Discussion

This is the first report showing that soil protists present in a forest soil can ingest MPs and store them within their food vacuoles. In addition, our observations using fluorescence microscopy clearly showed that the majority of large (>30 μm) phagotrophic protists, irrespective of taxon, could readily ingest MPs in any MP addition treatment. This finding was robust because green fluorescence (550 nm) was restricted to MP addition treatments, showing that any background fluorescent MPs were not detectable and that the protists’ organelles are not fluorescent at that wavelength. Approximately 75% of protists ingested MPs in the treatment with the lowest concentration MPs and a 10-fold increase in MP concentration resulted in nearly all protists ingesting MPs. These findings build on previous work (e.g., Fenchel, 1980) and can have profound ecological and environmental implications given the important role that protists play in the soil food web (Adl & Gupta, 2006). Motile phagotrophic protists can serve both as vectors in moving MPs in the soil matrix and in transferring them to higher trophic levels, thereby potentially amplifying MP pollution (Geisen et al., 2018, 2020). Furthermore, we could observe several ciliate and flagellate morphotypes, which suggests that the feeding behavior for MPs may not be species specific. Conversely, we anecdotally observed that some protist morphotypes seemed to show a greater propensity for ingesting MPs than others as indicated by the intensity of the fluorescence within their food vacuoles. Certainly, more experimentation is required to understand if protists show morphotype or taxa specific feeding behaviors regarding MP ingestion.
The results of trial 2 show that soil protists can readily ingest MPs in as little as 24 hours after these are introduced. In addition, in the treatment with 1% MPs (w/w) nearly all protists ingested MPs. This is consistent with data for other soil biota such as bacterial-feeding nematode species using similar methods with fluorescent microspheres (Mueller et al., 2020). This technique could potentially be utilized in future experiments to quantify MP ingestion and to source track MPs.
We detected a significant overall temporal difference in protist abundance in both trials across treatments, which is explained by declines after approximately a week. This was expected since microcosms are closed systems. However, the hypothesis that the overall abundance of phagotrophic soil protists would be reduced by the addition of MPs particles to soil was not strongly supported by the data. Nevertheless, MP addition had a marginally significant effect on protist abundance in trial one. These results are interesting given that most protists were observed carrying MPs within their food vacuoles which would indicate, at least within short-time scales, that they may not be detrimental to the organisms’ overall health. This is consistent with studies showing that MPs do not seem to cause considerable mortality in earthworms at environmentally relevant concentrations. However, mortality has been reported at relatively high concentrations (Jacques & Prosser, 2021). Likewise, the nematode Caenorhabditis elegans appears to be highly susceptible to high concentrations of MPs. In contrast, other species of nematodes may be more tolerant to MPs of similar polymer composition to those used in this study (Mueller et al., 2020). It is possible that the commercial microspheres used in this study are indeed inert and their ingestion may not substantially reduce the protist’s food intake. More research using different soils and a wider diversity of MPs types (e.g., varying the parameters shape (Lozano et al., 2021), polymer type (Waldman & Rillig, 2020), weathering status and additives (Kim et al., 2020)) will be required to clarify this.
If MPs particles were in fact safe to soil protists, given their high abundance in soils and important role as nutrient cyclers (Wood & Bradford, 2018), perhaps they may be able to further physically breakdown MPs. However, while they could probably use some of the additives, debris, or biofilms in the plastic particles it is unlikely that protists would be able to utilize the long carbon-backbone chains that makeup most plastics. The fact is, the MPs used in this experiment, although utilized in other MP-biota interactions research (Bringer et al., 2020), do not represent the average MP particle found in soil (Wang et al., 2019). Future studies could utilize other MPs commonly found in soil environments, such as polyacrylic fibers or polyethylene fragments. However, appropriate MP detection methods for those polymers (Shan et al., 2018) would be required in combination with methods to extract protists from soil and sediments (Alongi, 2018). Overall, our results show that, in general, large phagotrophic protists appear to have the ability to ingest MPs. More research is needed to verify to what degree MPs can in fact affect the abundance and community composition of soil protists and understand the effect of MPs on soil food webs.

Acknowledgements

This research was conducted in Robinson-Huron Treaty territory and the traditional territory of the Anishnaabeg, specifically the Garden River and Batchewana First Nations, as well as Métis People. The work was funded through a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) and a Canada Research Chair awarded to P.M. Antunes.

References

Adl, M. S. & V. V., SR. Gupta (2006): Protists in soil ecology and forest nutrient cycling. - Canadian Journal of Forest Research. Journal Canadien de La Recherche Forestiere, 36(7): 1805–1817. https://doi.org/10.1139/x06-056
Adl, S. M., A. G. B. Simpson, M.A. Farmer, R.A. Andersen, O.R. Anderson, J.R. Barta, S.S. Bowser, G. Brugerolle, R.A. Fensome, S. Fredericq, T.Y. James, S. Karpov, P. Kugrens, J. Krug, C.E. Lane, L.A. Lewis, J. Lodge, D.H. Lynn, D.G. Mann, & M. F. J. R. Taylor (2005): The new higher level classification of eukaryotes with emphasis on the taxonomy of protists. - The Journal of Eukaryotic Microbiology 52(5): 399–451. https://doi.org/10.1111/j.1550-7408.2005.00053.x 
Alongi, D. M. (2018): Extraction of protists in aquatic sediments via density gradient centrifugation. In Handbook of Methods in Aquatic Microbial Ecology (pp. 109–114). https://doi.org/10.1201/9780203752746-14 
Andrady, A. L. (2011): Microplastics in the marine environment. - Marine Pollution Bulletin, 62(8): 1596–1605. https://doi.org/10.1016/j.marpolbul.2011.05.030 
Azzarello, M. Y. & E. S.Van Vleet (1987): Marine birds and plastic pollution. - Marine Ecology Progress Series 37: 295–303. https://doi.org/10.3354/meps037295
Bringer, A., J. Cachot, G. Prunier, E. Dubillot, C. Clérandeau, & H. Thomas (2020): Experimental ingestion of fluorescent microplastics by pacific oysters, Crassostrea gigas, and their effects on the behaviour and development at early stages. - Chemosphere 254: 126793. https://doi.org/10.1016/j.chemosphere.2020.126793
Chamas, A., H. Moon, J. Zheng, Y. Qiu, T. Tabassum, J.H. Jang, M. Abu-Omar, S.L. Scott & S. Suh (2020): Degradation Rates of Plastics in the Environment. - ACS Sustainable Chemistry & Engineering 8(9): 3494–3511. https://doi.org/10.1021/acssuschemeng.9b06635
de Souza Machado, A. A., W. Kloas, C. Zarfl, S. Hempel & M.C. Rillig (2018): Microplastics as an emerging threat to terrestrial ecosystems. - Global Change Biology, 24(4): 1405–1416. https://onlinelibrary.wiley.com/doi/abs/10.1111/gcb.14020
de Souza Machado, A. A., C.W. Lau, J. Till, W. Kloas, A. Lehmann, R. Becker & M.C. Rillig (2018): Impacts of Microplastics on the Soil Biophysical Environment. - Environmental Science & Technology 52(17): 9656–9665. https://doi.org/10.1021/acs.est.8b02212
Fenchel, T. (1980): Suspension feeding in ciliated protozoa: functional response and particle size selection. Microbial Ecology, 6(1): 1-11. https://doi.org/10.1007/BF02020370
Foissner, W. (1992): Estimating the species richness of soil protozoa using the “non-flooded petri dish method.” Protocols in Protozoology, Society of Protozoologists, Lawrence. P. B-10. 1–B-10. 2. http://www.wfoissner.at/data_prot/Foissner_1992_B-10-1.pdf 
Fuller, S. & A. Gautam (2016): A Procedure for Measuring Microplastics using Pressurized Fluid Extraction. - Environmental Science & Technology, 50(11): 5774–5780. https://doi.org/10.1021/acs.est.6b00816 
Geisen, S., E. Lara, E.A. Mitchell, E. Völcker & V. Krashevska (2020): Soil protist life matters!. - Soil Organisms 92(3):189-196. https://doi.org/10.25674/so92iss3pp189 
Geisen, S., E.A.D. Mitchell, S. Adl, M. Bonkowski, M. Dunthorn, F. Ekelund, L.D. Fernández, A. Jousset, V. Krashevska, D. Singer, F.W. Spiegel, J. Walochnik & E. Lara (2018): Soil protists: a fertile frontier in soil biology research. - FEMS Microbiology Reviews, 42(3): 293–323. https://doi.org/10.1093/femsre/fuy006 
Helcoski, R., L.T. Yonkos, A. Sanchez & A.H. Baldwin (2020): Wetland soil microplastics are negatively related to vegetation cover and stem density. - Environmental Pollution 256: 113391. https://doi.org/10.1016/j.envpol.2019.113391 
Hidalgo-Ruz, V., L. Gutow, R.C. Thompson, & M. Thiel (2012): Microplastics in the marine environment: a review of the methods used for identification and quantification. - Environmental Science & Technology 46(6): 3060–3075. https://doi.org/10.1021/es2031505 
Huang, Y., Y. Zhao, J. Wang, M. Zhang, W. Jia & X. Qin (2019): LDPE microplastic films alter microbial community composition and enzymatic activities in soil. - Environmental Pollution 254(Pt A): 112983. https://doi.org/10.1016/j.envpol.2019.112983 
Jacques, O & R.S. Prosser (2021): A probabilistic risk assessment of microplastics in soil ecosystems. - The Science of the Total Environment 757: 143987. https://doi.org/10.1016/j.scitotenv.2020.143987 
Johansen, J. L., R. Rønn & F. Ekelund (2018): Toxicity of cadmium and zinc to small soil protists. - Environmental Pollution 242(Pt B): 1510–1517. https://doi.org/10.1016/j.envpol.2018.08.034 
Kim, S. W & Y.-J. An (2020): Edible size of polyethylene microplastics and their effects on springtail behavior. - Environmental Pollution 266(Pt 1): 115255. https://doi.org/10.1016/j.envpol.2020.115255 
Kim, S. W., Kim, D., Jeong, S.-W., & An, Y.-J. (2020). Size-dependent effects of polystyrene plastic particles on the nematodeCaenorhabditis elegans as related to soil physicochemical properties. Environmental Pollution , 258, 113740. https://doi.org/10.1016/j.envpol.2019.113740 
Kim, S. W., W.R. Waldman, T.-Y Kim & M.C. Rillig (2020): Effects of different microplastics on nematodes in the soil environment: Tracking the Extractable Additives Using an Ecotoxicological Approach. - Environmental Science & Technology 54(21): 13868–13878. https://doi.org/10.1021/acs.est.0c04641 
Lambert, S., C. Sinclair & A. Boxall (2014): Occurrence, degradation, and effect of polymer-based materials in the environment. - Reviews of Environmental Contamination and Toxicology 227: 1–53. https://doi.org/10.1007/978-3-319-01327-5_1 
Lei, L., S. Wu, S. Lu, M. Liu, Y. Song, Z. Fu, H. Shi, K.M. Raley-Susman & D. He (2018): Microplastic particles cause intestinal damage and other adverse effects in zebrafish Danio rerio and nematodeCaenorhabditis elegans . - The Science of the Total Environment 619-620: 1–8. https://doi.org/10.1016/j.scitotenv.2017.11.103 
Liu, P., X. Zhan, X. Wu, J. Li, H. Wang & S. Gao (2020): Effect of weathering on environmental behavior of microplastics: Properties, sorption and potential risks. – Chemosphere 242: 125193. https://doi.org/10.1016/j.chemosphere.2019.125193 
Lozano, Y. M., T. Lehnert, L.T. Linck, A. Lehmann & M.C. Rillig (2021): Microplastic shape, polymer type, and concentration affect soil properties and plant biomass. - Frontiers in Plant Science 12: 616645. https://doi.org/10.3389/fpls.2021.616645 
McKeen, L. W. (2013). The Effect of UV Light and Weather on Plastics and Elastomers. William Andrew. https://play.google.com/store/books/details?id=IqCo2mZcDx4C 
Mueller, M.-T., H. Fueser, S. Höss & W. Traunspurger (2020): Species-specific effects of long-term microplastic exposure on the population growth of nematodes, with a focus on microplastic ingestion. - Ecological Indicators 118: 106698. https://doi.org/10.1016/j.ecolind.2020.106698 
Pischedda, A., M. Tosin & F. Degli-Innocenti (2019): Biodegradation of plastics in soil: The effect of temperature. - Polymer Degradation and Stability 170: 109017. https://doi.org/10.1016/j.polymdegradstab.2019.109017 
Prokić, M. D., B.R. Gavrilović, T.B. Radovanović, J.P. Gavrić, T.G. Petrović, S.G. Despotović & C. Faggio (2021): Studying microplastics: Lessons from evaluated literature on animal model organisms and experimental approaches. - Journal of Hazardous Materials 414: 125476. https://doi.org/10.1016/j.jhazmat.2021.125476 
Ren, X., J. Tang, L. Wang & Q. Liu (2021): Microplastics in soil-plant system: effects of nano/microplastics on plant photosynthesis, rhizosphere microbes and soil properties in soil with different residues. - Plant and Soil. https://doi.org/10.1007/s11104-021-04869-1 
Rillig, M. C. (2012): Microplastic in terrestrial ecosystems and the soil? - Environmental Science & Technology 46(12): 6453–6454. https://doi.org/10.1021/es302011r 
Rillig, M. C., & M. Bonkowski (2018): Microplastic and soil protists: A call for research. In Environmental Pollution (Vol. 241, pp. 1128–1131). https://doi.org/10.1016/j.envpol.2018.04.147 
Rillig, M. C., E. Leifheit & J. Lehmann (2021): Microplastic effects on carbon cycling processes in soils. - PLoS Biology 19(3): e3001130. https://doi.org/10.1371/journal.pbio.3001130 
Ritchie, H. & M. Roser (2018): Plastic pollution. Our world in data. ”Plastic Pollution”. Published online at OurWorldInData.org. Retrieved from: ’https://ourworldindata.org/plastic-pollution’ [Online Resource]
Setälä, O., V. Fleming-Lehtinen & M. Lehtiniemi (2014): Ingestion and transfer of microplastics in the planktonic food web. In Environmental Pollution (Vol. 185, pp. 77–83). https://doi.org/10.1016/j.envpol.2013.10.013 
Shang, X., J. Lu, C. Feng, Y. Ying, Y. He, S. Fang, Y. Lin, R. Dahlgren & J. Ju (2020): Microplastic (1 and 5 μm) exposure disturbs lifespan and intestine function in the nematode Caenorhabditis elegans. - The Science of the Total Environment 705: 135837. https://doi.org/10.1016/j.scitotenv.2019.135837 
Shan, J., J. Zhao, L. Liu, Y. Zhang, X. Wang & F. Wu (2018): A novel way to rapidly monitor microplastics in soil by hyperspectral imaging technology and chemometrics. - Environmental Pollution 238: 121–129. https://doi.org/10.1016/j.envpol.2018.03.026 
Strungaru, S.-A., R. Jijie, M. Nicoara, G. Plavan & C. Faggio (2019): Micro- (nano) plastics in freshwater ecosystems: Abundance, toxicological impact and quantification methodology. In TrAC Trends in Analytical Chemistry (Vol. 110, pp. 116–128). https://doi.org/10.1016/j.trac.2018.10.025 
Verni, F. & P. Gualtieri (1997): Feeding behaviour in ciliated protists. In Micron (Vol. 28, Issue 6, pp. 487–504). https://doi.org/10.1016/s0968-4328(97)00028-0 
Waldman, W. R. & M.C. Rillig (2020): Microplastic research should embrace the complexity of secondary particles. - Environmental Science & Technology, 54(13): 7751–7753. https://doi.org/10.1021/acs.est.0c02194 
Wang, J., X. Liu, Y. Li, T. Powell, X. Wang, G. Wang & P. Zhang (2019): Microplastics as contaminants in the soil environment: A mini-review.- The Science of the Total Environment 691: 848–857. https://doi.org/10.1016/j.scitotenv.2019.07.209 
Wood, S. A. & M.A. Bradford (2018). Chapter 4 - Leveraging a New Understanding of how Belowground Food Webs Stabilize Soil Organic Matter to Promote Ecological Intensification of Agriculture. In B. K. Singh (Ed.), Soil Carbon Storage (pp. 117–136). Academic Press. https://doi.org/10.1016/B978-0-12-812766-7.00004-4 
Yuan, J., Ma, J., Sun, Y., Zhou, T., Zhao, Y., & Yu, F. (2020): Microbial degradation and other environmental aspects of microplastics/plastics. - The Science of the Total Environment, 715, 136968. https://doi.org/10.1016/j.scitotenv.2020.136968 
Zantis, L. J., E.L. Carroll, S.E. Nelms & T. Bosker (2021): Marine mammals and microplastics: A systematic review and call for standardisation. - Environmental Pollution 269: 116142. https://doi.org/10.1016/j.envpol.2020.116142 
Zhang, M., Y. Zhao, X. Qin, W. Jia, L. Chai, M. Huang & Y. Huan (2019): Microplastics from mulching film is a distinct habitat for bacteria in farmland soil. - The Science of the Total Environment 688: 470–478. https://doi.org/10.1016/j.scitotenv.2019.06.108