6167-4012

Mariana Santos Costa

Universidade Estadual Paulista, Departamento de Biologia Geral e Microbiologia, Instituto de Biociências, Rio Claro, São Paulo, Brasil

https://orcid.org/0000-0002-5981-1583

Vera Lucia Scherholz Salgado de Castro

Embrapa Meio Ambiente, Laboratório de Ecotoxicologia e Biossegurança, Jaguariúna, São Paulo, Brasil

https://orcid.org/0000-0002-5045-4540

Maria Aparecida Marin-Morales ^**Correspondence: marin.morales@unesp.br; Tel.: +55-19-997866505 (M.A.M.M.).

Universidade Estadual Paulista, Departamento de Biologia Geral e Microbiologia, Instituto de Biociências, Rio Claro, São Paulo, Brasil

https://orcid.org/0000-0003-3890-0648

*Correspondence: marin.morales@unesp.br; Tel.: +55-19-997866505 (M.A.M.M.).

Editor-in-Chief:

Paulo Vitor Farago

Associate Editor:

Ana Cláudia Barana

Conflicts of Interest:

The authors declare no conflict of interest.

Substance	Concentration	Effect	Reference
Immunotoxicity
Cypermethrin (pesticide)	<3 mg/L	Induce the transcription of genes related to cellular apoptosis and immunotoxicity	[³⁵35 Jin Y, Zheng S, Fu Z. Embryonic exposure to cypermethrin induces apoptosis and immunotoxicity in zebrafish (Danio rerio). Fish Shellfish Immunol. 2011;30:1049-54. https://doi.org/10.1016/j.fsi.2011.02.001. https://doi.org/10.1016/j.fsi.2011.02.00... ]
Graphene oxide	1, 5, 10 and 50 mg/L	Significant increase in the expression of cytokines TNF-alpha, IL-1 and IL-6	[³⁰30 Chen M, Yin J, Liang Y, Yuan S, Wang F, Song M, et al. Oxidative stress and immunotoxicity induced by graphene oxide in zebrafish. Aquat. Toxicol. 2016; 174: 54-60. https://doi.org/10.1016/j.aquatox.2016.02.015. https://doi.org/10.1016/j.aquatox.2016.0... ]
Atrazine (pesticide) and metabolites	30, 100 and 300 μgL⁻¹	Altered expressions of cytokines TNF-alpha, IL-1; IL-6 and IL-8	[³¹31 Liu Z, Fu Z, Jin Y. Immunotoxic effects of atrazine and its main metabolites at environmental relevant concentrations on larval zebrafish (Danio rerio). Chemosphere. 2017;166:212-20. https://doi.org/10.1016/j.chemosphere.2016.09.100. https://doi.org/10.1016/j.chemosphere.20... ]
Butchlor (herbicide)	0, 4, 6, 8 and 12 µM	Expression of cytokine genes IL-lß, CXCL-C1c, CC-chem, and IL-8) in early development	[³⁶36 Tu W, Niu L, Liu W, Xu C. Embryonic exposure to butachlor in zebrafish (Danio rerio): Endocrine disruption, developmental toxicity and immunotoxicity. Ecotoxicol Environ Saf. 2013;89:189-95. https://doi.org/10.1016/j.ecoenv.2012.11.031. https://doi.org/10.1016/j.ecoenv.2012.11... ]
Genotoxicity
Sewage effluents	samples from 6 sites	DNA damage measured by Comet Assay	[³⁹39 Babić S, Barišić J, Višić H, Sauerborn Klobučar R, Topić Popović N, Strunjak-Perović I, et al. Embryotoxic and genotoxic effects of sewage effluents in zebrafish embryo using multiple endpoint testing. Water Res. 2017;115:9-21. https://doi.org/10.1016/j.watres.2017.02.049. https://doi.org/10.1016/j.watres.2017.02... ]
Cd and carbon nanotubes	Cd alone and in co-exposure with oxidized multiwalled carbon nanotubes	DNA damage measured by Comet Assay	[⁴¹41 Morozesk M, Franqui LS, Pinheiro FC, Nóbrega JA, Martinez DST, Fernandes MN. Effects of multiwalled carbon nanotubes co-exposure with cadmium on zebrafish cell line: Metal uptake and accumulation, oxidative stress, genotoxicity and cell cycle. Ecotoxicol. Environ. Saf. 2020;202:110892. https://doi.org/10.1016/j.ecoenv.2020.110892. https://doi.org/10.1016/j.ecoenv.2020.11... ]
4-nitroquinoline-1-oxide (model genotoxicant)	0, 0.1, 0.3, 1.1, and 2.9 µg/l	Induced an unscheduled DNA synthesis (UDS test); DNA fragmentation; reduced cell viability (Alkaline filter elution technique); DNA fragmentation in gills and hepatocytes (Comet Assay); increase of micronuclei (Micronucleus test)	[⁴²42 Diekmann M, Waldmann P, Schnurstein A, Grummt T, Braunbeck T, Nagel R. On the relevance of genotoxicity for fish populations II: Genotoxic effects in zebrafish (Danio rerio) exposed to 4-nitroquinoline-1-oxide in a complete life-cycle test. Aquat. Toxicol. 2004;68:27-37. https://doi.org/10.1016/j.aquatox.2004.01.019. https://doi.org/10.1016/j.aquatox.2004.0... ]
Aquatic mixture	22 Water samples from the Danube River	Significant micronucleus induction	[⁴⁴44 Shao Y, Xiao H, Di Paolo C, Deutschmann B, Brack W, Hollert H, et al. Integrated zebrafish-based tests as an investigation strategy for water quality assessment. Water Res. 2019;150:252-60. https://doi.org/10.1016/j.watres.2018.11.039. https://doi.org/10.1016/j.watres.2018.11... ]
Naturally occurring metals in groundwater	Fe (0.8 and 1.3 mg/L); Mn (0.2 and 0.4 mg/L). Groundwater collected from deep tube wells with Fe (0.8/Mn 0.2 mg/L) and Fe (1.3/Mn 0.4 mg/L)	Increase in the frequency of micronucleus in all samples	[⁴⁵45 Marins K, Lazzarotto LMV, Boschetti G, Bertoncello KT, Sachett A, Schindler MSZ, et al. Iron and manganese present in underground water promote biochemical, genotoxic, and behavioral alterations in zebrafish (Danio rerio). Environ. Sci. Pollut. Res. 2019; 26: 23555-23570. https://doi.org/10.1007/s11356-019-05621-0. https://doi.org/10.1007/s11356-019-05621... ]
Ulexite	40 mg/L	Genotoxic damage observed by the Micronucleus assay and Oxidative DNA demage	[⁴⁶46 Alak G, Özgeriş FB, Yeltekin AÇ, Parlak V, Ucar A, Caglar O, et al. Hematological and Hepatic Effects of Ulexite in Zebrafish. Environ. Toxicol. Pharmacol. 2020;80. https://doi.org/10.1016/j.etap.2020.103496. https://doi.org/10.1016/j.etap.2020.1034... ]
Formalin	T1: 0.45 mg L⁻¹; T2: 4.57 mg L⁻¹ T3: 22.86 mg L⁻¹	Increase in the average number of micronuclei in peripheral blood erythrocytes	[⁴⁷47 Resendes AS, dos Santos DS, França FM, Petesse ML, Badaró-Pedroso C, Ferreira CM. Acute toxic and genotoxic effects of formalin in Danio rerio (zebrafish). Ecotoxicology. 2018;27:1379-86. https://doi.org/10.1007/s10646-018-1993-6. https://doi.org/10.1007/s10646-018-1993-... ]
Anticancer drugs	Cyclophosphamide (CP) and ifosfamide (IF) individually and in a mixture with 5-fluorouracil and cisplatin	CP and IF induced DNA break and genomic instability at high, concentrations (environmentally irrelevant); Mixture induced an increase in DNA break at low concentrations (for environmental contamination)	[⁴⁸48 Novak M, Žegura B, Modic B, Heath E, Filipič M. Cytotoxicity and genotoxicity of anticancer drug residues and their mixtures in experimental model with zebrafish liver cells. Sci. Total Environ. 2017; 601-602: 293-300. https://doi.org/10.1016/j.scitotenv.2017.05.115. https://doi.org/10.1016/j.scitotenv.2017... ]
Spent Pot Liner (SPL)	0.32; 0.64; 0.95 g L⁻¹	DNA fragmentation Frequency of micronuclei and damaged nucleoids increased with increasing SPL concentration	[⁴⁹49 Castro TFD, Paiva IM, Carvalho AFS, Assis IL, Palmieri MJ, Andrade-Vieira LF, et al. Genotoxicity of spent pot liner as determined with the zebrafish (Danio rerio) experimental model. Environ. Sci. Pollut. Res. 2018; 25: 11527-35. https://doi.org/10.1007/s11356-018-1404-9. https://doi.org/10.1007/s11356-018-1404-... ]
Epigenotoxicity
Bisphenol A	10 µM	Changes in methylation patterns of nervous system-related genes	[⁵⁷57 Olsvik PA, Whatmore P, Penglase SJ, Skjærven KH, D’Auriac MA, Ellingsen S. Associations between behavioral effects of bisphenol A and DNA methylation in zebrafish embryos. Front. Genet. 2019; 10: 1-18. https://doi.org/10.3389/fgene.2019.00184. https://doi.org/10.3389/fgene.2019.00184... ]
Fipronil (inseticide)	100; 200; 400 800 μg/L	Hypermethylation of genes related to D. rerio development	[⁵⁸58 Qian Y, Wang C, Wang J, Zhang X, Zhou Z, Zhao M, et al. Fipronil-induced enantioselective developmental toxicity to zebrafish embryo-larvae involves changes in DNA methylation. Sci. Rep. 2017; 7: 1-11. https://doi.org/10.1038/s41598-017-02255-5. https://doi.org/10.1038/s41598-017-02255... ]
Methylmercury (MeHg)	1; 3; 10; 30; 100 nM	Generations not exposed to the contaminant showed both phenotypic effects and epigenetic changes in male gametes (through the increase of differentially methylated regions)	[⁶⁰60 Carvan MJ, Kalluvila TA, Klingler RH, Larson JK, Pickens M, Mora-Zamorano FX, et al. Mercury-induced epigenetic transgenerational inheritance of abnormal neurobehavior is correlated with sperm epimutations in zebrafish. PLoS One. 2017; 12. https://doi.org/10.1371/journal.pone.0176155. https://doi.org/10.1371/journal.pone.017... ]
Bisphenol A	5; 10; 20 μg/L	Deregulation of reproduction-related genes due to histone modification	[⁶²62 Santangeli S, Maradonna F, Gioacchini G, Cobellis G, Piccinetti CC, Dalla Valle L, et al. BPA-Induced Deregulation of Epigenetic Patterns: Effects on Female Zebrafish Reproduction. Sci. Rep. 2016; 6: 1-11. https://doi.org/10.1038/srep21982. https://doi.org/10.1038/srep21982... ]
Bisphenol A	100; 2000 mg/L	Transmission of histone hyperacetylation patterns from the paternal epigenome to the progeny, impairing embryo development	[⁶³63 González-Rojo S, Lombó M, Fernández-Díez C, Herráez MP. Male exposure to bisphenol a impairs spermatogenesis and triggers histone hyperacetylation in zebrafish testes. Environ. Pollut. 2019; 248: 368-79. https://doi.org/10.1016/j.envpol.2019.01.127. https://doi.org/10.1016/j.envpol.2019.01... ]
Atrazine	0.3; 3; 30 ppb	Affected the expression of miRNA-126, related to the angiogenesis process	[⁵²52 Schlotman KE. An Epigenetic Look at Atrazine Toxicity. J. Purdue Undergrad. Res. 2014; 4: 48-57.]

Substance	Concentration	Effect	Reference
Cardiotoxicity
Pyrimethanil (fungicide)	2, 4, and 6 mg/L	Changes in the transcription of apoptosis-related genes (p53, Bax, Bcl2, Casp 9, and Casp6l1) and heart development-related genes (Tbx2b, Gata4, Myh6, Vmhc, Nppa, Bmp2b, Bpm 4 and Bpm 10)	[⁶⁹69 Meng Y, Zhong K, Xiao J, Huang Y, Wei Y, Tang L, et al. Exposure to pyrimethanil induces developmental toxicity and cardiotoxicity in zebrafish. Chemosphere. 2020; 255: 126889. https://doi.org/10.1016/j.chemosphere.2020.126889. https://doi.org/10.1016/j.chemosphere.20... ]
Deltamethrin	50; 25; 10; 1; 0.1 mg/L	Damage to the cardiovascular system	[⁷²72 Li M, Liu XY, Feng XZ. Cardiovascular toxicity and anxiety-like behavior induced by deltamethrin in zebrafish (Danio rerio) larvae. Chemosphere. 2019; 219: 155-64. https://doi.org/10.1016/j.chemosphere.2018.12.011. https://doi.org/10.1016/j.chemosphere.20... ]
Triclosan and Polycyclic Aromatic Hydrocarbons (PAHs)	0.4; 40; 400 μg TCS/L	Pericardial edema, altered heart structure and regurgitation (backflow across the heart valve)	[⁷³73 Saley A, Hess M, Miller K, Howard D, King-Heiden TC. Cardiac Toxicity of Triclosan in Developing Zebrafish. Zebrafish. 2016; 13: 399-404. https://doi.org/10.1089/zeb.2016.1257. https://doi.org/10.1089/zeb.2016.1257... ]
Neurotoxicity
Triclosan	62.5; 125; 250 μg/L	Neurotoxicity for oligodendrocyte precursor cells (OPCs)	[⁷⁷77 Ling Y, Sun L, Wang D, Jiang J, Sun W, Ai W, et al. Triclosan induces zebrafish neurotoxicity by abnormal expression of miR-219 targeting oligodendrocyte differentiation of central nervous system. Arch. Toxicol. 2020; 94: 857-71. https://doi.org/10.1007/s00204-020-02661-1. https://doi.org/10.1007/s00204-020-02661... ]
Oxybenzone	10; 100; 1000 μg L⁻¹	Reduced locomotion, decreased anxiety-like behavior, less time near/interacting with the shoal, fewer interactions with the mirror image, and decreased exploration of the novel arm in the T-maze test	[⁸¹81 Moreira ALP, Luchiari AC. Effects of oxybenzone on zebrafish behavior and cognition. Sci. Total Environ. 2022; 808. https://doi.org/10.1016/j.scitotenv.2021.152101. https://doi.org/10.1016/j.scitotenv.2021... ]
Buspirone; Chlordiazepoxide; Caffeine	25; 50; 100 mg/L 0,1; 1; 10 mg/L 25; 50; 100 mg/L	Anxiety-like larval behavior	[⁸²82 Varga ZK, Zsigmond Á, Pejtsik D, Varga M, Demeter K, Mikics É, et al. The swimming plus-maze test: a novel high-throughput model for assessment of anxiety-related behaviour in larval and juvenile zebrafish (Danio rerio). Sci. Rep. 2018; 8: 1-11. https://doi.org/10.1038/s41598-018-34989-1. https://doi.org/10.1038/s41598-018-34989... ]
Hepatotoxicity
Acetaminophen, Aspirin, Tetracycline HCl, Sodium valproate, Cyclophosphamide, Erythromycin and two non-hepatotoxic compounds: Sucrose and Biotin	Four concentrations: 1/10 Maximum non-lethal concentration (MNLC); 1/3 MNLC; MNLC and LC₁₀ of each drug or compound	Hepatotoxic drugs induced liver degeneration, reduced liver size and delayed yolk sac absorption in larval zebrafish. Non-hepatotoxic compounds did not have observable adverse effect on zebrafish liver	[⁹¹91 He J, Guo S, Zhu F, Zhu J, Chen Y, Huang C. Journal of Pharmacological and Toxicological Methods Original article A zebra fish phenotypic assay for assessing drug-induced hepatotoxicity. J. Pharmacol. Toxicol. Methods. 2013; 67: 25-32.]
Tebuconazole	1/10 MNLC, 1/3 MNLC, MNLC, LC₁₀)	Alterations in liver histoarchitecture. Increases in reactive oxygen species levels.	[¹⁰⁴104 Li S, Jiang Y, Sun Q, Coffin S, Chen L, Qiao K, Gui W, Zhu G. Tebuconazole induced oxidative stress related hepatotoxicity in adult and larval zebrafish (Danio rerio). Chemosphere. 2020; 241: 125129. https://doi.org/10.1016/j.chemosphere.2019.125129. https://doi.org/10.1016/j.chemosphere.20... ]
Thiophanate-methyl (pesticide)	6.25; 12.5 and 25 mg/L	Disturbed liver metabolism, activated caspase-3 and induced oxidative stress	[¹⁰⁵105 Jia K, Cheng B, Huang L, Xiao J, Bai Z, Liao X, Cao Z, Shen T, Zhang C, Hu C, Lu H. Thiophanate-methyl induces severe hepatotoxicity in zebrafish. Chemosphere. 2020; 248: 125941. https://doi.org/10.1016/j.chemosphere.2020.125941. https://doi.org/10.1016/j.chemosphere.20... ]
Polystyrene microplastics	5µm and 70nm	Inflammation and accumulation of lipids in the animals’ livers. Increase in the activity of oxidative stress enzymes	[¹⁰⁶106 Lu Y, Zhang Y, Deng Y, Jiang W, Zhao Y, Geng J, Ding L, Ren H. Uptake and Accumulation of Polystyrene Microplastics in Zebrafish (Danio rerio) and Toxic Effects in Liver. Environ. Sci. Technol. 2016; 50: 4054-4060. https://doi.org/10.1021/acs.est.6b00183. https://doi.org/10.1021/acs.est.6b00183... ]
Flutolanil (fungicide)	0.25; 50; 1000 μg/L	Activity decreases of oxidative stress enzymes, such as catalase and pathological changes including hepatic vacuolization	[¹⁰⁸108 Teng M, Zhou Y, Song M, Dong K, Chen X, Wang C, Bi S, Zhu W. Chronic Toxic Effects of Flutolanil on the Liver of Zebrafish (Danio rerio). Chem. Res. Toxicol. 2019; 32: 995-1001. https://doi.org/10.1021/acs.chemrestox.8b00300. https://doi.org/10.1021/acs.chemrestox.8... ]
Nephrotoxicity
Microcystin-LR	1; 5; 25 μg/L	Histopathological lesions (renal tubules filled with eosinophilic casts, abnormal renal tubules, intertubular space decrease, and blood infiltration)	[¹¹¹111 Wang CC, Si LF, Guo SN, Zheng JL. Negative effects of acute cadmium on stress defense, immunity, and metal homeostasis in liver of zebrafish: The protective role of environmental zinc dpre-exposure. Chemosphere. 2019; 222: 91-97. https://doi.org/10.1016/j.chemosphere.2019.01.111. https://doi.org/10.1016/j.chemosphere.20... ]
Acetaminophen (n-acetyl-para aminophenol, paracetamol)	22.5 and 45 mM	Kidney malformation (curved and cystic pronephric tube, pronephric duct, and cystic and atrophic glomerulus). The effects were dose-dependent	[¹¹²112 Peng H, Wang Y, Wen C, Wang W, Cheng C, Chen Y. Comparative Biochemistry and Physiology , Part C Nephrotoxicity assessments of acetaminophen during zebra fish embryogenesis. Comp. Biochem. Physiol. Part C. 2010; 151: 480-486. https://doi.org/10.1016/j.cbpc.2010.02.004. https://doi.org/10.1016/j.cbpc.2010.02.0... ]
Aristolochic acid (AA)	0.5 μM	Acute injury (led to a loss of function in the glomerular filtration barrier)	[⁸⁴84 Dasgupta S, Simonich MT, Tanguay RL. Vol 2474, Zebrafish Behavioral Assays in Toxicology. In High-Throughput Screening Assays in Toxicology. Methods in Molecular Biology, Humana, New York; 2022. https://doi.org/10.1007/978-1-0716-2213-1_11. https://doi.org/10.1007/978-1-0716-2213-... ]
Endocrine system disruptions
Water samples from a wastewater dominated stream	Complex mixture of Endocrine Active Compounds (EAC)	Hormonal imbalances affecting testicular growth, reproduction, inducing feminization of male fish, and consequent reduction of population	[¹²⁰120 Lozano N, Rice CP, Pagano J, Zintek L, Barber LB, Murphy EW, Nettesheim T, Minarik T, Schoenfuss HL. Concentration of organic contaminants in fish and their biological effects in a wastewater-dominated urban stream. Sci. Total Environ. 2012; 420: 191-201. https://doi.org/10.1016/j.scitotenv.2011.12.059. https://doi.org/10.1016/j.scitotenv.2011... ]
Sulfentrazone	0.0100; 0.0400; 0.400 mg/L	Influenced steroid biosynthesis	[¹²²122 Jiang J, Wang L, Zhang C, Zhao X. Chemosphere Health risks of sulfentrazone exposure during zebrafish embryo-larvae development at environmental concentration. Chemosphere. 2022; 288: 132632. https://doi.org/10.1016/j.chemosphere.2021.132632. https://doi.org/10.1016/j.chemosphere.20... ]
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)	50 pg/ml	Feminization of the population (appearance of fish with clearly female bodies, yet carrying testes in place of ovaries)	[¹²⁴124 Baker TR, Peterson RE, Heideman W. Early dioxin exposure causes toxic effects in adult zebrafish. Toxicol. Sci. 2013; 135: 241-250. https://doi.org/10.1093/toxsci/kft144. https://doi.org/10.1093/toxsci/kft144... ]
Bisphenol A	0.01; 0.1; 1 mg/L	Increased egg production, and reduced rate of fertilization. Altered the transcription of genes involved in reproductive function and epigenetic processes.	[¹²⁵125 Laing LV, Viana J, Dempster EL, Trznadel M, Trunkfield LA, Uren Webster TM, et al. Bisphenol A causes reproductive toxicity, decreases dnmt1 transcription, and reduces global DNA methylation in breeding zebrafish (Danio rerio). Epigenetics. 2016; 11: 526-538. https://doi.org/10.1080/15592294.2016.1182272. https://doi.org/10.1080/15592294.2016.11... ]
Bisphenol A	2, 10 and 100 μg/L	Lower sperm motility and alterations on velocity parameters of spermatozoa	[¹²⁶126 Silveira CR, Varela Junior AS, Corcini CD, Soares SL, Anciuti AN, Kütter MT, Martínez PE. Effects of Bisphenol A on redox balance in red blood and sperm cells and spermatic quality in zebrafish Danio rerio. Ecotoxicology. 2019; 28: 913-922. https://doi.org/10.1007/s10646-019-02091-5. https://doi.org/10.1007/s10646-019-02091... ]

[1] *Correspondence: marin.morales@unesp.br; Tel.: +55-19-997866505 (M.A.M.M.).

Brasil

Brasil

The Zebrafish as an Alternative Animal Model for Ecotoxicological Research and Testing

Abstract

HIGHLIGHTS

GRAPHICAL ABSTRACT