Asio otus

Key words: Asio otus View in CoL , wildlife, food chain, extraction process, microplastics

Since the 1930s, there has been a rapid increase in plastics production, with annual output steadily rising ( Kye et al., 2023). Microplastics, with sizes of <5 mm, have either been deliberately manufactured by various sectors, such as cosmetics, or have broken down over time through various processes, eventually entering the environment as waste ( Wagner et al., 2017). Due to the detrimental impact of plastic consumption on the survival and reproductive capabilities of numerous animal species (e.g., Foekema et al., 2013; Winkler et al., 2020; Başaran Kankılıç et al., 2023), microplastic pollution has become a global environmental health problem ( Shim and Thomposon, 2015). Nevertheless, there is relatively limited knowledge regarding the accumulation of microplastics in birds of prey such as hawks, eagles, owls, or vultures ( Washburn et al., 2014). This is important as it highlights the impact of microplastics on wildlife at higher trophic levels. Owls, as nocturnal carnivorous birds, prey on small mammals, reptiles, and small birds ( Bontzorlos et al., 2005; Dziemian Zwolak et al., 2012), which can lead to significant microplastic ingestion ( Washburn et al., 2014) due to

* Correspondence: unyazgan@gmail.com their foraging habits. While many studies have focused on microplastic accumulation in aquatic (e.g., Çevik et al., 2021) or terrestrial ecosystems (e.g., Chae and An, 2018), only four to date have examined its impact on birds of prey, which belong to higher trophic levels ( Carlin et al., 2020; Ballejo et al., 2021; Nessi et al., 2022; Pietrelli et al., 2023). However, each of these four studies employed different methods to extract microplastics from pellets. To ensure consistency across studies and more effectively aid wildlife conservation efforts, it is essential to process pellet samples for both microplastics and prey remains with the same methodology. A consistent and comprehensive approach will allow for a more accurate assessment of the potential ingestion of microplastics by owls. The objective of this study was to identify the most effective procedure for extracting microplastics and prey remains from owl pellets without causing damage, considering time, efficiency, and cost.

In this study, we tested different treatments to identify the optimal methodology for effectively extracting microplastics and prey items from owl pellets while ensuring their integrity ( Figure 1 View Figure 1 ). We conducted a review of recent global literature on owl pellet analysis using the keyword “owl pellet” in the Web of Science (WoS) database, including studies from Türkiye ( Tables 1 and 2). Additionally, the keywords “microplastics” and “bird” were included in the search for a more comprehensive evaluation. Using 100 pellet samples from long-eared owls ( Asio otus View in CoL ) from different habitats in Ankara, we optimized factors such as time, temperature, and chemicals with three replicates to improve microplastic and prey remain detection ( Table 3). In this process, to preserve plastic materials, the temperature should not exceed 60 °C ( Cowger et al., 2020; Lusher et al., 2020a, 2020b).

In each treatment (n = 25), we observed varying challenges related to prey remains or microplastic items, such as impractical sorting, difficulty in identifying microplastics, damage to prey items, and time consumption ( Table 3). As a result of these trials, the most optimal extraction methods that can be applied without damaging or losing prey items and microplastics are as follows: i) for prey items, soaking the pellet in a water bath at 60 °C for 10 min; and ii) for microplastics, utilizing sodium hydroxide (10% NaOH, 55 °C, 75 min), potassium hydroxide (10% KOH, 55 °C, 75 min), or Fenton reagent (H 2 O 2 30 % + FeSO 4, 50 °C, depending on the reaction) ( Figures 2 View Figure 2 and 3 View Figure 3 ). Ultimately, while both NaOH and KOH are suitable for organic material removal, KOH proved to be more effective than NaOH due to its superior ability to dissolve hair at a higher rate. Moreover, Fenton reagent necessitated variable process times and quantities depending on pellet size. Consequently, compared to KOH, Fenton reagent is not economically advantageous due to the higher volumes required ( Table 4).

Based on the findings of the trials, it was determined that a sequential approach is advisable to effectively extract both prey items and microplastics without causing damage or sample loss. The following steps outline a comprehensive pellet analysis process that may be most suitable for this purpose:

- Preparing 10% KOH solution;

- Placing the pellet sample to be examined in a glass container and adding sufficient distilled water to moisten the pellet;

- Submerging the moistened sample in a hot water bath at 60 °C for 10 min;

- Transferring the sample from the water bath to a beaker and adding the 10% KOH solution to a separate container nearby, and prey items can then be sorted in this KOH solution;

- Placing the cleaned prey items on blotting paper and allowing them to dry;

- After sorting, transferring the remaining fur back into the same glass beaker used for initial soaking (as microplastic samples may still be present in the beaker);

- Pouring the 10% KOH solution to cover the fur in the glass beaker and adding the KOH solution used for cleaning the prey items;

- Keeping the glass beaker in a hot water bath at 55 °C for 75 min;

- After completing the removal process, filtering the samples and transferring them to petri dishes for further microplastic identifications under a microscope.

- All of these processes should be conducted under a fume hood to prevent contamination.

SEYFE et al. / Turk J Zool

Fenton reagent (H

2

O

2

30% + FeSO

4

) Potassium hydroxide (10% KOH)

Pellet was soaked in a glass beaker and kept in a hot water bath at Pellet was soaked in a glass beaker and kept in a hot water bath at 60 °C for 10 min 60 °C for 10 min Prey items were removed from the pellet Prey items were removed from the pellet 30 mL of hydrogen peroxide (H 2 O 2) was poured onto the pellet in 10% KOH solution was poured onto the pellet in the glass beaker the glass container, and 30 mL of Fenton reagent was added in the

to cover the pellet same way to make the solution 1:1 Solution was mixed at 500 rpm at 50 °C on a magnetic stirrer Kept in a hot water bath at 55 °C for 75 min To sustain the reaction, an additional 30 mL of H 2 O 2 was poured onto the mixture; after about 1 h, the liquid solution containing the dissolved pellet turned light yellow, with prey items accumulated on

After 75 min, the pellet sample in KOH solution was taken out of the liquid; depending on the pellet’s structure and size, the addition

the hot water bath of H 2 O 2 could be necessary to maintain the reaction; after about 40 min, the liquid solution bubbled, indicating completion of the reaction The hairs dissolved and disappeared The hairs dissolved and disappeared The entire process took an average of 250 min The entire process took an average of 120 min The amount of chemicals used and the time spent varied depending The amount of chemicals used was constant and total time did not on pellet size vary according to pellet size With the H O process, only one magnetic stirrer can be used for a

2 2 With the KOH process, more than one pellet can be processed at single pellet; in other words, each pellet is processed individually in

the same time at least this much time. This process is more costly in terms of time and cost This process is more economical in terms of time and cost

Acknowledgments

We would like to thank Gökben Başaran Kankılıç, Atılgan Aygün, Şirin Bahar Karahasan, Ecem Mercan, and Alihan Eğmen for laboratory and field assistance. We would like to thank Eti Ester Levi for language editing. This study was part of the PhD dissertation of the first author (Institute of Science, Çankırı Karatekin University, Türkiye).