1 Introduction

Microplastics are among the most widespread anthropogenic pollutants in aquatic environments [1, 2] and potentially threaten many aquatic organisms [3]. While it is widely recognized that microplastics are a contaminant of concern, questions remain about their source, fluxes, environmental entry points, transport mechanisms, and their resultant spatial distribution in aquatic environments.

Plastics are typically derived from the polymerization of monomers extracted from oil or gas (though bio-derived plastics have the same chemistry as those generated from oil and gas) that have been mass-produced for items such as clothes, bottles, food packaging, and straws since the 1940s [4]. Owing to their extensive use, plastics can contaminate the environment due to mishandled waste and indiscriminately polluted water, soil, and air [5, 6]. In the past few decades, the environmental impact of plastic contamination has been a significant focus of global research [7], especially once plastics are broken down into smaller sizes (i.e., microplastics). Some microplastics are formed from the degradation of sizeable plastic material undergoing splintering and shriveling in marine systems with physical scuffing [8]. Sources of microplastic in many freshwater systems include and are not limited to textiles (e.g., manufacturing, laundering, and disposal industries; [9], personal care products (e.g., toothpaste [10]), tire wear, and road-wear-associated microplastics [11], artificial turfs (e.g., playgrounds; [12]), paint, and wastewater from washing machines [13].

Owing to their minute size, microplastics may have detrimental effects on organisms at varying degrees. Many researchers have demonstrated that microplastics can reduce food consumption, delay growth, alter behavior, and sometimes cause death [3]. Conversely, some previous research has shown that microplastics have no apparent effects on aquatic organisms. While the potential effects of microplastics are evident, microplastics in freshwater systems are one of the least studied environmental issues, with very limited data available in some ecosystems [14], especially rivers that flow along rural–urban habitats.

Rivers are recognized as a significant source of microplastics in the seas [15] but may simultaneously act as long-term sinks of microplastics in rivers [16]. Storms, wind, and other natural variables affect the long-term distribution of microplastics in freshwater rivers [17]. Sediment may act as a long-term sink; plastics may sink to the bottom, while smaller plastic debris may float on surface water [18]. Many researchers have demonstrated the spatial variability in surface water and sediment microplastics in a wide range of river systems (e.g [19]); however, the processes that control this spatial variability are tentative. Understanding the spatial variation in microplastics in rivers will allow for more accurate predictions of the full extent to which rivers contribute to global plastic fluxes in aquatic systems. There is growing recognition that, in addition to the spatial distribution of sources, the fate of microplastics is strongly linked to their transport, dispersal, and potential deposition and storage along river continuums. Microplastic particles can be deposited within riverbed sediment by various mechanisms, including granular filtration, settling in porewater, and retention in benthic and hyporheic biofilms [20]. Once microplastics are deposited and immobilized in the riverbed, they may be resuspended into the water column, chemically degraded, fragmented into smaller pieces, ingested by benthic and hyporheic fauna, or buried and stored for long periods [21]. High flow (high water velocity) in rivers commonly resuspends deposited microplastics when the bed sediment is remobilized [22]. Similarly, high stream flow (discharge) can move microplastics deeper into bed sediment and thus enhance long-term plastic burial in riverine sediment [22]. Several researchers have assessed the spatial and temporal changes in lotic systems’ surface water and sediment microplastics from upstream to downstream locations [23, 24]. Assessing these changes requires the removal of microplastics from the water through technologies such as filtration, density separation, froth flotation, and adsorption removal [25].

Currently, some researchers have demonstrated that in northern England, microplastics are abundant at all sites, with higher quantities documented downstream than upstream [26]. Elsewhere, in rivers in southern Thailand, microplastic abundance decreased predictably from upstream to downstream sites [27]. In North America (New Jersey; Raritan River), microplastics are also documented to decrease in a downstream direction [28]. Some studies have found no predictable changes in an upstream-downstream gradient. For instance, in South Wales, there was no specific trend of microplastic abundance between the upstream and downstream sites sampled [29]. In all the studies mentioned above, few have studied microplastics concurrently with other pollutants [30].

Lead, a particularly concerning heavy metal, is rarely studied in conjunction with microplastics. Its contamination originates from industrial waste, paints, gasoline, fertilizers, and pesticides. Due to their large surface area, microplastics can act as carriers for heavy metals, potentially increasing their concentrations in aquatic environments. Lead, a toxic pollutant, is frequently detected in North American freshwater systems, especially those with a legacy of paint, arsenical insecticide, and construction-related use. Though natural processes can release lead, historical human activities have substantially raised their levels. Investigating the combined effects of lead and microplastics may be essential for understanding their impact on freshwater ecosystems and informing management decisions [30]. Most studies on lead concentrations suggest that lead levels will be highest in areas nearer to industrial waste, high pesticide use, or lead-based paints [30]. Considering the possible links between heavy metals and microplastic abundance [30, 31], investigating concentrations of lead and microplastics in tandem will further our understanding of their synergistic effects on aquatic and riparian systems. This work is important because it provides baseline data that can then be used for making management decisions in lotic and lentic systems globally.

In this study, we investigated the spatio-temporal distribution of lead concentrations and microplastic abundances along a river that traverses a rural–urban gradient. Previous research has shown that both microplastics and heavy metals exhibit spatial variability based on land use and proximity to pollution sources. We hypothesized that lead and microplastic concentrations would be highest upstream, near agricultural and livestock activities in the headwaters, due to runoff from these areas contributing to elevated levels of both pollutants. Furthermore, given the documented correlation between microplastics and heavy metals, we predicted that higher microplastic abundance would be associated with elevated lead concentrations. We envisaged that this association may be driven by the ability of microplastics to adsorb lead from the aquatic environment, potentially increasing the overall concentration of lead in water systems where microplastics are prevalent.

2 Materials and methods

2.1 Study sites

The study was conducted along five sites along the Wolf River (35° 01′ 16.61″ N 89° 35′ 49.01″ W), an extensive perennial rural–urban river system in Memphis, Tennessee (Fig. 1) with varying land use activities across its reaches (Table 1). We denoted the name of the sites as FW1 upstream up to FW5 downstream (where FW denotes freshwater). The Wolf River ranges over 138.5 Km and flows through the city of Memphis and Shelby County in western Tennessee, draining a watershed of approximately 2000 km; flows northwesterly from Tippah County, Mississippi, to its mouth on the Mississippi River in Memphis, Tennessee. In Shelby County, the Wolf River flows through loose quaternary alluvium and a wide floodplain of Holocene sand overlain with clayey, silty bank deposits [32]. The Wolf River watershed is in a humid-temperate climate, with a rainy season from October to March and a dry season from April to September. Many human activities occur along this river, including kayaking, agricultural uses, and public beaches for leisure. Specifically, agriculture activities are more prevalent upstream (e.g., FW1 [33]). The middle reaches are famous for kayakers and boaters [34]. There are significant land use changes (e.g., construction of buildings on flood plains) in many of the middle and lower reaches that have resulted in collapsed riverbanks, paint residues, widening channels, and an abundance of sand and silt in the Wolf River [35]. We were particularly interested in studying lead because this would be a good indicator of the legacy of heavy paint use and construction activities along the Wolf River. Downstream reaches (i.e., FW5) that has a public beach that many people use for leisure and swimming. The Wolf River is expected to be used more by kayakers, with more accessibility and additional boat ramps expected by 2050 [34].

Fig. 1
figure 1

Location of study sites along the Wolf River, Memphis, Tennessee, USA, from the headwaters (FW1). The map was created using GPS coordinates dowloaded from a Garmin Fenix 6s Pro and edited in Photos on a computer (AppleMacbook Air)

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Table 1 Physical characteristics of study sites along the Wolf River
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2.2 Field collections

Water samples destined for microplastic analyses were collected via wading in the stream (about 4 ft away from the shoreline) using an 80 μm mesh plankton net that we held below the water surface (~ 15–20 cm depth) for 15 min. Three water samples were collected per site. We ensured the plankton net was stationary for the 15-min interval [36]. To prevent site-to-site contamination, we rinsed the plankton net and its corresponding cod-end three to five times before sampling. On each sampling day, three 1000 ml water bottles were collected in Corning™ Gosselin™ bottles. The bottles were sealed on-site to be transported back to Rhodes College (Department of Biology) for filtering and analysis. All samples for analysis were transferred to the laboratory and stored at − 20 °C freezer until further analysis.

Sediment samples destined for microplastic analyses were collected in wadeable portions of the river near where water samples were collected. Per site, three sediment samples were collected. Stainless steel trowels were used to collect the upper 2.5–5 cm of sediment, which was homogenized and transferred into pre-combusted aluminum foil.

At each of the five locations, we measured physicochemical parameters three times each in situ at three randomly selected locations within the 3-m reach about 1.2 m from the shore. We averaged values to characterize the study site. Specifically, we measured dissolved oxygen (DO) using the YSI Pro2030 Field Dissolved Oxygen Meter (YSI Incorporated, Yellow Springs, OH, USA). Subsequently, we measured pH, total dissolved solids (TDS), salinity, and conductivity with the Apera PC60-262 Z Smart Multi-Parameter Pocket Tester (Apera Instruments LLC, Columbus, OH, USA). In addition, we also collected water samples in 500 ml Corning™ Gosselin™ bottles for lead (Pb), Total Nitrogen (TN), and Total Phosphorus (TP).

2.3 Laboratory sample processing

2.3.1 Lead and nutrient analyses

Total nitrogen (TN), Total phosphorus (TP), and Lead (Pb) were measured using Hach kits TNT880 (range 0–16 mg/L), TNT843 (range 0.05–4.5 mg/L), TNT850 (range 0.01–2 mg/L) respectively; all according to the manufacturer’s instructions. We chose the aforementioned kits because they accurately measure the range of values typical of many riverine systems [37, 38] and use proprietary TNTplus® vials, which are pre-calibrated and verified to ensure no reagent blanks are needed. Samples destined for TN and TP analysis were digested using a DRB 200 Heating Block (Hach Company, Loveland, CO, USA). All sample values were read using a spectrophotometer (DR3900; Hach Company, Loveland, CO, USA). We measured the TN, TP, and Pb levels in triplicate for each of the five sites.

2.3.2 Microplastic analyses

Microplastic suspensions of water were vacuum filtrated (vacuum pump; Rocker-800 oil-free, Germany) using pre-combusted (450 °C for 5 h) and pre-weighed glass fiber filter (Whatman GF/A 47 mm, GE Healthcare Europe) kept in aluminum foil (to avoid contamination). Filters containing microplastics were then stained with Nile Red (CAS 7385–67-3, technical grade) in chloroform (extra pure, stabilized with amylene solution) in a glass petri dish and dried for 3–5 days. Chloroform was preferred over less toxic solvents such as acetone and n-hexane because Nile Red dissolved in chloroform was found to be more selective in binding plastics despite other biogenic materials than other solvents [39]. Nile Red dye was used to confirm microplastics as it exploits the hydrophobic properties of plastic by staining it and illuminating it under a fluorescent light. The volume and concentration of Nile Red solution were determined following tests from preliminary samples from the Wolf River. A priori, we prepared incremental amounts of Nile Red from 0.01 mg/L, 0.10 mg/L, 0.05 mg/L, and 0.5 mg/L using data from previous studies [40,41,42]. We ultimately chose 0.5 mg/L as this yielded better fluorescence under UV light for our samples. To confirm that the microplastic was, in fact, plastic and not organic matter, we separated the plastic using a needle test and/or inverted microscope to verify if the item was plastic or organic matter. The identified microplastics were quantified as the number of particles m−3 in water samples and particles kg−1 of dry weight (dwt) in the sediment samples.

The wet sediment (500 g) was dried at 60 °C for 3–5 days, subsequently weighed, homogenized (using pre-cleaned mortar and pestles), and sieved (500 μm sieve) to remove large plant materials and pebbles/stones. We conducted preliminary validation tests (using Potassium Chloride and Sodium Hypochlorite) to determine whether our sample needed prior chemical digestion. Given the low organic matter in most of the sand samples in the Wolf River, we did not use chemical digestions. We obtained a subsample of 50 g for all sediment analyses. The residual sediment destined for microplastic analysis was washed in supersaturated zinc chloride (ZnCl2) and vigorously shaken to concentrate microplastics (detailed in Rodrigues et al. [43]). The Nile Red staining and processing followed procedures similar to those of surface water samples.

To identify and quantify microplastics, we illuminated microplastic samples with ultra-violet (UV) light (GT700; Firefly Global Boston, MA, USA) at a wavelength of 365 nm, which is reported as an optimal wavelength for microplastic luminescence light [44]. Subsequently, microplastics were observed using a stereoscopic microscope (AmScope; 3.5x-180x trinocular stereomicroscope with LED ring light) and in a dark room to maximize the luminescence of microplastics. After staining the samples with Nile Red, the microplastics were counted and separated by morphological shapes (i.e., fragments, fiber, pellets). We did not consider the color of microplastics because perception is subjective and highly influenced by factors such as microscope illumination/background or personal characteristics [18]. For this study, we could detect microplastics > 20 μm with our microscope setup. Any piece of plastic with a larger width than 5 mm (except for fiber-shaped plastics that we estimated measured 3 mm to 15 mm) was not considered as these would be macroplastics. In all quantifications, we ensured there were more than two users to verify the microplastic type.

To prevent contamination, before all analyses, the entire laboratory was cleaned with all surfaces and equipment rinsed with ultrapure water (Milli-Q, Millipore, Burlington, MA, USA). All Petri dishes, filter papers, and forceps were examined underneath a dissection microscope before use to ensure no contamination was present. All work surfaces were wiped down with 95% ethanol thrice before work commenced. No air-conditioners or fans were utilized in the lab during the study to minimize the risk of potential air-borne microplastic particle transport. Similarly, other precautions included wearing cotton clothing and always wearing latex gloves.

2.4 Data analyses

We performed a two-way analysis of similarities (ANOSIM), which permits unbalanced replication between treatments [45], to describe differences in microplastic abundance. Site and sampling occasion (called season for simplicity) were classified as factors in all analyses, and resemblance matrices were based on Euclidean distances (9999 permutations). ANOSIM, a non-metric multivariate statistical method, has no underlying assumptions about the statistical distribution of the data (e.g., normality, variance equality) and creates an overall test statistic (R) that indicates if differences among taxa, sites, and years exist [45]. ANOSIM results in an R-value and a p-value. The R-value is scaled from − 1 to + 1. R-values = 0 indicate random grouping, R ≥ 0.3 shows that groups are slightly different but overlapping, and R ≥ 0.5 indicates well-separated groups [45]. Variations of microplastics across sites were visualized using non-metric multidimensional scaling (n-NMDS) based on Bray-Curtis similarity distances.

We used a Kruskal-Wallis test followed by a pairwise Dunn test to assess differences in physicochemical parameters among sites. Further, a Spearman’s non-parametric correlation coefficient was used to test relationships (correlation) between the concentration of microplastics and lead. All statistical analyses and graphing plotting were performed in R software (R Core Team 2023, version 4.3.1, Vienna, Austria) [46].

3 Results

3.1 Lead concentrations along the river continuum

We found that lead did not change predictably along the river continuum (Fig. 2). In the spring, lead levels were the highest at FW1 (0.22 ± 0.01 mg/L) and lowest at FW2 (0.06 ± 0.03 mg/L), which had the most minor lead level concentration. In the summer, the lead decreased at FW1 (0.09 ± 0.01 mg/L) but increased sharply at FW3 (0.24 ± 0.08 mg/L).

Fig. 2
figure 2

Lead concentrations (± SD; mg/L) along the Wolf River

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3.2 Microplastic concentrations along the river continuum

In support of our hypothesis, surface water microplastics decreased in a downstream direction (Fig. 3a). Summarily, in the spring surface water, microplastics were greatest upstream at site FW1 (1.05 ± 0.20 no. of particles. m−3) and decreased downstream at FW5 (0.09 ± 0.01 no. of particles. m−3). During the summer, microplastics in surface water were abundant at FW1 (1.86 ± 0.55 no. of particles. m−3) and decreased in a downstream direction (Fig. 3a). Both spring and summer microplastics in surface water at FW2 were consistently lower than in the other sampled sites (0.37 ± 0.09, 0.13 ± 0.08 no. of particles. m−3 ) (Fig. 3a).

Fig. 3
figure 3

Microplastic concentrations (± SD) in sediment and surface water of the Wolf River, FW1 is upstream, and FW5 is the downstream stream. Three samples were analyzed at each site

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Contrary to our hypothesis, we found no discernable trends in sediment microplastics during the spring and summer. In the spring, microplastics in sediment were more abundant at FW3 and FW5 compared to the other sampled sites (Fig. 3b). In the summer, microplastics in sediment were greatest at FW3 with the lowest abundances recorded at FW2 and FW5 (Fig. 3b)

The n-MDS ordination based on microplastic densities showed that water and sediment microplastics segregated slightly among systems (Fig. 4), with significant overlap across sampled locations. The greatest segregation was between FW1 (upstream) and FW5 (downstream). Microplastic variation was greatest at FW1 and FW5, while there was less variation at FW2, FW3, and FW4 (Fig. 4).

Fig. 4
figure 4

n-MDS ordination highlighting variation of microplastic densities across sites in the Wolf River. Data was pooled for all sampling occasions

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3.3 Relationship between lead and microplastics

We found a partial correlation between lead and microplastics in the sediment samples, with a general upward trend as microplastics and lead increased. In the surface water, there was no relationship between microplastics and lead samples (Fig. 5).

Fig. 5
figure 5

Spearman’s correlations between a Microplastics in water and heavy metals (Lead) and b Microplastics in sediment and heavy metals

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3.4 Physico-chemical parameters along the river continuum

The pH decreased from FW1 to FW5, ranging from 8.4 to 10.1. Conductivity increased from FW1 to FW5, starting at 23.4 ± 3.4 µScm−1 at FW1 and was greatest at 26.5 ± 0.8 µScm−1 at FW5. Total dissolved solids had a general increasing trend from FW1 to FW5, except for FW2, where the TDS value was greater at 18.2 ± 2.1 ppm than FW3 and FW4. Total phosphorous had no trend but was lowest in amount at FW3 where it was 0.02 ± 0.0 mgL−1and greatest in amount at FW5 where it was 0.41 ± 0.4 mgL−1. Total nitrogen also had no specific trend. It reached its highest amount at FW3 where it was at 1.08 ± 0.6 mgL−1 and its lowest amount at FW5 where it was 0.36 ± 0.2 mgL−1 (Table 2).

Table 2 Mean ± SD description of physicochemical parameters measured along the river continuum
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3.5 Dominant microplastic types along the river continuum

Fragments (Table S1 supplementary information; Fig. 6) were the most common type of plastic, and these varied across sites (ANOSIM; R = 0.65 indicates well-separated groups). Fibers (3 mm to 15 mm) overlapped but were moderately separated (ANOSIM; R = 0.45). Conversely, pellets were similar across many sites. Fibers and fragments varied by site and not by season (Table 3).

Table 3 Two-way analysis of similarity (ANOSIM) outputs with site and season as factors to determine differences in dominant microplastic types in the Wolf River
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4 Discussion

We studied lead and microplastic spatial variations in a North American temperate river. Overall, we found that microplastics and lead concentrations were greatest at upstream sites. This supported our hypothesis because concentrations were greatest at upstream reaches and lowest in downstream reaches. Our findings are in accordance with what has been documented by other researchers [27, 28, 47]. Our findings differed from those of other researchers [32, 72], who found that microplastic concentrations are greatest in downstream reaches. Our current research demonstrates the ubiquity of microplastics but also provides a baseline for further studies on microplastics ubiquity in rural–urban rivers.

In contrast to our hypothesis, lead concentrations did not change predictably along the river continuum. However, we found that lead levels were consistently high at FW1 (spring) and FW3 (summer), typical of sites associated with agricultural land. The lack of a specific trend in lead concentrations could be attributed to runoff additions, irregular bank erosion, rain patterns, or turbulent flow [48]. These can change lead distribution by increasing lead concentrations in areas affected, leading to no specific trends [49, 50]. Our results support findings by researchers [48, 51], who found that lead concentrations had no specific site distribution due to diffuse sources and mixed land usage. Considering that 30–90% of the total metal load of a river can be deposited in sediments, it is plausible that lead levels documented in our study may underestimate lead in the entire system. Studying the coupling between heavy metals in sediment and rivers is a promising future study.

Regarding our microplastic results, more microplastics were found in the sediment than in surface water. The abundance of microplastics in sediment could be due to microplastics accumulating for extended periods in the sediment [52]. Broadly, we found that microplastic abundance was highest upstream where agricultural activities are pervasive (FW1, FW3). The high microplastic abundance near agricultural land is supportable, considering that many organic fertilizers (e.g., manure) and irrigation can be significant sources of microplastics [53]. Agricultural landscapes may be particularly susceptible to microplastic contamination [54], and agricultural soils may be an important source of microplastics in rivers such as the Wolf River [54]. As demonstrated in the Wolf River, microplastics may be a contaminant of concern in many rural–urban rivers.

We found microplastics and lead to vary by site and season. The microplastic and lead variation is most likely attributed to human activities, agricultural runoff, and rainfall patterns [55, 56]. At FW3, for instance, there was extensive human activity alongside the river [34]. At FW1, agricultural and livestock usage is directly next to the site we sampled (S. Moyo and S. Lightman, personal observations). Researchers in austral aquatic systems have demonstrated that seasonal rainfall plays a significant role in microplastic variation [57]. Between the spring and summer months, there was an increase in precipitation in the Memphis area, which could have led to the re-suspension of sediment microplastics subsequently leading to changes in the distribution of microplastics [58]. As a result, current research indicates that rainfall patterns and anthropogenic activities influence the distribution of microplastic and lead concentrations in rivers. In many areas prone to flooding, such as our study river, flooding can export nearly 70% of the microplastic load stored in riverbeds [22].

This study demonstrated significant differences in microplastic concentrations between spring and summer. Considering we only sampled five river sampling stations, our data may under-represent the average microplastic concentration throughout the Wolf River annually. Our findings establish a microplastics baseline for spring and summer along a rural–urban gradient.

Microplastics and lead were correlated in microplastics found in sediment. A reason for this could be that microplastics have been found to serve as vectors for heavy metals [59].  This may be due to lead accumulating in biofilms, where microplastics adhere to surfaces in rivers, allowing biofilms to act as passive samplers for metals [60, 61]. Microplastics have a hydrophobic surface and, therefore, can bind to inorganic materials, including lead. As such, microplastics can accumulate lead in many riverine systems [60, 62]. The assertions above are in accordance with researchers who have demonstrated the link between heavy metal abundance and microplastic abundance in the northeastern portions of the Indian peninsula [63].

Microplastics found in the surface water had no direct relationship to lead concentrations (Fig. 5). These results are congruent to researchers who documented no specific congruency between surface microplastics and lead. This could be due to environmental factors such as flow rates of different areas and different anthropogenic influences on each site [64]. Microplastics also have been found to have a sorption behavior when they age, impacting their effect on attaching to heavy metals such as lead [65].

Fragments were the most dominant type of microplastic found at each site in sediment and surface water samples. Pellets were the second most common microplastic found in the sediment, with fibers being the least common, possibly as an artifact of visual quantification methods. Fibers were the second most common microplastic found in surface water, with pellets being the least commonly found. The dominance of the fragments could be attributed to paint particles, plastic litter, and agricultural runoff [66]. The high abundance of pellets could be related to an agrarian source such as fertilizer beads [67]. Plastic litter is common alongside and in the Wolf River due to illegal trash dumping and litter left from leisure activities [68]. According to the United States Department of Agriculture (USDA), In FW1 and parts of FW3, nearby agricultural and livestock fields also lead to pesticide and fertilizer runoff in our study system [69]. The dominance of fragments is comparable to studies in a Guangdong Province river, whereby fragments were found to be dominant in fish and the river [70]. Elsewhere, in the Atoyac River basin in Mexico, fragments comprised 22% of microplastics, with fibers making up 14.8% [71].

It is worth noting that comparisons with other studies worldwide can be challenging and require consideration of methodology and location since methodological protocols are not yet consistent across studies. For instance, previous work using neuston nets in freshwater ecosystems found lower values than those documented with grab samples [72, 73]. Greater concordance in methodological approaches among researchers will permit robust microplastic abundance comparisons among studies. In addition, the methods used can affect the quantification of microplastics. For instance, some of the drawbacks of physically counting microplastics (as in this study) can lead to underestimation of microplastics (e.g [74]). In future studies, integrating automated technologies [e.g., Fourier transform infrared (FTIR) and Raman] will yield better estimates of microplastic in the Wolf River. These technologies are particularly advantageous (nevertheless expensive) as they accurately and efficiently produce results without the arduous task of manually counting and identifying each microplastic. Our study is the first attempt in this region to provide baseline data for understanding microplastics and lead pollution in freshwater river systems.

The Wolf River was polluted by microplastics and lead relative to other freshwater rural–urban rivers. Due to microplastics being a relatively recent and understudied issue, further research needs to be done on the long-term effects of microplastics on the ecosystems of the river and how they affect organisms, including humans. As countries continue to industrialize and grow, understanding the issues of microplastics and lead and how they affect the environment will be important for further research.

In any study, there are inherent caveats in protocols, including collection methods and other analysis tools, which necessitate caution when interpreting results. We studied microplastic abundance using techniques similar to those employed by other researchers. However, different approaches may either overestimate or underestimate microplastic levels. We recommend that future studies incorporate FTIR and Raman microscopy, as these techniques could improve the accuracy of microplastic abundance estimates. As the field of microplastics research is relatively new and rapidly evolving, there are limitations, such as inconsistent methodologies across studies and the current lack of advanced technology to automatically count microplastics, requiring researchers to physically count them. Although we cannot definitively determine whether lead is adsorbed onto microplastics in our river system, we did observe a correlation between lead levels and microplastic concentrations. In the future, we plan to collect samples to investigate the potential adsorption of heavy metals onto microplastics.

5 Conclusion

In conclusion, microplastics were present at all sites in the Wolf River, in both surface water and sediment, across all seasons. These findings align with previous research, highlighting the widespread nature of microplastic pollution. Our study emphasizes the need for further investigations, particularly in rivers traversing both rural and urban regions. As microplastic contamination rises, the combined effects of microplastics and lead may pose severe risks to aquatic organisms, potentially disrupting population dynamics and species interactions. Such ecological disturbances could have far-reaching consequences for economies and societies in the future.