Development and Validation of Microplastic Extraction from Feces of the White-Throated Dipper (Cinclus cinclus)

Plastic pollution pervades marine, freshwater, and terrestrial ecosystems due to the ubiquitous presence and durability of synthetic polymers in the environment, leading to long-lasting negative impacts. Large plastic debris can fragment into microplastics (MPs) and nanoplastics (NPs), which are ingested by various organisms and accumulate in the food web, leading to sublethal effects on all trophic levels. The present work advances the understanding of MPs pollution by developing a MPs extraction method from feces of the white-throated dipper (Cinclus cinclus), a wellestablished sentinel species for assessing the effects of pollutants on freshwater systems. While the focus was on MPs extraction, the workflow was designed to enable future enumeration and chemical analysis of MPs using Fourier Transform Infrared Spectroscopy (FTIR). Developing the method required multiple trials to identify suitable techniques, due to the challenges of separating MPs from the complex sample matrix, which can hinder quantification and characterization. The optimized protocol consisted of two cycles of a Fenton reaction followed by a density separation with sodium bromide (NaBr). The method validation using metal-doped MPs enabled spiking with environmentally relevant polymers that were easily distinguishable from background MPs. The fecal samples were spiked with indium (In) doped polyethylene terephthalate (PET) and yttrium (Y) doped polypropylene (PP) in the size range of 63-125µm. MPs recovery rates for the final workflow were 72.45% for PET and 56.29% for PP, which ideally would be higher when applying the method to environmental samples. Measuring MPs recovery at different workflow stages showed that the density separation step was the primary contributor to MP loss. While recovery for PP was lower, preliminary tests indicated that the Y might leach from the MPs during the Fenton reaction. Both aspects should be investigated in the future for further optimization of the developed method and the validation process. This work lays the foundation for future research to refine and standardize MPs extraction techniques from bird feces, allowing for accurate quantification of MPs burden on the white-throated dipper and other bird species across ecosystems.