Cellular Responses of Chlorococcum Sp. Algae Exposed to Zinc Oxide Nanoparticles by Using Flow Cytometry

  • Abdallah OukarroumEmail author
  • Ilias Halimi
  • Mohamed SiajEmail author


Evaluation of 50 nm zinc oxide nanoparticles’ (ZnO-NPs) effects on the microalgae Chlorococcum sp. growing in high salt growth medium (HSM) was investigated by using flow cytometry parameters (cell size (FSC), granularity (SSC), chlorophyll a fluorescence (FL3), and formation of reactive oxygen species (ROS)). Algal cells in exponential growth were exposed to 0–100 mg/L of ZnO-NPs and their physiological responses were measured after 24 and 96 h of treatment. Behavior of ZnO-NPs was analyzed in HSM and results indicated that ZnO-NPs formed agglomeration with a large distribution. Total soluble Zn concentration increased when initial ZnO-NP concentration increased. Significant negative effect on algal cells was observed after 96 h exposition and at high ZnO-NP concentration. This negative impact was evaluated by the significant increase in ROS production, inhibition in the photosynthetic electron transport, and reduction in cell growth. In this study, using flow cytometry multi-parameters might help to prevent and evaluate inhibitory effect of oxide nanoparticles on aquatic photosynthetic microorganisms.


Chlorococcum sp. Zinc oxide nanoparticles ZnO-NPs Reactive oxygen species Flow cytometry 


Author Contributions

Conceived and designed the experiments: AO. Performed the experiments: AO, IH. Analyzed the data: AO, IH, MS. Wrote the paper: AO, MS.

Funding Information

This work was supported through funding from the Natural Science and Engineering Research Council of Canada (NSERC), the Canada Research Chairs program (CRC), and Canada Foundation for Innovation.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflicts of interest.


  1. Aruoja, V., Dubourguier, H.-C., Kasemets, K., & Kahru, A. (2009). Toxicity of nanoparticles of CuO, ZnO and TiO2 to microalgae Pseudokirchneriella subcapitata. Science of the Total Environment, 407, 1461–1468.CrossRefGoogle Scholar
  2. Aubin-Tam, M.-E., & Hamad-Schifferli, K. (2008). Structure and function of nanoparticle–protein conjugates. Biomedical Material, 3, 034001.Google Scholar
  3. Brayner, R., Ferrari-Iliou, R., Brivois, N., Djediat, S., Benedetti, M. F., & Fievet, F. (2006). Toxicological impact studies based on Escherichia coli bacteria in ultrafine ZnO nanoparticles colloidal medium. Nano Letters, 6, 866–870.CrossRefGoogle Scholar
  4. Cakmak, I., & Marschner, H. (1988). Increase in membrane permeability and exudation in roots of zinc deficient plants. Journal of Plant Physiology, 3, 356–361.CrossRefGoogle Scholar
  5. Chang, Y.-N., Zhang, M., Xia, L., Zhang, J., & Xing, G. (2012). The toxic effects and mechanisms of CuO and ZnO nanoparticles. Materials, 5, 2850–2871.CrossRefGoogle Scholar
  6. Collier, J. L. (2000). Flow cytometry and the single cell in phycology. Journal of Phycology, 36, 628–644.CrossRefGoogle Scholar
  7. Dawson, K. A., Salvati, A., & Lynch, I. (2009). Nanotoxicology: nanoparticles reconstruct lipids. Nature Nanotechnology, 4, 84–85.CrossRefGoogle Scholar
  8. El Badawy, A., Silva, R. G., Morris, B., Scheckel, K. G., Suidan, M. T., & Tolaymat, T. M. (2011). Surface charge-dependent toxicity of silver nanoparticles. Environmental Science & Technology, 45, 283–287.CrossRefGoogle Scholar
  9. Franklin, N. M., Rogers, N. J., Apte, S. C., Batley, G. E., Gadd, G. E., & Casey, P. S. (2007). Comparative toxicity of nanoparticulate ZnO, bulk ZnO, and ZnCl2 to a freshwater microalga (Pseudokirchneriella subcapitata): the importance of particle solubility. Environmental Science & Technology, 41, 8484–8490.CrossRefGoogle Scholar
  10. Franqueira, D., Orosa, M., Torres, E., Herrero, C., & Cid, A. (2000). Potential use of flow cytometry in toxicity studies with microalgae. Science of the Total Environment, 247, 119–126.CrossRefGoogle Scholar
  11. Fujino, T., & Itoh, T. (1998). Changes in the three dimensional architecture of the cell wall during lignification of xylem cells in Eucalyptus tereticomis. Holzforschung, 52, 111–116.CrossRefGoogle Scholar
  12. Fujiwara, K., Suematsu, H., Kiyomiya, E., Aoki, M., Sato, M., & Moritoki, N. (2008). Size-dependent toxicity of silica nano-particles to Chlorella kessleri. Journal of Environmental Science and Health. Part A, Toxic/Hazardous Substances & Environmental Engineering, 43, 1167–1173.CrossRefGoogle Scholar
  13. Goodman, C. M., Mc Cusker, C. D., Yilmaz, T., & Rotello, V. M. (2004). Toxicity of gold nanoparticles functionalized with cationic and anionic side chains. Bioconjugate Chemistry, 15, 897–900.CrossRefGoogle Scholar
  14. Gottschalk, F., & Nowack, B. (2011). The release of engineered nanomaterials to the environment. Journal of Environmental Monitoring, 13, 1145–1155.CrossRefGoogle Scholar
  15. Ji, J., Long, Z., & Lin, D. (2011). Toxicity of oxide nanoparticles to the green algae Chlorella sp. Chemical Engineering Journal, 170, 525–553.CrossRefGoogle Scholar
  16. Kaegi, R., Ulrich, A., Sinnet, B., Vonbank, R., Wichser, A., Zuleeg, S., Simmler, H., Brunner, S., Vonmont, H., Burkhardt, M., & Bolier, M. (2008). Synthetic Ti02 nanoparticle emission from exterior facades into the aquatic environment. Environmental Pollution, 156, 233–239.CrossRefGoogle Scholar
  17. Keller, A. A., Wang, H. T., Zhou, D. X., Lenihan, H. S., Cherr, G., Cardinale, B. J., Miller, R., & Ji, Z. X. (2010). Stability and aggregation of metal oxide nanoparticles in natural aqueous matrices. Environmental Science & Technology, 44, 1962–1967.CrossRefGoogle Scholar
  18. Klaine, S. J., Alvarez, P. J. J., Batley, G. E., Fernandes, T. F., Handy, R. D., Lyon, D. Y., Mahendra, S., McLaughlin, M. J., & Lead, J. R. (2008). Nanomaterials in the environment: behavior, fate, bioavailability, and effects. Environmental Toxicology and Chemistry, 27, 1825–1851.CrossRefGoogle Scholar
  19. Krug, H. F., & Wick, P. (2011). Nanotoxicology: an interdisciplinary challenge. Angewandte Chemie, International Edition, 50, 1260–1278.CrossRefGoogle Scholar
  20. Lee, W. M., & An, Y. J. (2013). Effects of zinc oxide and titanium dioxide nanoparticles on green algae under visible, UVA, and UVB irradiations: no evidence of enhanced algal toxicity under UV pre-irradiation. Chemosphere, 91, 536–544.CrossRefGoogle Scholar
  21. Lichtenthaler, H. K. (1987). Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. In L. Packer & R. Douce (Eds.), Methods in enzymology Vol. 148 (pp. 350–382). London: Academic Press.Google Scholar
  22. Manzo, S., Miglietta, M. L., Rametta, G., Buono, S., & Francia, G. D. (2013). Toxic effects of ZnO nanoparticles towards marine algae Dunaliella tertiolecta. Science of the Total Environment, 445–446, 371–376.CrossRefGoogle Scholar
  23. Misra, S.K., Dybowska, A, Berhanu, D., Luoma, S.N., Valsami-Jones, E. (2012). The complexity of nanoparticle dissolution and its importance in nanotoxicological studies. Science of the Total Environment, 438, 225–232.Google Scholar
  24. Mukherjee, S. P., Davoren, M., & Byme, H. J. (2010). In vitro mammalian cytotoxicological study of PAMAM dendrimers - towards quantitative structure activity relationships. Toxicology In Vitro, 24, 1169–1177.CrossRefGoogle Scholar
  25. Nabeshi, H., Yoshikawa, T., Matsuyama, K., Nakazato, Y., Arimori, A., Isobe, M., Tochigi, S., Kondoh, S., Hirai, T., Akase, T., Yamashita, T., Yamashita, K., Yoshida, T., Nagano, K., Abe, Y., Yoshioka, Y., Kamada, H., lmazawa, T., Itoh, N., Tsunoda, S. I., & Tsutsumi, Y. (2010). Size-dependent cytotoxic effects of amorphous silica nanoparticles on Langerhans cells. Pharmazie, 65, 199–201.Google Scholar
  26. Oukarroum, A., Polchtchikov, S., Perreault, F., & Popovic, R. (2012). Temperature influence on silver nanoparticles inhibitory on photosystem II photochemistry in two green algae, Chlorella vulgaris and Dunaliella tertiolecta. Environmental Science and Pollution Research, 19, 1755–1762.CrossRefGoogle Scholar
  27. Oukarroum, A., Samadani, M., & Dewez, D. (2014). Influence of pH on the toxicity of silver nanoparticles to the green algae Chlamydomonas acidophila. Water, Air, and Soil Pollution, 225, 2038.CrossRefGoogle Scholar
  28. Oukarroum, A., Zaidi, W., Samadani, M., Dewez, D. (2017). Toxicity of nickel oxide nanoparticles on freshwater algal strain of Chlorella vulgaris. Biomed Research International,
  29. Peng, X., Palma, S., Fisher, N. S., & Wong, S. S. (2011). Effect of morphology of ZnO nanostructures on their toxicity to marine algae. Aquatic Toxicology, 102, 186–196.CrossRefGoogle Scholar
  30. Perreault, F., Oukarroum, A., Melegari, S. P., Matias, W. G., & Popovic, R. (2012). Polymer coating of copper oxide nanoparticles increases nanoparticles uptake and toxicity in the green alga Chlamydomonas reinhardtii. Chemosphere, 87, 1388–1394.CrossRefGoogle Scholar
  31. Reddy, K. M., Feris, K., Bell, J., Wingett, D. G., Hanley, C., & Punnoose, A. (2007). Selective toxicity of zinc oxide nanoparticles to prokaryotic and eukaryotic systems. Applied Physics Letters, 90, 2139021–2139023.Google Scholar
  32. Saison, C., Perreault, F., Daigle, J. C., Fortin, C., Claverie, J., Morin, M., & Popovic, R. (2010). Effect of core-shell copper oxide nanoparticles on cell culture morphology and photosynthesis (photosystem II energy distribution) in the green alga, Chlamydomonas reinhardtii. Aquatic Toxicology, 96, 109–114.CrossRefGoogle Scholar
  33. Sakai, N., Matsui, Y., Nakayama, A., Tsuda, A., & Yoneda, M. (2011). Functional-dependent and size-dependent uptake of nanoparticles in PC 12. Journal of Physics Conference Series, 304, 012049.CrossRefGoogle Scholar
  34. Salvatia, A., Nelissen, I., Haase, A., Åberg, C., Moya, S., Jacobs, A., Alnasser, F., Bewersdorff, T., Deville, S., Luch, A., & Dawson, K. A. (2018). Quantitative measurement of nanoparticle uptake by flow cytometry illustrated by an interlaboratory comparison of the uptake of labelled polystyrene nanoparticles. NanoImpact, 9, 42–50.CrossRefGoogle Scholar
  35. Suman, T. Y., Rajasree, S. R. R., & Kirubagaran, R. (2015). Evaluation of zinc oxide nanoparticles toxicity on marine algae chlorella vulgaris through flow cytometric, cytotoxicity and oxidative stress analysis. Ecotoxicology and Environmental Safety, 113, 23–30.CrossRefGoogle Scholar
  36. Wang, B., Feng, W. Y., Wang, M., Wang, T. C., Gu, Y. Q., Zhu, M. T., Ouyang, H., Shi, J. W., Zhang, F., Zhao, Y. L., Chai, Z. F., Wang, H. F., & Wang, J. (2008). Acute toxicological impact of nano- and submicro-scaled zinc oxide powder on healthy adult mice. Journal of Nanoparticle Research, 10, 263–276.CrossRefGoogle Scholar
  37. Wang, Z., Li, J., Zhao, J., & Xing, B. (2011). Toxicity and internalization of CuO nanoparticles to prokaryotic alga Microcystis aeruginosa as affected by dissolved organic matter. Environmental Science & Technology, 45, 6032–6040.CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  1. 1.AgroBioSciencesUniversity Mohammed VI PolytechnicBen GuerirMorocco
  2. 2.Department of ChemistryUniversity of Quebec in MontrealMontrealCanada

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