Study on Pollution Characteristics and Ecological Risks of Microplastics in Ningyuan River

: A survey was conducted on the surface water samples of Ningyuan River in Hainan Province. The samples were collected through on-site filtration and concentration methods, and pretreated through oxidation digestion and density separation. The microplastics were statistically analyzed by confocal micro-Raman spectrometer, elucidating the abundance, type, particle size, and shape characteristics of the microplastics in the water samples. Seasonal differences were analyzed, and the main conclusions were as follows: microplastics were detected at all sampling points, with the abundance range of 66.67 items/m³ to 1106.67 items/m³; polyethylene terephthalate (PET), polypropylene (PP), polyethylene (PE), and polystyrene (PS) are the main types of microplastics; 100-1000 μm is the main particle size distribution range; the proportion order of microplastics with different shapes is fiber> fragment> film> particle. In terms of spatial distribution, there are significant differences due to the influence of functional zones along the river banks, while in terms of seasonal distribution, there is no significant difference between dry and rainy seasons; The overall level of microplastic pollution belongs to a medium to low risk.


Introduction
Microplastics usually refers to plastic particles with a particle size less than 5 mm, and was first proposed as a term in 2004 [1]. Due to its small density, stable properties and large specific surface area, microplastics are easy to migrate in the environment, and can be eaten by organisms and transmitted through the food chain and food web [2]. In addition, microplastics have an adsorption effect on pollutants such as heavy metals, organic pollutants, and pathogenic microorganisms [3][4][5], and can easily become carriers of other pollutants during migration, causing joint toxicity to organisms in the aquatic environment. Moreover, they can break down again to form nano microplastics (NPs) [6], causing more severe cell oxidative damage, cell inflammation, and even genetic toxicity [7]. Therefore, microplastic pollution has become an important environmental issue of global concern.
In recent years, with the establishment of Hainan Free Trade Port, downstream areas of Ningyuan River are undergoing vigorous development and construction, with a large number of enterprises and population influx. The issue of microplastic pollution deserves further attention, but relevant data on microplastic pollution in Ningyuan River is still lacking.

Study area overview
Hainan Province has strong sunlight intensity, developed agriculture and tourism, which are favorable social and natural conditions for the production of microplastics. Ningyuan River is the fourth largest river in Hainan Province, originating from Sugarcane Mountain in Baoting County and flowing into the South China Sea at the Central fishing port of Yazhou district. It has a total length of 94 kilometers and a catchment area of 1030 km 2 . The upstream is the central region of Hainan Island, with many tropical rainforests and a small population. The middle reaches flow through the northern hilly areas of Sanya City, with few residential areas. The main water supply source in the western part of Sanya is Dalong Reservoir, with a good ecological environment. The downstream mainly flows through Yazhou District, making it a valley plain agricultural area, and it's also a residential area of Yazhou residents.
Due to the sparse population in the mountainous area of the upper reaches of the Ningyuan River, this study set up a total of 12 sampling points from the upstream of the Dalong Reservoir to the end of the estuary, based on the classification principles of functional areas, tributaries, reservoirs, and estuaries. Among them, N-1 and N-2 are located in the Dalong Reservoir, N-3 to N-7 mainly flow through agricultural areas, and N-8 to N-12 are mainly residential areas and tidal river sections. Sample collection and analysis of microplastic pollution characteristics were conducted during the dry and rainy seasons, with sampling points arranged as shown in Figure 1.

Sampling, pretreating, and evaluation methods
The sampling method is concentration sampling, using a portable water pump to extract 50 liters of water within 20 cm of the surface water at a distance of 1 meter away from the river bank, meanwhile, filter it on-site. The filter screen is made of 500 mesh stainless steel seamless mesh( φ= 30.8 μm) After filtration, collect the filter residue into a 500 mL wide mouthed glass sample bottle, take it back to the laboratory, and store it at 4℃. During the sampling process, basic information such as longitude and latitude, water temperature, turbidity, etc. of the sampling points are recorded, and the surrounding environmental characteristics are recorded.
The oxidation-digestion-flotation method was used to pretreat the samples. The sample is evaporated and concentrated in a 60℃ oven, and then 30 mL of H 2 O 2 is added. Then, the sample is heated in a 65℃ water bath for 24 hours, with clarification as the digestion endpoint. Otherwise, 10 mL of H 2 O 2 is added for digestion until clarification. After digestion, perform flotation treatment and choose to prepare ZnCl 2 (ρ= 1.78 g/cm 3 ) was used as a flotation solution [8]. After 24 hours of flotation, it was filtered and naturally dried. The microplastics were observed and identified using a laser confocal micro Raman spectrometer (Thermo Fisher, DXR3xi), with a wavelength of 532 nm, laser power of 4 mW to 12 mW, exposure time of 0.025 seconds, 500 scanning times, and a wavenumber range of 50 to 3400 cm -1 . confocal hole of 50 μm, objective magnification of 10/50, and spectral correlation matching mode was used. When the matching degree reaches 70%, it can be confirmed as a certain type of polymer [9]. Through the Terrain Mosaic function, microplastic images are obtained, and information such as the size, shape, and type of microplastics are measured and recorded.
After organizing the sample data, the commonly used Pollution Load Index (PLI) model and risk index (H) were selected to assess the pollution risk of microplastic pollution. The risk index (H) model uses polymer toxicity as a reference indicator and can reflect the composite effects of different microplastics [10]. The model composition is as follows: In the formula, H is the risk index, Pn is the percentage of different types of microplastics at the sampling point, Sn is the hazard score of microplastic polymers, and the scores of PET, PE, PP, and PS are 4, 11, 1, and 30, respectively [11]. H< 10 is Class I, 10-100 is Class II, 100-1000 is Class III, and H> 1000 is Class IV.
The PLI model proposed by Tomlinson et al. [12] is commonly used in heavy metal pollution risk assessment, and some scholars have also applied it to the risk assessment of microplastics [13]. This model evaluates the overall pollution situation based on the abundance of microplastics at sampling points. The model composition is as follows: In the formula, CF i is the microplastic pollution coefficient of each sampling point, C i is the measured abundance of microplastics at a single sampling point, and C 0i is the background reference value (in this study, the point N-1 with the lowest abundance of microplastics upstream was selected as the reference value); N is the number of sampling points, PLI zone is the pollution load index within the region, and when PLI< 1, the pollution level is mild pollution; When PLI is 1-2, the degree of pollution is moderate; When PLI is 2-3, the degree of pollution is severe.

Characteristics of microplastic pollution
The abundance and distribution patterns of microplastics at different sampling points are described in Figure 2. The presence of microplastics was found at all sampling points, with an average abundance of 162.67 ± 102.76 items/m³ (rainy season) and 284.67 ± 333.23 items/m³ (dry season) respectively. With Nandu River, the largest river in Hainan (with an average abundance of 406 ± 223 items/m³) compared to being at the same level of magnitude [14] RDA analysis of pH, dissolved oxygen, turbidity, water salinity, and temperature revealed that turbidity was the most relevant factor. The microplastic abundance conforms to the normal distribution as Figure 2 described, and the paired sample T test of the microplastic abundance in dry and rainy seasons shows that p> 0.05 (p= 0.349), indicating that there is no significant difference in the microplastic abundance of the river water in dry and rainy seasons in the Ningyuan River basin. In the experiment, it was found that the minimum particle size of microplastics was 61 μm. The maximum particle size of plastic exceeded 5 mm, as the fiber diameter smaller than the mesh(used for prefiltration, φ= 5 mm) and was collected. In terms of particle size distribution in Figure 3, the main particle size distribution range is 100-1000 μm. The main particle size range varies slightly between dry and rainy seasons, with the rainy season ranging from 100-300 μm and 300-1000 μm in dry season. As shown in Figure 4, the average proportion of microplastics with different shapes in dry and rainy seasons is fiber > fragments > film > particles. Overall, the proportion of fibrous microplastics exceeds 50%, while the proportion of granular microplastics is less than 5%. The main type of fibrous microplastics is PET (accounting for over 70%), which is widely used in various fabrics. Laundry wastewater is one of the important sources of microplastics in water [15], so it is speculated that domestic sewage is the main source of fibers in rivers. In terms of the composition of microplastics, four types of microplastics were mainly monitored, namely PE, PS, PP, and PET, as shown in Figure 5. Among them, the proportion of PET and PP is relatively high, with the proportion exceeding 80%. PET and PP are widely used in various packaging and fabrics [16], further speculating that the main source of microplastics in rivers is domestic and production wastewater. In addition to the four types of plastics mentioned above, a large amount of cellulose derived from plants, a small amount of ethylene vinyl acetate copolymer (EVA), and poly (1,4-butylene terephthalate) particles (PBT) were also found.

Risk assessment of microplastic pollution
As shown in Figure 6, the risk index H of Ningyuan River is at a relatively low value, and only two sampling points are classified as Level II risks during the dry season, while the remaining points are classified as Level I risks. The H value is closely related to the type composition of microplastics, with PET and PP accounting for a higher proportion in the composition of microplastics in Ningyuan River, so their toxicity scores are lower resulting in a lower risk index H. After calculation, the PLI index during the dry and rainy seasons is shown in Table 1, with 1<PLI<2. The PLI index is closely related to the abundance of microplastics. The Ningyuan River is moderately polluted during both the dry and rainy seasons. Due to certain differences in water flow during the dry and rainy seasons, the PLI index during the dry season is slightly higher than that during the rainy season. Overall, the risk of microplastic pollution in the Ningyuan River is relatively low.

Conclusion
This study collected water samples from the Ningyuan River in Hainan Island and conducted the first investigation on its microplastic pollution characteristics. The differences in microplastic characteristics between the dry and rainy seasons of the Ningyuan River were analyzed, and the possible sources of microplastic pollution were analyzed in combination with industry and environment. The pollution risk of microplastics in the Ningyuan River was preliminarily evaluated, providing data support for understanding the current pollution status and ecological risks of microplastics in rivers entering the sea in Hainan Province.