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Exposure protocol for ecotoxicity testing of microplastics and nanoplastics

Nature Protocols volume 18, pages 3534–3564 (2023)Cite this article

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Abstract

Despite the increasing concern about the harmful effects of micro- and nanoplastics (MNPs), there are no harmonized guidelines or protocols yet available for MNP ecotoxicity testing. Current ecotoxicity studies often use commercial spherical particles as models for MNPs, but in nature, MNPs occur in variable shapes, sizes and chemical compositions. Moreover, protocols developed for chemicals that dissolve or form stable dispersions are currently used for assessing the ecotoxicity of MNPs. Plastic particles, however, do not dissolve and also show dynamic behavior in the exposure medium, depending on, for example, MNP physicochemical properties and the medium’s conditions such as pH and ionic strength. Here we describe an exposure protocol that considers the particle-specific properties of MNPs and their dynamic behavior in exposure systems. Procedure 1 describes the top-down production of more realistic MNPs as representative of MNPs in nature and particle characterization (e.g., using thermal extraction desorption-gas chromatography/mass spectrometry). Then, we describe exposure system development for short- and long-term toxicity tests for soil (Procedure 2) and aquatic (Procedure 3) organisms. Procedures 2 and 3 explain how to modify existing ecotoxicity guidelines for chemicals to target testing MNPs in selected exposure systems. We show some examples that were used to develop the protocol to test, for example, MNP toxicity in marine rotifers, freshwater mussels, daphnids and earthworms. The present protocol takes between 24 h and 2 months, depending on the test of interest and can be applied by students, academics, environmental risk assessors and industries.

Key points

  • In nature, micro- and nanoplastics (MNPs) occur in various shapes, sizes and chemical compositions. Each of these properties can affect both their dynamic behavior and their toxicology and should be considered when performing ecotoxicology experiments to assess their risk.

  • Here, MNPs are generated from plastic waste by using either a ball or centrifugal mill. Their physicochemical properties are measured. Ecotoxicology experiments in soil and aquatic systems are described.

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Fig. 1: An overview of the procedures.
Fig. 2: A comprehensive workflow.
Fig. 3: Ion chromatogram.
Fig. 4: Size-dependent toxicity of MNPs.
Fig. 5: Size-dependent toxicity of MNPs on ROS and oxidative signaling.
Fig. 6: Testing homogeneity in MNP distribution in soil.
Fig. 7: Nanoplastic uptake by plants.

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Data availability

All the data that support the plots within this paper have been previously published21,22,50,51,52.

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Acknowledgements

This work received financial support from the UEF Water research program, which is jointly funded by the Saastamoinen Foundation, the Wihuri Foundation and the Olvi Foundation. The study was also partially funded by the European Union’s Horizon 2020 research and innovation program, via the projects PLASTICSFATE (Grant Agreement number 965367) and POLYRISK (Grant Agreement number 964766). N.T. acknowledges the Canada Research Chair program and the Killam Research Fellowship.

Author information

Authors and Affiliations

  1. Department of Chemical Sciences, University of Padua, Padua, Italy

    Fazel Abdolahpur Monikh

  2. Department of Environmental and Biological Sciences, University of Eastern Finland, Joensuu, Finland

    Fazel Abdolahpur Monikh, Raine Kortet, Jarkko Akkanen & Jussi V. K. Kukkonen

  3. Department of Environmental and Biological Sciences, University of Eastern Finland, Kuopio, Finland

    Fazel Abdolahpur Monikh, Raine Kortet, Jarkko Akkanen & Jussi V. K. Kukkonen

  4. Department of Environmental & Resource Engineering, Technical University of Denmark, Kongens Lyngby, Denmark

    Anders Baun & Nanna B. Hartmann

  5. Department of Biological Sciences, College of Science, Sungkyunkwan University, Suwon, South Korea

    Jae-Seong Lee

  6. State Key Laboratory of Estuarine and Coastal Research, East China Normal University, Shanghai, China

    Huahong Shi

  7. UK Centre for Ecology and Hydrology, Crowmarsh Gifford, UK

    Elma Lahive

  8. SIB Labs, University of Eastern Finland, Kuopio, Finland

    Emilia Uurasjärvi

  9. Department of Chemical Engineering, McGill University, Montreal, Quebec, Canada

    Nathalie Tufenkji

  10. Federal Institute of Material Research and Testing (BAM), Berlin, Germany

    Korinna Altmann & Yosri Wiesner

  11. Department of Plankton and Microbial Ecology, Leibniz Institute for Freshwater Ecology and Inland Fisheries, Stechlin, Germany

    Hans-Peter Grossart

  12. Institute for Biochemistry and Biology, Potsdam University, Potsdam, Germany

    Hans-Peter Grossart

  13. Institute of Environmental Sciences (CML), Leiden University, Leiden, the Netherlands

    Willie Peijnenburg

  14. National Institute of Public Health and the Environment (RIVM), Center for Safety of Substances and Products, Bilthoven, the Netherlands

    Willie Peijnenburg

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Contributions

F.A.M. designed the protocol, and conceptualized and supervised the protocol development. All the co-authors contributed to developing, writing, editing and reviewing the protocol.

Corresponding author

Correspondence to Fazel Abdolahpur Monikh.

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Nature Protocols thanks Win Cowger and Xianfei Huang for their contribution to the peer review of this work.

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Key references using this protocol

Jeong, C.-B. et al. Environ. Sci. Technol. 50, 8849–8857 (2016): https://doi.org/10.1021/acs.est.6b01441

Abdelsaleheen, O. et al. Sci. Total Environ. 798, 149196 (2021): https://doi.org/10.1016/j.scitotenv.2021.149196

Abdolahpur Monikh, F. et al. Nano Today 46, 101611 (2022): https://doi.org/10.1016/j.nantod.2022.101611

Xu, E. G. et al. Environ. Sci. Technol. 54, 6859–6868 (2020): https://doi.org/10.1021/acs.est.0c00245

Altmann, K. et al. Appl. Res. e202200078 (2023): https://doi.org/10.1002/appl.202200078

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Abdolahpur Monikh, F., Baun, A., Hartmann, N.B. et al. Exposure protocol for ecotoxicity testing of microplastics and nanoplastics. Nat Protoc 18, 3534–3564 (2023). https://doi.org/10.1038/s41596-023-00886-9

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