Identification and Analysis of Plastic Microparticles in the Inlet and Outlet of the Wastewater Treatment Plant and Investigation of the Relationship between Different Seasons of the Year with the Amount of Production and Emission of Particles

was performed in three seasons: spring, summer, and autumn, to determine the season with the most microplastic production. Ten, the acid washing using the 30% hydrogen peroxide (H 2 O 2 ) and 0.05M divalent iron was performed on the prepared samples. Also, since this method is based on creating density gradient, the sodium iodide (NaI) salt was used. Notably, the performance of the studied wastewater treatment plant in terms of the microplastic removal efciency was also evaluated using this method. Accordingly, the wastewater treatment plant was able to remove microplastics by 94–96%, which means that about 5% of the residual microplastics are drained into the aquatic and terrestrial environments. Moreover, it was revealed that microfbers have a lower percentage of removal in wastewater treatment process than microparticles. Te result of the method used has shown that more than 95% of the particles can be detected.


Introduction
Among various contaminating sources, plastics and their derivatives play an important role in environmental issues. A considerable portion of these plastics is composed of the micro-and nanoplastics. Plastics entering aquatic environments have diferent sizes ranging from micrometers to meters, among which microplastics are defned as particles smaller than 5 mm. Based on this, plastic particles were classifed according to size, which is shown in Table 1.
As shown in Table 1, microplastics are very small particles whose presence in wastewater, seawater, and oceans has already been proven [1,2]. One of the main ways for these particles to enter the sea water is through conventional wastewater treatment plants that are not able to completely remove these particles [3,4]. Since these particles are invisible due to their very small size, little attention has been paid to this type of pollution [5,6].
Te existence of these particles (microplastics) in the production efuent of diferent countries is diferent according to the amount and pattern of consumption and lifestyle, and it is also a matter of discussion.
Since Iran ranks ffth in plastic consumption in the world, conducting this research is very important. Based on this concern, the present study is dedicated to determining a method for identifying and extracting particles from sewage and efuent streams. Multiple research studies have been performed to determine a high-yield method to detect and separate microplastics from efuents. Some of the methods to detect microplastic particles were performed on seawater and some others were performed on municipal sewage [7][8][9]. Meanwhile, each method has its own steps and techniques, which were discussed further. Many authors have investigated this problem.
A study titled Detection and Removal of Microplastics in Wastewater: Evolution and Impact was conducted by Tuhin et al.; this paper provides a critical analysis of existing and newly developed methods for detecting and separating microplastics from discharged wastewater, which are the ultimate challenges in the microplastic treatment systems. A critical study on the efect of microplastics on aquatic organisms and human health is also discussed. Tus, this analysis provides a complete understanding of entire strategies for detecting and removing microplastics and their associated issues to ensure a waste discharge standard to minimize the ultimate potential impact in aquatic environments [10].
A new method was reported in a study for analyzing the characteristics of microplastic fbers using the Fouriertransform infrared spectroscopy (FTIR) by Corami et al. based on this method, a new quantifying procedure and simultaneous detection of fber polymers using the FTIR analysis were developed. Simulating the washing process was done using the commercially available domestic products and wastes were fltered using the GF/F (0.7 µm) or 0.2 µm flters to collect the smallest fbers. In addition, a new method for wastewater treatment was also proposed. After that, flters were investigated using the scanning electron microscope (SEM) to confrm the length and width of fbers. Tis method allows better release of fbers and identifcation of fber polymers [11]. Also, the existing methods for removing microplastics were comparatively evaluated by Tielea et al. in this study; the existing techniques were compared using the hydrogen peroxide, proteinase, trypsin, and potassium hydroxide. Also, the refning ability, digestion efect, ability of recovering microplastics, and polymer detection were investigated using the Raman spectroscopy and matching software [12].
A study entitled determining an improved method for dispersing microplastics in the secondary treatment was performed by Raju et al. In this work, an improved method was reported to determine the size, form, polymer type, and particles sharing using the combination of oxidation, fuorescent color, and attenuated total refectance FTIR (ATR-FTIR) analysis to detect particles smaller than 20 µm in efuent [13].
A study entitled transportation and destination of microplastics during the ordinary activated sludge process in a wastewater treatment plant in China was performed by Liu et al. Based on that, the main purpose of this study was to investigate the transportation and destination of microplastics in a wastewater treatment plant based on the ordinary activated sludge process and also to determine the amount of microplastics stored in the plant sludge and waste [14].
A study entitled Identifcation and Quantifcation of Microplastics in Efuents of Wastewater Treatment Plant by Diferential Scanning Calorimetry (DSC) was performed by Joaquín et al. In this research, the presence of microplastics was detected through a diferential scanning calorimetry (DSC) analysis of three wastewater treatment plants. One of these plants applied only a preliminary treatment stage while the others applied up to a secondary treatment stage to evaluate their efectiveness. Te results showed the presence of polyethylene (PE), polystyrene (PS), polypropylene (PP), and polyethylene terephthalate (PET), which were classifed as fragments, fbers, or granules. During the evaluation of the plants, it was determined that the preliminary treatment did not remove more than 58% of the microplastics, while the plants applying up to a secondary treatment with activated sludge achieved microplastic removal efectiveness between 90% and 96.9% [15].
A research was conducted under the title of Investigating the Method of Detecting and Separating Microplastics in Wastewater Treatment Plants Based on the Density Difference and Determining the Contribution of the Treatment Process in Removing Particles by Darvishi et al. In the mentioned research, sodium chloride salt was used to create density diference, which separated fewer polymers compared to sodium iodide salt. Also, in that research, the smallest particle that was extracted was 20 microns, but the smallest particle extracted in this research is 7 microns [16].
However, the source of transporting particles to seas is the most important problem [17]. Terefore, numerous studies have been oriented to investigate microplastics source of production and entrance to aquatic and marine environments. Most of these studies have concluded that the output waste of the wastewater treatment plants is one of the major sources of this environmental problem [18,19]. For example, as a result of various studies performed in USA, it was revealed that the waste stream from wastewater treatment plants has increased the concentration of microplastics in the Chicago River [20].
Because there are other organic and inorganic compounds in efuents along with microplastics and microfbers, their separation is of great importance [21][22][23].
A study was performed by Ziajahromi et al. in Australia entitled "Wastewater Treatment Plants as a Pathway for Microplastics: Development of a New Approach to Sample Wastewater-Based Microplastics." Te main purpose of this study was establishing a validation method for sampling microplastics existing in efuent waste and using a method for determining and measuring microplastics in waste streams. Te acid washing method using hydrogen peroxide and sodium iodide salt was utilized in this study, which is based on creating density gradient between particles to separate them. With the method and materials used in their study, the extraction of particles was done through an optimized procedure. Te minimum considered size for the particles was 25 μm [24].
Contrary to the previous methods performed on efuent samples, a diferent and optimized acid washing method and a diferent salt for creating density gradient were used in the present study. Tese changes are not limited to these cases and new materials, techniques, and parameters were also applied during our investigations. Also, the methods of sampling and preparation of wastewater samples were done in such a way that can be cited to be a reliable criterion to a large extent in terms of the statistical population. As a result, it was demonstrated that how diferences between performing methods can afect the efciency of particles detection.

Materials and Methods
According to the aforementioned content and the previous studies conducted in this feld on various samples, such as seawater, the present study is focused on human wastewater with its specifc characteristics, which will be discussed later in detail. First of all, the conditions of the sampling location related to the samples used in this study as the human wastewater were presented. Te studying wastewater treatment plant is located in Iran, which is important to be studied due to its vicinity with aquatic environments that can result in disposing contaminated waste streams to the agricultural lands. Tis plant has a capacity of 20,000 m 3 /day that supplies the drinking water to about 120,000 people. Tis plant is one of the most advanced and equipped centers in the country. Te present study not only performed the separation of microplastic particles but also determined that what percentage of particles can be removed by a typical treatment system in existing in efuent and what percentage needs a more advanced treatment system to be removed. Also, the devices and equipment used in this research include a stereomicroscope device (Nikon SMZ-1500), Magnetic Stirrer, oven, sieve, beaker, and pipette.

Sampling Methodology.
Sampling method is of great importance in any research. Sampling must be done in such a way that provides an acceptable and universal statistical population. Terefore, sampling in this study was conducted in such a way that microplastic particles can be estimated to a reasonable level. Sampling from the waste streams of the plant was performed on both the input and output streams. Te sampling was performed in three seasons of spring, summer, and autumn, 10 consecutive days in each season, and 100 L in each day, which resulted in 1 m 3 for each season. Te reason for sampling in diferent seasons was to investigate the relationship between seasons and production of microplastic particles. Te sampling was also performed on the input efuent and output waste to evaluate the performance of the plant in removing these particles. Finally, the samples were passed through sieves to separate other components existing in the waste. Ten, the materials and particles were washed by distilled water and sent to the laboratory for the next stages.

Microplastic Particles Identifcation.
Due to the presence of microparticles other than microplastics in municipal sewage, their identifcation and separation are challenging and are of great importance. Te presence of other particles along with microplastics makes it difcult to observe and detect them and it cannot be claimed with complete certainty that the observed or removed particles were microplastics or not. Terefore, prior to perform any experiment to separate and remove microplastic particles, these particles must be segregated to determine the amount of them in the input efuent and output waste; this can also get the removal percentage. Based on that, determining any method for the waste treatment and separation of microplastic particles from waste stream needs a precise detection. Waste was frst passed through the prepared sieves to separate the waste ingredients and then thoroughly washed for the further laboratory processes. It should be mentioned that because the consumption pattern varies by season, this stage was performed on the samples in three seasons of spring, summer, and autumn. 5 sieves with grades of 100, 200, 270, 400, and 500 with mesh opening diameters of 150, 75, 53, 38, and 25 µm, respectively, were used in this study to classify existing particles in term of size, by passing the waste stream through these specifed sieves.
After passing the waste and efuent, the samples on the sieves mesh were washed with 400 ml distilled water (the amount of washing water is optional) and then oven dried at 90°C for 24 h to reduce its amount to about 50 ml. From this point on, the acid washing process was performed. First, 25 mg of 0.05 M divalent iron was poured into a beaker containing the distilled water resulted from washing the input efuent and output waste. Afterwards, the beakers were placed on magnetic stirrers to obtain a complete mixture. Ten, the 30% hydrogen peroxide solution was added by the amount of 50 and 30 ml to the beakers containing the input efuent (due to the high concentration of organic compounds) and output waste, respectively, which is illustrated in Figure 1.
At this point, due to the presence of acid, interactions occur that lead to the production of foam and its possible overfow from the beaker. However, this problem can be  [23]. After the solution has calmed down, the beaker is placed on the magnetic stirrer again. Te temperature of the magnetic stirrer should be fxed at 70°C and the mixing process should be done for 45 min.
All the processes performed up till now were aimed to remove organic compounds in order to inhibit the formation of precipitates. As the separation mechanism is based on density gradient, 7-8 gr NaI was added to the beaker solution per each 20 ml of samples to increase the density. Ten, the solution was allowed to further mix at 70 o C to form a complete mixture with NaI.
After the NaI particles were completely dissolved in the mixture, the magnetic stirrer was turned of and the beaker was remained immobilized for 24 h to complete the separation process by particles precipitation. Ten, as can be seen in Figure 2, the heavier particles were precipitated after 24 h and microplastic and microfber particles were remained suspended on the surface.
At this stage according to Figure 3, the liquid in the beaker was collected from the surface and intermediate depth of the solution using laboratory pipette and was washed with water by passing through a sieve. Ten, the size of microplastic particles was determined using stereo microscope.
After the collection and extraction, the distilled water (containing microplastic particles) was evaporated in the ambient temperature and the dried sample was prepared for observation, detection, and counting of particles.

Detection of Suspicious Particles.
Given the possibility of existence of any particles in the efuent, detection, and separation of microplastics and microfbers from other materials are of great importance to avoid any mistakes in their detection. Based on that, to detect suspicious microplastics, the Rose Bengal coloring method was applied [24]. In this method, natural particles such as fbers, which are similar in appearance to plastic fbers, are colored in a way that can be observable with naked eyes and separable. To do so, the strainer containing suspicious microplastics was colored by 5 ml of the 0.2 mg/L Rose Bengal solution for 5 min at the ambient temperature. After that, the strainer was further washed with distilled water under the vacuum condition to remove the residual color. Ten, the strainer was oven dried at 60°C for 15 min to get prepared for further investigations by stereo microscope and spectroscopy.

Observation and Measurement
Steps. Te method of measuring microplastics and determining its types consists of two parts. Stereo microscope is a kind of optical microscopes that usually works based on the refected light from an object and applied to observe samples in more magnifcation. Tis device shines light on the object from two separate paths and magnifes objects up to about 160 times through optical lenses. Also, the separated particles were later detected using the FTIR analysis to obtain the types and spectra of microplastics present in the sample. Te results of this spectroscopy are given in the next chapter.

Results and Discussion
After performing the separation and extraction of particles, the samples were prepared for the observation and counting. Ten, the polymeric structure of microplastic particles was determined using the spectroscopy method. Based on that, the primary version of efuent was observed using stereo microscope at the beginning and before performing any extraction, to demonstrate the diference before and after the extraction steps. Tis approach has clearly revealed that the separation and performing a suitable method can substantially reduce the error of detecting particles and destroying materials that can be considered as microplastic by mistake.

Results of the Observing Samples and Spectroscopy of
Particles. According to the previously mentioned points and observations, the results are illustrated in this subsection. In the fgures depicted in Figure 4, which is related to the primary version of the efuent, it is evident that there are various particles and materials that make it challenging to detect microplastic particles from others.
Afterwards, the acid washed samples were observed by the stereo microscope, which are depicted in Figure 5. Accordingly, microplastic particles were successfully segregated from sludge carcasses by acid washing and microplastic particles are clearly visible.
Terefore, by comparing Figure 4 with Figure 5, it can be concluded that the error possibility in observing and detecting microplastics mitigates by determining a suitable separation/extraction method. Another noteworthy point is that the primary and secondary treatments in the wastewater treatment plant cannot completely separate particles from the efuent. In addition, it was proved by the FTIR analysis that the extracted particles are microplastics and based on that, types of polymers were determined and illustrated in Table 2. Figure 6, by dividing the plate surface into four identical parts, the number of microplastics was counted using the stereo microscope. Te device used in this research is Nikon (SMZ-1500) type.

Counting Particles. As shown in
Particles counting was performed by considering their appearance and the results for the input efuent and output waste of the treated stream for all three seasons are presented in Tables 3-5.
According to the results obtained from the input efuent and output waste, the efciency of the treatment process in removing microplastic particles from the efuent can be evaluated. On average, 95% of these particles were separated in the treatment process and disposed of with sludge. Based on the results depicted in Tables 3-5 and Figure 7, the abundance of particles in diferent seasons is clearly visible and illustrates the quantity of particles.
Accordingly, in terms of the amount of microplastic particles, summer has the highest abundance compared to spring and autumn. Also, in terms of geometric shape, the number of rounded particles was more than sharp-corner particles and microfbers.
Given that microplastics are one of the key components used in the production of detergents, it should be noted that the release of microplastics in the summer is infuenced by the rise in air temperature, which causes an increase in the use of bathrooms, detergents, and washing machines. Based on this explanation, the emission of these particles is less in the spring than in the summer, and it decreases drastically in the cold seasons of the year (autumn and winter). Cosmetics are another product where microplastic particles are heavily present. Changes in the decrease and increase of microplastic particle emissions are infuenced by the relationship between the use of cosmetics and the seasons of the year. For instance, the use of cosmetics rises and their residues are released into the environment in substantial quantities during the summer due to holidays, a surge in weddings, and other events. Also, the usage of nylon bags and disposable plastic containers, among other things, has a meaningful impact on the discharge of microplastic particles throughout the year.
Also, particle measurements were performed for each of the sieves. Based on this, the surface of each sieve was washed   Figure 8. Based on that, the abundance percentage of microplastics in the inlet and outlet of the treatment plant was calculated separately for the shape of the particles, which is illustrated in Table 6. In addition, the removal percentage of microparticles and microfbers through the wastewater treatment process was demonstrated.
It can be deduced from the results presented in Table 6 and Figure 9 that during the usual treatment process in the treatment plant, the removal percentage of microparticles is higher than microfbers.
According to the obtained results and determined number and form of particles, per capita microplastic particles production can be estimated by considering the population and the volume of produced efuents per day in the city or country that the wastewater treatment plant is located in. Te covering population is about 120,000 and the volume of produced efuent is 20,000 m 3 /day. It should be noted that the per capita determination was based on both incoming efuent and outgoing waste stream. Also, calculations were conducted based on the summer data as the time of maximum particle production. Initially, the per capita production was determined based on the sample of         Advances in Materials Science and Engineering 7 inlet efuent of the treatment plant and the results are illustrated in Table 7.
After that, the daily per capita of each person was calculated based on the particles obtained from the output waste as shown in Table 8, which indicates that how many microplastic particles pass through the treatment plant output waste and enter the aquatic and terrestrial accepting environments.

Evaluating the Separation of Particles Smaller than 25 μm
Using the Existing Method. As mentioned in the sampling section, the largest sieve used for washing the waste to obtain samples was the grade of 500 with the mesh opening diameter of 25 μm. In this step of the research, for validating and evaluating the efciency of the proposed procedure, the waste stream passed through the aforementioned sieve was washed by acid to determine the size range of particles that can be extracted. As discussed in the introduction section, previous methods have been able to extract particle sizes with a minimum range of 25 μm. According to the method proposed in this study, by use of the divalent iron, diferent salt to create density gradient, altering the sampling method, optimizing the use of hydrogen peroxide, and the performed trial and error, it is possible to detect particles smaller than 25 μm in size. According to the obtained results using this optimized method, smaller particles were detected in the output waste stream and the removal efciency was also enhanced, which can be seen in Figure 10. Also, the number of extracted microplastics smaller than 25 μm in size was calculated to be 129 particles/m 3 , of which the majority portion was microfbers. Overall, the size of these particles was in the range of 7-22 μm.

Discussion.
It should be noted that the diference in salts had a signifcant efect on the separation of particles and formed precipitates. According to the conducted experiments, using sodium iodide salt has resulted in more acceptable results in making density gradient compared with other salts. Notably, the type of salt should be selected based on the density of polymers existing in the wastewater, which is determined using the spectroscopy tests. Based on that, sodium iodide was selected for this study according to the aforementioned considerations. Up till now, various methods have been used to extract particles from seawater and municipal sewage, each of which has included diferent parameters and instructions. In the cases reviewed in the introduction section, the acid washing process was performed on the dried sample of efuent or seawater, and in some other cases, the chemical extraction process was performed on the liquid sample upto 100 ml. With the trial and error conducted in this study, it was concluded that the acid washing method on dry samples is suitable only if microplastic particles are existing in large sizes. It was also found that in liquid samples, the lower the amount of liquid volume, the better the acid washing operation and consequently better the detection of microplastics from other organic matter, because the precipitates will be formed better. Besides this issue, there are other things that make the result of this research diferent. Te type of materials used, changes in the mixing time and temperature, changes in the amount of material utilized in the incoming and outgoing efuent, and the sampling technique all contribute to this variance. In the frst section, sampling plays a crucial role as the frst step in examining the identifcation of microplastics, and it should be carried out as accurately as possible (in terms of sampling time, number of sampling times, and comprehensiveness, as well as sample preparation for transfer to the laboratory). It implies that it ought to be statistically regarded as an appropriate portion of the entire wastewater produced by the treatment plant.
In the second section of this study, diferent results from other studies have been achieved by altering the test materials and parameters. As you are aware, diferent salts are utilized to produce a diference in density, and the diference of density between these salts and micropolymer particles greatly infuences the ability to recognize various forms of microplastics. For example, because of their higher density than NaCl salt, polymers like polyethylene terephthalate or polyvinyl chloride, if present, cannot be separated using  NaCl salt. In this study, NaCl salt was used to identify polyethylene terephthalate based on its high density, which can be useful in future research on treatment plants containing such polymer in their streams. In the third section, the use of 0.05 M Fe (II) aqueous solution in wastewater samples, which was done in this research to catalyze the reaction before sedimentation, is a signifcant method that has not been used in previous studies. Tis method was able to catalyze the reaction before sedimentation, which ultimately signifcantly improved the results. Finally, it can be mentioned that all the changes employed on the method used in this study, have led to signifcant outcome, which is the identifcation and separation of smaller particles compared to the previous research studies. Te smallest particle identifed and separated in previous study has been 25 μm. Tis research has successfully separated smaller particles with a size of less than 25 μm (in the range of 7 μm) with the changes made to the test method. Te amount of these particles is about 130 particles per cubic meter, which is signifcant due to the large volume of the output from the wastewater treatment plants.

Conclusions
Given the importance of the subject and the threats that microplastics pose to aquatic and terrestrial environments, using appropriate methods to detect and extract particles is essential. In the current study, an attempt has been made to provide an optimal and appropriate method compared with other methods, through which microplastic particles can be well detected and extracted. Up till now, various methods have been used to extract particles from seawater and municipal sewage, each of which has included diferent parameters and instructions. Also, by determining an appropriate amount of hydrogen peroxide injection in the input efuent, which contains a lot of organic matter, the sedimentation efciency was substantially increased and particle extraction was achieved to a desirable level, which shows better performance compared with the previous research. Considering the abovementioned cases, it was found that the results of the optimized method in this research are extremely suitable and it has been able to separate the microplastic particles in the wastewater from its organic materials. Also, optimizing the consumption, time, and temperature parameters and innovation in using the 0.05 M iron solution have resulted in higher efciency in detecting smaller particles upto 10 μm). It should be mentioned that the conventional process of the treatment plant was found to dispose 94-96% of microplastic particles with sludge. Te noteworthy point is that a fraction of particles, which their number was determined, was not separated by the conventional treatment process and entered the aquatic and terrestrial environments. To separate these particles, advanced approaches should be evaluated. Based on the seasonal sampling performed in this study, the more abundance of microplastics was related to summer. It was also found those diferences in salts, the use of divalent iron, and changing the volume of efuent in order to perform acid washing, have a signifcant efect on oxidation, sediment formation, and fnally particle detection.

Data Availability
Te data used and obtained are fully included in the article. Tese data include the following: number of microplastic particles in municipal wastewater, spectrophotometry of microplastic particles in municipal wastewater, per capita microplastic particle production, number of microplastic particles leaving the treatment plant per day, emission rate of microplastic particles in diferent seasons, and other cases. Te data in this article does not require a license and can be freely accessed. Also, previous data are not used in this Table 7: Per capita production of microplastic particles according to the input efuent.

Types
Amount of production (in m 3 ) Amount of production (in day) Per capita production (for person per day)  Microparticles  27180  543,600,000  4530  Microfber  4325  86,500,000  720.83  Total  31505 630,100,000 5250.83  Advances in Materials Science and Engineering 9 article. Te data are completely new and have been obtained by sampling and analysis. Finally, the use of the data in this article is open to the public and all the necessary information is included in this article.

Conflicts of Interest
Te authors declare that they have no conficts of interest.