Toxicity of zinc oxide nanoparticles to zebrafish embryo: a physicochemical study of toxicity mechanism


The biological impact of engineered nanomaterials released into the aquatic environment is a major concern. In this work, the properties of ZnO nanoparticles (nano-ZnO, 30 nm) were characterized in a water suspension (E3 medium), and a zebrafish 96-h post fertilization (hpf) embryo–larval test was performed to assess the toxicity of nano-ZnO suspension. Nano-ZnO was found to readily form aggregates with different sizes; small aggregates (142.4–517.7 nm) were still suspended in E3 medium, but large aggregates (>1 μm) quickly deposited on the bottom of 24-well plates; nano-ZnO was partially dissolved to Zn species (Zn(dis)) in E3 medium. In the nano-ZnO suspension, small aggregates, Zn(dis), and large aggregates might jointly exert influence on the development of zebrafish embryos. The embryo toxicity test revealed that nano-ZnO killed zebrafish embryos (50 and 100 mg/L), retarded the embryo hatching (1–25 mg/L), reduced the body length of larvae, and caused tail malformation after the 96 hpf exposure. Zn(dis) only partially contributed to the toxicity of nano-ZnO. This research highlights the need to further investigate the ecotoxicity of nano-ZnO in the water environment.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6


  1. Adams L, Lyon DY, Alvarez PJJ (2006) Comparative eco-toxicity of nanoscale TiO2, SiO2, and ZnO water suspensions. Water Res 40(19):3527–3532

    Article  CAS  PubMed  Google Scholar 

  2. Beckett WS, Chalupa DF, Pauly-Brown A, Speers DM, Stewart JC, Frampton MW, Utell MJ, Huang LS, Cox C, Zareba W, Oberdörster G (2005) Comparing inhaled ultrafine versus fine zinc oxide particles in healthy adults: a human inhalation study. Am J Respir Crit Care Med 171(10):1129–1135

    Article  PubMed  Google Scholar 

  3. Berube DM (2008) Rhetorical gamesmanship in the nano debates over sunscreens and nanoparticles. J Nanopart Res 10:23–37

    Article  CAS  Google Scholar 

  4. Brand M, Granato M, Nüsslein-Volhard C (2002) In: Nüsslein-Volhard C, Dahm R (eds) Zebrafish: a practical approach. Oxford University Press, Oxford, Chapter 1, pp 7–37

  5. Brayner R, Ferrari-Iliou R, Brivois N, Djediat S, Benedetti MF, Fievet F (2006) Toxicological impact studies based on Escherichia coli bacteria in ultrafine ZnO nanoparticles colloidal medium. Nano Lett 6(4):866–870

    Article  CAS  PubMed  ADS  Google Scholar 

  6. Brunner TJ, Wick P, Manser P, Spohn P, Grass RN, Limbach LK, Bruinink A, Stark WJ (2006) In vitro cytotoxicity of oxide nanoparticles: comparison to asbestos, silica, and the effect of particle solubility. Environ Sci Technol 40(14):4374–4381

    Article  CAS  PubMed  Google Scholar 

  7. Cheng JP, Flahaut E, Cheng SH (2007) Effect of carbon nanotubes on developing zebrafish (Danio rerio) embryos. Environ Toxicol Chem 26(4):708–716

    Article  CAS  PubMed  Google Scholar 

  8. Degen A, Kosec M (2000) Effect of pH and impurities on the surface charge of zinc oxide in aqueous solution. J Eur Ceram Soc 20(6):667–673

    Article  CAS  Google Scholar 

  9. Environmental Protection Agency (2007) Science Policy Council, Nanotechnology Workgroup Nanotechnology White Paper. Available

  10. Federici G, Shaw BJ, Handy RD (2007) Toxicity of titanium dioxide nanoparticles to rainbow trout (Oncorhynchus mykiss): Gill injury, oxidative stress, and other physiological effects. Aquat Toxicol 84(4):415–430

    Article  CAS  PubMed  Google Scholar 

  11. Franklin NM, Rogers NJ, Apte SC, Batley GE, Gadd GE, Casey PS (2007) Comparative toxicity of nanoparticulate ZnO, bulk ZnO, and ZnCl2 to a freshwater microalga (Pseudokirchneriella subcapitata): the importance of particle solubility. Environ Sci Technol 41(24):8484–8490

    Article  CAS  PubMed  Google Scholar 

  12. Fraysse B, Mons R, Garric J (2006) Development of a zebrafish 4-day embryo-larval bioassay to assess toxicity of chemicals. Ecotoxicol Environ Saf 63(2):253–267

    Article  CAS  PubMed  Google Scholar 

  13. Gojova A, Guo B, Kota RS, Rutledge JC, Kennedy IM, Barakat AI (2007) Induction of inflammation in vascular endothelial cells by metal oxide nanoparticles: effect of particle composition. Environ Health Perspect 115(3):403–409

    Article  CAS  PubMed  Google Scholar 

  14. Hallare A, Nagel K, Kohler HR, Triebskorn R (2006) Comparative embryotoxicity and proteotoxicity of three carrier solvents to zebrafish (Danio rerio) embryos. Ecotoxicol Environ Saf 63:378–388

    Article  CAS  PubMed  Google Scholar 

  15. Inohaya K, Yasumasu S, Araki K, Naruse K, Yamazaki K, Yasumasu I, Iuchi I, Yamagami K (1997) Species-dependent migration of fish hatching gland cells that commonly express astacin-like proteases in common. Dev Growth Differ 39(2):191–197

    Article  CAS  PubMed  Google Scholar 

  16. Lam HF, Conner MW, Rogers AE, Fitzgerald S, Amdur MO (1985) Functional and morphologic changes in the lungs of guinea pigs exposed to freshly generated ultrafine zinc oxide. Toxicol Appl Pharmacol 78(1):29–38

    Article  CAS  PubMed  Google Scholar 

  17. Lee KJ, Nallathamby PD, Browning LM, Osgood CJ, Xu XHN (2007) In vivo imaging of transport and biocompatibility of single silver nanoparticles in early development of zebrafish embryos. ACS Nano 1:133–143

    Article  CAS  PubMed  Google Scholar 

  18. Lin DH, Xing BS (2007) Phytotoxicity of nanoparticles: inhibition of seed germination and root growth. Environ Pollut 150(2):243–250

    Article  CAS  PubMed  Google Scholar 

  19. Lin WS, Xu Y, Huang CC, Ma Y, Shannon KB, Chen DR, Huang YW (2009) Toxicity of nano- and micro-sized ZnO particles in human lung epithelial cells. J Nanopart Res 11:25–29

    Article  CAS  Google Scholar 

  20. Long TC, Tajuba J, Sama P, Saleh N, Swartz C, Parker J, Hester S, Lowry GV, Veronesi B (2007) Nanosize titanium dioxide stimulates reactive oxygen species in brain microglia and damages neurons in vitro. Environ Health Perspect 115(11):1631–1637

    Article  CAS  PubMed  Google Scholar 

  21. Rawson DM, Zhang T, Kalicharan D, Jongebloed WL (2000) Field emission scanning electron microscopy and transmission electron microscopy studies of the chorion, plasma membrane and syncytial layers of the gastrula-stage embryo of the zebrafish Brachydanio rerio: a consideration of the structural and functional relationship with respect to cryoprotectant penetration. Aquac Res 31:325–336

    Article  Google Scholar 

  22. Reichle RA, McCurdy KG, Hepler LG (1975) Zinc hydroxide: solubility product and hydroxy-complex stability constants from 12.5–75°C. Can J Chem 53(24):3841–3845

    Article  CAS  Google Scholar 

  23. Samson JC, Shenker J (2000) The teratogenic effects of methylmercury on early development of the zebrafish, Danio rerio. Aquat Toxicol 48:343–354

    Article  CAS  PubMed  Google Scholar 

  24. Schulte C, Nagel R (1994) Testing acute toxicity in the embryo of zebrafish, Brachydanio rerio, as alternative to the acute fish test: preliminary results. Altern Lab Anim 22(1):12–19

    Google Scholar 

  25. Strehlow WH, Cook EL (1973) Compilation of energy band gaps in elemental and binary compound semiconductors and insulators. J Phys Chem Ref Data 2:163–193

    CAS  ADS  Google Scholar 

  26. Usenko CY, Harper SL, Tanguay RL (2007) In vivo evaluation of carbon fullerene toxicity using embryonic zebrafish. Carbon 45(9):1891–1898

    Article  CAS  PubMed  Google Scholar 

  27. von Westernhagen H (1988) Sublethal effects of pollutants on fish eggs and larvae. In: Hoar W, Randall DJ (eds) The physiology of developing fish. Fish physiology II. Academic Press, New York, pp 253–346

    Chapter  Google Scholar 

  28. Wang ZL (2004) Zinc oxide nanostructures: growth, properties and applications. J Phys Condens Matter 16:829–858

    Article  CAS  ADS  Google Scholar 

  29. Wang B, Feng WY, Wang M, Wang TC, Gu YQ, Zhu MT, Ouyang H, Shi JW, Zhang F, Zhao YL, Chai ZF, Wang HF, Wang J (2008) Acute toxicological impact of nano-and submicro-scaled zinc oxide powder on healthy adult mice. J Nanopart Res 10(2):263–276

    Article  CAS  Google Scholar 

  30. Yang ZH, Xie CS (2006) Zn2+ release from zinc and zinc oxide particles in simulated uterine solution. Colloid Surf B 47(2):140–145

    Article  CAS  Google Scholar 

  31. Zhang LL, Jiang YH, Ding YL, Povey M, York D (2007) Investigation into the antibacterial behaviour of suspensions of ZnO nanoparticles (ZnO nanofluids). J Nanopart Res 9(3):479–489

    Article  CAS  Google Scholar 

  32. Zhang Y, Chen YS, Westerhoff P, Hristovski K, Crittenden JC (2008) Stability of commercial metal oxide nanoparticles in water. Water Res 42(8–9):2204–2212

    Article  CAS  PubMed  Google Scholar 

  33. Zhu XS, Zhu L, Li Y, Duan ZH, Chen W, Alvarez PJJ (2007) Developmental toxicity in zebrafish (Danio rerio) embryos after exposure to manufactured nanomaterials: buckminster fullerene aggregates (nC60) and fullerol. Environ Toxicol Chem 26(5):976–979

    Article  CAS  PubMed  Google Scholar 

  34. Zhu XS, Zhu L, Chen YS, Tian SY (2008a) Acute toxicities of six manufactured nanomaterial suspensions to Daphnia magna. J Nanopart Res 11:67–75

    Article  CAS  Google Scholar 

  35. Zhu XS, Zhu L, Duan ZH, Qi RQ, Li Y, Lang YP (2008b) Comparative toxicity of several metal oxide nanoparticle aqueous suspensions to zebrafish (Danio rerio) early developmental stage. J Environ Sci Health A 43(3):278–284

    Article  CAS  Google Scholar 

Download references


The authors thank the National Zebrafish Resources of China, Peking University, for kindly providing zebrafish strains. This research is supported by National Natural Science Foundation of China (Grant No. 10505024, 20707027, 10875136), the Knowledge Innovation Program of the Chinese Academy of Sciences (Grant No. KJCX3.SYW.N3), and the Ministry of Science and Technology of China (Grant No. 2006CB705605).

Author information



Corresponding author

Correspondence to Zhiyong Zhang.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Bai, W., Zhang, Z., Tian, W. et al. Toxicity of zinc oxide nanoparticles to zebrafish embryo: a physicochemical study of toxicity mechanism. J Nanopart Res 12, 1645–1654 (2010).

Download citation


  • ZnO nanoparticles
  • Solubility
  • Aggregate
  • Toxicity
  • Zebrafish embryo
  • Environment
  • EHS