Evidences of Microplastic in Air and Street Dust: A Case Study of Varanasi City, India

Microplastics (MPs) are ubiquitous in our environment. Its presence in air, water and soil makes it a serious threat to living organisms. The present study aimed to assess the availability of MPs in air and street dust of a metropolitan city Varanasi, India. Suspended dust samples and street dust samples were collected from various sampling sites. The assessment of MPs was conducted by for physical identi�cation binocular microscopy, �uorescence microscopy and Scanning Electron Microscopy (SEM), while elemental analysis done by Energy Dispersive X-Ray Analysis (EDX). and �nally, Fourier-transform infrared spectroscopy (FTIR) was used for functional group analysis. the presence of MPs in both suspended dust and street dust samples of all selected sampling sites was con�rmed by results. MPs of different color with the shape of Fragments, Films, Spherules and Fibers were observed in the study. However, most of the MPs were less than 1mm in size. The MPs identi�ed in our study were majorly polypropylene, polystyrene, polyethylene, polyethylene terephthalate, polyester, and polyvinyl chloride. EDX analysis showed presence of trace elements like aluminum, cadmium, magnesium, sodium, and silicon apart from carbon and oxygen, which indicates the presence of additives or adsorption capacity of MPs. Con�rmation of MPs in the air of a locality of Varanasi explains the need of deep research in this concerned �eld to protect our future from negative impacts of breathing MPs.

. Studies clearly shows that the problem of presence of MPs in our surrounding is a big issue.
Many researches also tried to quantify the reach and impacts of MPs. Fine MPs present in air can easily pass through our respiratory system and reach deep in the bronchioles. Pauly et al. (1998) got bers of up to 250µm in the deep part of the lung in their study (Pauly et al. 1998). But still potential risk of ingested airborne MPs to human organ is not known completely till date but it is assumed that the risk associated with it depends on many factors like particle size, adsorbed chemical, concentration, deposition and clearance rate (Teuten et al. 2007, Teuten et al. 2009). Mainly, ability of MPs to absorb toxic chemicals on their surface because of their large surface area and hydrophobic property make them chemical toxic. Pollutants like heavy metals (Kumari et al. 2021, Li et al. 2013, Mohanraj et al. 2004) and polycyclic aromatic hydrocarbons (Akhbarizadeh et al. 2021) got detected in atmospheric particulate matters. Microplastics can also act as a medium to carry pathogens or microorganisms to lungs and possibly result in an infection to the humans (Prata 2018). Adsorbed microbial bio lm on MPs could also be responsible to adsorb and transport heavy metals with it as it assists as chelating agents for metals . Considering all the reviews, our study was designed with the objective to identify and classify microplastic in atmospheric air and dust over Varanasi region.

Study area
Atmospheric suspended particulate matter and street dust samples were collected from some selected sites of Varanasi city, India (Fig. 1). The city of Varanasi is situated on the Indo-Gangetic plains of North India and has a population of 1,198,491 and an area of 112.26 km 2 . The present study was conducted in June, 2019 and September, 2019.
For suspended dust samples, Site (S1), Banaras Hindu University (Institute of Environment and Sustainable Development, 25.2677°N, 82.9913°E), was chosen. It is an institutional setting surrounded by a typical mixed urban environment characterized by congested roads and a variety of commercial and residential activities. For street dust samples, six speci c sites (S2, S3, S4, S5, S6, and S7). were selected representing the urban environment within Varanasi (25°19'0.05"N, 83°0'3748"E). The details of the sampling sites are given below in the Fig. 1 and Table 1 respectively. . To minimize plastic contamination, a local anti-static wooden brush and a metal pan was used to gently sweep the material straight into an airtight bag made of low-density polyethylene (PE) which was rinsed with Milli-Q water and dried between subsequent samplings. All collected samples of street dust were then brought to the laboratory and unnecessary material such as gravels, leaves, small pieces of bricks and concrete etc., were removed from the samples. Then each sample was sieved using a 5-mm sieve and all particles collected on 5mm sieve also discarded. To remove mistakes caused by lingering particles on the road surface and particle disruption in the ambient air during the sweeping process, the MPs concentration was given as the number of MPs debris per 15g mass of dust rather than the number of MPs per sq. meter of street dust (Dehghani et al. 2017).

Atmospheric suspended dust
Over the roof of IESD-BHU, particulate matter (PM10) samples were collected on 10x8 inch glass ber lters (GF/A Whatman) with the help of respirable dust sampler (APM-BL 460, Envirotech) that isolates aerodynamic dust particles of desired aerodynamic diameters (≤10 micron) at an average ow rate of 1.2 m3/minute. Before and after sampling, all lters were desiccated for 24 hours. To avoid local disturbances like, from automobiles, the high-volume sampler was installed at a height of 7.5 meter.
Following ltering, lters were carefully removed from the sampler and quickly transferred to clean airtight, LDPE bags using stainless steel tweezers and were brought to the laboratory for MPs extraction. The desiccated lter papers were weighted using electronic microbalance with 0.01 mg resolution. The difference among the initial weight and nal weight of the lters was used to compute the aerosol mass (g/m 3 ), and then the concentration was estimated by dividing the aerosol mass by the total volume of air (m 3 ).

Extraction of MPs from street dust and air-suspended dust
The reagents used for this study were-30% hydrogen peroxide and ZnCl 2 provided from thermo sher scienti c India Pvt. Ltd., and de-ionized water (Milli-Q) were used throughout all the experiments. To avoid MPs loss because of the intense reaction that occurs after 20 min of adding hydrogen peroxide, 500 ml glass beaker was used. After digestion process, the content was ltered through Whatman lter paper (Whatman grade 42, diameter 42.5 mm, pore size 2.5 micron) and has been washed using Milli-Q water to collect all particles which has been sticked to the beaker wall and to eliminate remaining hydrogen peroxide on the dust particles. After that, the samples were dried for 24 hours in an oven at 50 degrees (Dehghani et al. 2017).

For Suspended dusts:
Each sample collected on the lter paper was carefully removed and kept in the air-tight low density PE bag and brought to the laboratory for further experiment. The contents of the lter paper were washed with ltered deionized water afterwards cautiously transferred to a clean beaker as much as possible using a clean metal spatula. Then, the content was kept in the oven at 70-80 degree Celsius for drying completely. After the content was completely dried, it was transferred to another beaker and then 35 ml of H 2 O 2 was added and is kept for approximately 8-10 days until the air bubbles coming out from the beaker stops (Abbasi et al. 2017, Abbasi et al. 2019). This was done to remove all the organic matter or biogenic matter present in the samples. Then, the content was ltered out on Whatman lter paper (Whatman 42, 2.5µm pore size). After ltration washed with Milli-Q water to remove the remaining hydrogen peroxide adhered to the sample and to transfer all the MPs from the beaker and again kept at 50 degree for 24 hours for drying.

Microplastic separation
A Zinc chloride (ZnCl 2 ) solution of density 1.6 g/cm 3 was prepared and 100 ml of this solution was mixed with each sample and the content was shaken at 350 rpm for 5 min and left to settle approx. 1 to 2 hours. The supernatant was then centrifuged at 4000 rpm for 4 minutes and then vacuum ltered through a cellulose nitrate lter (Axiva Sichem Pvt. Ltd., pore size 0.45 µm). The ltered contents were then washed with Milli-Q water to restrict the formation of ZnCl2 crystals. Density separation, centrifugation, and ltration processes were repeated three times on the same lter paper to capture all MP. These lter papers were dried for few days at room temperature until it completely dried and then transferred to a petri dish.

Microplastics identi cation
For identi cation of MPs on the lters from the street dust samples and suspended particulate matter, uorescence microscopy, binocular microscopy, SEM, EDX and FTIR analysis were performed.

Binocular Microscopy
MPs were observed and identi ed on the basis of visual characteristics mainly shape, color and size using Binocular microscope. Apart from this, particles were identi ed as MPs considering certain criteria described by (Chubarenko et al. 2018) like: 1. There should be no evidence of cellular or biological structure.
2. Fibers should be uniformly thick along their length and not tapering at the end.
3. Colored particles must be uniform in color.
4. Fibers should not be segmented and should not look as twisted at ribbons.
In this study, a binocular microscope (Catscope) was used to identify and separate MPs on lter paper from road and aerial dust samples, and images were taken with a digital camera (Catymage) equipped with a microscope.

Fluorescence microscopy
During the different process of synthetic textile and plastic industries a wide range of dyes, uorescent pigments, optical brightening and whitening chemicals are used (Christie 1994). Hence, uorescence microscopy was used for identi cation of the extracted MPs using uorescence microscope (Nikon eclipse 90i) with x100 magni cation under ultra violet lter. The corresponding images were taken with the Nikon digital camera which was equipped with the uorescence microscopy (Dehghani et al. 2017).

SEM-EDX analysis
The morphological characteristics and elemental content of MPs were investigated using SEM-EDX. The selected samples were prepared on double sided carbon conductive tape and were xed on a 10mm diameter SEM stub and were analyzed under an accelerating voltage (Dehghani et al. 2017). These samples were also examined for their compositional characteristics using an EDX detector. In this study, ZEISS EVO 18 scanning electron microscope was used for better and clearer morphological characteristics.

FTIR analysis
FTIR is widely used analytical approach to identify MPs samples. We have also used FTIR for the analysis of our desired samples. FTIR spectroscopy method has a long history of use in the investigation and characterization of synthetic polymers and their products. The vibrations of a sample's molecules are stimulated and detected by vibrational spectroscopy, resulting in unique spectrum ngerprints in the FTIR. Which helps in characterization on the basis of comparison of polymeric chemical structure with known reference spectra. Here, in our study, PerkinElmer Spectrum version 10.4.3 was used for sample analysis and a free software named essential FTIR software was used for comparison of the unknown FTIR spectra. During FTIR spectroscopy the sample was treated with Infrared light (wavenumber range 400-4000 cm-1 for Mid-IR). A part of this Infrared radiation is absorbed by the sample and nally measured in transmission or re ection mode.
Quality control and assurance of experiment All equipment and glassware were pre-cleaned with milli-Q and distilled water to prevent the contamination of external plastic/ ber with the required test setup throughout the test. All the chemicals and the reagents used were also ltered through the Whatman 42. Working areas were carefully wiped with ethanol, and closed environment was maintained by closing windows and door shut, as much as possible. Single use non-latex nitrile gloves were used and samples or the containers were also covered with aluminum foil to avoid any contamination. Also, blank was run throughout the experimental period to observe any contamination of MPs presence from surrounding environment or from the apparatus used, and the same procedure of double ltration was followed as for the samples analyzed. The resulting lter paper obtained after was also observed under the microscope revealed that there was no detectable MPs contamination during the analysis.

Presence of Microplastics in Varanasi
MPs were found in the samples of street dust at all the sites of Varanasi and also in all the replicates of suspended dust samples at IESD-BHU. These MPs were of different size, shape and color. The observed color of the Microplastics were Pink, yellow, green and red etc., and very few transparent MPs were observed. At all sites the presence of MPs was observed indicating the contamination of lower atmospheric environment with MPs.

Binocular microscopy
From the experimental observation, varieties of MPs in the atmosphere were found which were of different size, shapes and colors. This study recorded bers and fragments as the dominant type of MPs. Fig. 2 and Fig. 3 exempli es the types of few MPs that were commonly observed in the present study by Binocular Microscopy. Fragmented MPs were the dominant type of MPs followed by the ber in the overall study. Another study from India found that bers were the most common form in roadside suspended dust from Nagpur's urban and rural environments (Narmadha et al. 2020  In the samples of MPs extracted from the street dust from the different locations of Varanasi, bers of different color and different shapes, i.e., brous, fragmented and Film-like MPs were observed (Fig. 3). In another study from Chennai, India, mostly fragments and bers were resulted from the street dust samples

Fluorescence Microscopy
Fluorescence Microscopy con rmed the presence of MPs. In uorescence, the MPs mainly absorb UV light at 300-400 nm and radiate Blue (450-480 nm) or Purple (400-450 nm) uorescence (Lei et al. 2006). Most of the visible particles include fragments and bers which give uorescence, followed by rare lms, spherules, and pellets as shown in the pictures of various MPs shown in Fig. 4 and Fig. 5 In Fig. 4, MPs from the atmospheric fallout has been shown while, Fig. 5 shows the MPs obtained from the street dust. Morphology and elemental analysis SEM-EDS is considered as a signi cant technique for assessment of the surface morphology and composition of MPs (Rocha-Santos &Duarte 2015). Therefore, this technique is better for obtaining a high-resolution image of the surface properties of the selected MP, with qualitative information and results of the elemental composition shown in Fig. 6.
The image shows a fairly smooth surface without cracks. It also shows the mark of mechanical and chemical weathering as aking, pits, grooves and jagged edges. The elemental result is that MP is composed primarily of carbon and oxygen (and Nitrogen for Spherules), the presence of other elements may re ect the contamination by foreign solids, chemicals, dust and soil (Al, Ca, Si and Mg) or materials In present study, major particles were weathered in street dust samples and Al and Si were the most common elements identi ed which may be possibly originate from nearby soil ground. However, Ca and Mg majorly found to attached with rough surfaced tiny particles shown in (Fig. 6(e) The spectra of the selected samples are shown in the given Fig. 7. Using IR spectrum, (Fig. 7a, b, and c) sample 1, 2 & 3 obtained from street dust of Sir Sunderlal hospital backyard, chemical engineering department and Durgakund respectively, could be classi ed as a synthetic of a common PE. The function PE absorbance bands are positioned at 2914, 2847, 1470 & 718cm −1 . The wide band around 1000cm −1 possibly detected because of inorganic impurity (presumably a silicate) on the particle surface. The typical PE bands at 1034, 1100, 1449, 2852 & 2920cm −1 were clearly seen. Further bands especially at 530, 690, 1385 & 1460cm −1 , not existing in pure PE, were also visible. Also, in the atmospheric suspended dust (Fig. 8), spectra of the sample show almost the same trend and peaks, thus it is also classi ed as PE.

Conclusion And Future Insight
Plastic particles became commonly found in abundance in our surroundings still less understood and investigated in urban suspended and settled dust. In the current study, presence of MPs in Street and suspended dust of Varanasi was investigated. It is clear that MPs deposited and in the air are potentially important pollutants in urban environments. This study shows that road dust and airborne dust from all sampling points contain MPs of numerous shapes and colors, and most MPs are less than 1 mm in size. Mostly found particles were particles of bers, spherules, fragments, and lms of different color and size that may come from domestic, vehicular and other commercial sources. The MPs found were dominated by fragmented and brous shape MPs followed by spherules and lms. The observed color of the MPs was Pink, yellow, green and red etc., and very few transparent MPs was observed, this may be due to the reason that transparent MPs are not easily visible or identi ed. It has also been observed that MPs gives uorescence under blue lter. SEM images showed that grooves, pits and fractures were the common pattern of degradation in MPs particles. The EDX analysis has proved that all the MPs are made up of carbon and oxygen majorly and in trace amount Al, Ca, Mg, Na and Si. These elements indicate presence of additives or adsorbed debris on the MPs surface. The identi cation of MPs using FTIR has been performed for some samples and their spectra reveals the presence of PE with the characteristic peak resemblance with the PE. Majorly detected PE, could be due to the widespread use various PE made items such as toys, milk and shampoo bottles, pipes, packaging lms, grocery bags and other bags in our dayto-day life. A better and accurate result can be found on more analysis of samples with pyrolysis GC-MS technique for their more accurate quanti cation and identi cation of types of MPs is in progress. Zinc Chloride was used throughout the experiment for MPs extraction since its high density helped to obtain maximum MPs from samples. A further detailed study is required of the street dust and atmospheric suspended dust for a long duration in Varanasi city. Identi cation and quanti cation of atmospheric and street dust contaminants adsorbed on MPs are needed to further investigation in detail. Also, standardized or reliable methods for the analysis of airborne MPs is required to develop.

Declarations Funding
The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.
Competing Interests Figure 1 Geographic Map of the study area and respective sampling sites.