The research was carried out in the estuarine system of Huizache–Caimanero (22.831685°, 106.030473° to 23.092533°, 106.291138°), a shallow intermittent estuary heavily influenced by freshwater inputs from the Presidio and Baluarte rivers ( Figure 1 ) (Amezcua et al., 2019). These rivers are connected to coastal lagoons through narrow tidal channels surrounded by mangrove forests. This coastal system receives ample freshwater during the rainy season (June to November), which mixes with the seawater from the ocean, creating a typical estuarine circulation pattern, and making the average area of the system 175 km², which during the dry season reduces to 65 km². Both rivers receive discharges from rural, agricultural, semi-urban, and aquaculture sources (Valencia-Castañeda et al., 2022). This ecosystem is also under human impact as it supports diverse small-scale fisheries (Ramírez-Rodríguez et al., 2014), and the landscape has undergone significant environmental changes due to the expansion of agriculture and aquaculture in the floodplain and margins of the lagoon in recent decades (Valencia-Castañeda et al., 2022).

A network of stations was established along the estuarine system ( Figure 1 ) to study the connection between anthropogenic particles in water and those ingested by fish. At each station, water and fish samples were taken during morning hours and high tide between October 2022 and November 2023 at every one of the three climatic seasons in the area: dry cool season (DCS, from December 1st to March 31st), dry warm season (DWS, from April 1st to June 30th), and humid warm season (HWS, from July 1st to November 30th), as defined by Amezcua et al. (2019), using skiffs equipped with 15 hp outboard engines, a submersible pump, a gillnet with a mesh size of 3.5 inches, and a cast net with a mesh size of 0.6 inches. Water samples were obtained using a submersible pump and filtered through a 63-μm metallic sieve on-site. The filtered water was then transferred to glass jars and transported in a cooler to the laboratory for analysis. Fish were captured by local fishers holding a fishing concession for this estuarine system issued by the Ministry of Rural Development and Agriculture, and the National Commission for Fisheries and Aquaculture of Mexico. Gillnets were used in stations exceeding a depth of 1.5 m, and it was left adrift at each station for 60 minutes. In stations with depths below 1.5 m, a cast net was used. The captured fish were placed in labeled plastic bags and transported in a cooler.

Necessary precautions were taken to prevent contamination from external sources of Potential Anthropogenic Particles (PAP) before analyzing water samples and the gastrointestinal tract of fish to extract PAP. The following measures were implemented: a) Access to the work area was restricted; b) Plastic materials were avoided near the samples, and wearing cotton lab coats was required; c) Glassware, caps, and Whatman^® filters (Whatman International Ltd., Mainstone, UK) underwent pre-treatment in an oven at 400°C for 4 hours. In addition, weighing dishes and dissection equipment were cleaned with methanol, acetone, and distilled water filtered through a 1.2 μm Whatman^® GF/C glass fiber filter; d) Clean Petri dishes containing distilled water were placed in the work area for each sample set as a blank at each step. These dishes remained open during sample exposure to the environment.

In total, five control blanks were processed at different time points during sample handling within the restricted area of the laboratory. These include dissection, filtration, and filter inspection under a stereomicroscope. The number of particles detected and their corresponding materials are listed in Supplementary Table 1 .

Each fish specimen was measured for total length (mm) and body mass (g) with an accuracy of 1 mm and 0.1 g, respectively. The stomach and intestinal organs of each individual were carefully removed through a longitudinal incision from the cloaca to the esophagus, weighed, and stored for future analysis and digestion. The gastrointestinal tract contents were placed on a Petri dish and examined visually using a stereomicroscope (Zeiss Stemi 508). Any PAP were isolated, and prey items were counted and identified to the most specific taxonomic level possible. If items were too digested to be counted but still recognizable as belonging to a large taxonomic group, they were categorized as ‘remains’ and weighed together. For partially consumed prey, individual numbers were estimated based on countable parts like claws, legs, otoliths, or beaks, considering size and shape to determine that they came from the same individual (Muro-Torres et al., 2023). They were then categorized based on their ecological characteristics. The stomach and intestinal lining were scraped and washed to collect suspected plastic particles and prey items. The materials were then placed on a heating plate for further analysis. The trophic position (TP) of the most abundant species caught was determined using references specific to the study area (Amezcua et al., 2015; Muro-Torres et al., 2019, 2022). Feeding guilds were assigned based on TP and food preferences (dominant items in the diet) following the methodology outlined by Dantas et al. (2020), and TP and trophic guilds were taken from the study of Muro-Torres et al. (2020), which was undertaken in an estuarine system next to our studied area. Six species with different TP, feeding habits, and feeding guilds were chosen for this study, each with a minimum sample size of 20 individuals. The sample size exceeds the recommended number to prevent bias from a small sample size (Markic et al., 2020).

The prey items were separated, and each gastrointestinal tract was placed in a glass jar for chemical digestion. Each water sample and each gastrointestinal tract of fish were digested to remove all organic matter using 20% (w/v) potassium hydroxide (KOH) in a 3:1 ratio. The samples were then incubated at 40°C ± 5°C for 24 hours. Afterward, 30% hydrogen peroxide (H₂O₂) (5 to 10 ml) was added at room temperature and left for an additional 12 hours to clarify the sample and remove any remaining organic matter. The digested samples were filtered using a vacuum pump filtration system with Whatman^® GF/C 1.2 μm glass fiber filters. The filters were dried at 45°C ± 5°C in glass Petri dishes covered with aluminum foil. Finally, the filters were examined under a stereomicroscope (Zeiss Stemi 508, Carl Zeiss Microscopy GmbH, Jena, Germany) to separate and characterize AP by type (fragment or fiber), color, and size.

The material composition of all the AP found in both water and the gastrointestinal system of fish was analyzed using Fourier-transform infrared spectroscopy (FTIR) with a Thermo Scientific™ Nicolet™ iN™10 infrared microscope. Sample readings were taken at a pressure of 15–25 psi and an aperture of 50 × 50 µm. The FTIR-ATR spectrum was an average of 16 scans from 650 to 4000 cm⁻¹ with a resolution of 4 cm⁻¹. Microplastics were identified by comparing them to standard polymers in the FTIR spectrometer library, with a minimum match threshold of 75% for all samples.

One-way analyses of variance (ANOVA) were used to test for differences in the mean number and length of AP in water samples from the two analyzed lagoons (Huizache and Caimanero), to determine spatial differences, and the mean number and length of the AP per fish species. Before the analysis, the homogeneity of variances was assessed using Cochran’s C test. Post-hoc pairwise mean comparisons were conducted using Tukey’s HSD test, in case differences exist.

To test the null hypothesis (H₀) that prey and microplastic ingestion do not vary by species, locality, feeding habits, TP, and trophic guild, a permutational Multivariate Analysis of Variance (PERMANOVA) was conducted (Anderson et al., 2008). Two matrices were created, one with the relative abundance of prey items and the other with the relative abundance of microplastic polymers as rows, and each fish individual as columns. To account for variations in gut content among individuals, the data were sample-standardized (Malthaner et al., 2024). The standardized data were then expressed as a percentage composition of each prey and microplastic category, with each column totaling 100. Bray–Curtis similarity matrices were then generated with species, trophic guild, and TP as factors.

Principal coordinates analysis (PCO) was used in the case of significant differences (Anderson et al., 2008). This method is very flexible as it can be based on any resemblance matrix. However, it is a projection of the points onto axes that minimizes residual variation in the space of the chosen resemblance measure. APs were overlaid as vectors on the PCO to assess their importance. The trajectory of the vectors indicates the significance of each polymer in the different fish species’ diets. The axes range from -n to n, with the centroid at 0, 0 representing no difference in prey or microplastic items based on established factors (Malthaner et al., 2024). Fish with empty stomachs were excluded from the analysis. Statistical analyses were performed in Primer 7 with the PERMANOVA add-on (License number 7875).

A total of 1,162 AP particles were identified in the water samples, all displaying typical AP shapes observed under a stereomicroscope, with 61% being fragments and 39% fibers ( Figure 2 ). Concentrations ranged from 0 to 110 AP/L, with an average of 50.5 ± 28.1 AP/L. The highest concentration of AP was found in the northern lagoon (Caimanero) with an average of 51.9 particles, compared to 47.4 in Huizache Lagoon. However, there were no significant differences in the mean number of AP particles between the two lagoons (F1,21 = 0.117, p>0.05). The average length of AP particles was larger in Huizache lagoon (0.64 mm) compared to Caimanero lagoon (0.45 mm), but these differences were not statistically significant (F1,21 = 0.676, p>0.05).

A total of 133 stomach contents of six selected fish species from the estuarine system of Huizache-Caimanero were analyzed. A minimum of 20 individuals per species were analyzed, following the recommendations of Markic et al. (2020), for a meaningful analysis. The fishes were demersal and benthopelagic species with different trophic and feeding guilds, as well as different TP according to Muro-Torres et al. (2020), from the first-order consumer persistent anchovy (Anchoa walkeri, Baldwin & Chang) to the system top predator, the orangemouth weakfish (Cynoscion xanthulus, Jordan & Gilbert). Trophic guilds included planktivorous (persistent anchovy), carnivorous of different orders (Carnivore 1: Tete sea catfish, Ariopsis seemanni (Günther); Carnivore 2: Yellowfin snook, Centropomus robalito, Jordan & Gilbert; Carnivore 3: orangemouth weakfish), and the omnivore white mullet (Mugil curema, Valenciennes). Feeding guilds included mainly zoobenthivores (tete sea catfish, yellowfin snook, and the orangemouth weakfish), and from these two were also considered piscivores (yellowfin snook and orangemouth weakfish), one planktivore (persistent anchovy), and one detritivore (white mullet) ( Table 1 ). All these species have commercial importance in the area (Ramírez-Rodríguez et al., 2014)

In total, 235 APs were found. Of these, 214 were fibers (approximately 91%), and 21 were fragments (almost 9%) ( Figure 2 ). APs were present in 93 fishes out of 133 (69.9%) and all the analyzed species. The APs ranged in length from 0.07 mm to 11.2 mm with a mean length of 1.25 mm, indicating that the majority can be considered microparticles. The abundance per individual varied from 0 to 11, with a mean number of AP per individual of 1.8. The frequency of occurrence per species varied from 60.7 in the orangemouth weakfish to 100 in the Tete sea catfish, and in all the species, AP were found.

The results of the one-way ANOVA testing for differences in mean abundance and mean length of the different species indicated that the detritivore/omnivore white mullet had a higher average particle ingestion compared to the Tete sea catfish (F4,122 = 3.73, p<0.05; Figure 3A ). However, no significant differences were found between any of the other species. In terms of length, the planktivore persistent anchovy ingested the largest AP on average (F_4,122 = 3.2, p<0.05; Figure 3B ), and these differences were significant only with the mean length of AP ingested by the Tete sea catfish, which ingested the lowest number and the smallest AP on average.

Results from the FTIR spectra show the presence of five types of APs in water and five in fish ( Figure 4 ) ( Table 2 ). In water, three polymers accounted for 94% of the particles found. Processed cotton was the most prevalent polymer with 615 particles (53%), all of which were fibers, followed by PET with 251 particles (22%), which were also fibers. Then it was followed by alkyd resin, which came in the shape of fragments, with 228 particles (20%). The remaining 6% was composed of cellulose fibers with 46 particles (4%), and finally fragments of diallyl phthalate resin, with 23 particles (2%).

In fish, three polymers were the most common types of plastics found, making up 76% of the total. Processed cotton was the most prevalent, with 83 particles (35%). Nylon was the second most common, with 50 particles (21%), and it was the only polymer found in all species. PET ranked third, with 46 particles (20%). The remaining two polymers accounted for 24% of the total, with polyethylene having 29 particles (12%) and acrylic being the least common with 27 particles (11%).

PERMANOVA indicated that the diet of the analyzed organisms was significantly different according to the species, feeding guilds, and TP (Species pseudo-F_4,132 = 130.5, p<0.05; feeding guild pseudo-F_3,132 = 83.1, p<0.05; TP pseudo-F_2,132 = 107.4, p<0.05). These differences can be seen in the Principal Coordinates plot ( Figure 5A ), in which four clear-cut groups formed, each containing a feeding guild. Zoobenthivores and piscivores species, i.e., the snook and the weakfish, which are carnivores 2 and 3, are grouped. These results indicate that the selected species effectively have different eating habits and feeding guilds, and agree with the previous results presented for a neighborhood system (Muro-Torres et al., 2020).

When analyzing the presence of AP in the digestive tract of these species, PERMANOVA also indicated differences regarding the species, feeding guild, and TP (Species pseudo-F_4,126 = 29.1, p<0.05; feeding guild pseudo-F_3,126 = 34.2, p<0.05; TP pseudo-F_2,126 = 107.4, p<0.05). However, the PCO indicates only two groups ( Figure 5B ). One with the majority of the species in which the overlaid vector indicates the importance of the AP PET, processed cotton, and nylon to those species, and another group formed only by the anchovies, in which the most important APs were acrylic and polyethylene. Pairwise comparisons using a pseudo-t test confirmed that the ingestion of plastics differed in anchovies, a pelagic species, compared to all other species, and therefore AP ingestion also differed in planktivores of TP 1, concerning other feeding guilds and trophic position. In the case of demersal fish, which remain associated with the bottom, no differences were found among them for the ingestion of APs.