Skip to main content
SpringerLink
Log in

Early Life Stage Assays in Zebrafish

  • Protocol
  • First Online:
Toxicity Assessment

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2240))

Abstract

Fish embryo toxicity (FET) test using zebrafish (Danio rerio) has been established as an alternative assay to animal experimentation. The FET assay enables the assessment of multiple morphological endpoints during the development of zebrafish early life stages, showing high impact to the field of ecotoxicology on risk assessment of chemicals and pollutants. Moreover, it is also applied to screening drug-induced toxicity and human diseases, due to the high genetic and physiological orthology between zebrafish and humans. Here, we describe FET test, with all steps and several adaptations involved in the methodological procedures. To demonstrate the efficiency of this method, results using the reference substance 3,4-dichloroaniline (DCA) were included to demonstrate sublethal and teratogenic malformations on zebrafish embryos. Thus, there is a strong tendency for using FET tests as a replacement strategy of traditional tests in toxicology and ecotoxicology.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 179.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. OECD (2013) Test no. 236: fish embryo acute toxicity (FET) test. OECD guideline for the testing of chemicals, section 2. OECD publishing, Paris, France

    Google Scholar 

  2. Braunbeck T, Bottcher M, Hollert H et al (2005) Towards an alternative for the acute fish LC50 test in chemical assessment: the fish embryo toxicity test goes multi-species—an update. ALTEX 22(2/05):87–102

    PubMed  Google Scholar 

  3. Scholz S, Fischer S, Gündel U, Küster E, Luckenbach T, Voelker D (2008) The zebrafish embryo model in environmental risk assessment—applications beyond acute toxicity testing. Environ Sci Pollut Res 15:394–404. https://doi.org/10.1007/s11356-008-0018-z

    Article  CAS  Google Scholar 

  4. Lammer E, Kamp HG, Hisgen V, Koch M, Reinhard D, Salinas ER, Wendler K, Zok S, Braunbeck T (2009) Development of a flow-through system for the fish embryo toxicity test (FET) with the zebrafish (Danio rerio). Toxicol In Vitro 23:1436–1442. https://doi.org/10.1016/j.tiv.2009.05.014

    Article  CAS  PubMed  Google Scholar 

  5. Embry MR, Belanger SE, Braunbeck TA, Galay-Burgos M, Halder M, Hinton DE, Léonard MA, Lillicrap A, Norberg-King T, Whale G (2010) The fish embryo toxicity test as an animal alternative method in hazard and risk assessment and scientific research. Aquat Toxicol 97:79–87. https://doi.org/10.1016/j.aquatox.2009.12.008

    Article  CAS  PubMed  Google Scholar 

  6. Strähle U, Scholz S, Geisler R, Greiner P, Hollert H, Rastegar S, Schumacher A, Selderslaghs I, Weiss C, Witters H, Braunbeck T (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. https://doi.org/10.1016/j.reprotox.2011.06.121

    Article  CAS  PubMed  Google Scholar 

  7. Knöbel M, Busser FJM, Rico-Rico A, Kramer NI, Hermens JLM, Hafner C, Tanneberger K, Schirmer K, Scholz S (2012) Predicting adult fish acute lethality with the zebrafish embryo: relevance of test duration, endpoints, compound properties, and exposure concentration analysis. Environ Sci Technol 46:9690–9700. https://doi.org/10.1021/es301729q

    Article  CAS  PubMed  Google Scholar 

  8. Scholz S, Sela E, Blaha L, Braunbeck T, Galay-Burgos M, García-Franco M, Guinea J, Klüver N, Schirmer K, Tanneberger K, Tobor-Kapłon M, Witters H, Belanger S, Benfenati E, Cretonm S, Cronin MTD, Eggen RIL, Embry M, Ekman D, Gourmelon A, Halder M, Hardy B, Hartung T, Hubesch B, Jungmann D, Lampi MA, Lee L, Léonard M, Küster E, Lillicrap A, Luckenbach T, Murk AJ, Navas JM, Peijnenburg W, Repetto G, Salinas E, Schüürmann G, Spielmann H, Tollefsen KE, Walter-Rohde S, Whale G, Wheeler JR, Winter MJ (2013) A European perspective on alternatives to animal testing for environmental hazard identification and risk assessment. Regul Toxicol Pharmacol 67:506–530. https://doi.org/10.1016/j.yrtph.2013.10.003

    Article  PubMed  Google Scholar 

  9. EU. 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. Off. J. Eur. Union L 276/33

    Google Scholar 

  10. Postlethwait JH, Yan YL, Gates MA, Horne S, Amores A, Brownlie A, Donovan A, Egan ES, Force A, Gong Z, Goutel C, Fritz A, Kelsh R, Knapik E, Liao E, Paw B, Ransom D, Singer A, Thomson M, Abduljabbar TS, Yelick P, Beier D, Joly JS, Larhammar D, Rosa F, Westerfield M, Zon LI, Johnson SL, Talbot WS (1998) Vertebrate genome evolution and the zebrafish gene map. Nat Genet 18:345–349

    Article  CAS  Google Scholar 

  11. Barbazuk WB, Korf I, Kadavi C, Heyen J, Tate S, Wun E, Bedell JA, McPherson JD, Johnson SL (2000) The syntenic relationship of the zebrafish and human genomes. Genome Res 10:1351–1358. https://doi.org/10.1101/gr.144700

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kettleborough RNW, Busch-Nentwich EM, Harvey SA, Dooley CM, Bruijn E, Eeden F, Sealy I, White RJ, Herd C, Nijman IJ, Fényes F, Mehroke S, Scahill C, Gibbons R, Wali N, Carruthers S, Hall A, Yen J, Cuppen E, Stemple DL (2013) A systematic genome-wide analysis of zebrafish protein-coding gene function. Nat Lett Res 496:494–497. https://doi.org/10.1038/nature11992

    Article  CAS  Google Scholar 

  13. Legler J, Velzen M, Cenijn PH, Houtman CJ, Lamoree MH, Wegener JW (2011) Effect-directed analysis of municipal landfill soil reveals novel developmental toxicants in the zebrafish Danio rerio. Environ Sci Technol 45:8552–8558. https://doi.org/10.1021/es201099s

    Article  CAS  PubMed  Google Scholar 

  14. Liu Y, Semina EV (2012) pitx2 deficiency results in abnormal ocular and craniofacial development in zebrafish. PLoS One 7(1):e30896. https://doi.org/10.1371/journal.pone.0030896

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Truong L, Gonnerman G, Simonich MT, Tanguay RL (2016) Assessment of the developmental and neurotoxicity of the mosquito control larvicide, pyriproxyfen, using embryonic zebrafish. Environ Pollut 218:1089–1093. https://doi.org/10.1016/j.envpol.2016.08.061

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Dzieciolowska S, Larroque AL, Kranjec EA, Drapeau P, Samarut E (2017) The larvicide pyriproxyfen blamed during the Zika virus outbreak does not cause microcephaly in zebrafish embryos. Nat Sci Rep 7:40067. https://doi.org/10.1038/srep40067

    Article  CAS  Google Scholar 

  17. Grisolia CK, Oliveira R, Domingues I, Oliveira-Filho EC, Monerat RG, Soares AMVM (2009) Genotoxic evaluation of different δ-endotoxins from Bacillus thuringiensis on zebrafish adults and development in early life stages. Mutat Res 672:119–123. https://doi.org/10.1016/j.mrgentox.2008.10.017

    Article  CAS  PubMed  Google Scholar 

  18. Zhu X, Tian S, Cai Z (2012) Toxicity assessment of iron oxide nanoparticles in zebrafish (Danio rerio) early life stages. PLoS One 7(9):e46286. https://doi.org/10.1371/journal.pone.0046286

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Pérez J, Domingues I, Monteiro M, Soares AMVM, Loureiro S (2013) Synergistic effects caused by atrazine and terbuthylazine on chlorpyrifos toxicity to early-life stages of the zebrafish Danio rerio. Environ Sci Pollut Res 20:4671–4680. https://doi.org/10.1007/s11356-012-1443-6

    Article  CAS  Google Scholar 

  20. Oliveira R, McDonough S, Ladewig JCL, Soares AMVM, Nogueira AJA, Domingues I (2013) Effects of oxytetracycline and amoxicillin on development and biomarkers activities of zebrafish (Danio rerio). Environ Toxicol Pharmacol 36:903–912. https://doi.org/10.1016/j.etap.2013.07.019

    Article  CAS  PubMed  Google Scholar 

  21. Oliveira GAR, Lapuente J, Teixidó E, Porredón C, Borràs M, Oliveira DP (2016) Textile dyes induce toxicity on zebrafish early life stages. Environ Toxicol Chem 35(2):429–434. https://doi.org/10.1002/etc.3202

    Article  CAS  PubMed  Google Scholar 

  22. Scholz S (2013) Zebrafish embryos as an alternative model for screening of drug-induced organ toxicity. Arch Toxicol 87:767–769. https://doi.org/10.1007/s00204-013-1044-2

    Article  CAS  PubMed  Google Scholar 

  23. Zhong TP, Rosenberg M, Mohideen MA, Weinstein B, Fishman MC (2000) Gridlock, an HLH gene required for assembly of the aorta in zebrafish. Science 287(5459):1820–1824. https://doi.org/10.1126/science.287.5459.1820

    Article  CAS  PubMed  Google Scholar 

  24. Beattie CE, Carrel TL, McWhorter ML (2007) Fishing for a mechanism: using zebrafish to understand spinal muscular atrophy. J Child Neurol 22(8):995–1003. https://doi.org/10.1177/0883073807305671

    Article  PubMed  Google Scholar 

  25. Chhetri J, Jacobson G, Gueven N (2014) Zebrafish—on the move towards ophthalmological research. Eye 28:367–380. https://doi.org/10.1038/eye.2014.19

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. OECD (2011) Validation report (phase 1) for the zebrafish embryo toxicity test: part I and part II. Series on testing and assessment no. 157. OECD, Paris

    Google Scholar 

  27. OECD (2012) Validation report (phase 2) for the zebrafish embryo toxicity test: part I and part II (annexes). Series on testing and assessment no. 179. OECD, Paris

    Google Scholar 

  28. McNabb A, Scott K, Ochsenstein E, Seufert K, Carl M (2012) Don't be afraid to set up your fish facility. Zebrafish 9(3):120–125. https://doi.org/10.1089/zeb.2012.0768

    Article  PubMed  Google Scholar 

  29. Paige C, Hill B, Canterbury J, Sweitzer S, Romero-Sandoval EA (2014) Construction of an affordable and easy-to-build zebrafish facility. J Vis Exp 93:e51989. https://doi.org/10.3791/51989

    Article  Google Scholar 

  30. Nagel R (2002) DarT: the embryo test with the zebrafish Danio rerio – a general model in ecotoxicology and toxicology. ALTEX 19(1/02):38–48

    PubMed  Google Scholar 

  31. ISO (1996) International Standards. Water quality—Determination of the acute lethal toxicity of substances to a freshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)]. ISO 7346-3: Flow-through method. http://www.iso.org

  32. OECD (2000) Guidance document on aquatic toxicity testing of difficult substances and mixtures. Series on testing and assessment no. 23. OECD, Paris

    Google Scholar 

  33. EU. European Union Risk Assessment Report, 2006. 3,4-dichloroaniline (3,4-DCA), CAS No: 95–76-1. EINECS No: 202–448-4. Summary risk assessment report. Institute for Health and Consumer Protection, European Chemicals Bureau, I-21020 Ispra (VA) Italy

    Google Scholar 

  34. EU. Commission Recommendation 2006/283/EC of 11 April 2006 on risk reduction measures for the substances: Dibutylphthalate; 3,4-Dichloroaniline; Di-‘isodecyl’ phthalate; 1,2-Benzenedicarboxylic acid, di-C9–11-branched alkyl esters, C10-rich; Di-‘isononyl’ phthalate; 1,2-Benzenedicarboxylic acid, di-C8–10-branched alkyl esters, C9-rich; Ethylenediaminetetraacetate; Methyl acetate; Monochloroacetic acid; n-Pentane; Tetrasodium ethylenediaminetetraacetate. Off. J. Eur. Union L 104/45

    Google Scholar 

  35. Safety Data Sheet 3,4-dichloroaniline (2012) Sigma-Aldrich Co. LLC Product number 437778. http://www.sigmaaldrich.com

  36. ZFIN. The Zebrafish Information Network. Wild-type lines. Available via https://zfin.org/action/feature/wildtype-list. Accessed 10 Jan 2017

Download references

Acknowledgments

We thank the financial support of São Paulo Research Foundation (FAPESP Processes No. 2013/14397-0, 2014/27009-0 and 2013/01509-4, São Paulo, Brazil), Fundação para a Ciência e a Tecnologia (FCT), and POPH/FSE (Programa Operacional Potencial Humano/Fundo Social Europeu) for the research contract of C. Gravato (IF/01401/2014). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001

Author information

Authors and Affiliations

  1. School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil

    Flávia Renata Abe, Klaus Alvaro Guerrieri Accoroni & Danielle Palma de Oliveira

  2. Faculdade de Ciências & CESAM, Universidade de Lisboa, Campo Grande, Lisbon, Portugal

    Carlos Gravato

  3. National Institute for Alternative Technologies ofremoval of Detection, Toxicological Evaluation and Removal of Micropollutants and Radioactives (INCT-DATREM), Unesp, Institute of Chemistry, Araraquara, Brazil

    Danielle Palma de Oliveira

Authors
  1. Flávia Renata Abe
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Klaus Alvaro Guerrieri Accoroni
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Carlos Gravato
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Danielle Palma de Oliveira
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Danielle Palma de Oliveira .

Editor information

Editors and Affiliations

  1. MitoLab, Department of Life Sciences, Center for Neurosciences and Cell Biology of the University of Coimbra, Coimbra, Portugal

    Carlos Manuel Marques Palmeira

  2. School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil

    Danielle Palma de Oliveira

  3. Department of Chemistry, Faculty of Philosophy, Sciences and Letters of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil

    Daniel Junqueira Dorta

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Abe, F.R., Accoroni, K.A.G., Gravato, C., de Oliveira, D.P. (2021). Early Life Stage Assays in Zebrafish. In: Palmeira, C.M.M., de Oliveira, D.P., Dorta, D.J. (eds) Toxicity Assessment. Methods in Molecular Biology, vol 2240. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1091-6_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1091-6_7

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1090-9

  • Online ISBN: 978-1-0716-1091-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation