Skip to main content
SpringerLink
Log in

Nile red staining in microplastic analysis—proposal for a reliable and fast identification approach for large microplastics

  • Published:
Environmental Monitoring and Assessment Aims and scope Submit manuscript

Abstract

A variety of methods concerning the identification of microplastics in environmental samples exist. While visual identification is often used, implemented easily, and cost-efficient but implying biased results, spectroscopic or chromatographic approaches are reliable but time-consuming and need specific equipment. Nile red staining is an available alternative and complement method for identifying microplastics. In this study, Nile red staining and subsequent photographing in a UV light photobox was tested on its reliability and feasibility. The approach was compared with a second identification process using again staining but a fluorescence microscope. Selected identified microplastic particles were analyzed by μ-Raman spectroscopy to prove their polymeric origin. The results show that the presented approach is faster compared with the use of a fluorescence microscope or μ-Raman spectroscopy. Furthermore, it is cost-effective as well as accurate for large microplastics > 0.63 mm and, therefore, may be applied when large sample volumes need to be analyzed.

Graphical abstract

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Anbumani, S., & Kakkar, P. (2018). Ecotoxicological effects of microplastics on biota: a review. Environmental Science and Pollution Research, 25, 14373–14396. https://doi.org/10.1007/s11356-018-1999-x.

    Article  CAS  Google Scholar 

  • Andrady, A. L. (2010). Measurement and occurrence of microplastics in the environment, in: Presentation at the 2nd research workshop on microplastic debris. Tacoma, WA.

  • Andrady, A. L. (2015). Persistence of plastic litter in the oceans. In M. Bergmann, L. Gutow, & M. Klages (Eds.), Marine anthropogenic litter (pp. 57–72). Cham: Springer International Publishing. https://doi.org/10.1007/978-3-319-16510-3_3.

    Chapter  Google Scholar 

  • Arthur, C., Baker, J., Bamford, H. (2009). Proceedings of the international research workshop on the occurrence, effects and fate of microplastic marine debris. NOAA marine debris program. Technical memorandum NOS-OR&R-30. Available: https://marinedebris.noaa.gov/proceedings-second-research-workshop-microplastic-marine-debris

  • Barnes, D. K. A., Galgani, F., Thompson, R. C., & Barlaz, M. (2009). Accumulation and fragmentation of plastic debris in global environments. Philosophical Transactions of the Royal Society, B: Biological Sciences, 364, 1985–1998. https://doi.org/10.1098/rstb.2008.0205.

    Article  CAS  Google Scholar 

  • Bonney, R., Cooper, C. B., Dickinson, J., Kelling, S., Phillips, T., Rosenberg, K. V., & Shirk, J. (2009). Citizen science: a developing tool for expanding science knowledge and scientific literacy. BioScience, 59, 977–984. https://doi.org/10.1525/bio.2009.59.11.9.

    Article  Google Scholar 

  • Cohen, J. (1988). In L. Erlbaum Associates (Ed.), Statistical power analysis for the behavioral sciences (2nd ed.). Hillsdale: N.J.

    Google Scholar 

  • Collard, F., Gilbert, B., Eppe, G., Parmentier, E., & Das, K. (2015). Detection of anthropogenic particles in fish stomachs: an isolation method adapted to identification by Raman spectroscopy. Archives of Environmental Contamination and Toxicology, 69, 331–339. https://doi.org/10.1007/s00244-015-0221-0.

    Article  CAS  Google Scholar 

  • Dris, R., Gasperi, J., Rocher, V., Saad, M., Renault, N., & Tassin, B. (2015). Microplastic contamination in an urban area: a case study in Greater Paris. Environment and Chemistry, 12, 592. https://doi.org/10.1071/EN14167.

    Article  CAS  Google Scholar 

  • Erni-Cassola, G., Gibson, M. I., Thompson, R. C., & Christie-Oleza, J. A. (2017). Lost, but found with Nile red: a novel method for detecting and quantifying small microplastics (1 mm to 20 μm) in environmental samples. Environmental Science & Technology, 51, 13641–13648. https://doi.org/10.1021/acs.est.7b04512.

    Article  CAS  Google Scholar 

  • Fischer, M., & Scholz-Böttcher, B. M. (2019). Microplastics analysis in environmental samples—recent pyrolysis-gas chromatography-mass spectrometry method improvements to increase the reliability of mass-related data. Analytical Methods, 11, 2489–2497. https://doi.org/10.1039/C9AY00600A.

    Article  Google Scholar 

  • Fischer, E. K., Paglialonga, L., Czech, E., & Tamminga, M. (2016). Microplastic pollution in lakes and lake shoreline sediments—a case study on Lake Bolsena and Lake Chiusi (central Italy). Environmental Pollution, 213, 648–657. https://doi.org/10.1016/j.envpol.2016.03.012.

    Article  CAS  Google Scholar 

  • Forrest, S. A., Holman, L., Murphy, M., & Vermaire, J. C. (2019). Citizen science sampling programs as a technique for monitoring microplastic pollution: results, lessons learned and recommendations for working with volunteers for monitoring plastic pollution in freshwater ecosystems. Environmental Monitoring and Assessment, 191(172). https://doi.org/10.1007/s10661-019-7297-3.

  • Galgani, F. (2015). Marine litter, future prospects for research. Frontiers in Marine Science, 2. https://doi.org/10.3389/fmars.2015.00087.

  • GESAMP. (2015). Sources, fate and effects of microplastics in the marine environment: a global assessment. Kershaw PJ (Ed.). Rep. Stud. GESAMP No. 90, 96 pp.

  • GESAMP. (2019). Guidelines or the monitoring and assessment of plastic litter and microplastics in the ocean. Kershaw P.J., Turra A., Galgani F. (Eds.), (IMO/FAO/UNESCO-IOC/UNIDO/WMO/IAEA/UN/UNEP/UNDP/ISA Joint Group of Experts on the Scientific Aspects of Marine Environmental Protection). Rep. Stud. GESAMP No. 99, 130p.

  • Geyer, R., Jambeck, J. R., & Law, K. L. (2017). Production, use, and fate of all plastics ever made. Science Advances, 3, e1700782. https://doi.org/10.1126/sciadv.1700782.

    Article  CAS  Google Scholar 

  • González, D., Hanke, G., Tweehuysen, G., Bellert, B., Holzhauser, M., Palatinus, A., Hohenblum, P., Oosterbaan, L. (2016). Riverine litter monitoring—options and recommendations (MSFD GESTG Marine Litter-Thematic Report No. EUR 28307), JRC Technical Reports.

  • Hartmann, N. B., Hüffer, T., Thompson, R. C., Hassellöv, M., Verschoor, A., Daugaard, A. E., Rist, S., Karlsson, T., Brennholt, N., Cole, M., Herrling, M. P., Hess, M. C., Ivleva, N. P., Lusher, A. L., & Wagner, M. (2019). Are we speaking the same language? Recommendations for a definition and categorization framework for plastic debris. Environmental Science & Technology, 53, 1039–1047. https://doi.org/10.1021/acs.est.8b05297.

    Article  CAS  Google Scholar 

  • Hengstmann, E., Tamminga, M., vom Bruch, C., & Fischer, E. K. (2018). Microplastic in beach sediments of the Isle of Rügen (Baltic Sea) - implementing a novel glass elutriation column. Marine Pollution Bulletin, 126, 263–274. https://doi.org/10.1016/j.marpolbul.2017.11.010.

    Article  CAS  Google Scholar 

  • Hidalgo-Ruz, V., Gutow, L., Thompson, R. C., & Thiel, M. (2012). Microplastics in the marine environment: a review of the methods used for identification and quantification. Environmental Science & Technology, 46, 3060–3075. https://doi.org/10.1021/es2031505.

    Article  CAS  Google Scholar 

  • Käppler, A., Fischer, D., Oberbeckmann, S., Schernewski, G., Labrenz, M., Eichhorn, K.-J., & Voit, B. (2016). Analysis of environmental microplastics by vibrational microspectroscopy: FTIR, Raman or both? Analytical and Bioanalytical Chemistry, 408, 8377–8391. https://doi.org/10.1007/s00216-016-9956-3.

    Article  CAS  Google Scholar 

  • Laforsch, C. (2017). Monitoring of plastics in freshwater environments in German federal states. In: Bänsch-Baltruschat, B., Brennholt, N., Kochleus, C., Reifferscheid, G., Koschorreck, J., 2017. Conference on plastics in freshwater environments. Berlin.

  • Lenz, R., Enders, K., Stedmon, C. A., Mackenzie, D. M. A., & Nielsen, T. G. (2015). A critical assessment of visual identification of marine microplastic using Raman spectroscopy for analysis improvement. Marine Pollution Bulletin, 100, 82–91. https://doi.org/10.1016/j.marpolbul.2015.09.026.

    Article  CAS  Google Scholar 

  • Löder, M. G. J., & Gerdts, G. (2015). Methodology used for the detection and identification of microplastics - a critical appraisal. In M. Bergmann, L. Gutow, & M. Klages (Eds.), Marine anthropogenic litter (pp. 201–227). Cham: Springer International Publishing. https://doi.org/10.1007/978-3-319-16510-3_8.

    Chapter  Google Scholar 

  • Lusher, A., Bråte, I. L. N., Hurley, R., Iversen, K., & Olsen, M. (2017). Testing of methodology for measuring microplastics in blue mussels (Mytilus spp) and sediments, and recommendations for future monitoring of microplastics (R&D-project). NIVA Report, 7215–2017. https://doi.org/10.13140/rg.2.2.24399.59041.

  • Maes, T., Jessop, R., Wellner, N., Haupt, K., & Mayes, A. G. (2017). A rapid-screening approach to detect and quantify microplastics based on fluorescent tagging with Nile Red. Scientific Reports, 7(44501). https://doi.org/10.1038/srep44501.

  • Mason, S. A., Garneau, D., Sutton, R., Chu, Y., Ehmann, K., Barnes, J., Fink, P., Papazissimos, D., & Rogers, D. L. (2016). Microplastic pollution is widely detected in US municipal wastewater treatment plant effluent. Environmental Pollution, 218, 1045–1054. https://doi.org/10.1016/j.envpol.2016.08.056.

    Article  CAS  Google Scholar 

  • Morét-Ferguson, S., Law, K. L., Proskurowski, G., Murphy, E. K., Peacock, E. E., & Reddy, C. M. (2010). The size, mass, and composition of plastic debris in the western North Atlantic Ocean. Marine Pollution Bulletin, 60, 1873–1878. https://doi.org/10.1016/j.marpolbul.2010.07.020.

    Article  CAS  Google Scholar 

  • MSFD Technical Subgroup on Marine Litter. (2013). Guidance on monitoring of marine litter in European seas. European Commission. In Joint Research Center. Luxembourg: Institute for Environment and Sustainability.

    Google Scholar 

  • Nuelle, M.-T., Dekiff, J. H., Remy, D., & Fries, E. (2014). A new analytical approach for monitoring microplastics in marine sediments. Environmental Pollution, 184, 161–169. https://doi.org/10.1016/j.envpol.2013.07.027.

    Article  CAS  Google Scholar 

  • Pinheiro, L. M., Monteiro, R. C. P., Ivar do Sul, J. A., & Costa, M. F. (2019). Do beachrocks affect microplastic deposition on the strandline of sandy beaches? Marine Pollution Bulletin, 141, 569–572. https://doi.org/10.1016/j.marpolbul.2019.03.010.

    Article  CAS  Google Scholar 

  • PlasticsEurope. (2018). Plastics – the facts 2018. An analysis of European plastics production, demand and waste data. Available: https://www.plasticseurope.org/ application/files/6315/4510/9658/Plastics_the_facts_2018_AF_web.pdf

  • Primpke, S., Lorenz, C., Rascher-Friesenhausen, R., & Gerdts, G. (2017). An automated approach for microplastics analysis using focal plane array (FPA) FTIR microscopy and image analysis. Analytical Methods, 9, 1499–1511. https://doi.org/10.1039/C6AY02476A.

    Article  CAS  Google Scholar 

  • Primpke, S., A. Dias, P., & Gerdts, G. (2019). Automated identification and quantification of microfibres and microplastics. Analytical Methods, 11, 2138–2147. https://doi.org/10.1039/C9AY00126C.

    Article  CAS  Google Scholar 

  • R Core Team. (2017). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/.

  • RStudio Team. (2016). RStudio: integrated development for R. RStudio, Inc., Boston, MA URL http://www.rstudio.com/

  • Shim, W. J., Song, Y. K., Hong, S. H., & Jang, M. (2016). Identification and quantification of microplastics using Nile Red staining. Marine Pollution Bulletin, 113, 469–476. https://doi.org/10.1016/j.marpolbul.2016.10.049.

    Article  CAS  Google Scholar 

  • Silva, A. B., Bastos, A. S., Justino, C. I. L., da Costa, J. P., Duarte, A. C., & Rocha-Santos, T. A. P. (2018). Microplastics in the environment: challenges in analytical chemistry - a review. Analytica Chimica Acta, 1017, 1–19. https://doi.org/10.1016/j.aca.2018.02.043.

    Article  CAS  Google Scholar 

  • Song, Y. K., Shim, W. J., Jang, M., Hong, S. H., Han, G. M. (2014) A Nile red staining method for microplastic identification and quantification. 제 52 회 한국분석과학회 춘계학술대회 143–143.

  • Song, Y. K., Hong, S. H., Jang, M., Han, G. M., Rani, M., Lee, J., & Shim, W. J. (2015). A comparison of microscopic and spectroscopic identification methods for analysis of microplastics in environmental samples. Marine Pollution Bulletin, 93, 202–209. https://doi.org/10.1016/j.marpolbul.2015.01.015.

    Article  CAS  Google Scholar 

  • Tamminga, M., Hengstmann, E., & Fischer, E. K. (2017). Nile red staining as a subsidiary method for microplastic quantification: a comparison of three solvents and factors influencing application reliability. SDRP Journal of Earth Sciences and Environment Studies, 2(2). https://doi.org/10.15436/JESES.2.2.1.

  • Tamminga, M., Stoewer, S.-C., & Fischer, E. K. (2019). On the representativeness of pump water samples versus manta sampling in microplastic analysis. Environmental Pollution, 254, 112970. https://doi.org/10.1016/j.envpol.2019.112970.

    Article  CAS  Google Scholar 

  • Wang, F., Wong, C. S., Chen, D., Lu, X., Wang, F., & Zeng, E. Y. (2018). Interaction of toxic chemicals with microplastics: a critical review. Water Research, 139, 208–219. https://doi.org/10.1016/j.watres.2018.04.003.

    Article  CAS  Google Scholar 

  • Zarfl, C. (2019). Promising techniques and open challenges for microplastic identification and quantification in environmental matrices. Analytical and Bioanalytical Chemistry, 411, 3743–3756. https://doi.org/10.1007/s00216-019-01763-9.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank the “Evangelisches Studienwerk Villigst” for their grant and Grace Swanson for the language revision of our manuscript.

Funding

This study was partly funded by “Evangelisches Studienwerk Villigst”.

Author information

Authors and Affiliations

  1. Center for Earth System Research and Sustainability (CEN), Universität Hamburg, Bundesstraße 55, 20146, Hamburg, Germany

    Elena Hengstmann & Elke Kerstin Fischer

Authors
  1. Elena Hengstmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Elke Kerstin Fischer
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Elena Hengstmann.

Ethics declarations

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(PDF 634 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hengstmann, E., Fischer, E.K. Nile red staining in microplastic analysis—proposal for a reliable and fast identification approach for large microplastics. Environ Monit Assess 191, 612 (2019). https://doi.org/10.1007/s10661-019-7786-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s10661-019-7786-4

Keywords

Search

Navigation