Source tracking microplastics pollution from Qarasu basin rivers to Gorgan Bay, Caspian Sea


 Concerns about the negative effects of MPs on human health have led to increasing attention to the occurrence of MPs in the aquatic environment. Recent studies have focus from the marine environment to inland waters, particularly the spatio-temporal distribution of MPs in rivers for residential and agricultural area. Qarasu river basin, watershed to Gorgan Bay, is the site of many permanent rivers and the levels of MP pollution in those rivers are unknown. This research was conducted in three different type of land use: forest, residential and agricultural along 8 different rivers. A total of 9 fish species were identified and the presence of MPs in 87% of fish was confirmed. The prevalence of MP among species was Liza sp.> C.gibelio> G.holbrooki> R.rutilus> N.melanostomus> C.carpio = V.vimba> R.frisii> Barbus sp. The highest frequency of MPs was observed in Qarasu river (1880 ± 251 n/kg) and Mohammadabad river (184 ± 1340 n/kg) in agricultural land use, the lowest frequency was related to Baghu and Ziarat rivers with 660 ± 77 and 600 ±91 n / kg was found in forest land use respectively. The highest type, color and size of MP sediments were fragment, black and 1-2 mm, respectively. The highest type, color and size of MP in fishes were fiber, black and 0.5-0.1mm respectively. Overally according to the source tracking result, agricultural land use was the main source of MP pollution of Gorgan Bay. The Bay is strongly affected by the incoming rivers, the Qarasu River Basin as the most important river supplying water.


Introduction
Annual plastic production increased from 1.5 million tons in the 1950s to 368 million tons in 2019 (Plastics Europe, 2019). Plastics are made of a wide variety of materials. MP particles (MP) are plastics smaller than 5 mm in size that result from primary and secondary MPs (Ryan et al., 2009). MP pollution is a global environmental concern due to its ubiquity in the environment and the dangers they pose to ecosystems (Rachman et al., 2018). In general, MPs cause the most damage to the natural environment, because they are readily available to higher species and are concentrated as they move upward in the biological food chain (Canesi et al., 2015). Since the beginning of present century, contamination from large disposable items such as plastic bags has attracted much attention, while smaller and more harmful MPs have only been studied (Browne et al., 2007).
The effects of MPs are present not only in surface waters, but also in deep water, sediments, soils and organisms (Zhang et al., 2018). MPs, with their speci c size and stable properties, are breeding grounds for microorganisms and carriers of contaminants (Zhang et al., 2018). Plastics, ame retardants and other chemicals are added to plastic products to improve their performance. These chemicals can be transported to the environment by MPs and pose complex environmental hazards (Abarghouei et al., 2021). MPs can be ingested by aquatic animals, seabirds or mammals; After accumulating in the food chain, they eventually enter the bodies of top predators, including humans (Liu et al., 2019). Given the presence of MPs in water, the importance of marine MP contamination research is obvious. However, freshwater areas are very important, most ocean MPs come from land, freshwater ecosystems play an important role in MP transport (Bagheri et al., 2020). It is useful to study the locations and characteristics of MP contamination in freshwater areas.
There are few studies here that focus on MP consumption by freshwater sh, Wagner (2014) shows that only ve studies have examined its occurrence in freshwater sh. Among these, we can refer to a study in China, where it was found that most of the particles eaten by freshwater sh are MP fragments (Jabeen, 2017

Materials And Methods
Qarasu River with a catchment area of 1762 Km 2 and a discharge of 54.9 million cubic meters, originates from the heights of Qaleh Maran Mountain and after a 160-kilometer route, ows into Gorgan Bay, Caspian Sea. These rivers ows along the east-west in the south of Golestan province, but the source of most of its branches, before joining the Qarasu river, is from the Alborz mountain range and in the south-north direction (Caspian Water Consulting Engineers, 1998). The average height of the basin is 624 meters above sea level, the minimum of which is -26 meters on the shores of the Caspian Sea and the maximum is about 3200 meters in the southern heights of the basin. Up to a height of about 1000 m, the slopes are covered with forest and gradually decreases in height with decreasing forest density and increasing agricultural activities (MasoomPour, 2005). Qarasu River, which ows into Gorgan Bay (southeast of the Caspian Sea) near Qarasu village (54° 02´ 23 east and 36° 50´ 52 north) (Fig. 1).
In the present study, 24 stations were sampled in three types of land use (forest, residential and agricultural) ( 200 g of precipitate was dried overnight at 60 ° C and then added by adding 400 ml of NaI solution at a density of 1.8 g per cubic centimeter. The materials were stirred again and the samples were centrifuged at 5000 rpm for 5 minutes, after which the samples were immobilized for one and a half hours. Finally, the solution was centrifuged for 10 minutes and the separated solution was ltered through a strainer. After the lters dried, the particles on the lter paper were transferred to a petri dish for microscopic examination. The sh were frozen immediately after catching and be defrosted at room temperature for testing.
The digestive organs of each sh were removed separately from the top of the esophagus and at the end of anus and immediately placed in plastic zippered bags. All used equipment were examined under a microscope to check for polymer contamination before the test. The wet samples were weighed before drying, then the samples were placed in pre-cleaned glass containers. Place the dishes in an oven at 60 ° C for 72 to 24 hours, after which the dry weight was measured again.
10% (w / v) KOH (Merck, Germany) was used for digestion and added to all organs and tissues. The product were placed in glass containers and in an oven at 40 ° C for 72 to 48 hours to complete the digestion process.
Once the viscera are completely dissolved, it will be ltered through lter paper (Whatman PLC 122 United Kingdom). The lter paper was transferred to sodium iodide solution (4/4 M) NaI (Merck, Germany) and then subjected to sonication and severe shock. The NaI solution was then centrifuged and ltered again, visual observation and recording were also performed under a stereomicroscope.
The extraction method used for sh was used by Karimi et al., (2017). All particles were analyzed by stereomicroscope (NOVEL, Ningbo Yongxin Opitics Co., Ltd., China) with IC80 HD camcorder and software (LAS). Totally 8800 certain plastic particle were detected from 72 sites, the maximum length and width of the

1-4 MP contamination in different river sediments
According to ANOVA analysis, a signi cant difference in MP abundance was observed between the sample areas (p <0.05). The highest frequency of MPs was observed in agricultural use site of Qarasu river (1880 ± 251 n / kg) and Mohammadabad river (720 (184 ± 1340 n / kg)), the lowest frequencies was also in forest use related to Baqo and Ziarat rivers with 660 ± 77 and 600±91 n/kg respectively (Table 1).  The highest size of 1-2 mm were observed in stations with residential use (214. 2180 n / kg) and the lowest size was in 3-5 mm in forest land use (160 ± 31 n / kg). In all sampling stations, most MPs were black (64.22%) and gray 16.89% (Figure 4). The lowest color variation were observed in Baqo and Mohammadabad rivers.

2-4 MP contamination in shes of different rivers
Morphometric measurements including the number and size of MPs in the species are presented in Table 1.
The results show that C.gibelio had the highest total length (10.23± 1.56 cm), standard length (7.15± 1.1 cm) and body weight (8.33±0.4 g) ( Table 2). The highest number of MPs in gastrointestinal tract of C.gibelio and Liza sp were recorded with a range of 1-7 MPs. The lowest number of MPs was reported in C. carpio. The smallest MP size between 130 and 1500 mm was recorded only in C.carpio. A total of 140 sh belonging to 9 species were analyzed in different habitats (forest, agriculture and residential), which shows 87% of the prevalence of MPs, that means only 13% of the sh samples were found without MPs (Fig.2a).
Highest MP with 15.71% and 15% were in Liza sp. and C.gibelio, while the lowest was recorded in Barbus sp. with 43.6%. Also, the prevalence of MP in different shes were Liza sp.> C.gibelio> G.holbrooki> R.rutilus> N.melanostomus> C.carpio = V.vimba> R.frisii> Barbus sp (Fig. 5b). It is important to note that the occurrence and prevalence of MPs directly re ects the number of sh samples (n) for each species. The most MPs observed in shes were related to Qarasu river with agricultural application (1340± 241). The lowest observed rate was also in MahammadAbad river (40±10) with residential use.
The frequency and size distribution of MPs in the sampling areas in the Qarasu Basin is shown in Figure 6.
Fiber was the most signi cant MP (55%) followed by fragment (40.71%), while bead was below 5% ( Figure   6b). The highest frequency of MPs were in agricultural and residential stations in ber type. The highest percentage of observed colors were belonged to black (56.93%) and gray (18.25%) and the lowest to blue  In the present study, 5 cm above the surface of MP sediments was collected so that the abundance of MPs in the agricultural area was signi cantly higher than forest sediments (upstream). Overall, the results showed that the MP pollution of Gorgan Bay is strongly affected by the incoming rivers, including the Qarasu river basin as the most important river supplying water of the Gorgan bay. In the study of 8 different rivers, Qarasu river was introduced as the most polluted river and agricultural use as the most polluting type of use. In the study of 9 different sh species, Liza sp. introduced as the most polluted species.
The most type, color and size of MP sediments were 1-2 mm, fragment and black respectively. The highest type, color and MP size of sh were ber, black, 0.5-0.1 mm, respectively. MPs have become widespread in the environment, leading to potential concerns about their impact on environmental health. Due to the importance of the seas and its role in human food needs, and considering that Gorgan Bay is a semienclosed environment, they are very vulnerable to pollution. As well as the outlet of residential and industrial wastewater that enters directly into the Gorgan bay, it can be used as an indicator of changes in the amount of plastic waste in certain areas and can also be presented as a suggested topic for future studies.