Environmental Drivers and Bioaccumulation Pathways of Microplastics in Freshwater Fish from the River Yamuna, India

Abstract

Microplastic (MP) contamination is an emerging threat to aquatic ecosystems. However, species-specific bioaccumulation patterns across trophic guilds in tropical river ecosystems remain scarcely understood. This study assessed the occurrence, organ-level distribution, polymer composition, and ecological risk of MPs in 220 fish representing 12 species, spanning across multiple trophic guilds, sampled from four sites along a pollution gradient of the river Yamuna, India. MPs were detected in all examined species, confirming extensive distribution across the river ecosystem. A total 1678 MPs were recovered, with significantly higher abundance in fish from the highly urban Delhi stretch than in those from upstream regions (Kruskal–Wallis, H = 11.03, p = 0.011). The highest species-specific MP load was recorded in omnivorous Oreochromis niloticus from Sonia Vihar (436 MPs), whereas the carnivorous species Xenentodon cancila exhibited the lowest accumulation (37 MPs). Surface- and mid-water herbivores and omnivores accumulated more MPs than benthic carnivores and detritivores. Nonetheless, spatial pollution gradients exerted a stronger influence on MP accumulation, compared to trophic guilds. The gastrointestinal tract exhibited the highest MP abundance (751 MP particles), followed by gills (605) and muscle tissues (322), confirming ingestion as primary uptake route, and suggesting possible tissue translocation. Fibers dominated in the assemblage (77.8%), while transparent (44%) and blue (19.5%) were most abundant colors. ATR–FTIR analysis confirmed 10 diverse polymers, with polyethylene (≈24%) and polypropylene (≈21%) together accounting for nearly half of the identified particles. The Polymer Hazard Index analysis classified the recovered MP mix as Category IV (high ecological hazard). These findings identify the Delhi stretch of the Yamuna as a high MP contamination zone and highlight the combined influence of urban pollution and fish ecology on MP bioaccumulation.

1. Introduction

Currently, reliance on plastic products has become indispensable owing to their sturdiness, affordability, durability and ease of handling. Hence, plastic pollution has increased substantially over the last 80 years, leading to the production of about 413.8 million tons of plastic products globally [1,2]. The extensive use of these materials is largely attributed to their favorable physico-chemical properties, such as resilience, thermostability, and greater standards across diverse environmental conditions [3].

Microplastics (MPs), defined as miniscule (<5 mm) plastic particles, are persistent and ubiquitous in the environment [4,5]. They are broadly classified into two distinct categories: primary microplastics, which are intentionally manufactured by plastic and cosmetic industries to be incorporated in their products, for example, microbeads in cosmetics or plastic pellets for industrial uses; and secondary microplastics, which form when larger plastic items get broken down due to fragmentation, UV-degradation, wave action, and the shedding of synthetic fibers from textiles [6]. Despite their widespread occurrence, a significant legislative gap persists globally in regulating MPs, with most existing frameworks limited to primary MPs, such as intentionally produced microbeads in personal care products (PCPs), while secondary MPs, such as from tire abrasion and synthetic textiles, remain largely under-addressed; additionally, the absence of standardized MP-detection methodologies further complicates regulatory enforcement [7]. They are omnipresent across terrestrial, aquatic and even atmospheric environments, and additionally pose hazardous threats to organisms through unintentional inhalation, ingestion, and bioaccumulation, which in turn indicates that these ubiquitous contaminants have reached the different levels of the food web [8]. The evidence indicates that rivers function as major conduits for the transport of plastic debris from terrestrial to marine ecosystems [9]. Microplastic pollution is progressively being documented as a serious potential threat to many biotic communities [10,11]. MP ingestion could pose substantial health risks to different fish species, including genotoxicity effects in Danio rerio [12] and Cyprinus carpio [13], as well as pronounced inflammation responses in O. niloticus [14], histopathological alteration in Clarias gariepinus [15] and Oncorhynchus mykiss [16], and neurotoxic effects in zebrafish [17,18] and Japanese medaka (Oryzias latipes) [19]. MP contamination also disrupts antioxidant ability in C. carpio and Carassius auratus and reproductive performance in zebrafish, tilapia and common carp [20,21,22]; imbalances reactive oxygen species (ROS); and damages cellular components like proteins, DNA, and mitochondrial functions [23,24]. The widespread occurrence of MPs has also raised concerns about current environmental laws and highlighted the need for unified policies to reduce the effects of their occurrence [7].

MPs uptake in fish primarily occurs through direct ingestion, wherein the fish might mistake MPs for their prey/food, as the material mimics the shape, size and color of their natural prey, or through non-selective filtering of water and sediments by omnivorous and filter feeder fishes. These ingestion patterns are strongly influenced by the fish’s trophic level and feeding ecology [25]. The filter feeders and omnivorous fish species often exhibit the highest MP ingestion rates, due to their broad dietary spectrum and frequent interaction with suspended particulates and benthic detritus, whereas predator species primarily acquire MPs via trophic transfer from contaminated prey, leading to progressive accumulation along the food web [26,27]. Furthermore, environmental factors played a role; urbanized sites exhibited higher MP accumulation in fish populations [28]. The dietary exposure to polylactic acid microplastics (PLA–MPs) caused retardations in growth, hemato-physiological indices, mineral composition and nutrient digestibility in the fingerlings of Labeo rohita [29]. Also, controlled exposure to MPs in freshwater fishes (D. rerio, Perca fluviatilis, Ctenopharyngodon idella, O. latipes, O. mykiss, C. carpio) can induce biochemical, physiological, and behavioral change disruptions in fish [30].

Globally, freshwater fish represent one of the most diverse vertebrate groups, with more than 19,000 species documented [31], while India alone harbors over 1239 freshwater fish species [32]. India is now the second-largest aquaculture producer worldwide, with aquaculture contributing markedly to national nutrition security and yielding a per capita fish consumption of ~8–9 kg/year, a figure steadily increasing with rising protein demand. This growing dependence on fish as an affordable animal protein source raises concerns regarding MP exposure, as contaminated fish tissues represent a direct dietary pathway for human consumption. In parallel, rapid industrialization, untreated sewage discharge, textile fiber release, packaging waste runoff and fragmented urban plastic waste have emerged as major MP-generating sources, intensifying ecological pressure on aquatic food webs.

Multiple studies across geographically diverse freshwater systems in India have confirmed microplastics ingestion by fish. Studies from north India showed the presence of MPs in gills, gastrointestinal tract (GI tract) and muscle tissue of different fish species, viz., Channa punctatus, L. rohita, Labeo bata, Salmostoma bacaila and Puntius amphibius, with contamination levels varying among the species [33]; 144 MPs were recovered from eight fish species, namely, L. rohita, L. bata, Mystus tengara, Ompak bimaculatus, Tenualosa ilisha, Pangasius pangasius, Scomberimorus guttatus, and Labeo catla, with fibers accounting for nearly 69% of the particles found in the Subarnarekha–Kharkai river system of the Chhota Nagpur region [34]. Similarly, MPs were detected in water, sediments, and the digestive tracts of fish from the Brahmaputra River, with ingestion rates ranging from 1 to 5.33 MP particles/individual [35]. In the Pawna River (Maharashtra), MPs abundance in GI tracts ranged from 4.2 ± 1.5–22.3 ± 5.6 in C. punctatus to 3.1 ± 1.2–18.9 ± 4.7 MP particles g⁻¹ in L. rohita.

Similarly, multiple studies on the contamination of MPs in fish have been reported along the Indian coastline: Nithin et al. [36] detected MPs in gills and GI tracts of commercially important fish species from the Vellar estuary, Parangipettai; Prusty et al. [37,38] reported that MPs contamination varied between 6.98 ± 6.73 MPs/g in gastrointestinal samples from the fishing harbors of Gujrat and Maharashtra; Mandal et al. [38] and Patel et al. [39] detected MPs in approximately 30% of the commercially important fish species from the eastern coast—the study reported a MPs burden ranging from 3.2 to 7.8 MPs/individual in sixteen fish species from the Adyar Estuary [40] and Ennore Creek, Chennai [41], and another study on four species present in Sal estuary in Goa also reported high levels of fiber across all samples [42].

The river Yamuna, a major tributary of the river Ganga and a vital freshwater resource for northern India, flows through densely populated and industrially intensive regions, making it preferentially susceptible to pollution, with possible toxicological implications. The river embodies one of the most anthropogenically stressed freshwater ecosystems in the Indian subcontinent, receiving continuous inputs from industrial effluents, storm run-off, urban wastewater drainage and legacy pollutants along its course, especially in densely populated regions [43]. Such sustained pressure accelerates the fragmentation of macroplastics and promotes the influx of polymeric fragments and textile-derived microfibers as well as other synthetic particulate debris, rendering the river Yamuna a MP accumulation zone. Recent studies have highlighted substantial legislative gaps in the management and control of microplastic contamination, emphasizing the need for robust scientific evidence to support policy development and environmental protection measures [7]. Given their multiple-trophic-level habitat and diverse feeding habits, freshwater fishes are sensitive bioindicators for the tracing of MP exposure pathways within the impacted ecosystems. Such data are essential not only for ecological risk assessment but also for supporting evidence-based management strategies and regulatory frameworks addressing microplastic pollution. In the present study, we therefore selected 12 different ecologically and taxonomically distinct fish species (n = 220) inhabiting different zones of the river to examine species-specific MP ingestion, retention and organ-level accumulation patterns. To evaluate the influence of ecological traits and provide an in-depth understanding of how ecological characteristics affect microplastic uptake and retention across the river’s pollution gradient, these species were decisively chosen in accordance with their varied feeding guilds (herbivores, insectivores, carnivores, omnivores and detritivores) and trophic positions. Despite the growing reports of MP pollution in marine ecosystems, species-specific evidence linking the trophic ecology, organ-level accumulation and polymer-specific ecological risk assessment in tropical freshwater river ecosystems remains limited, particularly for heavily anthropogenically urbanized rivers in developing countries such as India. In addition to this, comparatively fewer studies have simultaneously examined spatial pollution gradients, organ translocation patterns and polymer hazard profiles across multiple fish taxa from freshwater-inhabiting distinct ecological niches. In the present study, we hypothesized that (i) the freshwater fish species inhabiting highly urbanized downstream stretches of river Yamuna would exhibit a significantly higher MP load than the fish species sampled from relatively less-impacted upstream regions; (ii) the GI tract would contain highest MP load relative to those of gills and muscle tissues, which reflects ingestion as the primary uptake exposure route, followed by possible secondary organ translocation; (iii) MP accumulation would vary among the different fish species according to their habitats, feeding guilds and trophic behaviors, and (iv) the polymer composition would be dominated by one-time-use or consumer plastics.

Hence, the study aimed to generate robust baseline evidence on MP prevalence, spatial distribution, characterization, and polymer confirmation in freshwater fishes of the river Yamuna by quantifying the MP uptake and bioaccumulation in the freshwater fish species collected along the different pollution gradients of the river. To the best of our knowledge, this study is a pioneer in comprehensive studies conducted in the river Yamuna that simultaneously examine species-specific MP bioaccumulation, polymer composition, organ specific distribution and hazard-based ecological risk assessment across diverse freshwater fishes representing multiple trophic guilds. This study also advances an ecological assessment framework, which could further help in informing targeted management strategies for heavily stressed tropical river systems all over the world.

2. Materials and Methods

2.1. Fish Sampling and Collection Sites

The Yamuna River is a principal tributary of the Ganga River system, and originates from the Yamunotri glacier in the western Himalayan range at an elevation of approx. 6387 m. As it extends along its course (~1376 km), it drains a catchment area of nearly 366,000 km² across Uttarakhand, Himachal Pradesh, Haryana, Delhi, and Uttar Pradesh, encompassing diverse physiographic as well as different climatic zones, which helps in shaping its hydrological regime [43]. Geographically, the river Yamuna basin lies between 28°24′–31°31′ N and 77°00′–79°48′ E. Its downstream flow experiences intensive anthropogenic pressures due to unmanaged industrial discharges, urban run-off, and untreated municipal wastewater, which is drained directly into the river [44,45]. Previous studies have documented elevated sewage inputs, organic pollution, suspended solid wastes in the wastewater and suspended particulate matter loads leading to deteriorated water quality parameters in the Delhi stretch of the Yamuna River, particularly near Sur Ghat and Sonia Vihar [44,46]. In this study, 4 different sampling sites (Figure 1), viz., Yamuna Nagar (YNR) and Karnal (KRN), Haryana, and Sur Ghat (S) and Sonia Vihar (SV), Delhi (Table 1), were chosen, from which a total of 12 fish species (220 individuals) (Figure 2) were collected. Comprehensive and systemic ecological sampling was carried out in the months of February–March, 2023 and September–November, 2024, over a course of two years, to capture representative fish populations under distinct hydrological conditions, while avoiding the heavy monsoon flooding periods, which could noticeably alter the river water flow and fish distribution patterns. Multi-season sampling also improved the representativeness of MP exposure assessment across the studied Yamuna River stretches. The four sampling sites were selected to represent a spatial pollution gradient along the river Yamuna, encompassing the comparatively less-impacted upstream regions Yamuna Nagar (YNR—30°05′00.4″ N 77°21′50.8″ E) and Karnal (KRN—29°40′57.8″ N 77°08′41.4″ E) in Haryana, and in a highly anthropogenic–urbanized downstream stretch, Sur Ghat (S—28°42′36.1″ N 77°13′51.0″ E) and Sonia Vihar (SV—28°43′53.6″ N 77°14′08.3″ E), located in Delhi (Figure 1, Table 1). Upstream sampling locations were characterized by their relatively low population density, reduced industrial discharge and low sewage influx, whereas for the Delhi stretches, there was a substantial number of unmanaged solid waste inputs, significant municipal wastewater discharge, untreated sewage, textile effluents and stormwater run-off into the main channels, through multiple drainage systems. Therefore, this sampling-site selection framework enabled a more comprehensive and comparative assessment of MP contamination under varying anthropogenic contaminant pressure levels and facilitated evaluation of spatial trends in MP bioaccumulation across the ecologically distinct river segments. The number of fish species at each sampling point varied based on specific site conditions. The Karnal and Yamuna Nagar sites showed relatively higher species diversity, while the Delhi sites contained more pollution-tolerant species: O. niloticus and L. rohita. The fish samples were collected using cast and gill nets at each sampling site and immediately preserved by wrapping in a pre-cleaned aluminum foil to avoid secondary plastic contamination, as per standard protocols, and placed in insulated ice boxes to be brought back to the laboratory. Sampling was conducted under comparable field conditions to minimize the site-specific collection bias. The fish were then carefully placed in a −20 °C deep freezer until the experimental procedures were carried out, to avoid any contamination and degradation. Synthetic storage materials were strictly avoided during the sample handling and transport process.

Figure 1. Geographical map and pictorial images of sampling sites along the river Yamuna.

Table 1. Fish species, sampling locations, feeding habits and feeding zone for each sampling location.

Fish Common Name	Scientific Name	Location Code	Total No.	Sampling Location	Length (cm)	Weight (g)	Feeding Habit	Feeding Zone
Notopterus	Chitala chitala	KRN	10	Karnal, Haryana	20 ± 1	60 ± 10	Carnivore	Bottom
L. boggut	Labeo boggut	KRN	20	Karnal, Haryana	11 ± 1	6 ± 4	Herbivore	Mid
Needle Fish	X. cancila	KRN	10	Karnal, Haryana	16 ± 1	30 ± 15	Carnivore	Surface
Ticto Barb	Pethia ticto	KRN	10	Karnal, Haryana	5 ± 3	21 ± 2	Omnivore	Mid
Malli	Wallago attu	YRN	10	Yamuna Nagar, Haryana	34 ± 2	319 ± 50	Carnivore	Bottom
Pool Barb	Puntius sophore	KRN	10	Karnal, Haryana	5 ± 2	5 ± 4	Omnivore	Surface–Mid
Pool Barb	P. sophore	YNR	10	Yamuna Nagar, Haryana	5 ± 2	5 ± 2	Omnivore	Surface–Mid
Fine scale razorbelly minnow	Salmostoma phulo	YNR	10	Yamuna Nagar, Haryana	20 ± 2	7 ± 5	Omnivore	Surface–Mid
Battar	Labeo angra	YNR	20	Yamuna Nagar, Haryana	25 ± 1	250 ± 20	Detritivore	Bottom
Chilwa	Chela cachius	KRN	10	Karnal, Haryana	6 ± 2	6 ± 3	Insectivore	Surface
Chilwa	C. cachius	S	10	Surghat, Delhi	6 ± 2	8 ± 2	Insectivore	Surface
China Rohu	L. rohita	SV	20	Sonia Vihar, Delhi	32 ± 3	370 ± 10	Herbivore	Surface–Mid
China Rohu	L. rohita	KRN	20	Karnal, Haryana	30 ± 3	250 ± 50	Herbivore	Surface–Mid
Rohu	Labeo calbasu	YNR	10	Yamuna Nagar, Haryana	22 ± 1	270 ± 20	Omnivore	Mid–Bottom
Tilapia	O. niloticus	SV	20	Sonia Vihar, Delhi	15 ± 2	42 ± 5	Omnivore	Surface–Mid
Tilapia	O. niloticus	S	20	Surghat, Delhi	13 ± 2	36 ± 7	Omnivore	Surface–Mid

Location code—KRN = Karnal, YNR = Yamuna Nagar, SV = Sonia Vihar, S = Surghat.

Figure 2. Fish species collected from four different sampling sites along river Yamuna.

The selected distinct fish species represented varied trophic guilds, feeding behaviors, ecological habitats and water-column niches, thereby enabling comprehensive evaluation of species-specific as well as habitat-dependent MP accumulation patterns. The selection of sampled species was also based on their ecological significance and availability across the studied river stretches, ensuring a broad representation of the diverse freshwater fish community inhabiting the river Yamuna ecosystem.

2.2. Wet Peroxidation Reaction: Hydrogen Peroxide (H₂O₂) Treatment

After defrosting, the fishes were wiped with alcohol and muslin cloth, and then individuals of each fish species were measured for total length and weight. For the extraction of MPs, organs gills, gastrointestinal tract (GI tract), and muscles were dissected and placed in pre-cleaned beakers into which 30% H₂O₂ was added for a wet peroxidation reaction [47,48]. Then, the beakers were covered by aluminum foil to avoid any environmental contamination and kept for 24–72 h in a shaker incubator at 65 °C at 80 rpm to ensure complete digestion of the organic matter present in the samples.

2.3. Density Separation and Filtration

The subsequent step involved density separation; this was performed by adding filtered supersaturated saline solution [NaCl] to each beaker and then again covering the beakers with aluminum foil. Thereafter, the beakers were kept in a clean and dry place for around 2–3 weeks to allow effectual floatation, prompting as well a separation of the potential MPs based on buoyancy [49]. Prolonged settling ensured complete separation of low-density and high-density polymers. NaCl was used for the density separation and floatation as it has higher MPs extraction efficiency, extending up to 90% [47,50,51]. For efficient extraction of low-density and high-density MPs (such as PE, PP, PS, LDPE, HDPE, PET, and PVC), the protocol of Dong et al. [51] has been employed.

2.4. Extraction and Visual Observation of Microplastics

The supernatants obtained after density separation in each beaker were subjected to vacuum filtration using glass fiber filters (Whatman GF/C Membrane Filter, 47 mm diameter, 0.45 µm). A Borosil glass vacuum pump filtration unit was used to filter each sample separately. The filter papers were then recovered carefully with sterilized stainless-steel forceps, and subsequently placed in covered glass Petri plates and kept for 1–2 days in an isolated and dark place for them to dry out for further procedures. All the MPs that accumulated on the surface of the filter papers were further analyzed under an Olympus SZ61 Stereo Microscope (Olympus Corporation, Tokyo, Japan) and a Nikon Upright Microscope–Eclipse Ei Microscope (40–1000×) equipped with Digital Sight 1000 camera (Nikon Corporation, Tokyo, Japan). This step was also done to help in manual quantification of the MPs present in the samples.

2.5. Characterization and Polymer Group Identification of Microplastics

MP morphotypes were classified into five different categories (fibers, fragments, microbeads, film, and foam) and colors (transparent, blue, yellow, red, green, black, etc.) and were simultaneously recorded as per the guidelines [50]. The images were taken at various magnifications for successive identification of MPs. In addition, a JEOL JSM 6610LV Scanning Electron Microscope (SEM) (JEOL Ltd., Tokyo, Japan) coupled with Energy Dispersive X-ray Analysis (EDAX) (Ametek Inc., Mahwah, NJ, USA) was used to detect the elemental composition and associated potential contaminants on the microplastics’ surface [52]. For the precise visualization of surface topography enabling identification of structural deformities such as cracks, pits, scratches, fractures, tiny grooves and gouges, a scanning electron microscope (SEM) was used. These surface morphologies demonstrate how the environmental processes cause plastic particles to abrasively age as well as degrade, oxidatively and mechanically [53,54]. For SEM–EDAX imaging, MP particles retained on the filter membranes were carefully isolated and then mounted on aluminum stubs. The mounted samples were examined under high-vacuum conditions using SEM to obtain the detailed particle surface topography and physical alterations of the MPs, such as morphological degradation patterns and elemental composition, thereby offering insights into polymer weathering and adsorption of pollutants. At the same time, EDAX enabled elemental characterization, further supporting the assessment of surface modification and the environmental interaction of the recovered MP particles.

Polymeric confirmation of the recovered MP particles was performed using Fourier Transform Infrared Spectroscopy (FTIR). Particles of the size range ~200 µm to 5 mm were analyzed using a Nicolet iS50 FTIR Tri–detector equipped with a built-in diamond, in Attenuated Total Reflectance (ATR) mode (Thermo Fisher Scientific, MA, USA). Each spectrum was acquired in the mid-infrared range of 4000–400 cm⁻¹, with 30–100 scans per sample and a spectral resolution of 8 cm⁻¹ for polymer confirmation. The obtained spectra were matched against the Open Specy software’s known FTIR polymer reference spectral library [55], the Hummel Polymer Sample Library, FLOPP, and FLOPPe, with a minimum spectral match threshold of 80% against reference libraries was accepted as confirmation; synthetic polymers were also used to analyze and confirm FTIR peaks [56]. Polymer types were then confirmed through verification based on the characteristic absorption peaks and fingerprint regions. The particles that did not meet this threshold were excluded from the counts. Spectral peak matching and processing were conducted using Open Specy and the Hummel spectral libraries.

Atmospheric compensation and automatic baseline correction were applied prior to the spectral matching. Also, the spectra with poor or below signal-to-noise ratios, excessive atmospheric interference and flattened baselines were excluded. For the smoothing/noise reduction, a Savitzky–Golay filter was applied where necessary, to improve spectral clarity [56]. The false positive rate, which is defined as the proportion of visually identified particles subsequently not confirmed as a synthetic origin polymer by FTIR spectroscopy, was calculated from the validated data subset to provide a transparency of identification accuracy, aligning with the study done by Käppler et al. [57].

2.6. Ecological Risk Assessment

The Polymer Hazard Index (PHI) is an ecological risk assessment index that quantifies the measurement used to rank plastic polymers based on their inherent chemical hazard, incorporating factors like persistence, toxicity and harmful monomers or additives. These pose a greater risk to fish health, as they often leach out toxic additives as well, which can induce oxidative stress, physiological disruption and tissue damage. Polymers like polystyrene (PS) and polyvinyl chloride (PVC) archetypally exhibit higher PHI values owing to their associated carcinogenic or endocrine-disrupting components. In contrast, polymers such as polyethylene (PE) and polypropylene (PP) fall in lower hazard categories [58]. PHI provides a comprehensive and beneficial framework for assessment of the relative risks posed by different types of polymers detected in biological or environmental samples. It also helps in contextualizing polymer-specific contamination patterns and their potential ecological implications. Therefore, identifying high-hazard polymers that are ingested by fish species is critical, so that further evaluation can support determinations of potential toxic effects.

Herein, the PHI was calculated using the formula

$$\mathrm{PHI}=\sum (Pn\times Sn)$$

where Pn is percentage composition of a specific polymer and Sn is the hazard score that is assigned to each polymer type [58,59,60]. PHI was divided into 5 distinct levels, I (10, very low risk), II (10–100, low risk), III (100–1000, medium risk), IV (1000–10,000, high risk) and V (>10,000, very high risk) [58,61,62].

2.7. Quality Assessment and Control (QA/QC)

Rigorous, multi-tiered and standard laboratory and systematic QA/QC operating protocols with stringent quality controls were implemented throughout the experimental procedures to minimize contamination risks and ensure analytical replicability of data [55,63].

Laboratory contamination prevention:

All the glassware used in the experiments were precleaned by soaking in chromic acid solution, rinsed with filtered double-distilled water (0.45 µm filter membrane), and wrapped in aluminum foil between experiments. Linen and cotton laboratory aprons exclusively were worn throughout the experimental procedures to eliminate the risk of synthetic-fiber shedding. All the chemical solutions (H₂O₂ and NaCl) were also pre-filtered through 0.45 µm glass-fiber membranes prior to usage. Sample vessels and the filter papers were stored in Petri plates wrapped in aluminum foil and kept in an isolated, designated laboratory space away from air conditioning vents, general laboratory traffic, and synthetic textiles. To ensure analytical consistency, the handling procedures were maintained across all sampling sites. All the species specimens were processed using identical dissection, digestion, filtration and analytical workflows under standard conditions.

Atmospheric [Air–blank] control:

To quantify the potential airborne MPs deposition, open, pre-cleaned Petri plates containing untouched glass fiber filter papers were subsequently placed beside each active sample during the experimental procedures and left exposed for equivalent procedural durations. These airborne-control blanks were subsequently examined under the stereomicroscope, using experimental protocols identical to the ones for sample filters (controls). The mean numbers of airborne MP particles, which were next to negligible, deposited on the atmospheric blanks were recorded and these values were then subtracted from the corresponding sample counts to yield the correct MP concentrations [64].

Procedural blanks:

For each batch of the sample processed, a full, standardized procedural blank was run in parallel: 30% H₂O₂ was applied to an empty pre-cleaned beaker that contained no fish organ/muscle tissue; this was followed by complete density separation and a further filtration protocol under the identical environmental conditions. No MP particles were detected in any of the procedural blanks, indicating a low likelihood of MP contamination during sampling, pre-treatment and experimental procedures. These comprehensive and precautionary measures ensure the legitimacy and reliability of the research findings.

2.8. Statistical Analysis

Origin 2022 and OpenSpecy were used for FTIR data and graph visualization. Fish species were classified into different ecological taxa, feeding habits and habitats as per FishBase data [65]. Statistical analyses were performed to investigate the correlations between MP concentrations in different fish organ systems and to assess the variance among ecological categories and sample locations. Pearson and Spearman correlation coefficients were calculated to investigate linear and monotonic relationships between MPs within the GI tract, gills, and muscle tissues, and total MP load. The non-parametric Kruskal–Wallis test was conducted to examine differences in MP abundance between feeding guilds and sample sites. All the analyses were done in the standard statistical software RStudio version 4.2, JASP 0.17, and Microsoft Excel 2019, with the level of significance at p < 0.05. These statistical analyses were performed to test the study hypotheses concerning the spatial variation in MP abundance, organ-specific accumulation patterns and differences among trophic guilds.

3. Results and Discussions

3.1. Distribution and Abundance of Microplastics

A total of 220 fish specimens representing 12 diverse species were collected from different sampling locations and were analyzed to assess the distribution and abundance of MPs; the data are presented in Table 2. MP abundance exhibited substantial variability among species and across sampling sites. The findings of the study broadly supported the proposed hypotheses that anthropogenically strained urban river stretches and the tropic ecology strongly influence MP bioaccumulation patterns in the diverse freshwater fish species. A total of 1678 MP particles were recorded across all examined individuals, with the highest accumulation observed in O. niloticus from Sonia Vihar, Delhi (n = 20; 436 MPs), followed by O. niloticus from Surghat, Delhi (n = 20; 265 MPs), and the lowest abundance was detected in X. cancila from Karnal, Haryana (n = 10; 37 MPs). Spatial variation in total MP abundance per fish species across sampling sites (Figure S4) showed markedly high MP loads and variability at Sonia Vihar, Delhi compared to the upstream locations.

Table 2. Relative abundance of microplastic reported in different fish species from the river Yamuna.

Scientific Name	Total No.	Sampling Locations	GI Tract	Gills	Muscle Tissues	Total MPs	Average Mps/Fish
C. chitala	10	Karnal, Haryana	33	21	10	64	6.4
X. cancila	10	Karnal, Haryana	15	13	9	37	3.7
W. attu	10	Yamuna Nagar, Haryana	17	23	11	51	5.1
L. rohita	20	Sonia Vihar	123	64	18	205	10.25
L. boggut	20	Karnal, Haryana	31	23	11	65	3.25
L. rohita	20	Karnal, Haryana	25	22	12	59	2.95
P. ticto	10	Karnal, Haryana	12	23	10	45	4.5
L. calbasu	10	Yamuna Nagar, Haryana	13	21	11	45	4.5
P. sophore	10	Yamuna Nagar, Haryana	36	25	10	71	7.1
P. sophore	10	Karnal, Haryana	39	45	18	102	10.2
S. phulo	10	Yamuna Nagar, Haryana	17	13	10	40	4
O. niloticus	20	Sonia Vihar, Delhi	201	139	96	436	21.8
O. niloticus	20	Surghat, Delhi	100	94	70	264	13.2
L. angra	20	Yamuna Nagar, Haryana	36	21	10	67	3.35
C. cachius	10	Karnal, Haryana	20	22	3	45	4.5
C. cachius	10	Surghat, Delhi	33	36	13	82	8.2

Across all the species, MPs were most abundant in the gastrointestinal tract [~751 MPs] which confirms oral ingestion, likely through means of suspended MP particulates or contaminated prey; followed by gills [~ 605 MPs], which indicates the fish’s probable interaction with its surrounding habitat through water filtration or filter feeding, active movement, respiration, or foraging [47]; and was least abundant in the muscle tissues [~322 MPs], which supports the growing evidence of organ translocation across epithelial barriers via blood circulation, with implications for food safety and trophic transfer [63,66]. This pattern strongly indicates primary exposure through oral ingestion, either via contaminated prey or suspended particles, and secondary exposure via branchial filtration, reflecting interaction with the surrounding water column during respiration, feeding and active movement.

Among species, omnivores and herbivores from highly urbanized downstream regions exhibited the highest MP loads. Particularly, L. rohita from Sonia Vihar displayed elevated accumulation (205 MPs), whereas bottom-dwelling carnivores such as C. chitala and W. attu showed comparatively lower counts, 64 MPs and 51 MPs, respectively. These differences appear to be linked with the feeding habits and habitat zones, in which surface and mid-water feeders are likely to encounter high concentrations of suspended MPs than benthic species inhabiting deeper, less turbulent river sections. In the past, research on MP accumulation and biomagnification in organisms has largely focused on species across different trophic levels in the food chain [67]. However, associating MP consumption with trophic level yields ambiguous and imprecise results. Some studies propose a positive correlation, others a negative correlation, and some even suggest no association at all [68,69,70]. For instance, MPs were examined in two fish species, a pelagic feeder, Osmerus eperlanus, and a benthic feeder, Platichthys flesus. MPs were found in 75% of benthic feeders compared with 20% of pelagic feeders [68]. This suggests that benthic feeders are more exposed to MPs because sediments act as major sinks for MPs. Their feeding behavior also increases the likelihood of ingesting MPs directly from the sediments or indirectly through the contaminated prey. Therefore, feeding habitat and diet are the two main important factors influencing MP ingestion in fish species. Numerous studies indicate that an organism’s dietary habits may also affect MP accumulation [71]. Reports suggest that piscivorous fish are less affected by MPs [72], whereas omnivorous fish consume more MPs [73,74,75]. Ref. [73] also studied Larimichthys polyactis and Collichthys lucidus and indicated that inhabitation in higher contamination may not translate into MP consumption. This further indicates that trophic feeding strategies and habitat preferences play active roles in MP uptake in both of the fish species. Other studies have also suggested that MPs can bioaccumulate due to their availability, persistence, and chronic toxicity, which may present a risk of biomagnification throughout the food chain [76]. In the Liaohe estuary in China, a significant correlation (R² value of 0.7) was observed between MP abundance and the trophic levels of aquatic animals [53], indicating that MPs may become accessible to higher trophic levels once the MPs have penetrated the food chain in freshwater systems [77].

In findings consistent with these observations, omnivorous fish species in the present study exhibited the highest average MP burden (4.19 MPs/fish), whereas the carnivore and detritivore fishes exhibited comparatively lower MP loads. On the other hand, trophic guild differences were not statistically significant, indicating that the local pollution intensity and the habitat-specific exposure may exert even stronger influences on the MP accumulation than that exerted by the trophic position alone. Spatially, downstream stretches of Delhi (Sonia Vihar and Surghat) showed the greatest microplastic loads, contributing a major proportion of total MPs detected, whereas upstream sites (Karnal and Yamuna Nagar, Haryana) recorded lower abundances. This spatial gradient reflects the influences of urban wastewater discharge, textile effluents and increased suspended particulate load in Delhi’s metropolitan section.

On average, per individual fish, 44.75% of the MPs were detected in the GI tract, 36.05% of the MPs in gills and 19.19% of the MPs in muscle tissues, confirming a consistent organ-level accumulation pattern across all examined species. Evidence of MPs in muscle tissues, although comparatively lower, suggests there could be a potential translocation across epithelial barriers, raising concerns regarding systemic distribution, cellular interaction and potential ecological implications for trophic exposure or possible ecological relevance for trophic interactions [63,66,78].

Conversely, the upstream sites (Karnal and Yamuna Nagar, Haryana) had left-shifted ECDFs with relatively lower MP loads and distributions, which denotes lower levels of contamination pressure. The steeper slopes also denote lower levels of dispersion and higher exposure levels among the sampled population; this denotes consistent urban inputs. The distributional trends in the present investigation denote a strong spatial gradient of microplastic contamination along the Yamuna river, where the downstream urban sections denote consistent exposure hotspots.

3.2. Morphotype and Color of Microplastics

Across all examined fish species, fibers represent the predominant microplastic (MP) morphotype in all dissected organs, accounting for more than 1306 particles (77.8%), followed by fragments, which accounted for 348 particles (20.7%). Other morphotypes occurred at much lower frequencies, including microbeads (13 MP particles, 0.77%), film (11 MP particles, 0.65%), and foam (9 MP particles, 0.53%) (Figure 3C). Organ-wise analysis of MPs showed that fibers were most dominant in the GI tract (~585 MP particles), followed by gills (~468 MP particles) and muscle tissues (~253 MP particles). Fragment-morphotype MPs were primarily detected in the GI tract (~142 MP particles) and gills (~121 particles), with comparatively fewer in the muscle tissues (~85 MP particles). Spatially, fish species collected from Delhi sampling sites (Sonia Vihar and Sur Ghat) exhibited substantially higher MP abundance and morphotypic diversity compared to the upstream sites (Karnal and Yamuna Nagar, Haryana). For example, O. niloticus from Sonia Vihar recorded 436 MPs, while the same species from Sur Ghat recorded 265 MPs, whereas upstream species such as X. cancila from Karnal contained only 37 MPs. Similarly, P. sophore from Sonia Vihar contained 102 MPs, compared to 45–67 MPs in fish from upstream locations. Upstream sites consistently exhibited fewer MP morphotypes, predominantly fibers and fragments, with rare or no occurrence of film, foams or microbeads. L. rohita (SV), from the Delhi sampling location, reflected strong influences from effluents, unmanaged sewage discharge, textile industries and urban run-off [44,45].

Figure 3. (A) Abundance of microplastics per fish organ, (B) Average number of microplastics per feeding habit of fish, (C) Morphotypic composition of microplastics in different fish species, (D) Heatmap and bar graph depicting different colored microplastics in fish species.

The predominance of fibers (Figure 3A) across organs suggests that the major MP sources are likely associated with textile shedding, domestic washing effluents and abrasion of synthetic clothing, which are known to release large quantities of microfibers into urban wastewater streams [79,80,81]. The co-occurrence of fragments, albeit in lower quantities, indicates degradation of larger plastic products and packaging debris within the river system [82,83,84]. Notably, species collected from Delhi sites exhibited both higher total MP loads and greater morphotype diversity (Figure 4), which is consistent with inputs from dense residential clusters, unmanaged waste run-off and varying-effluent-rich drains. In contrast, upstream regions showed fewer morphotypes and lower overall abundance, reflecting reduced anthropogenic pressure and limited industrial discharge. These spatial patterns, when considered alongside morphotype prevalence, point to a strong influence of local pollution intensity and wastewater pathways in shaping the microplastic profile across the Yamuna river.

Figure 4. Microscopic images of distinct morphotypes of microplastic particles from different organs ((A–C)—GI tract; (D–F)—Muscle tissues, (G–I)—Gills) of the fish specimens (Scale bar = 50 µm).

MP color analysis revealed 13 distinct colors across the samples (Figure 3). Transparent MP particles were the most abundant (44%, ~739 particles), followed by blue (19.5%, ~329 particles), black (16.3%, ~275 particles), red (5%, ~84 particles), pink (4.4%, ~74 particles), white (3.8%, ~64 particles), grey (2.1%, ~35 particles), brown (1.9%, ~32 particles), and amber (1.4%, ~24 particles), while purple, green, orange, and yellow were around (~0.7% each or less) (Figure 3D). Species from Delhi sampling sites, particularly O. niloticus (SV, S), P. sophore (SV) and L. rohita (SV), exhibited the widest color diversity, whereas upstream fishes from Karnal and Yamuna Nagar were largely dominated by transparent MP particles, with minimal color diversity.

Color-wise investigation across organs showed that transparent MPs were the most abundant in all organ systems, representing approx. 44% of MPs in the GI tract, 42% in gills and 14% in muscle tissues [67,85]. This was followed by blue (~19–21% across all organs) and black (~15–17%); while red, pink, white, and grey occurred in lower proportions (~6%); and brown, amber, purple, green, orange, and yellow particles together accounted for less than 5% in each of the organ analyzed (Figure 3D). Fish species from Delhi sampling sites depicted greater diversity of MP colors across all organs, whereas upstream fishes were largely dominated by transparent and blue.

Overall, this spatial contrast among both, morphotype and color distributions reflects a clear spatial gradient, with higher MP abundance, greater morphotype diversity, and broader color profiles in downstream and polluted Delhi sites, compared to upstream locations, due to anthropogenic pressure; this is due to the fact that the Delhi stretch receives intense inputs from domestic wastewater, textile effluents, stormwater drains and packaging waste, a pattern consistent with documented deterioration of water quality indices [44,45,86]. In comparison, upstream regions showed simpler color profiles and fewer morphotypes, which is indicative of reduced effluent complexity and lower urban influence. The MP contamination observed in this study is consistent with the growing national evidence of widespread MP bioaccumulation in Indian freshwater fish species. L. rohita and C. punctatus repeatedly serve as sentinel species across geographically distinct basins [33,34,35]. Detection of MPs in edible muscle can indicate GIT translocation [33], which is linked to severe oxidative stress and human health hazard quotients above safety thresholds [87]. This makes the findings of our research work within a wider toxicological and food-safety concern and highlights the need for pathway-resolved monitoring in urbanized Indian rivers. This concern is not regionally associated; globally, the collective evidence confirms that MP contamination of freshwater fish poses a threat to ecosystem and food safety, with ingestion rates reaching 100% in heavily impacted ecosystems and the occurrence of MP translocation into tissues demonstrated across diverse species [88]. The tissue-level translocation of microplastics reported globally underscores that freshwater fish consumption represents an increasingly documented pathway of human exposure to microplastics [89]. A global review on MP contamination in fishes from 44 countries found contamination with polyethylene to be the most dominant polymer type, fibers to be the most dominant shape, the 0–1 mm size class the most abundant, and blue the most commonly reported color [90].

Similar study has been carried out on fishes from the upper Himalayan stretch of the river Ganga, which exhibited an average MP abundance of 29.38 MPs/individual in the GI tract [91]; the Han River, Korea, 17.39 MPs/individual in the GI tract [92]; and the Meghna River in Bangladesh, which had 14.63 MPs/individual in the GI tract [93]; in addition to Lake Michigan, USA, with 11.5 MPs/individual [94]. Amini–Birami et al. [63] detected much higher MP loads in the GI tract (3.6–9.8 MPs/individual) than in gills (2.1–5.5 MPs/individual) and less in muscle tissues (0.6–2.3 MPs/individual) from the sampled freshwater fish species inhabiting the Chishui River, China [60]. The elevated organ-wise MP burdens reported in these studies correspond closely with environmental MP concentrations documented in urban river systems (Table 3). Within China, the rate of MP ingestion by fish was noted to be 100% in fish collected from the main stem of the Yellow River as compared to only 41.5% in freshwater fishes of the Thames River. The concentrations of MPs detected within the intestines of 41 wild species collected from the upper Yangtze ranged between 7.17 and 606.67 item/g in 33 different polymers [88]. In Africa, the presence of MPs in the Nile River was recorded for the first time through their recovery from Nile tilapia, selected as a bioindicator, in which 567 MP particles were recovered from only 30 specimens, mostly secondary fibers in smaller sizes verified through Raman spectroscopy analysis [95].

Table 3. Current determinations of microplastic ingested by different fish species across the globe.

Country	River	Fish species (n = Total no. of Individuals)	Organ System	Average Microplastic Abundance (MPs/Individual)	Main Shapes	Main Colours	Major Polymers	Reference
India	Yamuna River	12 (n = 220)	GI tract, Gills & Muscle tissues	7.63	Fiber > fragment > sphere	Transparent, blue, black	PE, PS, PP, PET, PC, Nylon–6	Present study
India	Ganga River (Upper Himalayan Region)	4 (n = 96)	GI tract	29.375	Fiber > fragment > film	Not mentioned	PET & PS	[91]
Republic of Korea	Han River	22 (n = 106)	GI tract	17.39	Fragment	Not mentioned	PP, PE & PTFE	[92]
Bangladesh	Meghna River	3 (n = 30)	GI tract	14.63	Fiber > film > fragment	Blue, red, and black	PP, PE, PET, Nylon 6 & PS	[93]
USA	Lake Michigan	11 (n = 74)	GI tract	11.5	Fiber	Blue, transparent, white, black	PE, PAN, POM, PVAc & PET	[94]
China	Lijiang River	4 (n = 84)	GI tract and Gills	0.6	Flake > film > fiber	Colored, white, transparent, black	PET	[96]
China	North & West rivers of Guangdong province	2 (n = 74)	GI tract and Gills	2.105	Fragment > fiber	White, black, blue	Not mentioned	[97]
China	Dafeng River	33 (n = 122)	GI tract	2	Fiber > film > fragment	Blue	PET, Rayon & PBT	[98]
			Gills	0.7
Turkey	Karasu River Erzurum	3 (n = 78)	GI tract	2.974	Fiber > fragment > pellet	Black, blue	PE, Polyester, Poly (vinyl stearate), PET, PP & Cellulose	[99]
India	Deepor Beel of Assam	2 (n = 18)	GI tract	3.45	Fragment > fiber > sphere	Blue, black	PP, PVC, PE, ABS, PC & PS	[100]
Japan	Tokyo Bay	1 (n = 64)	GI tract	2.35	Fragments > beads	Blue, transparent	PE, PP & PS	[101]
Bangladesh	Rivers, canals and lakes surrounding Dhaka	18 (n = 48)	GI tract	3.05	Fiber > fragments	Transparent	HDPE, PP–PE copolymer	[102]
India	Alaknanda River	5 (n = 15)	GI tract	12.67	Fiber > fragment > film	White, black, blue, pink, red	HDPE, PP & Polyester	[103]

Abbreviations: PE—Polyethylene, PS—Polystyrene, PAN—Polyacrylonitrile, POM—Polyacetal (Polyoxymethylene), PVAc—Polyvinyl Acetate, PET—Polyethylene Terephthalate, PP—Polypropylene, PTFE—Polytetrafluoroethylene, PBT—Polybutylene terephthalate, ABS—Acrylonitrile Butadiene Styrene, PC—Polycarbonate, and HDPE–High Density Polyethylene.

A low but persistent MP burden in the GI tract of fish has been consistently reported across diverse aquatic ecosystems. In studies in several Chinese river systems, GI-tract MP concentration ranged from 0.6 to 2.11 MPs per individual in the Lijiang, Dafeng and Guangdong Rivers [99,100,101]. Similarly low MP load was detected in fish gills (0.7 MPs/individual), indicating continuous but moderate exposure through both ingestion and aeration.

Comparable abundance ranges have also been documented in geographically distant freshwater systems, including the Karasu River, Turkey (2.97 MPs/individual) [66]; Tokyo Bay, Japan (2.35 MPs/individual); Deepor Beel, India (3.45 MPs/individual) [100]; and Dhaka, Bangladesh (3.05 MPs/individual) [102]. These concordant observations suggest that chronic, low level MP exposure is a pervasive feature of freshwater and estuary fish habitats, rather than an anomaly associated with heavily polluted environments. In comparison, fish examined in the present study exhibited a mean MP abundance of 7.63 MPs/individual across multiple organ systems, positioning the Yamuna River within the globally reported range for urban-impacted freshwater ecosystems.

This MP presence and retention have been linked with oxidative stress, inflammation, altered metabolism and histopathological injury in Mullus barbatus and Alosa immaculata [66], adult zebrafish (D. rerio) [104] and other freshwater fish species [30,105], implying potential sub-lethal impacts for the fish species inhabiting contaminated segments. The combined morphotype and color data reveal that MP exposure in the Yamuna River is strongest in urbanized–downstream sites, posing risks to fish health in addition to raising concern for food-web transfer and human consumption.

3.3. Relation Between Abundance of MPs and Weight/Length of Fish Species

Across all sampling locations, clear patterns emerged between fish body size, river segment and MP accumulation. Fish samples from downstream sites in Delhi (SV and S) consistently exhibited the highest MP burdens, irrespective of their total length or weight. O. niloticus from Sonia Vihar was moderate in body size (15 ± 2 cm; 42 ± 5 g), but accumulated the highest total load of 436 MPs, whereas L. rohita from the same site, a comparatively larger species (32 ± 3 cm; 370 ± 10 g), accumulated 205 MPs. This indicated that local pollution intensity and feeding behaviors outweighed body size effects. A similar trend was observed in P. sophore, in which individuals from Sonia Vihar (5 ± 2 cm; 5 ± 2 g) accumulated 102 MPs, whereas conspecifics from Sur ghat accumulated only 71 MPs, despite their similar sizes.

In contrast, upstream fish species from Karnal and Yamuna Nagar, despite being larger in body dimensions, had significantly lower MP load. L. calbasu (22 ± 1 cm; 270 ± 20 g) accumulated only 45 MPs, markedly lower than smaller downstream omnivores. O. niloticus from Sonia Vihar (15 ± 2 cm; 42 ± 5 g) recorded the highest overall abundance, 436 MPs, which therefore suggests that body mass is not the sole predictor in explaining MP accumulation; rather, it seems to be an interactive function of the two parameters. Many large-sized upstream fish species, such as L. angra from Yamuna Nagar (25 ± 1 cm; 250 ± 20 g; 67 MPs) and L. calbasu at Karnal (22 ± 1 cm; 270 ± 20 g; 45 MPs), showed far lower MP particle loads compared to the smaller or comparably sized species from the sampling sites of Delhi, indicating the overriding effect of pollution intensity. Overall, the length of sampled fish ranged between 5 and 34 cm, and MP loads varied between 37 and 436 MP particles, with larger fish from less-polluted upstream sites often showing lower concentrations than smaller fish from polluted sites. This pattern has been also been reported in other freshwater fish species along pollution gradients [30,60].

Feeding guild further modulated exposure risk, as evident in the surface- and mid-water omnivores (O. niloticus, P. sophore, L. rohita) consistently exhibiting highest MP accumulation, whereas benthic carnivores and detritivores (C. chitala, W. attu, L. angra) showed lower burdens, even when larger in size (Figure 3B). Differences in the MP abundance among the feeding guilds were comparatively feebler than the spatial variations, as illustrated in Figure S3. This aligns with observations that broad dietary breadth and frequent interaction with suspended particulates increase ingestion likelihood [26,27]. Thus, body size alone did not predict MP abundance, but rather the combination of habitat exposure, feeding mode and pollution intensity at sampling sites.

Collectively, these findings indicate that fish from the Delhi sites accumulated approximately 1.5–4 times more MPs than conspecifics of similar size from upstream regions, driven by higher suspended particle loads, household wastewater influx and textile-derived fibers entering the river [41,79]. The elevated MP abundance may also reflect hydrological and environmental conditions previously reported for this particular river stretch, which includes high particulate loads, reduced water flow velocity and sediment accumulation as well as substantial waste influx [44,45,46]. Such conditions may enhance the aggregation, retention and biological availability of MP particles within aquatic environments, thereby increasing the exposure of the aquatic organism. This relationship demonstrates that MP burden is an emergent property of site-specific contamination pressure and ecological traits, rather than a simple function of body length or mass. The resulting variability underscores the importance of spatial gradients in MP exposure, which is consistent with observations from urban freshwater systems [28,63,106].

The mean MP abundance recorded in the study (7.63 MPs/individual) falls within the global range reported for freshwater fishes, and is comparable to other urban systems influenced by wastewater and textile inputs [80,81,107]. Higher averages have been reported from the stretch of the river Ganga (29.4 MPs/individuals) and Han River, Korea (17.39 MPs/individual), whereas considerably lower values occur in less contaminated rivers of China, Turkey and Assam, India (0.6–3.45 MPs/individual). Across studies (Table 3), a consistent pattern emerges: fibers dominate the MP assemblage and polymers such as PE, PP, PS and PET are common, reflecting the pervasive role of domestic effluents, laundry waste and packaging debris. The fiber-dominated morphotypic profile observed in Yamuna River fish species supports their internal presence, indicating that the Delhi stretch functions similarly to other densely populated urban rivers, for which wastewater-driven contamination governs MP exposure.

Smaller and moderate-sized species inhabiting highly polluted regions accumulated substantially higher MP loads than the larger conspecifics from upstream sites, reinforcing the dominance of spatial pollution gradients over morphometric predictors. This pattern reflects similar findings from both freshwater and marine ecosystems, in which MP ingestion has been shown to correlate more stalwartly with ambient MP availability, and is more consistent with the hydrodynamic conditions and feeding ecology than with fish size or mass [63,106]. In marine ecosystems, surface and pelagic omnivores similarly exhibited elevated MP loads due to prolonged exposure to buoyant fragments and fibers within the water column, whereas piscivorous or benthic species often show lower ingestion rates despite their larger body size [27]. P. sophore of Karnal, Haryana, had 102 MPs, and the same species collected from Yamuna Nagar, Haryana, had accumulated only 71 MPs, while C. cachius from Sonia Vihar, Delhi (SV), had 45 MPs, whereas Sur Ghat, Delhi (SG), had a higher number, at around 82 MPs in total. Species-specific feeding traits further modulated MP accumulation, where omnivores and mid-water feeder fishes O. niloticus, P. sophore, and L. calbasu exhibited elevated MP loads relative to benthic-carnivores as well as detritivores. This disproportionately elevated MP load in O. niloticus from SV, Delhi, compared to its moderate body size, points toward a higher exposure risk for surface- and mid-water. This may reflect its omnivorous feeding behaviors and frequent interaction with suspended MP particulates in the regions receiving heavy domestic, wastewater and industrial discharges. The fiber-dominated MP profiles and prevalence of common consumer polymers (PE, PS, PP, and PET) observed in river Yamuna fishes further corroborates the role of wastewater effluents, urban runoff and laundry discharge as MP sources [81], a mechanism widely reported across urban sites of the river and costal zones [80,107]. Together, these findings highlight that MP bioaccumulation in fish is an emergent outcome of site-specific contamination intensity and habitat use, as well as dietary breadth, rather than a linear function of body weight or length. Such spatially structured exposure to persistent pollutants emphasizes the importance of integrating pollution gradients and trophic behavior into MP risk assessment for freshwater biota, particularly in the densely populated urban river systems like the river Yamuna.

The Pearson and Spearman correlation analysis revealed strong positive correlations between MP concentrations in GI tract, gills, muscle tissues and total MP load (r/ρ = 0.635–0.958, p < 0.001), indicating that internal distribution and MP ingestion in fish species are coordinated (Figure S2). Pearson and Spearman tests deduced that the fish species with higher MP gut contamination presented larger MP loads in gills and muscle tissues. These relationships are further illustrated through pairwise regression analyses (Figure S5). MP abundance differed significantly among sampling sites (Kruskal–Wallis, H = 11.03, df = 3, p = 0.011), with the highest values observed in the Delhi stretch. Fewer apparent changes were found among the feeding guilds, showing that MP accumulation seems more affected by the site-specific levels of pollution and less affected by the dietary habits of the fish. The ECDF-based assessment supports the pragmatic spatial differences in the MPs’ abundance (Figure S1), in agreement with the significant sampling site-wise variations detected by the Kruskal–Wallis test.

The empirical cumulative distribution functions [ECDFs] were employed to investigate site-wise differences in the total number of microplastics per fish (Figure S1). The ECDF plots for fish species collected from the Delhi area of the Yamuna River evidently indicate a rightward shift compared to the upstream sites, suggesting higher MP values in the fish. For instance, the ECDF plots for Sonia Vihar and Sur Ghat illustrate that more fish had higher microplastic loads/concentrations, even at lower cumulative probabilities. In contrast to the use of central tendency measures, the characterization of the total distribution of microplastic loads, instead of using only central tendency measures, makes the empirical cumulative distribution function (ECDF) analysis of microplastic exposure patterns in fish populations informative. The consistent rightward displacement of the ECDF curves for Delhi locations suggests that high microplastic loads are not a rare condition in individual fish but are a widespread phenomenon in the majority of fish in the river stretches.

The statistical analysis showed that in MP abundance variation among trophic guilds, the omnivorous fishes had the highest average MP burden (4.19 MPs/fish), followed by the insectivorous (2.00 MPs/fish), herbivorous (1.89 MPs/fish), carnivorous (1.67 MPs/fish), and finally the detritivore fishes (1.33 MPs/fish) (Figure S6). However, the Kruskal–Wallis analysis was not able to give profound and significant difference among MP abundance in the different trophic guilds (χ² = 5.27, df = 4, p = 0.261). This may have happened because of the high levels of variability within the group, particularly in the omnivorous group, as demonstrated by the O. niloticus. On the other hand, location wise differences in MP abundance were observed to be highly significant (Kruskal–Wallis χ² = 14.26, df = 3, p = 0.0026). Dunn’s post hoc analysis established significant differences in MP abundance in comparing Delhi-related sites, namely Sonia Vihar and Surghat, to Karnal and Yamuna Nagar. The statistical analysis of organ-wise differences in MP accumulation was also found to be significant (Kruskal–Wallis χ² = 11.03, df = 2, p = 0.004). Using Dunn’s post hoc analysis, it is clear that there was significantly less MP abundance in muscle tissue samples compared to values for both gut and gill (p < 0.05). There was no notable difference in MP abundances when comparing gut and gill samples. The above observations point towards ingestion and respiration of MPs in respective exposure organs without any translocation to tissues. Friedman repeated measures analysis provided strong statistical support for within-fish differences in MP abundance in different organs (χ² = 17.29, df = 2, p < 0.001) (Figure S7).

The DHARMa Residual Diagnostics reveal that the negative binomial GLMM is a statistically sufficiently valid model to interpret ecologically. The QQ-plot of residuals links to the expected value, and the Kolmogorov–Smirnov test confirms that there is notable deviation from uniformity (KS test, p = 0.5299) (Figure S8). Therefore, the data can be considered to be reasonably well-distributed; as a result, there is no clear indication of overdispersion based on the dispersion test (p = 0.744), which supports the suitability of application of the negative binomial regression model. An outlier test showed no significant outlier or influential observations in the dataset. The DHARMa plot demonstrates slight deviation from the red quantile line, revealing slight heterogeneity in the distribution of residuals. MP abundance in fish depends significantly on tissue type and sampling location (p < 0.001). Specifically, tissue samples have significantly lower MP abundance than the direct exposure-associated organs (p < 0.001), which indicates the slow translocation of MPs into the internal tissues of fish. Our results indicate that fish sampled from Sonia Vihar, Delhi, have a significantly higher MP abundance (p < 0.001), and those from Surghat, Delhi, demonstrated elevated MP abundance (p < 0.05). These findings show the anthropogenic effect on MP presence. At the same time, trophic guild, fish length, and weight were not directly correlated with MP abundance. These results might indicate that environment is the key factor, rather than fish feeding behavior, in determination of MP abundance.

The contamination patterns of MPs were analyzed using permutational multivariate analysis of variance (PERMANOVA), based on Bray–Curtis dissimilarity matrices. The results indicate that there were no significant multivariate differences between organ types (F = 0.084, R² = 0.0037, p = 0.993). This suggests that the type of organ has little to no impact on the ecological variation of MP contamination. The analysis also revealed that there are significant notable differences between sampling locations (F = 4.46, R² = 0.293, p = 0.001), which revealed that the location from which the sample has been obtained has a stronger influence on the distribution of MPs. Finally, multivariate analysis of contamination of trophic guilds shows significant differences (F = 5.94, R² = 0.356, p = 0.001). Therefore, feeding ecology is another important factor affecting MPs accumulation in fish. PERMANOVA requires several important assumptions, including homogeneous dispersion. The betadisper analysis was used for the assessment of this assumption. The analysis indicates that there are no significant differences between dispersions in the organ groups (p = 0.984). In contrast, location groups show marginal differences in dispersions (p = 0.059). The visualization was done using a non-metric multidimensional scaling (NMDS) ordination. The NMDS produces a very low stress level (stress = 0.026), which means that it provides an excellent estimation of ecological distances among samples. NMDS plots indicate high levels of overlap between groups of organs. Indeed, PERMANOVA did not identify significant multivariate differences between organs, which explains the NMDS results (Figure S9). Analysis showed partial overlap among organ samples, indicating a relatively similar MPs distribution pattern in gut and gill, clustered more closely as compared to tissue, which was widespread. Sampling location-based analysis exhibited close clustering in Delhi-associated locations, particularly Sonia Vihar and Sur Ghat, whereas Haryana sites showed comparatively broader dispersion and partial separation within ordination space.

3.4. Surface Screening of Microplastics Under SEM–EDAX

Representative SEM micrographs revealed distinct weathering characteristics that included surface cracks, grooves, abrasions and pits marks on both fiber and fragment particles recovered from the fish tissues (Figure 5). The analyzed MP particles were recovered from the GI tracts, gills and muscle tissues of sampled fish species and were then subsequently subjected to EDAX analysis. Scale-calibrated SEM micrographs enabled visualization of particle dimensions and degradation features associated with persistent environmental exposure. SEM–EDAX analysis confirmed a strong carbon-rich peak, identifying their synthetic plastic–polymer origin (Figure 5) [53]. Significant oxygen levels indicated surface oxidation and photo-degradation of environmentally exposed microplastics [108]. Detection of Na and Cl in several spectra were attributed to surface-adhered environmental salts rather than polymer-bound halogens like PVC, while Si and Al peaks suggested the adhesion of silicate minerals and sediments, and elevated nitrogen (N) levels may indicate the presence of amide-based polymers or biological residues from microbial biofilms (Figure 5). Oxidized surfaces could reveal prolonged exposure to sunlight and reactive oxygen species (ROS), while nitrogenous and silicate attachments suggest concurrent interactions with biofilms and sediments. These modifications can affect buoyancy, enhance MP particle adhesion, and increase the potential for ingestion of MPs through both feeding and respiration [109,110].

Figure 5. SEM micrographs and corresponding EDAX spectra of representative MP particles recovered from different organ systems of the freshwater fish species of river Yamuna. The micrographs illustrate fiber and fragments isolated from the GI tract, gills and muscle tissues of sampled fish species. Surface features include cracks, pits, abrasions and groove marks that indicate the environmental aging and weathering of MP particles. Corresponding spectra show dominant carbon (C) and oxygen (O) peaks, confirming their rich polymer nature, together with minor elemental signatures (Si, Na, AI, N and Cl) often associated with oxidation, environmental weathering, and biofilm interactions.

SEM–EDAX confirmed the carbon-rich composition of these particles, but due to the inability to differentiate specific polymer classes complementarily spectroscopic validation was done. FTIR analysis was employed to identify the polymers conclusively, showing PE, PP, PET, PA, and other common consumer plastics. Similar methodological integration of FTIR and SEM–EDAX has been widely adopted in MP studies to distinguish between the polymer composition and environmentally induced surface modifications and degradation [111]. As for the elemental signatures determined, these observations on the enrichment of oxygen and the incorporation of nitrogen and mineral-associated elements like silicon and aluminum are taken to be in line with observations of environmentally aged MPs that have undergone oxidative weathering, biofilm colonization, and sediment interaction in aquatic environments [112,113,114]. Such surface modifications have been shown to alter particle density, surface charge, and hydrophobicity; these, in turn, affect buoyancy, residing time in the water column, and biological availability of microplastics to aquatic organisms [115]. Furthermore, biofilm-coated and mineral-encrusted microplastics are known more to enhance adhesion to gill epithelia and digestive tissues, thereby increasing the probability of uptake via both feeding and respiratory pathways [110,116,117]. Collectively, these findings indicate that fish in the Yamuna River are exposed not only to pristine plastics but to environmentally weathered MPs, the altered surface properties of which reflect prolonged environmental exposure and intricate physico-chemical interactions. Such weathered microplastics are increasingly recognized as more biologically interactive and potentially more toxic than virgin particles. This may emphasize their ecological relevance within freshwater systems impacted by urban and wastewater-derived pollution [63,106].

3.5. Identification and Confirmation of Microplastic Polymer Using FTIR

Fourier Transform Infrared Spectroscopy equipped with Attenuated Total Reflectance (ATR–FTIR) analysis spectra of selected MPs were analyzed to identify polymer types. All the particles provisionally identified as MPs through visual examination and further examination under stereomicroscope were characterized by their morphotype and color prior to their confirmation. Out of 1678 visually identified particles, around 890 particles (≥50%) were subjected to ATR–FTIR confirmation, including all ambiguous morphotypes, which were subjected to ATR–FTIR analysis. These particles were selected to proportionally represent all morphotypes, colors, fish species, organs and sampling locations. The spectra, collected over the mid-infrared range (400–4000 cm⁻¹) and compared with the reference spectra from Open Specy software [55], Hummel Polymer Sample Library, FLOPP, and FLOPP–e, confirmed ten distinct types of polymers, dominated by common consumer plastics, viz., polyethylene (PE) ~23% and polypropylene (PP) ~22%, followed by poly(ethylene terephthalate) (PET) ~19% and polystyrene (PS) ~14%. Small but measurable percentage fractions comprised polyamides (PA) ~6%, Nylon–6 ~4%, poly(vinyl) acetate (PVAc) ~4%, polycarbonate (PC) ~4%, polyimide (PI) ~3%, and trace amounts of polysiloxanes ~1% (Figure 6). The predominance of PE, PP, PS and PET reflect widespread use and environmental persistence [118,119], while the detection of polymers such as PA, PC and PI, typically associated with industrial or specialized applications, suggest multiple and diverse MP pollution sources [120,121]. The minor presence of polymers like polysiloxanes highlights the complexity of MP inputs and the need for source tracking approaches. Cumulatively, this polymeric diversity underscores the importance of polymer-specific risk assessments rather than reliance on bulk MP counts alone.

Figure 6. (A) FTIR–ATR spectra of different polymers identified based on the spectral library of OpenSpecy software, and (B) a pie chart depicting different polymer composition distribution.

Spectra corresponding to PE exhibited the diagnostic vibrational bands, including strong aliphatic C–H stretching peaks at ~2915 cm⁻¹ asymmetric and ~2848 cm⁻¹ symmetric, a prominent CH₂ bending at ~1465 cm⁻¹ and distinctive CH₂ rocking vibration near ~720–730 cm⁻¹; these four bands form the unique fingerprint of polyethylene (PE) (Figure 6). Also, PP showed characteristic aliphatic C–H stretching vibrations at around ~2950–2840 cm⁻¹ and CH₃ bending modes around 1455–1375 cm⁻¹, along with strong rocking and wagging modes in 900–800 cm⁻¹ region. PET polymer was confirmed by a strong ester (C=O) stretching peak near 1715–1730 cm⁻¹, accompanied by a prominent aromatic carbon (C=C) vibration (~1600–1500 cm⁻¹) and multiple C–O stretching bands between 1240–1100 cm⁻¹. PS was confirmed by pronounced aromatic carbon overtones and C–H out–of–plane blending near 700–750 cm⁻¹, together with an aromatic carbon (C=C) stretching in the 1600–1500 cm⁻¹ region. Two different spectra, PA and Nylon–6, showed characteristic N–H stretching vibrations around 3300–3290 cm⁻¹ and depicted a strong amide I band at ~1650 cm⁻¹ and a pronounced amid II band near 1540 cm⁻¹, along with C–N stretching peaks between 1300 and 1200 cm⁻¹. In contrast, PVAc was identified by its ester (C=O) peak at ~1740 cm⁻¹ and strong C–O stretching bands. PC displayed a sharp carbonyl absorption around 1770–1740 cm⁻¹, while PI was confirmed by symmetric and asymmetric imide carbonyl bands (1770–1710 cm⁻¹) and imide ring vibrations in the lower wavenumber region.

FTIR confirmed the polymer profile, which indicates that MPs ingested by fish come mainly from widely used consumer plastic products, reflecting strong anthropogenic inputs along the studied river stretch. The dominance of PE and PP, together accounting for about half of all the identified polymers, suggests major contributions from single-use carry bags, packaging, general household items, waste generated from municipal effluents and surface runoff. The substantial presence of PET and PS further points toward the contributions of textile fibers, single-use beverage bottles and food containers, and disposable items, which is characteristic of urban waste mismanagement in densely populated areas like Sur Ghat and Sonia Vihar, Delhi. While the proportion is smaller, the detection of PAs, Nylon–6, PVAc, PC, and PI indicates contributions from fishing gear, synthetic textiles, industrial waste materials, adhesives, paint chips, and electronic wires and parts. Consequently, this reflects shared impacts from unmanaged domestic wastewater and industrial discharge, and degradation of single- and multi-use plastic products. A broad range of polymer classes with their confirmed FTIR fingerprints suggests diverse urban and peri-urban activities with variable influx into the river, which further contributes to a heterogeneous MP burden. Overall, the distribution of various polymers agreed with the observed pollution gradient, showing the most significant contributions to the load of MP polymers in the aquatic environment to be those of the urbanized downstream sections.

3.6. Ecological Risk Assessment of Microplastics

The PHI value was calculated based on identified polymers in fish organs (GI tract, gills, and muscle tissues) and showed the significantly high hazard potential of the MPs assemblage in the Yamuna River. While PE and PP represented a large proportion of the polymers identified, their contributions to the overall hazard index were minimal, since they had very low intrinsic hazard scores (Sn = 1). However, the fractions of hazard-associated polymers, even in small quantities, substantially raise the total hazard index load. Among these, PC, with an extremely high hazard score of Sn = 1000, was the single most dominant contributor to higher PHI, followed by PI (Sn = 100) and moderately hazardous polymers such as PET, PS, PA, and Nylon–6 with Sn = 10, collectively increasing the hazard risk profile. The cumulative PHI value was higher, defining the polymer mixture as Hazard Category level IV (Table 4), indicating a high hazard score, according to [58]. This indicates that MPs-associated risks are not driven merely by abundance but rather by the toxicological profiles of specific polymer types. High PHI values are driven by the presence of small fractions of high-hazard polymers such as PC and PI. Thus, the compositions of high-hazard polymers with greater capacities to leach toxic additives, absorb persistent pollutants and induce oxidative or inflammatory responses in these fish species indicate the presence of polymers categorized as relatively higher-hazard polymer classes, according to PHI-based screening frameworks. These results emphasize the need for polymer-specific risk assessment frameworks as well as targeted regulatory strategies that prioritize the bio-monitoring and mitigation of high-hazard polymers, rather than focusing solely on overall microplastic abundance. The PHI framework provides a more relative hazard-based screening approach as to polymer composition but doesn’t directly quantify the chemical exposure, additive leaching, bioavailability, or organism-level potential toxicological effects.

Table 4. Polymer Hazard Index (PHI) for microplastic polymers found in fish species from the river Yamuna.

S.No.	Polymer	Hazard Level	Hazard Grade	Hazard Class (Category)
1.	Polypropylene [PP]	I	1	Explosives [Div 1.3, 1.5] Pyrophoric liquids & solids Flammable aerosols, gases, and liquids [mostly cat. I] Organic peroxide [Type A, B] Self-reactive blend/material [Type A, B] Oxidizing liquids & solids
2.	Polyethylene [PE]	I	1	Flammable aerosols, gases, and liquids [mostly cat. I] Pyrophoric solids and liquids Self-reactive blend/material [Types A and B] Type A and type B organic peroxide Explosives [Div 1.3, 1.5] Oxidizing liquids & solids
3.	Polyamides (Poly–lactams) [Nylon]	II	10	Skin corrosion/irritation [cat. II] Exposure to specific target organ toxicity [cat. III] Severe eye discomfort/injury [cat. II] Dangerous to aquatic life [Chronic cat. III] Acute toxicity [category IV – inhalation, cutaneous, and oral]
4.	Nylon–6	II	10	Skin corrosion/irritation (cat. II) Exposure to specific target organ toxicity [cat. III] Acute toxicity [category IV– inhalation, oral & cutaneous] Dangerous to aquatic life [chronic cat. III] Severe eye distress/injury [cat. II]
5.	Poly (Ethylene terephthalate) [PET]	II	10	Exposure to specific target organ toxicity [cat. III] Acute toxicity [category IV– inhalation, oral & cutaneous] Detonators [Div 1.2] Dangerous to aquatic ecosystem [chronic cat. III] Skin corrosion/irritation [cat. II] Severe eye irritation/damage [cat. II]
6.	Polystyrene [PS]	II	10	Acute toxicity [category IV– inhalation, oral & cutaneous] Bombs [Div 1.2] Toxicity to a specific target organ—single exposure [cat. III] Dangerous for the aquatic ecosystem [chronic cat. III] Skin corrosion/irritation [category II] Severe eye irritation/damage [cat. II]
7.	Poly (Vinyl Acetate) [PVA]	I	1	Flammable aerosols, gases & liquids [mostly cat. I] Pyrophoric solids/liquids Self-reactive material/mixture [Type A, B] Oxidizing solids/liquids Explosives [Div 1.3, 1.5] Organic peroxide [Type A, B]
8.	Polycarbonate [PC]	IV	1000	Aquatic ecosystem-threatening [chronic cat. I, IV] Respiratory & skin sensitization [cat. I] Repeated exposure to specific target organ toxicity [cat. I] Exposure to specific target organ toxicity [cat. I] Mutagenicity of germ cells [cat. II] Acute toxicity [categories I and II– inhalation, oral & cutaneous]
9.	Polyimide [PI]	III	100	Skin corrosion/irritation [cat. II] Severe eye distress/injury [cat. II] Toxicity to a specific target organ—repeated exposure [cat. II] Respiratory & skin sensitization [cat. I] Aquatic environments are at risk [chronic cat. II, III]
10.	Polysiloxanes [o–si–o]	I	1	Irritation of the skin [cat. III/none] Aquatic toxicity [chronic cat. III, primarily for low-molecular weight siloxanes] Inflammation of the eyes [cat. II (B)/none] Inflammable liquids [cat. III depending on side groups]

4. Conclusions

This study demonstrates that MP contamination in freshwater fish from the Yamuna River is pervasive and omnipresent. The widespread occurrence of MPs across fish species indicates consistent exposure in the studied river stretch and highlights the need for further environmental and ecotoxicological assessment. Species from the urbanized Delhi stretch consistently exhibited the highest MP burdens, demonstrating that local pollution intensity strongly influences MP exposure in freshwater ichthyofauna. The dominance of fibers and transparent coloring; the presence of multiple polymer classes, including high-hazard polymers; and strong inter-organ correlations jointly reveal that detection of MPs suggests potential translocation within fish tissues beyond primary exposure organs, posing risks of systemic physiological stress. By integrating species-level accumulation patterns with polymer-specific hazard profiling, this study underlines microplastic pollution as a potential ecological threat capable of impairing fish health, altering trophic interactions, and compromising food-web resilience. Distribution-based analyses further validate the sustained and population-level MP exposure in fish species from the urbanized Delhi stretch of the Yamuna River. These findings emphasize the urgent requirement for targeted mitigation strategies, strengthened wastewater management, and polymer-specific monitoring to protect the ecological integrity of the Yamuna River and the safety of communities reliant on its fisheries.

5. Environmental Implications

The high microplastic loads, dominance of environmentally persistent fibers, and occurrence of high-hazard polymers in edible freshwater fish from the Yamuna River indicate significant environmental and public-health concerns. The accumulation of MPs across trophic guilds suggests that contamination is now integrated into the river inhabitants’ food web, with potential repercussions for ecosystem functioning, nutrient cycling, and predator–prey dynamics. The detection of MPs in muscle tissues further highlights direct exposure risks for human consumers relying on these fish species as a protein source. Given the clear pollution gradient and the strong influences of urban wastewater and textile effluents, these findings point out the urgent needs for better management of solid waste, advanced treatment of wastewater, and better control of the use of plastics to reduce further ecological degradation and safeguard riverine biodiversity.