Accumulation of plastics in the aquatic environment is a global ecological threat and estimated that 4.8-12.7 million metric tons (MMT) of plastic waste enter the world oceans [1]. Due to their chemical composition and non-biodegradable nature, they make plastic waste as a great pollutant of varied dimensions and chemical composition affecting the life forms. Microplastics are fragments with a diameter < 5 mm as defined by USA National Oceanic and Atmospheric Administration are available in different shapes and colors. Primary microplastics are discharged from textiles and personal care products while secondary particles are derived from the manufactured macro plastics (>5 mm) and degraded within the ecosystem where they are deposited. The aquatic microplastics dominated by polymers such as Polyethylene, Polyethylene terephthalate, Polystyrene, Polypropylene, Polyvinyl chloride, nylon, and polyester films, foam and pellets found in sediments and water. Microplastic concentration found along the coasts of India particularly fishing and landing beaches of East coast and West coast of India. Given the growing evidence of the threats caused by plastics on marine organisms, reported that the Gulf of Kachchh and Gulf of Khambhat are the two potential plastic accumulation zones as Gujarat state is the highest producer and consumer [2-4]. Moreover, in the marine sediments along the ship-breaking yard of Alang, detected prominent level of Polyurethane (PU), nylon, PS, polyester, which are commonly used polymers in the construction of ships and the making of associated components such as insulating materials, fabrics. Packaging, etc. Microplastic ingestion in marine fish is well documented, with field studies reporting more microplastic ingestion by carnivores than omnivores [5]. Incidence of microplastics on marine invertebrates suggests their impacts through biomagnification to human via transfer of residues through marine food chains. There is evidence that nano plastics can move from a fish’s stomach to its muscle tissue and can disrupt marine food webs as they are found in the guts of fishes, seabirds, sea turtles and many invertebrates. Microplastics are harmful to the endocrine system, and cause internal injuries, and leads to stress for human consumers. Thus, a regular assessment of the bioaccumulation of microplastics in various dietary species of the marine environment became necessary to ensure food quality and maintain consumer preference [6-9]. In the present study, a baseline qualitative and quantitative study on the occurrence of microplastics on the alimentary tract of commercially important fishes has been undertaken from the Gulf of Kachchh [10-15].

Collection and Preparation of Sample

The present study was conducted on 16 species that belongs to two habitats viz., eight pelagic and twelve demersal fish samples were collected from Kandla, Mundra, Mandvi, and Pingleshwar coasts of Gulf of Kachchh, Gujarat (Fig.1). The specimens were immediately preserved in iceboxes for transportation to the laboratory and stored at -20°C until further process for the analyses. Followed by the identification, the feeding type, habitat and trophic level of the fishes were obtained from published literature and the online database Fish Base.

Fig1: Fish Sample Collection Locations along the Coast of Gulf of Kachchh.

Microplastics Extraction and Detection

The fish were thoroughly washed with distilled water to remove any surface contaminants, measured the total length and weight before dissecting them with sterile instruments. The stomach, intestine and gills were digested with KOH (10 % w/v, three times tissue volume) at 60? to get a clear solution [16]. The solution is filtrated using GF/C grade 47 mm glass filter paper in a vacuum system. After drying, the filters were examined under stereo-zoom microscope and subsequently analyzed under a fluorescence microscope (ZEISS Axioscope 5) to detect for the presence of microplastics based on their fluorescent signal and morphology [17-20]. Images of the identified microplastics were recorded for further characterization.

Fish specimens were collected from four distinct locations along the coastal region: Kandla, Mundra, Mandvi, and Pingleshwar. A total of 20 fish specimens representing 10 families were systematically documented and analyzed. Each specimen was identified to the species level following standard taxonomic keys and classified according to their respective families. Morphometric measurements were recorded for each specimen, including total length (cm) and body weight (g) using standard measuring techniques. Habitat preferences were categorized as either demersal or pelagic based on species' known ecological behaviors. Feeding habits were classified into four distinct categories: carnivore, omnivore, herbivore, and filter feeder, determined through literature review and gut content analysis. The sampling yielded diverse collections across locations: Kandla (5 species: Acanthopagrus arabicus, Thryssa purava, Nibea soldado, Mugil cephalus, and Arius jella), Mundra (5 species including Mugil parsia and Epinephelus coioides), Mandvi (3 species including Strongylura leiura), and Pingleshwar (7 species including Tenualosa ilisha and Ellochelon vaigiensis). Notable variations in species composition were observed across locations, with specimens ranging in length from 16 cm (A. arabicus) to 39.5 cm (S. leiura), and weight from 47.6 g to 382 g. Family representation varied significantly, with Mugilidae and Ariidae showing the highest representation (4 species each), followed by Sparidae (3 species), while other families were represented by single species (Table 1).

Table 1: List of Collected Fish Species.

The gut contents of the fish samples filtered on the GF/C filter paper were observed through the microscope for the presence of the microplastics. The presence of red, violet, blue, sky-blue, green and black filaments, beads and fragments were observed (Fig 4, 6). The particles were visible under stereomicroscope, however very small particles more visible under the fluorescent microscope. The size of the particles ranged between <100μm and >1000μm; in which, <100μm sized 71% of the particles were noticed in the gut of fish samples examined (Fig. 2 & 3).  The plastic fibre and pellets constituted 43% and 29% respectively and the fragments formed 19.2% of the microplastic recorded from the gut content of the fishes (Fig. 4).  While considering the colour of the particles, the dark blue was more common forming 41% followed by sky blue 20%, black 15%, green 11% and violet 9% (Fig. 6). The maximum number of microplastics were collected from the gut of Mugil cephalus followed by Arius jella. However, both pelagic and demersal fish showed the presence of microplastics in their stomach content. Quantitative assessment of the plastic contaminants and the possible impacts have been reported from the marine environment. Ingestion of plastic debris, large and small, has been demonstrated across food webs from zooplankton to large predatory fish and marine mammals [21]. There are several methodologies adopted to identify the microplastics in the soil, water and in the tissues of animals. Acid digestion followed by microscopic examination as well as spectroscopy techniques are the familiar methods for the extraction of micro plastics.

The highest levels of microplastic contamination were observed in two dominant species. Arius jella, a demersal catfish, exhibited the maximum contamination at 15%, followed closely by Mugil cephalus at 14%. These findings are particularly significant as these species represent different habitat preferences and feeding strategies, suggesting multiple pathways of microplastic accumulation in marine organisms. A considerable number of species showed moderate levels of microplastic occurrence. Tenualosa ilisha and Eleutheronema tetradactylum each demonstrated 7.5% contamination, and Thryssa purava displayed 5.63%. Additionally, Epinephelus coioides showed 5.33% occurrence, while Nibea soldado, Arius maculatus, and Thryssa setirostris each demonstrated 5% microplastic presence. Several species exhibited relatively lower levels of microplastic contamination. Acanthopagrus arabicus and Ellochelon vaigiensis each showed 4.16% occurrence, followed by Scatophagus argus at 3.33%. The lowest levels of microplastic contamination were observed in Strongylura leiura and Cynoglossus sp., each registering 2.5% occurrence (Fig.5).

Fig 2: Microplastic Particles Identified by Light Microscope.

Fig 3: Microplastic Particles Identified by Fluorescent Microscope.

Fig 4: Percentage of Different Microplastics.

Fig 5: Percentage Occurrence of Microplastics (%) in Fish Species.

Fig 6: Percentage Composition of Coloured Particles.

The distribution of microplastic contamination showed interesting patterns related to habitat preferences. Demersal species, particularly Arius jella, showed higher contamination levels, possibly due to their bottom-dwelling nature and feeding habits. Pelagic species demonstrated varying levels of contamination, suggesting that water column position might influence microplastic exposure. The variation in microplastic occurrence appears to correlate with feeding strategies. Filter-feeders and bottom-feeders generally showed higher contamination rates, suggesting that feeding behavior plays a crucial role in microplastic ingestion. Carnivorous species showed moderate to high contamination levels, while omnivorous species demonstrated variable contamination rates.

The microscopic examination of the gut content of the fishes revealed on the prevalence varied shapes and sizes and types of microplastics in the alimentary tract of common food fishes caught from the Gulf of Kachchh during the pre-monsoon season. The microplastics were identified from the stomach content of both pelagic and demersal fishes.  Mugil cephalus is widely found in the creek environments and exhibit diurnal feeding habit and omnivore. This fish consumes more microplastics like the shallow bottom feeding omnivore, Arius jella which consume detritus, worms and microcrustaceans. There was no significant difference in the number of microplastics ingested between demersal and pelagic fish [22-24]. The carnivorous fishes in the study showed less microplastics. Microplastics are ingested by zooplankton, fishes, crustaceans and Molluscs which are filter feeders and may incorporate into marine food webs through trophic transfer.

The analysis of microplastic types and their relative abundance in the gut contents of the collected fish species provides further insights into the nature of plastic pollution affecting the marine ecosystem. The predominant form of microplastics observed were fibers, comprising 43.32% of the total microplastics found. This suggests that the fish are ingesting a significant amount of textile-derived fibers, likely originating from wastewater discharge or fragmentation of larger textile items. Beads and pellets were the second most common type, accounting for 29.22% of the microplastics. These small, spherical or cylindrical microplastics are often associated with personal care products and industrial processes. Fragmented pieces of larger plastic items made up 19.22% of the microplastics, indicating the breakdown of larger plastic waste in the marine environment. Finally, thin plastic films represented the smallest proportion at 8.33%, potentially originating from packaging materials or other sheet-like plastic sources (Fig.4).

Microscopic examination applied for the rapid screening of the particles-to report the shape, color, and size of the plastics in the water, sediment, and gut content samples are characterized. Stereomicroscopy of microplastics revealed blue, pink, and green color filaments, irregular shaped pieces and films having   size varying between >50 μm to <1000 μm in the gut samples of the selected fishes. The presence of microplastics in the gut contents of the Bombay-duck (Hargadon nehereus) and Sardinella gibbosa and Cyprinus carpio following alkali digestion protocol and reported the predominance of fibers less than 0.5 mostly green, blue, and black colour [6]. The contamination of fish and fish food products by microplastics has been reported from [2] the processed fish meal is greater than the whole fish. Similarly cultured organisms harvested in the wild showed microplastics in their guts. The occurrence of microplastics in the planktivorous fish Decapterus muroadsi is associated with its nature of feeding by seeing the prey and capturing them. Hence long-term extensive studies including the spatio-temporal distribution in environment and the biota of various trophic levels are essential.

Microplastics are of environmental concern as their small size makes them available to a wide range of marine biota through trophic transfer.  Microplastic ingestion has been demonstrated in fish and those ingested ranged between 0.01 and 1.6 mm [23,25]. Filamentous micro plastics are generally dominated among the ingested micro plastics as observed in the study and there were blue and red filaments in the gut content. The presence of plastic particles in the stomach of Mugil cephalus has been reported [26,27] and the findings indicate that the amount varies greatly among species.

The study reveals a complex pattern of microplastic contamination in marine fish species, with contamination levels ranging from 2.5% to 15%. The variation in contamination levels appears to be influenced by both habitat preferences and feeding strategies, suggesting that multiple factors contribute to microplastic accumulation in marine organisms. The diversity of microplastic forms encountered, ranging from fibers and fragments to beads and films, underscores the complex nature of plastic pollution in the coastal waters. This variety of microplastic types highlights the multiple pathways through which these synthetic particles can enter the marine food web and ultimately be consumed by fish species.      

Acknowledgement

The authors are thankful to the Director and Management of the Gujarat Institute of Desert Ecology, Bhuj, for providing all the laboratory facilities to carry out the study. One of the authors wishes to express his gratitude to the Department of Fishery Science, Alagappa University for permitting to complete the study and all the staff at GUIDE for their encouragement and cooperation during the study period.

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