Microplastics Monitoring in Environmental Matrices of an Alpine River originating from Hindu Kush Mountain Range

Microplastics (MPs) are cosmopolitan in distribution and an emerging threat to life and the environment. These particles are not restricted to human-inhabited lands but also found in different mountains and glaciers where the human population is relatively low. These MPs make their way to the river ecosystem from glaciers, rains, and municipal and industrial euents. The current study was designed to highlight MPs' pollution in water, sediments, and shes of the Swat River: originating from the Hindu Kush Mountain Range. These samples were collected from eight different sites across the river. The average MPs concentrations were 305.79 ± 289.66 MPs/m 3 , 588.29 ± 253.95 MPs/kg, and 12.54 ± 8.02 MPs/individual the water, sediments, and sh samples, respectively. The highest MPs concentrations (753.71 ± 330.08 MPs/m 3 ) in water were recorded near Mingora City, whereas MPs concentrations (834.0 ± 367.21 MPs/kg) were the highest on the same site. Among the selected sh species, the highest number of MPs was observed in Shizothorax plagiostomus. In contrast, the lowest in Wallago attu being 17.08 ± 8.27 Among the selected sh species, the highest number of MPs was observed in Shizothorax plagiostomus, whereas the lowest in Wallago attu being 17.08 ± 8.27 MPs/individual and 5.0 ± 2.36 MPs/individual, respectively. Fibres were the most prevalent MPs in all the matrices representing 80%, 92%, and 85% of the total MPs count in water, sediments, and sh samples. These ndings highlighted that the high concentration of MPs prevailing in the river originating from high-altitude freshwater sources and awareness, education, ecotourism, sustainable reduction in plastic use, and strict rules and regulations could be helpful to prevent the anthropogenic menace.


Introduction
It has been estimated that about 368 million tons of plastics are used per year worldwide, and this amount is increasing daily at an alarming rate (Plastics Europe 2020). Most plastic products are disposable and thrown away after their single-use (Barnes et al.2009;Hopewell et al.2009). Extensive usage, limited recycling, and durability of plastic polymers are the most critical factors for plastic waste accumulation in the environment ). The problem of plastic pollution aggravates when the macroplastic items disintegrate into smaller particles due to different physical, chemical, and environmental factors (GESAMP 2015). The large-sized plastics (> 5mm) are Macroplastics, while the small-sized plastic particles termed micro (1-5mm), meso (0.1µm-1mm), and nano-plastics (< 0.1µm) based on their size ranges. The MPs can also be terms as primary or secondary, where the term primary refers to particles produced in the micro size and secondary refers to MPs produced by the breakdown of MPs (Hanslik, 2020;Napper et al. 2015). However, there is no such research on freshwater sh from Pakistan, so the current study is the rst to establish a correlation between MPs in freshwater sh, sediments, and surface water in the country. Moreover, the Swat River is the remote river at a high altitude in the Northern mountains of Pakistan. Only a few studies have been reported globally to assess the MPs pollution in remote regions.
This study can be a valuable addition to present MPs literature to establish a relationship between MPs pollution level and ingestion in different sh species. It can also be signi cant to indicate potential sources of plastic pollution in such remotely located freshwater bodies.

Study area
The Swat River is a 240 km long river located in Khyber-Pakhtunkhwa (KPK), Pakistan. This perennial river has a basin area of 14,000 km 2 and receives plastic waste mainly due to shing and tourism activities in the catchment area before joining Kabul River, near Sardaryab (Charsadda-KPK). A total of eight sampling sites (n = 8) were selected for the water, sediments, and sh sampling, including six sites located along with River Swat (n = 6; Kalam, Bahrain, Madyan, Mingora, Chakdara, Charsadda) and two locations (n = 2) along with the Con uence point of River Swat and Kabul ( Figure.

Sampling protocols
A total of 24 samples (n = 24) were collected from eight sampling sites, and triplicate samples were performed for the surface water and sediments and Passive surface water from each site. The MPs in water were collected from each site using a manta trawl with a mesh size of 300 µm (Vermaire et al. 2017). The stationary manta trawl net was deployed thrice and xed at a position along the river's width (left-side, midstream, right-side) for one hour. The trapped anthropogenic material was separated from the collecting pock of Manta trawl, further rinsed with distilled water into a 500 ml amber glass jar to get maximum material. Three sub-samples of Sediment samples collected from 1cm depth from the shoreline of the same area, from where MPs collected the surface water following the pre-de ned methodology. The shoreline sediments sampled using a stainless-steel quadrate (30 cm) and a spatula, the collected sample stored in a Glass Jar (Irfan et al. 2020a; Klein et al. 2015). A total of 24 sh samples (n = 24) belonging to 4 different species were collected using rods and Nylon hand shing nets with the help of local shermen. The collected samples were weighed, labelled, and stored in the icebox and transported to the Environmental Toxicology laboratory, College of Earth and Environmental Sciences, University of the Punjab, Lahore, Pakistan. The sh samples were stored in a refrigerator while water and sediment samples stored kept at room temperature before further analysis.

Preparatory steps
The water samples digested using hydrogen peroxide along with Fenton reagent. After digestion of organic matter, MPs were segregated using density separation techniques. placed the beaker on a water bath for heating up at 75°C until the digestion of the organic content. The supernatant was then ltered through 300, 150, and 50 µm sieves and transferred to lter papers as described earlier.
Fish samples were thawed for 1-2 hours and dissected to collect their guts. Each gut sample of sh was weighed and digested in 250 ml glass bottles using 10% KOH solution with a 5:1 ratio with the sample.
Then, incubate the sample at 55oC for 36 hours. Sodium Chloride (3:1 v/v) was added to the digested material immediately and thoroughly stirred for 20 minutes before being left to settle for 2 hours (Pellini et al. 2018). The supernatant layer was collected and ltered through 300, 150, and 50 µm sieves to get three different fractions. The solid contents of each fraction from sieves then backwashed and transferred to lter paper (MCE 0.45 µm pore size and 47 mm diameter) using a vacuum ltration assembly. The walls of the ltration assembly cup washed twice, and the lter papers containing solids dried up for one day in a Petri dish covered with Aluminum foil before detection.

Identi cation and classi cation of MPs
After drying, the solid material at lter papers analyzed under a stereomicroscope with a digital camera.
Then examined coarse range of analyte on lter papers (prepared from 300 µm sieves) under the microscope. At the same time, observation for a ne range of MPs made on the lter papers (from 150 and 50 µm sieves) under the microscope with a digital camera (Irfan et al. 2020a). The physical shape and colour of MPs were used for the identi cation of MPs. These MPs further classi ed into bres, sheets, fragments, foams, and beads. Internationally recognized units viz; MPs/individual, MPs/m 3 , and MPs/kg for the sh, water, and sediment samples, respectively (Hidalgo-Ruz et al. 2012).

Statistics and computations
Descriptive statistics, ANOVA, and Pearson correlation were calculated by SPSS 18. The volume of water ltered through the manta trawl net was calculated using a modi ed ow rate formula as given below:

Laboratory Contamination Control
All the precautionary measures considered to prevent material and laboratory contamination. A dedicated portion in the laboratory was used for MPs samples to avoid cross-contamination. The tools and glassware used during the sampling and analysis were carefully rinsed with distilled water and covered with aluminium foil when not being used. Reagents and distilled water were also ltered using and covered with aluminium foil to avoid atmospheric contamination. A few lter papers were placed on random locations in the lab for 72 hours to measure the suspended load of MPs from the laboratory environment then analyzed under the stereomicroscope. Six such lter papers were examined during the whole analysis and kept as a control. MPs on the lters were subtracted from the sample to avoid the atmospheric contamination from the laboratory environment.

Shapes and Size Of MPs
Among the water samples, the relative proportion of the bres remained the highest (80%), and the fragments were minimum (6%). Comparable relative dominancy of bres also observed by Jian et al. Following the similar trend as observed in water and sediments, bers were also dominant in shes (88%) followed by sheets (7%), fragments (5%), and foams (0%). In the present study, the sheets ranked as the second most ingested form of MPs because they keep oating below the water's surface for being lighter in weight than fragments, which increases their chance of ingestion during sh feeding. Recently, Kuśmierek and Popiolek, (2020) described the similar trend of the highest proportion of bres (99.8%) in the gastrointestinal tract of freshwater sh. Zhang et al. (2020 b) and Wang et al. (2020) also reported that the bres with the highest concentration in native freshwater sh belonging to different trophic levels from Pearl River South China. The possible reason for the highest proportion of bres in sh species may be its di culty to excrete with faeces as bres stick with intestinal walls rmly compared to other shape MPs. Further comparison among relative abundance of MPs shapes given in Table 2. Phytophagous species may ingest the bres accidentally as these bres may resemble algae and phytoplankton. activities by local inhabitants and tourists, such as using nets for shing and the breakdown of plastic disposable being left at the bank of the river or directly thrown into the river. In sh samples, the overall relative abundance of MPs followed a similar trend as in the surface water, and prominent size MPs were dominant (43%), followed by small size MPs (29%) and medium-sized MPs (28%). MPs with sizes up to 1000 µm reported being highest (70.5%) in a study by Sun et al. (2020), whereas Wang et al. (2020) reported that 80% of the detected MPs belonged to the size range of 500-1000 µm. The relative proportion of ingested MPs sizes also varied according to the sh feeding habit. Large-sized MPs contributed 76% in carnivorous, 75% in lter-feeding, 59% in omnivorous, and 53% in herbivorous sh. Furthermore, the dominance of larger MPs particles and the lower proportion of smaller sizes particles in the gut contents may be due to sh inability to excrete along with faeces.
Larger size MPs (300µm) were also dominant in the sediment samples (44%), followed by medium and small-sized MPs in an equal proportion. A similar trend in MPs ratio reported from Veeranam Lake India,

Conclusion
The present research ndings showed that the contributions of large-sized MPs (300µm) were the highest in all the surface water, sediments, and sh samples from a higher altitude to the plain reaches of Swat River, Pakistan. Based on MP types, the bres were in high proportions in the overall samples. The present study indicated that Swat River is suffering from indiscriminate use of plastic in the catchment. Due to the high in ux of tourists, MPs pollution has been increased, resulting in a large volume of plastic waste directly or indirectly disposed into the river. It is highly recommended to start an organized campaign to educate the local community and tourists to minimize plastic and stringent enforcement of plastic reduction laws to manage this problem and make it an eco-friendly tourist destination.

Declarations
Compliance with ethical standards Con ict of Interest: The authors declare that they have no con ict of interest.
Ethical approval: Not Applicable