The development of a reliable digestion method for MP extraction from multi-tissues of cetaceans

A reliable digestion method for the extraction of MPs from multi-tissues of cetaceans with heterogeneous properties and different fat content should have the advantages of easy operation (high tissue digestion rate, high filter membrane permeability (FMP)), high MP recovery rate, and low damage level of MPs. Three categories (14 types) of digestion techniques of alkaline, enzyme, and HO-based methods with different digestion temperatures and times were investigated (Supplementary Table 1).

The recovery rates of PP and PVC MPs exceeded 98.9% across all methods (Fig. 1a). There is no significant difference in the MP recovery rate among the three categories of digestion methods (H = 0.466, df = 2, p > 0.05). The results of the analysis of 14 digestion methods for PP and PVC revealed significant variations in the recovery rates of PP (H = 27.664, df = 13, p < 0.05). However, no significant differences were observed in the recovery rates of PVC across different digestion method groups (H = 20.160, df = 13, p > 0.05). Overall, all methods demonstrated acceptable recovery rates.

While the alkaline digestion method has superior digestion efficiency than the enzyme (40.38%) and oxidant (28.32%) methods (Fig. 1b), with the overall digestion rate exceeding 80.45% (H = 66.989, df = 2, p < 0.01), helping to observe MPs more clearly and reduce misjudgments in actual sample analysis. Specifically, the T1-T4 digestion rates for both lungs and liver exceeded 94.87% (average 95.68%). The alkaline digestion method can digest an average of 65.21% of the skin. Although the skin digestion rate is low, no substances that hinder observation will remain on the filter membrane after digestion (Supplementary Fig. 1). The alkaline digestion methods can digest an average of 60.17% of the digestive tract, but the T4 digestion rate exceeds 97.59%. And for all methods, the type of tissue also significantly affects the dissolution rate (H = 65.168, df = 3, p < 0.01). The observed variations in digestion efficiency across different tissues and methods may be attributed to differences in keratin, cartilage, ligament distribution, and fat content within the tissues (Supplementary Discussion (1)). In short, the T4 method demonstrated the best tissue digestion rate.

And the alkaline digestion method also has a higher filter membrane permeability (1.69 ± 0.99) than the enzyme (1.17 ± 1.90) and oxidant (0.59 ± 0.04) methods (H = 32.319, df = 2, p < 0.01), demonstrating shorter-time experimental procedures. Due to the ineffectiveness of enzyme methods for skin digestion (digestion rate 0.00%), an abnormally high FMP was observed, which is not helpful for the selection of digestion methods. Therefore, the skin group of enzymes was not included in the differential analysis (Fig. 1c). Especially, the FMP of the T4 method shows the highest level as 2.35 ± 1.14, demonstrating significantly higher FMP compared to the T1 and T2 (H = 7.954, df = 3, p < 0.05). In summary, the T4 method demonstrated the highest FMP.

Different digestion methods may result in some degree of surface morphology and composition changes of MPs. XRD results indicate no obvious change in the peak width of XRD diffraction, suggesting that different digestion methods have no evident effect on the crystallinity of MPs (Fig. 2a). XPS results show these methods can affect the oxygen-to-carbon (O/C) ratio after digestion, influenced by different mechanisms (Fig. 3b, c, Supplementary Discussion (2)). SEM results show, for rough and loose-surfaced PP MPs, the digestion methods employed can cause the expansion of surface voids (Fig. 2b). While under exposure to a 10% KOH solution (T1-T4), the surface of PVC MPs did not exhibit obvious changes across various digestion times and temperatures (Fig. 2c). However, under other digestion methods, varying degrees of cracks and wrinkles appeared. It should be noted that although alkaline methods may cause changes at the level of MPs, the degree of changes to MPs in materials such as PVC is smaller. These changes are acceptable at the level of MPs at the micron-level, while for future surveys that are conducted on the quantity of nanoplastics at the nanoscale, those methods require cautious evaluation before use. In short, the application of alkaline digestion methods is also supported from the perspective of minimizing damage to MPs.

Given the aforementioned considerations, the T4 method (10% KOH, 60 °C for 48 h) is considered the optimal digestion method for extracting MPs from multiple tissues of cetaceans with a high recovery rate, easy operation, and acceptable surface changes. To verify the reliability of the overall process, spiked experiments were conducted by adding 2 mm and 500 microns of purchased PVC (under environmental levels, 10 items, Feihong Plasticization) in four different tissues for digestion. The T4 digestion method under the entire analysis process ensures a recovery rate of 98.75% (Supplementary Fig. 4). There is no significant difference in the recovery rate of MPs of different particle sizes (t-test, p > 0.05) or different tissues (One-way ANOVA, p > 0.05) in this analysis process, indicating that the current digestion method and analysis process can be used for actual sample analysis. Research on MPs has faced criticism for the lack of rigorous methodologies, and currently, studies that disclose spiked recovery rates remain limited. The high recovery rate of 98.75% achieved through this method ensures the effectiveness of the digestion process for extracting MPs from multiple tissues of cetaceans. Thus, the T4 method was applied for our case study to survey MP accumulation levels in multi-tissues in wild and captive S. attenuata individuals.

To understand the true pollution situation of cetaceans, multi-tissue analysis is essential. MPs were detected in the skin, lungs, liver, and gut of both captive and wild S. attenuata (Supplementary Fig. 5), with the detection rate of MPs in different tissues is between 80.00 and 100.00% (Fig. 3a). A significant correlation was observed between the abundance and detection rate of MPs (Spearman test, p < 0.05), and the model fitting yielded a strong logistics relationship (adj. R > 0.90, Fig. 3b). According to the fitting formula, when the detection rate of MPs reaches 100%, the corresponding abundance of MPs is approximately 0.196. This suggests that once the abundance of MPs in a tissue exceeds this threshold, they are likely to be ubiquitously distributed within the tissue. This discovery emphasizes the widespread presence of MPs in various tissues of dolphins at relatively low levels. Consequently, a multi-tissue study, rather than the traditional focus on the digestive tract, is necessary to reveal the full scope of MP pollution in cetaceans. When considering the average abundance gram, MPs in the digestive tract account for 26.67% (for wild individuals) to 30.11% (for captive individuals) of all tissues (Fig. 3a). Therefore, traditional studies are limited to MPs in the digestive tract, which may lead to an underestimation of MP levels in cetaceans (Supplementary Table 2). Tissues significantly affect MP abundance (H = 10.412, df = 3, p < 0.05), thus multi-tissues investigation is crucial for understanding the level of MP accumulation in cetaceans.

In addition, the abundance of MPs in S. attenuata is also influenced by body length (H = 26.873, df = 10, p < 0.01), and grounding years (H = 16.243, df = 6, p < 0.05) significantly affect MP abundance instead of sex (Mann-Whitney U = 1938.5, p > 0.05). This indicates that the abundance of MPs in cetaceans is influenced by a complex interplay of factors, contrary to the traditional review that it is unaffected by body length. Captive individuals face a higher level of MPs than wild individuals, and survival mode significantly influences MP abundance (Mann-Whitney U = 2266.5, p < 0.01). In wild individuals, MP abundance is found to be highest in the skin (0.31 ± 0.15 items g), followed by the gut (0.20 ± 0.12 items g), liver (0.13 ± 0.08 items g), and lungs (0.11 ± 0.07 items g). The captive individuals show a similar pattern, with the highest MP abundance in the skin (0.60 ± 0.23 items g), followed by the gut (0.53 ± 0.30 items g), liver (0.44 ± 0.16 items g), and lungs (0.19 ± 0.25 items g). The MP pollution in these tissues indicates that MPs in diet, air, and water environments are important sources of MPs in cetaceans, a finding not previously revealed (Supplementary Discussion (3)). The captive environment presents several potential sources of MPs. Intensive human activities have resulted in high levels of MPs in the atmosphere, textiles are considered an important source of indoor MPs, wear and tear on walls, ceilings, floors, etc. can also produce MPs. The use of air conditioning can also cause the resuspension of MPs. Focusing on the aquarium, costumes for keepers and spectators, waterproof paint for the venue, and continuously open air conditioning are potential sources of MPs in captive individuals. These sources of MPs can be inhaled or adhered to the skin of captive cetaceans (when MPs settle in the water or jump up during cetaceans' performances), thus the higher MPs in the lungs and skin of captive individuals than wild. Additionally, the dietary differences between wild and captive individuals may further account for this disparity. Wild individuals directly catch prey, but the food of captive individuals inevitably introduces more MP pollution through processes such as fishing (adhering from fishing gear), transportation (detached from plastic packaging), thawing (atmospheric deposition), and feeding by trainers (from clothing). Moreover, providing freshwater to captive cetaceans often involves using ice cubes, which have been shown to contain MPs at concentrations ranging from 19 ± 4 to 178 ± 78 L . This factor contributes to higher MP levels within the gut (Mann-Whitney U = 14.000, p > 0.05) and significantly higher MPs in the liver (Mann-Whitney U = 35.000, p < 0.01) (transferred from the gut, and see later) of captive individuals. However, considering the type Ⅱ errors caused by sample size, we cannot conclude that the digestive tract of captive individuals is not significantly different than that of wild individuals. A higher abundance of MPs means that captive individuals may face a higher risk of disease, and it has been determined in human studies that MP abundance may be associated with the progression of inflammatory bowel disease. In mouse studies, it has also been shown that MPs alter intestinal tissue structure and increase intestinal permeability through erosion of villi, reduction of crypt numbers, and extensive infiltration of intestinal inflammatory cells. One possible explanation is that the high MP levels in the gut of captive individuals increase intestinal permeability, resulting in more MPs being transferred to the liver than in wild individuals. Additionally, due to the possible difficulty in metabolizing MPs in the liver, significantly higher levels have been achieved. In a meta-analysis of the effects of MPs on the liver in vertebrates, it has been revealed that MPs can damage the liver by altering liver morphology, inducing oxidative stress, producing intracellular toxicity, altering biotransformation processes, and interfering with lipid metabolism. Therefore, captive individuals may face higher levels of MP stress. To address these issues and promote animal welfare and health, it is essential to implement measures that reduce MPs exposure. This includes establishing a safe distance between visitors and cetaceans, ensuring rigorous quality control of their feed, and providing MPs-free water (It is even possible to remove 100% of MPs from water through physical and chemical methods). These steps are crucial in mitigating the risks associated with MP pollution and ensuring the well-being of captive cetaceans.

Wild individuals suffer from broader sources of MPs than captive individuals. The divergent sources of MPs in cetaceans are reflected in their distinct characteristics. The diversity of MPs was higher in wild individuals (range: 0.84-0.50, mean: 0.67 ± 0.12) than in captive individuals (range: 0.67-0.44, mean: 0.59 ± 0.09) according to shape and color. Fiber is the predominant form of MPs observed in the tissues of S. attenuata. Captive individuals exhibited fewer shape types (Fig. 4a), with fragments and films exclusively detected in the liver and lungs, while only fiber MPs were identified in the skin and gut. While only fiber-based MPs were identified in the skin, other tissues of wild S. attenuata exhibited MPs in various forms, including fragments, films, foams, and microbeads (Supplementary Fig. 6). A total of eight colors of MPs were identified in S. attenuata tissues, with evident variations in color distribution among tissues (Fig. 4b). Specifically, seven colors of MPs were detected in the guts of wild S. attenuata, five in the lungs of wild individuals, and five in the livers of captive individuals. Other tissues exhibited fewer colors, typically 2-4. In terms of both morphology and color, wild individuals exhibit greater diversity in MPs, which may stem from a wider range of MP sources, including diverse food types and the presence of MPs in food for wild individuals. While intensive human activities introduce MPs to captive environments, these are typically limited to specific types (as point source pollution). In contrast, wild individuals are influenced by complex non-point sources of pollution, such as riverine input, atmospheric deposition, maritime transportation, abandoned fishing gear, and ocean power transport. Consequently, wild individuals exhibit greater diversity in the morphology and color of MPs. Morphology also provides corresponding evidence: foam is mainly used as a fishing gear float, while microbeads are mainly used as friction agents, such as in cosmetics, so these two forms have not been found in individuals in captive environments. Fragments and films mainly come from painting peeling and packaging bags, and fibers are the most common form of MPs worldwide, so it is normal to find them in both captive and wild environments. The diversity of MPs in wild environments has also been supported by other studies: S. attenuata consumes over 80 different species, which may contribute to the observed diversity of MPs. Furthermore, a study conducted in the Beibu Gulf, adjacent to this research area, detected MPs in 93.7% of 32 fish species, showcasing notable diversity in MP characteristics. Some fish may actively consume brightly colored MPs, mistaking them for food, which explains the wide range of colors observed in the digestive tracts of wild individuals with diverse diets. Finally, wild S. attenuata inhabiting open ocean environments are additionally exposed to MPs introduced through atmospheric pathways. MP pollution is prevalent in the atmosphere of the South China Sea, and the color distribution of MPs aligns with those identified in the lungs of wild individuals.

MPs with large particle sizes (2111.98-16.00 μm) are present in the livers of cetaceans, but their particle sizes are relatively smaller compared to other tissues (shorter than 500 μm > 69.49%, while >23.52% for skin, 25.80% for lung, and 64.44% for gut). MPs in the livers of captive individuals were significantly shorter than those in other tissues (H = 28.359, df = 3, p < 0.01, Fig. 4c). While no significant differences were observed in wild individuals (H = 2.642, df = 3, p > 0.05), this may be attributed to data number limitations. Relatively smaller MPs are more conducive to their tissue-to-tissue transfer. Earlier studies have suggested that MPs in the digestive tracts of fish may be transferred to the liver through some channel due to the presence of shorter MPs. The results of non-metric multidimensional scaling and Cluster analysis based on MPs characteristics indicate that MPs in the digestive tract and liver have more similar characteristics, supporting the hypothesis that MPs transfer from the digestive tract to the liver (Supplementary Discussion (4), Supplementary Fig. 7). Whereas there is controversy in traditional research regarding the particle size of MPs that can accumulate in the liver. It is generally believed that MPs with a particle size less than 150 μm can cross the intestinal mucosal barrier and transfer to the liver. However, in actual investigations, many examples refute this particle size threshold. MPs measuring 438 μm were found in the liver of Engraulis encrasicolus in the Mediterranean. MPs up to 567 μm in length were found in the liver of freshwater fish Squalius cephalus in the rivers of Paris. Recent studies have shown that the particle size range of MPs in the liver of different fish species is between 999.5 and 18.7 μm, exhibiting inter-species differences. On the other hand, long-term exposure to large particle size MPs can also exceed the threshold of 150 μm. For example, when using 0.1 to 1 mm MPs to expose Mugil Cephalus for 7 days, liver tissue sections confirmed the presence of 200-600 μm MPs. After exposing Uca Rapax to 180-250 μm MPs of different densities in surrounding sediments for 2 months, polystyrene fragments within this particle size range were found in the liver and pancreas of 100% individuals. For large animals, it is easier to accumulate larger particle size MPs in the liver. In the liver studies of cattle and sheep, 0.14 ± 0.23 and 0.06 ± 0.03 items g MPs were detected, and mainly distributed in the 500-1000 μm and 100-500 μm ranges, respectively. Especially for cows, MPs exceeding 500 μm account for over 80% of MPs in the liver. Therefore, for large organisms such as S. attenuata, on the one hand, their pipelines may be wide, which is more conducive to the transfer of large particle MPs to the liver. On the other hand, their longer lifespan may further increase the probability of this accidental entry. In the future, empirical work is still needed to understand the transfer and mechanism of MPs in the liver. For cetaceans, studying large animals such as cows or sheep may help to understand the entry of MPs into cetaceans' liver.

The material composition of MPs in cetaceans exhibits limited diversity. Specifically, 20 out of 27 MPs from wild individuals and 21 out of 38 MPs from captive individuals were identified as plastic materials. Both wild and captive individuals only had Polyamide (PA) and Polyethylene terephthalate (PET) detected (Fig. 4d). However, due to the lack of material confirmation for all MPs in this study, some potential MPs materials with relatively small proportions may have been underrepresented. Despite this limitation, the proportion analyzed is considered sufficient. In this study, there were only two types of MP materials in cetaceans, and one possibility for this phenomenon is that MPs of these two materials are common in the living environment of cetaceans in this study. This observation aligns with the PET MPs material being the most common (>30%) in seawater of the South China Sea. Earlier studies have also identified PA and PET as mainly components of MPs in the South China Sea. PA and PET are the most common MP materials in 18 fish species in the Xisha Islands Sea near the research area, accounting for 77% and 11% respectively. 38% of MPs were identified as PA in 481 fish species from 24 species in the Beibu Gulf adjacent to the study area. The limited diversity observed in this study may be attributed to another factor as the poor health conditions of the selected individuals. Wild individuals that are stranded often undergo rescue efforts, while captive individuals typically receive treatment in controlled environments after their health deteriorates. These circumstances may result in exposure to a restricted range of MP materials. PA and PET are commonly used plastics in textiles and engineering, and accidental contamination during the treatment process is a possibility. Another potential factor is that cetaceans may struggle to excrete specific components of MPs. These two materials account for 86.91% of all MPs in the digestive tract contents of stranded finless porpoises in Fujian, which provided side evidence for this hypothesis. This hypothesis provides a plausible explanation than hypothesis of ingestion during treatment for the observed phenomenon where wild cetaceans exhibit greater diversity in the forms and colors of MPs compared to their captive counterparts.

Cetaceans greatly contribute to global ecosystem functioning and maintaining marine ecology. Pollution is considered one of the major threats to cetaceans, and MPs as an emerging pollutant that may cause multi-level stress. Recognizing the target tissues of MPs in cetaceans and clarifying the differences in MPs between wild and captive dolphins can help promote cetacean conservation and improve animal welfare. The key contributions of this study are: