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Microplastics in Digestive System of Little-black cormorant (Phalacrocorax sulcirostris) in Pulau Rambut Sanctuary

Microplastics have been found in many seabirds in various areas in the world, but there has been no report from Indonesia. The objective of this research was to discover whether there were microplastics in the Indonesian Seabirds, represented by the Little-black cormorant (Phalacrocorax sulcirostris). The study site was Pulau Rambut Wildlife Sanctuary, a small island in Jakarta Bay, where a big colony of the cormorants’ nests together with other waterbirds. The gastrointestinal tract of a sample Little-black cormorant was collected to find macroplastic, mesoplastic, or microplastic. Microplastics were detected through NaCl dilution, followed by observation using 10x10 magnification and SRCC. There was no macroplastic and mesoplastic found in the sampled bird. As for microplastic, 16 microplastic particles of different types, sizes, and colours were found, giving an estimation of 320 particles/bird. Microplastic film type were dominant (75.0%), followed by fiber (18.7%) and fragment (6.3%). The size of the microplastic was mostly (68.7%) between 100-1,000 μm. Color-wise, more than half (56.2%) were transparent. Other colors found were red (18.7%), black (12.5%), yellow and blue (6.2% each), no brown or green.


Introduction
Plastic is lightweight, resistant to water, and has low prices, making it ideal for packaging and furniture. Global plastic production has increased from 1.5 million cubic tons per year in 1950 to 359 million cubic tons in 2018 [1]. Plastic waste has also increased with the discovery of a much plastic waste around us. In fact, plastic is the most abundant waste in waters, including in the marine environment. About 4.8 to 12.7 million cubic tons of plastic waste originating from land have been known to enter the oceans [2]. Plastic waste in the sea is commonly referred to as marine litter [3], which is usually grouped into five types according to their size: megaplastics (>100 cm), macroplastics (2.5 cm -100 cm), mesoplastics (5 mm -2.5 cm), microplastics (<5 mm) and nanoplastics (<1 μm) [4]. The plastics in the oceans have been shown to have a negative impact on marine life. More than 260 species, including invertebrates, turtles, seabirds, mammals, and fish, accidentally entangled and ingested plastics [5]. Several studies have proven the existence of plastics in the digestive tract of animals including seabirds, for example around 58% in the stomach of 1,295 Fulmarus glacialis in the North Sea [6], 13 out of 65 seabirds in Ireland [7], and 113 seabirds in the Catalan coast (Mediterranean) [8]. Furthermore, plastics were also found in the species Ciconia ciconia, Larus fuscus, and Larus michahellis in Portugal [9].
Pulau Rambut Wildlife Sanctuary -a small island located in Jakarta Bay, about 3 km off the coastline -also facing a plastic problem. The presence of plastic waste in the waters around Pulau Rambut cold has been polluted marine life, including birds. The island is a heronry of 13 waterbird species. Of these waterbirds, cormorants (Little-black cormorants Phalacrocorax sulcirostris and Pygmy cormorants P. pygmaeus) forage for food at sea, although these species are also able to fish in the freshwater wetlands in mainland java. Fish and insects water are the main food for cormorants [10,11]. Cormorants may accidentally ingest plastic when fishing in the sea water and/or ingest the plastic-contaminated prey fish.
The plastic consumed by animals can indirectly harm them and may lead to death in the long term. In the short term, plastics ingested by animals, including birds, may cause blockage of the digestive tract, decrease secretion of enzymes, decrease levels of steroid hormones, and disrupt reproduction [12]. Research on the impact of plastic on bird especially seabirds in Indonesia, unfortunately, has not been done. This study aims to identify plastics in the digestive system of Little-black cormorants roosting/nesting in Pulau Rambut Wildlife Sanctuary. The Little-black cormorants were selected as the research object due to their habit of fishing on sea water and due to their abundant population on the island. The data that had been obtained can be used as a reference in conservation management, especially seabirds and their habitat at Pulau Rambut Sanctuary.

Methods
Due to its status as a wildlife sanctuary, the local authority (Balai Konservasi Sumberdaya Alam DKI Jakarta) gave permits only to 3 individuals of the Little-black cormorant for this research. The birds were planned to be captured at night at their roosting tree. Fortunately, on the captured day, there was one individual bird found recently dead, and the body was quickly transported by using a cool box to the temporary holding in Taman Mini Indonesia Indah Bird Park in Jakarta. A few days after obtaining the samples, the sanctuary was locked down due to Covid-19, and remained locked down for the next year or so. Thus, no additional samples were able to be used for this research. We just used one sample Little-black cormorant.
Laboratory analysis was carried out in the Faculty of Fisheries and Marine Science, IPB University. Bird sample dissection followed the standard collection and dissection procedures [16]. The digestive organ from the esophagus to the intestine near the cloaca was removed for further analysis [16]. The contents of the digestive organs were liquified with 100 ml of saturated NaCl solution. Plastic particles that were directly visible to the naked eye (i.e., mesoplastic and macroplastic) were separated directly. Microplastics were identified and enumerated using a monocular microscope (magnification 10x10) and SRCC (Sedgewick Rafter Counting Cell) with five repetitions [17]. Plastics found then were grouped by type, size, and color.

Plastics in Little-black Cormorant
There were no macroplastic and mesoplastics found in the sampled bird. For microplastic, a total of 16 microplastic particles were found, having different types, colors, and sizes. The abundance of microplastics in the digestive tract was estimated at 320 particles. The number of microplastics found in the Little-black cormorants was lower than to other studies elsewhere. The number of microplastics found in the Little-black cormorant was lower than previous research in Florida found 1,189 microplastic particles in 63 samples with the fragment, fiber, and microbeads type [18]. Fewer microplastic particles were found in Fulmarus glacialis and Uria lomvia in the Canadian Arctic Region, as many as 34 particles in 57 individual birds [19]. On the Catalan coast of the Western Mediterranean Sea, 1,168 microplastic particles were found in 113 individual birds [8], while in Ireland, 92 pellets were found inside the Great Cormorant [12].
The types of microplastics found in the study area were mostly film (75%), followed by fiber (18.75%) and fragments (6.25%). A study of microplastics at the Labrador Sea on 30 Fulmar also found microplastics with film, fiber, and pellet types, as well as foam and rubber [21]. In Shanghai, fiber was the type of microplastic that dominate (54.9%) of the 17 sample birds [21]. The fiber was also the predominant type in bird samples observed in Florida at 94% [18]. Other previous studies of microplastics that have focused on contamination of water, sediment, and tissue in animals have shown that fiber was the dominant type [5,23,224].
The type of film that dominated microplastics in this study was related to the number of plastic bags and packaging found in the marine habitat, especially the waters surrounding Pulau Rambut. These plastics have undergone degradation into microplastics which can then be swallowed by the cormorants. As for fiber, it might originate from fishing activities around Pulau Rambut. Fiber is microplastics derived from nylon, polypropylene (clothing fibers, carpets, ropes, and fishing nets), and polyvinyl alcohol, all are common substances for fishing lines [25,4,23].
The color of microplastics has become an important research topic because many animal species forage for food by color [17]. The microplastic found in the sample Little-black cormorant consisted of 5 colors: transparent (dominant, 56.2%), black, red, yellow, and blue (Figure 3; left). Transparent colors were found in the microplastic fiber (3 particles) and film types (6 particles), while fragments (1 particle) were only found in yellow. Apart from transparent colors, films were also found in black (2 particles), red (3 particles), and blue (1 particle).  Plastic with transparent colors was commonly used for various products. Color might be added to polymers during the plastic manufacturing process, and over time these colors may be faded away due to environmental conditions [26]. The result of this research on microplastic color was different from other previous research. In the research on seabirds in the South China Sea, blue was the dominant color (91%), followed by dark and white [27]. Blue and transparent colors dominated two raptor birds, Pandion haliaetus and Buteo lineatus in Central Florida [17]. Furthermore, blue microplastic particles predominate in the seabirds Fulmarus glacialis and Uria lomvia, followed by red and white in Arctic Canada [19]. The different types or colors of microplastics found in animals can be caused by the abundance of microplastics depends on the environment [28]. Birds may ingest transparent or blue microplastic when looking for food or nesting material because they were mistaken to recognize plastic as prey or nest material [29].
Microplastics size of less than 5 μm were easily consumed by small-sized organisms such as phytoplankton and zooplankton, and later on, becomes a pathway for transfer to the food chain [30]. The size of the microplastics commonly was grouped into 8 groups, ranging in size from 0-5,000 μm. Microplastics found in the Little-black cormorant in this study were found in 5 of the 8 size groups. The most common size group was 101-1,000 μm, with a dominating size of 101-500 μm (37.5%, 6 particles), followed by the group size of 501-1,000 500 μm (31.2%, 5 particles; Figure 3b). This bird species catches prey underwater by swimming and diving, by using their large and webbed feet, assisted by the membranes on the feet and the nictitating membranes in the eyes [11]. These are the example of microplastics that were found in the Little-black cormorant digestive tract, fiber type with red color, and film type with blue color (Figure 4).
The Little-black cormorant might ingest microplastics by mistake of detecting plastics as food. Microplastics might accidentally enter the digestive system of the cormorants in the study area through sea water, while diving and swimming for fish and collect nest material, as also observed elsewhere [22]. The discovery of microplastics in the sample of the Little-black cormorant has shown the water pollution from plastic waste already has an impact on waterbirds nesting and roosting in Pulau Rambut. It is possible that microplastics can also be found in other waterbird species as well, either through direct accidental consumption from the sea water or through the food chain or trophic transfer, as reported by other researchers [31,24]

Environmental Impact
Plastic pollution has a negative impact on the environment and animals, especially marine life. The entanglement and accidental ingestion of macroplastics by animals has occurred, especially in vertebrates [32]. In this present study, no bird was found entangled by macroplastic, either in the sea water or while on the nest. However, the results of laboratory analysis have found microplastics in the digestive tract of the sampled water bird. The accumulation of plastic in the digestive tract can have an effect on bird health [12,33]. The effects of plastic consumption would not happen in immediately, and likely happen in long term.
Previous studies have shown that polybrominated diphenyl ethers (PBDE) compounds were found in the fatty tissue of the Short-tailed Shearwater (Puffinus tenuirostris) [34]. PBDE is one of the compounds contained in plastics and has persistent and toxic properties. Experimental research on the testing of butyl benzyl phthalate (BBP) in fish (Danio rerio) and bisphenol-A (BPA) in crustaceans (Acartia tonsa) and amphibians (Xenopus laevis ) showed that BBP and BPA could affect the development and reproductive disorders of various species of aquatic species [35]. This can also happen to other species that accidentally ingest plastic, including the Little-black cormorant in the study area, and might lead to the decline in the population of this species In conclusion, microplastics (320 items) were already found in the digestive tract of the Little-black cormorant, with the type of film, fragment, and fiber. The two dominant colors of the microplastics were transparent and black, while the dominant sizes were dominated by the 101-1,000 μm. This is the first evidence of the existence of microplastics in a wild bird. The results of this study are expected to help the conservation of seabirds, especially the little black cormorant on Pulau Rambut and in managing their habitat. Further research involving larger samples and various fish-eating species is needed, as a human has the same trophic level as a bird in fish consumption.

Acknowledgments
We would like to thank the Balai Konservasi Sumber Daya Alam DKI Jakarta for permit to conduct this study. We appreciate the assistance provided by Dicky, Warsajaya, Budi, Masrul, Iskandar, Hendra, Coki, Suli, Regia and Maya during the data collection, and by drh. M. Piter Kombo and Taman Burung Taman Mini Indonesia Indah for bird dissection process and laboratory support.