New Methods for the Quantification of Ingested Nano- and Ultrafine Plastics in Seabirds.

Plastic ingestion has been documented in a plethora of taxa. However, there is a significant gap in the detection of nano- and ultrafine particles due to size limitations of commonly used techniques. Using two Australian seabird species as case studies, the flesh-footed shearwater (FFSH) Ardenna carneipes and short-tailed shearwater (STSH) A. tenuirostris, we tested a novel approach of flow cytometry to quantify ingested particles <70 µm in the fecal precursor (guano; colon and cloacal contents) of both species. This method provided the first baseline data set for these species for plastics in the 200 nm-70 µm particle size ranges and detected a mean of 553.50 ± 91.21 and 350.70 ± 52.08 plastics (count/mg fecal precursor, wet mass) in STSH and FFSH, respectively, whereas Fourier transform infrared spectroscopy (FT-IR) provided accurate measurements of polymer compositions and quantities in the size range above 5.5 × 5.5 µm2. The abundance of nano- and ultrafine particles in the guano (count/mg) was not significantly different between species (p-value = 0.051), suggesting that foraging distribution or prey items, but not species, may contribute to the consumption of small plastics. In addition, there was no correlation between macroplastics in the stomach compared to the fecal precursor, indicating that small particles are likely bioaccumulating (e.g., through shedding and digestive fragmentation) and/or being directly ingested. Combining flow cytometry with FT-IR provides a powerful quantitative and qualitative analysis tool for detecting particles orders of magnitude smaller than that are currently explored with wider applications across taxa and marine environments.

Foraging strategy influences the quantity of ingested micro- and nanoplastics in shorebirds.

Coastlines, including estuaries, mudflats, and beaches, are particularly susceptible to plastic pollution, which can accumulate from both marine and terrestrial sources. While numerous studies have confirmed the presence of microplastics (1-5 mm) along coastlines, few have focused on very small particles (<1 µm) or quantified exposure within the organisms that inhabit these areas, such as shorebirds. Here, we quantified small plastics (200 nm-70 µm) in two resident shorebird species in Tasmania, and compared this to quantities found in the surrounding sediments in order to investigate the potential exposure and transfer of particles within these ecosystems. Analysis was performed using a combination of flow cytometry for quantification of micro- and nanoplastics (200 nm-70 µm), and µm-FT-IR for validation and polymer identification of particles >5.5 × 5.5 µm. Micro- and nano-plastics were detected in 100% of guano samples from surface-feeding Eastern Hooded Plovers (Thinornis cucullatus) and 90% of Australian Pied Oystercatcher (Haematopus longirostris) guano, a species that forages for coastal invertebrates at 60-90 mm depth, and 100% of beach sediments. Hooded Plover guano contained 32 × more plastics, on average, than Pied Oystercatcher guano. Interestingly, the abundance of plastic particles within sediments collected from shorebird foraging sites did not appear to have a significant effect on the number of plastics the birds had ingested, suggesting the difference between species is likely a result of other variables, such as prey selection. The results of this study highlight the importance of including techniques that provide quantitative data on the abundance and size of the smallest possible particle sizes, and demonstrate the significant proportion of small plastics that are 'missed' using standard analysis tools.