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Environ Eng Res > Volume 29(1); 2024 > Article
Nafea, Al-Maliki, and Al-Tameemi: Sources, fate, effects, and analysis of microplastic in wastewater treatment plants: A review


Microplastics (MPs) are tiny pieces of plastic smaller than 5mm that have raised concerns to aquatic organisms and human health due to their ubiquitous presence. Wastewater Treatment Plants (WWTPs) are a significant point source for aquatic MPs pollution, as millions of MPs with different characteristics reach WWTPs through the Sewage. Even though, WWTPs are not specifically designed to remove MPs, researchers report that more than 90% of MPs can be removed in WWTPs. However, given the huge volumes of effluent discharge into natural aquatic bodies, even small concentrations of MPs in the effluent would be significant. A typical WWTP consists of three key stages: primary, secondary and tertiary. Removal of MPs in these stages is reviewed. In addition, plastics can adsorb toxic chemical and biological pollutants on its surface and can lead to spread of toxic pollutants in the environment. Therefore, in this review we aim to provide comprehensive knowledge about the sources of MPs in wastewater, their fate and removal in WWTPs, their interactions with different chemical and biological pollutants, and their effects on environment. In addition, it also highlights the different methodologies used for sampling, sample preparation and physical and chemical identification of MPs found in the WWTPs.

1. Introduction

Throughout the history, water has always been a very important factor for human civilization and wellbeing. However, in the recent times water pollution has become one of the major problems worldwide [1]. Microplastics (MPs) are one of the most recently identified water pollutants. They are ubiquitous in nature and their presence is mainly due to human activity. MPs are tiny plastic particles with size less than 5 mm in diameter [2, 3]. MPs have been detected in almost all the ecosystems including marine [4], freshwater [5, 6], lakes [7], atmosphere [8, 9] and in soil [10, 11]. They are formed from a wide range of sources such as polymer manufacturing, synthetic fibres from clothing and textile industry, processing industries, cosmetics, and personal care products and due to breakdown of larger plastic products [12].
MPs could be of primary or secondary origin. The MPs manufactured intentionally to be used in makeup and personal care products are primary MPs. The MPs formed naturally due to the breakdown of larger plastics by exposure to heat and ultraviolet rays from the sun or abrasion by wind and rain are secondary MPs [13, 14]. It has been estimated that in the European Union about 4360 tons of MPs are used annually in personal care and cosmetic products [15, 16]. However, secondary MPs are a much bigger source compared to primary MPs. The most common types of MPs found in aquatic environment are polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET), Polymethyl methacrylate (PMMA) and nylon (NY) [16].
MPs pollution is the cause of serious problems that has gained very high contemplation in the past decade since it was first reported by Thompson et al. [17]. MPs in the environment have increased dramatically due to an increase in utility, production, and mismanagement of plastics. MPs can be found commonly in water bodies like rivers [18], lakes [7], estuaries, coastlines [19] and marine ecosystem [15]. Effects of MPs on aquatic organisms have been reported in many studies, it includes genotoxicity, growth delay, oxidative stress, and sometimes death [20, 21]. Studies investigating effects of MPs on fish show that MPs can enter the brain, circulatory system and other organs making them more sluggish [21]. This infers that the MPs can enter the food chain via fish and other seafood and may eventually affect human health. Additionally, several studies have demonstrated that MPs can built-up and transfer organic and inorganic toxic chemicals as well as other pathogens such as bacteria and viruses. Chemical pollutants like polycyclic aromatic hydrocarbons (PAHs), organochlorine pesticides, polybrominated diphenyl ethers and metal ions present in wastewater can build up on the MPs due to its large surface area and hydrophobicity [22, 23]. Any plastic smaller than 150 microns can pass into the animal digestive system and release these harmful chemicals inside the animal body [24].
Wastewater treatment plants (WWTPs) are identified as major source of MPs in the aquatic environment [25]. MPs present in numerous cosmetic and personal care products that are generally used on daily basis are rinsed directly from the household drains end up in the WWTP. In addition, synthetic fibres like nylon and polyesters from clothing may shed several of its fibres into wastewater during washing [26]. Moreover, any other plastic products such as bags, bottles, foams etc., flowing down the sewer can breakdown to MPs and reach WWTPs. As the treatment technologies currently employed at the WWTPs are not designed to remove MPs in wastewater, WWTPs can be a point source of MPs emission into the natural aquatic environment [27]. In addition, the MPs can interact with chemical and biological toxicities present in the wastewater that can lead to spread of these impurities in the aquatic environment. The large surface area combined with hydrophobic nature of the MPs can attract many organic and inorganic chemicals from the wastewater to be adsorbed on its surface. The adsorption of organic chemicals on the MPs surface can further lead to the formation of biofilm containing various microorganisms such as bacteria, protozoa, viruses etc. that can lead to spread of toxicities in the natural aquatic environment.
The sampling and analytical methodologies for MPs vary with studies due to lack of a universally accepted standardized sampling and analysis procedure. Currently different types of sampling methods such as surface sampling, grab sampling etc., are used. Quantitative analysis is generally carried out by observing samples under a microscope and manually counting the MPs particles. The particles identified as MPs are then subjected to chemical characterization to confirm the polymer type. It is done by analytical methods such as spectroscopy, thermal degradation coupled with mass spectrometry or chromatography with spectrometry etc. However, the spectroscopic methods including the Fourier transform infrared spectroscopy (FTIR) and Raman are most commonly used.
This paper focuses on reviewing the main sources of MPs, their occurrence, and fate in wastewater treatment plants. In addition, the interactions of MPs with different chemical and biological toxicities present in the wastewater and their effect on the environment is reviewed. Moreover, it also reviews the different sampling, pre-treatment and analytical methodologies used for MPs analysis in wastewater. This review is aimed to fill the literature gaps and to provide updated paper for treating MPs in WWTPs. The review is concluded by highlighting the future prospective of research.

2. Sources of Microplastics in Wastewater System

Plastic pollution has been an issue of global concern for some time now. However, it is only since the last couple of decades that MPs are classified as a pollutant on their own right [28]. MPs are a collection of tiny plastic particles of different shapes, polymers, and colours. MPs can have different origin based on the polymer type and shape. MPs are classified as primary or secondary based on their origin. Primary MPs are intentionally manufactured for their commercial applications. One of the most common sources of primary MPs in wastewater is microbeads that are manufactured to be used in the cosmetics and personal care products for their exfoliating property and to control the viscosity of the product.
Microbeads can be made of different polymers such as PE, PP, polytetrafluoroethylene (PTFE), polyurethane (PU), nylon-6 (NY-6), nylon-12 (NY-12) and polylactic acid (PLA) [27, 29]. However, PE and PP microbeads are the most commonly used and they together form up to 90% of the total microbeads associated with cosmetic and personal care products [13]. The United Kingdom is reported to have at least 80 body and facial scrub products containing PE microbeads that are used by millions of people [28]. It is estimated that every single use of an exfoliating personal care product or toothpaste can release between 4000 to 95500 microbeads into the water [30, 31].
Secondary MPs are those formed due to the breakdown of larger plastics such as plastic bags, bottles, clothes, foams, packaging materials, tyres etc. [32, 33]. The larger plastics can undergo degradation by biotic and abiotic mechanisms or a combination of both. In biotic degradation, the plastics break up into smaller particles due to the actions of different types of organisms such as bacteria, fungi, and some insects that consume plastics by breaking them into smaller pieces. Whereas, in abiotic degradation, the plastics are broken down into smaller particles due to various actions such as thermal, radiation and photo chemical oxidation from the sun, mechanical abrasions due to wind, rain or human activity [34].
Furthermore, microfibers that are a type of MPs are considered to be of both primary and secondary in origin. For instance, synthetic fibres such as polyester (PE) and nylon (NY) used in making clothes, carpets and other textile products are manufactured in the microscale, it can end up in water during the cutting, washing or weaving process, these are considered primary MPs [19, 35]. Whereas synthetic microfibers that are released into the water during laundering of synthetic textiles in the washing machine can be considered secondary MPs. It is estimated that at least 100 microfibers are present per litre of laundry wastewater that eventually reaches WWTPs [9]. Another study estimates that a laundry load of about 5 to 6 kg releases about 60,000 to 70,000 synthetic fibres. However, it should be noted that number of microfibers released depends on many factors such as the type of cloths, condition of washing machine, type of detergent and softener used etc. [36, 37]. Therefore, laundries and synthetic fibre manufacturing industries have huge contributions in the presence of MPs in WWTPs [38].
Some recent studies have identified atmospheric fallout of airborne MPs as a potential source of MPs in the aquatic environments such as rivers, lakes etc. Therefore, MPs particles present in the air can also be identified as a potential source of MPs in wastewater, as the WWTPs are open systems and atmospheric fallout of the MPs can occur at any point in the WWTP [39]. However, atmospheric fallout has not yet been studied as a substantial source of MPs in the WWTPs and further research must be carried out to study its influence on MPs concentration in WWTPs.
Moreover, MPs entering into the aquatic environment can broadly be determined to have originated from a point source or diffuse source (non-point). Diffuse or non-point sources consist of MPs from agriculture runoff, MPs escaped from plastic manufacturing industry and other industrial areas, microfibers from textile industries, and MPs deposited from the atmospheric fallout [39, 40]. Point sources of MPs emission include WWTPs and Combined Effluent Treatment Plants (CETP) that release a very large amount of MPs into the receiving waters (CETPs are WWTPs that treat municipal and industrial wastewater combined). It has been estimated that annually, about 1.15 to 2.41 million tons of plastics is dumped into the oceans by the rivers [41], and the largest portion of the MPs in the river comes from the effluents of WWTPs. Therefore, WWTPs are considered an important point source of MPs [25].
Many factors affect the MPs quantity in aquatic environment. Such as, human population density in the proximity of the water source, urban centres near the water body, type of treatment technology employed in the WWTPs, and amount of effluent released into water body, industrial activities and tourism [7]. Additionally, physical forces such as wind, UV radiations from the sun and extent of rainfall also affect the quantity as these factors accelerate the degradation of large plastics into MPs [42]. Studies have characterised the correlation between the human activities through waste loads and input of pollutants into environment and it is evident that, highly populated urban centres and communities will have higher MPs in their wastewater and therefore can be considered as major contributors for release of MPs into environment [43]. In addition, in most of the low and medium income countries about 90% of all the wastewater is discharged into environment without any treatment, which further increases the level of MPs release into environment [44].

3. Fate of Microplastics in WWTP

Currently none of the WWTPs are designed specifically to remove MPs but depending on the type of unit operations employed in WWTP, it may remove certain amount of MPs. WWTPs with primary and secondary treatment system may remove up to 99% of the MPs [45, 46]. A study at the University of Leeds evaluated MPs concentrations up and downstream of six WWTPs, it reported that all the WWTPs were responsible for an increase in the MPs concentration in the rivers, on average up to three folds higher in downstream water [42]. Several studies have tried to determine the efficiency of WWTPs in removing the MPs particles from wastewater. Estimated microplastics flow across WWTPs with primary, secondary, and tertiary treatments obtained from different studies is summarized in Table 1.
Murphy et al. [47] studied MPs abundance in different stages of wastewater treatment and reported that, bulk of the MPs were removed during the primary and secondary settling stages. However, 0.25 (±0.04) MP/L effluent were detected in the later stages of treatment including final effluent discharged. By extrapolation, it can be estimated that about 65 million MPs could be discharged into the receiving water body per day. Another study in Germany evaluated samples collected from 12 WWTPs and the results suggested that about 9×107 to 4×109 MP particles are discharged annually [48]. A study in Denmark estimated that, ten of the largest WWTPs in the country discharge about 3 tons of MPs annually into the natural water systems in the size range 10 to 500 μm [22]. The treatment technologies employed in the WWTP is one of the major factors in determining the MPs concentration in the effluent. In general, WWTPs employing any type of tertiary treatment had very high MP removal with an average of only 0.21–8 particle/L in the effluent. However, some studies have observed that MPs concentration do not decrease in tertiary treatment stage [49]. It is believed that most of MP removal occurs during the primary treatment of wastewater, with about 80–90% of the MPs elimination. In the primary treatment, the heavier MPs like Polyvinyl chloride (PVC), polyethylene terephthalate (PET), and polystyrene (PS) can be removed by sedimentation and the lighter MPs that float on the surface can be removed by skimming it off along with the fats, oils, and grease layer. However, the studies by Dris et al. [9], Murphy et al. [47] and Ziajahromi et al. [50], have reported only 50, 45 and 70% of MPs were removed respectively. Furthermore, in secondary treatment, it is possible that, the MPs can be stuck with the unstable flocks of microorganisms and when the bacterial flocks are removed, MPs are removed. In addition, some studies suggest that use of flocculants such as Ferric sulphate in the secondary sedimentation can further help in the MPs removal by aggregating the particulate matter into flocs, which can also include the attached MPs [47]. It is also possible that some of the bacteria and other microorganisms in the flock can biodegrade the MPs during the secondary treatment. However, the interactions between the MPs and the microorganism flocs, and their role in the removal of MPs from wastewater is not yet determined. Therefore, effects of microorganisms used in secondary treatment on the MPs degradation must be further investigated. Most of the studies report that about MPs removal during secondary settling to be about 90 to 99.9%. However, some studies have also reported removal efficiencies less than 90% [9, 21, 50]. Furthermore, when an Anaerobic Membrane Bio Reactor (AnMBR) was used as a secondary treatment, the MPs removal efficiency was much higher than conventional treatment methods with primary and secondary stages [51]. However, some studies have reported contradictory observations. For example, Leslie et al. [52] observed that MPs removal efficiency of a MBR was similar to that of a conventional WWTP employing secondary treatment and Lee and Kim [53] reported the MPs removal efficiency of MBR to be much lower at 44.7%. These variations in the MPs removal efficiencies are not yet fully understood. There could be many reasons for these variations, MPs removal efficiency can depend on many factors, such as, treatment technologies used, the flow system in the WWTP, retention time, age, and actual efficiency of WWTPs, the initial load of MPs, and also the atmospheric deposition that may occur over the area. Therefore, more studies should be carried out to investigate the MPs removal efficiencies of different treatment technologies such as Activated sludge process (ASP) and Anaerobic Membrane Bio reactor (AnMBR).
Furthermore, some studies have also reported inefficient MPs removal in other tertiary treatment technologies. For instance, tertiary treatment technologies such as gravity sand filtration (GSF) and biological aerated filter (BAF) had no impact on further MPs removal from the effluent [31, 48, 54]. In South Korea, Hidayaturrahman and Lee [55] investigated the MPs removal efficiency of three different types of tertiary treatment methods with a common secondary treatment and reported overall MPs removal efficiency of 99.2%, 99.0% and 98.9% for ozonation, membrane disk filter and rapid sand filter respectively. More recently, Pittura et al. [56] reported MPs removal efficiency of 99.5% for secondary Up-flow Anaerobic Sludge Blanket (UASB) and tertiary Anaerobic Membrane Bioreactor (AnMBR) and an efficiency of 85.7% for conventional ASP with tertiary disinfection [56]. Table 1 summarizes the MPs concentrations, their removal efficiency and the treatment technologies employed at different locations.
In addition to this, the Hydraulic Retention Time (HRT) is also an important factor for the removal of MPs in WWTPs. When the HRT is low, the wastewater takes less time to pass through the tank and therefore, MPs have less time to settle or to be removed from the top also during lower HRT water flows faster which can also lead to resuspension of the MPs. However, when the HRT is high water flows slower and MPs have higher time to settle down [57].
Another factor that must be considered is that most of these results are obtained by analysing a finite volume of wastewater sample and extrapolating the results obtained to the whole volume of wastewater flowing through WWTPs and the MPs are not uniformly distributed in the WWTPs, this can also lead to inconsistent results. Moreover, the sampling, and analytical methods employed for identification and quantification of MPs and limit of detections of each method employed in different studies play a vital role in determining the final MPs abundance.
Most of the studies have reported MPs removal efficiencies are more than 90%, but, despite the high reduction ability, WWTP may act as large point source of microplastics into the aquatic environment because volumes of effluents released into the aquatic bodies are very large. Even with a MP removal efficiency of 90%, several millions to billions of MP particles can be discharged from WWTPs into natural aquatic environment [58]. Fig. 1 represents a schematic diagram of a typical WWTP with primary, secondary, and tertiary treatment. It also indicates MPs removal in WWTPs with 100% entering in the WWTP influent followed percentage removal in each treatment stage of WWTP.

4. Microplastics Interaction with Pollutants in Wastewater

Wastewater contains large quantities of organic, inorganic, and biological pollutants. Ashton, Holmes, and Turner [67] studied the interactions between MPs and heavy metals for the first time. Consequently, several studies have shown that MPs can interact with organic and inorganic pollutants present in wastewater. Moreover, they can also act as carriers of wastewater contaminants such as, pesticides, pharmaceutical pollutants, hormones, pathogenic microorganisms, and metal ions etc. that can result in the modification of the environmental fate of these pollutants. MPs are known to interact with metal ions including Al, Cu, Cd, Mn, Fe, Zn, Pb, Ni and Ag among others [11, 58, 67, 68]. Interactions between MPs and metal ions occurs because of adsorption of organic matter from the wastewater on the MPs surface. When the organic matter adsorbs on the surface of MP, it acquires a negative charge from the functional group of the organic matter, and these negatively charged MPs then easily react with positively charged metal ions [69]. However, as most of the water treatment plants use Al or Fe for coagulation-flocculation process, interactions between MPs with these metal ions could be beneficial for MPs removal. However, MPs removal using chemical coagulants such as Fe or Al has not previously been studied in the municipal wastewater matrix and needs further investigation [70].
In addition, MPs can also accumulate the contaminants that are present at low concentrations in the wastewater [14, 71]. For instance, the organic and inorganic trace pollutants such as hormones, pharmaceutical chemicals etc. present in wastewater as trace contaminants can be accumulated on the MPs surface, thereby increasing their concentration in wastewater. Sorption of these trace organic pollutants to MPs can occur by either adsorption and/or absorption mechanisms. As these sorption mechanisms are dependent on the physical and chemical characteristics of both MPs and the organic pollutants, different types of organic pollutants have different sorption capacities on different MPs [27].
Adsorption is the main sorption mechanism of organic pollutants on MPs. It occurs either through the interactions between organic pollutant and hydrophobic surface of MPs [46] or by specific interaction (hydrogen bond and electrostatic bond) [39]. Just like cellulosic nanofibers, the active sites of adsorption in MP include carboxyl, hydroxyl, amino, ester etc. groups which are correlated to the adsorption capacity [72]. The adsorption capacity of a MP is also directly proportional to the surface area of MP that is available for adsorption [50, 68]. Therefore, as the size of MP decreases, the surface area increases and thus, higher is the sorption capacity [27].
The small size and large surface area of MPs can alter the behaviour of organic contaminants in wastewater by dynamic sorption and desorption equilibrium mechanism [73]. Poly aromatic hydrocarbons (PAHs) are the most widely detected organic pollutants adsorbed to MPs. Concentration of PAHs can reach as high as 119μg/g of MP, 44.8μg/g and 5615 μg/g MP in seawater, beaches, and sediments respectively [74, 75]. When these contaminated MPs are exposed to clean water, the contaminants can desorb within a short span of 24 hours and potentially increase the toxicity of the surrounding seawater, endangering the surrounding ecosystem [76].
Furthermore, MPs are one of the effective substrates for the colonization of the microorganisms on their surface. Their tiny size, the rough and hydrophobic surface and long half-life, makes it an ideal candidate for the microorganisms to attach and grow [77]. MPs in wastewater attract various microbial organisms including bacteria, viruses, fungi, and protozoa. These microorganisms together with algae and diatoms form a layer on the surface of MPs called biofilms [78]. The MPs in wastewater first adsorb a layer the organic and inorganic pollutants available [79]. The contact process of MPs with microorganisms begins with the electrostatic attraction and repulsion between the cell wall of microorganisms and the coated surface of MP. Extracellular Polymeric Substances (EPS) are secreted from the microorganisms, that starts forming a stable biofilm [80]. Contents of the biofilm varies with the aqueous environments in which it is formed. The flow state, nutrient availability of the aqueous environment, pH and seasonal changes are all important factors that dictate the biofilm formation on the MPs. During the low flow rate (High HRT), the rate of biofilm colonization is faster, resulting in denser biofilms. During high flow (Low HRT) the adhesion of microorganisms on the MPs surface is lower forming thinner biofilms. The availability of nutrients such as carbon, nitrogen and phosphorous in the wastewater is another important factor for growth biofilms on the MPs. Biofilms are much thinner and uniform when there is unstable carbon availability. It is also observed that, during the summer when the temperatures higher, days are longer and dissolved oxygen is lower the biofilms grow faster on the MPs surface due to higher enzyme activity and metabolism speed [81].
In the absorption mechanism, the organic pollutants diffuse inside the MP polymer and therefore, it mainly depends upon the molecular structure of the MP polymer [68]. Polymers such as PE and PP that exhibit a rubbery structure show a higher sorption capacity for trace organic pollutants like polycyclic aromatic compounds (PACs), than the polymers that have glassy structure such as PVC. [82, 83].
Very little research is done for understanding the sorption behaviour and chemical fate of MPs in WWTP. Therefore, further research is needed in understanding the effects of MPs in altering the concentrations of trace organic pollutants such as pesticides, pharmaceutical, and hormones in WWTPs effluents.

5. Effect of Microplastics on Environment

The effects of MPs on the environment can be due to direct exposure of MPs themselves or due to the release of the contaminants that are associated with MPs [84]. There is a significant need to remove MPs from wastewater before discharge as they pose a threat to the health of aquatic animals. Aquatic organisms consume MPs confusing it to be food, which can then enter into their vital organs and circulatory system, causing several abnormalities like genotoxicity and retarded growth [20]. MPs can also cause physical damage to an organism due to its accumulation in the digestive tract [85]. The risk of damage caused due to direct exposure of MPs is based on the type of organism [86], type of MP polymer, duration of exposure [19], concentration of exposure and shape and size of the MP [85]. Another risk for aquatic organisms due to MPs exposure can be due to presence of microorganisms like bacteria, viruses, and other pathogens on the surface of MPs, which can lead to health effects of aquatic organisms [45].
Most of the studies on Eco toxicological effects of MPs are performed mainly on crustaceans followed by fish and mollusks respectively. Crustaceans are generally the primary consumers, and they often consume MPs confusing it for food [87]. Fishes are often top or intermediate predators and therefore ingest MPs directly or indirectly [54]. Effects of MPs on fish include neurotoxicity [21], reduction in the predatory performance and efficiency [54], and genotoxicity. The effects of MPs observed in mollusks and crustaceans include retarded growth and fertility [88], increased energy consumption, neurotoxicity, genotoxicity and ultimately death [88, 89].
Apart from effects of direct MPs ingestion, several studies have also identified the combined effects of MPs with other environmental pollutants such as, hormones, pharmaceutical pollutants, metal ions, PAHs etc. [90]. Most of these studies are performed on the fish and the results of these studies documented several effects such as, genotoxicity, reproductive and behavioural health effects in the fish D. rerio. Studies have demonstrated that presence of PE MPs can lead to significant increase in the Cr (IV) toxicity of marine fish compared to Cr alone [90].
MPs have a high adsorption capability for many pollutants. The biofilm formation on its surface further increases the ability of MPs to adsorb persistent organic pollutants, heavy metals, pharmaceutical, hormonal, and antibiotic pollutants. In addition, the biofilms may also contain microorganisms and algae that are harmful for environment and humans, and they also act as carriers in spreading these harmful microorganisms. Accumulation of MPs and their associated contaminants in the WWTP effluents can lead to a potential risk of contaminant accumulation for higher-level organisms and contaminants transfer through food chain that could ultimately cause health effects in terrestrial animals including humans.

6. Detection and Analysis of Microplastics in Wastewater Samples

6.1. Sampling and Pre-Treatment of Wastewater Samples

There is a lack of a standardized and generally accepted method for sampling MPs from WWTPs, which makes it difficult for the results obtained from these inconsistent sampling techniques to be accurately compared with each other. Microplastics collection can be performed by various methods including direct sampling in a container [49, 91], auto sampler collection [11, 83], surface filtration [45], and separate pumping and filtration [48, 50, 58]. The volume of water sample collected can vary between some millilitres [5, 45, 50] to few litres [13, 83] or even few cubic meters of water [45, 92], depending upon the point in WWTP where the sample is collected [93]. For instance, while analysing tertiary effluent of a WWTP, Carr, Liu, and Tesoro [31] collected 1.89×105 to 2.23×105 L sample from tertiary treatment effluent, while Tagg et al. [83] collected 2 to 285 L from secondary treated effluent. As effluents of WWTPs contain significantly lower organic matter, it is sampled by pumping large volume of effluents over a filter. On the contrary, influents have higher organic matter that would clog the filter membrane; therefore, smaller volume of the influent is collected directly into a container. Therefore, separate pumping and filtration method can be described as best suited for sampling effluents of WWTP, and auto sampler or direct sampling in a container as best suited for sampling WWTP influents.
Carr, Liu, and Tesoro [31] designed a surface filtration system for sampling the surface water at the final discharge point of WWTP effluents. In this method, the volume of sample collected further increases to thousands of m3. However, since the sampling is done at surface of water in an open channel, there is a high risk of sample contamination due to atmospheric fallout of MPs. In addition; high-density MP polymers may not be sampled at the surface water that can lead to error in total MPs count. After the samples are collected, they are filtered to increase the MPs concentration in the sample. However, the pore-size/mesh size of the filters used is not standardized and different studies have used different mesh sizes ranging from 1 to 500μm. Some studies have also used a stack of sieve pans with different mesh sizes [45]. This method can help in studying the size distribution of MPs in the wastewater sample.
The next step is the sample pre-treatment where, organic, and inorganic impurities are removed from the sample. Methods like oxidation [7, 45, 48, 49] wet peroxide treatment [92], enzymatic degradation [53, 94] and acid and alkaline treatment [50, 73] are used for removal of organic pollutants present in the wastewater. The inorganic impurities such as sand, clay, rust particles can be removed by density separation method [53, 94].
The most commonly employed method of purification is wet peroxide oxidation, where the sample is treated with hydrogen peroxide (H2O2) or sodium chlorate (NaClO) for oxidizing organic impurities [95]. Studies have demonstrated that wet peroxide oxidation can remove up to 83% organic matter without altering the plastic debris [49]. Hydrogen peroxide oxidation is the most commonly used method for organic matter removal where the samples are mixed with (30%) H2O2. Bubbles are formed during to oxidation of the organic matter and when the oxidation is complete, no more bubbles are formed. The drawback of this method is that it takes long time, i.e., an average of 7 days for achieving complete oxidation. However, recently Fenton’s reagent, which is a solution of hydrogen peroxide with iron (II) sulphate (FeSO4) catalyst, is used for oxidation, it can reduce the oxidation time to just 10 minutes [96]. Alternatively, Sodium hypochlorite solution (NaClO) with a strength of 6–14% can be used for oxidation, it requires 24h for complete oxidation [97]. Other oxidizing chemicals that can be used for organic compound digestion include potassium hydroxide (KOH), and nitric acid (HNO3).
Enzymatic degradation is another emerging method for removal of organic pollutants from wastewater samples. In this purification method, MPs samples are submerged in a mixture containing several enzymes such as, lipase, amylase, proteinase, chitinase and cellulose [98]. These enzymes degrade carbohydrates, proteins and lipids from the MP sample leaving the MPs unaffected. A multi-step enzymatic degradation method has been demonstrated to remove organic contaminants while leaving the MPs unaffected [94]. In this method a combination of enzymes such as proteases, lipases, and celluloses with sodium dodecyl sulphate (SDS, 5% w/vol) and hydrogen peroxide (H2O2, 35%) was used. However, the limitations of this method include it may take more than 13 days for complete degradation and the enzymes are expensive to acquire.
Apart from these, alternative purification methods such as, acid treatment and alkali treatment could also be used. These methods are demonstrated to be good at removing the organic pollutants. However, they can also severely damage the MPs polymers in the sample and therefore, extra care should be taken when employing these methods for purification of sample [98]. Acid treatment is usually performed while heating the sample for up to 120°C, whereas it has been observed that some of the MP particles can melt at 90°C [45].
After organic matter removal, some studies employ further treatment to remove inorganic impurities. Density separation is widely used for separation of inorganic impurities form MPs. In this method, the sample is mixed with a solution of known density and centrifuged, and then allowed to settle. Depending on the density of the particles, they will float, sink, or suspend in the liquid. As the MP particles are usually very less light, they float on the solution whereas, impurities and dense particles settle. Zinc chloride (ZnCl2), sodium iodide (NaI), sodium bromide (NaBr), and zinc bromide (ZnBr2) are some of the commonly used solutions for organic matter removal [99]. Fig. 2 summarizes and provides a graphic representation of the procedure for extraction and analysis of MPs from wastewater samples.

6.2. Microplastics Analysis and Characterization

Microplastics analysis can be carried out by characterizing it physically or chemically. Physical characterization refers to distinguishing the MPs based on their size and other physical parameters such as shape and colour. Chemical characterization refers to MP classification based on its composition [86].
For physical characterization, visual identification by naked eye or by microscope are commonly used to measure the size, distinguish the shapes and to specify MPs count.
This allows the initial identification of suspected MPs thereby reducing the number of non-plastic particles in further analysis [100]. However, it has been estimated that up to 70% of the particles characterized as MPs by visual identification are not confirmed as MPs by spectroscopic analysis [84]. It is very difficult to differentiate between natural fibres and synthetic fibres just by looking into a microscope. Other limitations of visual identification include human dependency, microscope property, and difficulty in accurate identification of smaller particles (<500 μm) [84]. Fig. 3 shows suspected MPs from wastewater samples as observed by light microscopy. To improve the MPs identification during visual analysis some studies have dyed the sample (e.g., Nile Red) prior to analysis as allows a high throughput detection of MPs. In this method, the MPs samples are dyed with Nile Red (10 μg/ml) in 10% dimethyl sulfoxide solution and incubated for 10 minutes followed by observing and counting under a microscope. However, due to different sizes, shapes and types of MPs present in the sample it not possible to dye the MPs consistently and therefore, the accuracy of this method is very low, about 78% of the particles identified as MPs in Nile Red method are not confirmed as MPs in FTIR method [101]. This error ratio increases with decrease in the size of the microplastics [102]. For instance, natural fibres and synthetic fibres cannot be differentiated just by visual inspection using microscope [51]. However, some studies have reported that the Nile Red staining method was appropriate for differentiating PP MPs fragments from sand particles that may be present in the sample [103].
UV-Visible measurement method can also be used for quantitative measurement of MPs in water sample. In this method Beer’s law can be employed for the UV-Vis readings to calculate the number moles of solute in a sample [104]. However, qualitative analysis is not possible and if the positions of MPs in the sample changes, it can lead to errors in the results. In an attempt to reduce the error of physical characterization, some studies have used petri dish with numbered grids [45, 105]. While others have used a standardized MPs selection criterion for better counting and to avoid misidentification of MPs [52, 106]. To reduce the error of physical characterization Norén [69] used a standardized MPs selection criterion, where fibres and plastic particles are identified as MPs if:
  1. The organic or cellular structures are not visible through microscope.

  2. If coloured particles are present, then they must be evenly coloured.

  3. Thickness of the particles or fibres should be uniform throughout the length of the particle.

  4. Fibres or particles should not be segmented or appear flat or as twisted ribbon.

  5. Fibres or particles do not break when they are pressed.

Only the particles that fulfilled the above-mentioned criteria were identified as MPs particles. This method introduced a standard for visual identification and reduced the identification error in different studies.
Chemical characterization is used to identify the chemical composition of the MPs, thereby improving the efficiency of MPs identification. The chemical analysis can be carried out by spectroscopic, thermal decomposition coupled with spectrometry, chromatography with spectroscopy and sometimes Scanning Electron Microscope (SEM).
Fourier Transform Infrared Spectroscopy (FTIR) and Raman Spectroscopy are the two most common methods used for chemical characterization of MPs [71, 94, 107113]. In FTIR analysis, MPs samples are exposed to Infrared (IR) radiations to obtain a characteristic spectrum that is specific to chemical bonds between the atoms, the spectrum can be used to identify the composition of the MPs. However, a reference spectrum of every type of plastic that might be present in the MP sample is required. The most recent development in FTIR spectroscopic analysis is the Focal Plane Array (FPA) based micro-FTIR imaging which can evaluate the spectra of individual particles present in a sample more effectively [68]. μ-FTIR can be used to identify MPs smaller than 11 μm [114]. Fourier-transform near infrared (FT-NIR) spectroscopy is one of the latest developments in FTIR that is faster spectroscopic method compared to conventional FTIR. It uses a fibre-optic refection probe for quicker analysis. This method was used by Paul et al. [115] to determine the presence of PET in soil sample. However, it requires at least 1% mass contents, which is very high for environmental samples.
The limitations of this method include inability to analyse smaller MPs of size 10μm-20μm and for identification of fibres, the imaging technique is stretched to its limits [48, 113]. However, FTIR is still one of the most preferred analytical methods for MPs analysis. Raman spectroscopy is also frequently used for analysis of MPs in atmospheric MPs sample. Here, a higher frequency laser (usually 532 nm) is used to excite the surfaces of the materials until it emits photons, only few of the photons (1 in every 7–10) are emitted at right angles by Raman scattering, while others are emitted in line with the laser. Raman scattering can be identified as a vibrational spectrum used to identify the composition of the MPs present in the sample [33]. Advantages of Raman spectroscopy include, high reliability, high throughput screening and environmentally friendly, also Raman analysis has one major advantage over FTIR, that is its ability to identify particles up to 500 nm in size [116]. Drawbacks include long measurement time and vulnerable to spectral distortion induced by fluorescence and very small organic, inorganic, or microbial impurities in the sample can cause fluorescence interference, which can further lead to errors in the analysis [112, 117]. Even though results obtained by Raman spectroscopic method are almost as consistent as FTIR, very few studies have used it. However, acceptance of Raman spectroscopy is on the rise in recent studies.
μ-Raman spectroscopy can chemically analyse MP particles with diameter as small as 0.25μm compared to μFTIR at 10 μm, providing a higher resolution analysis of MP particles [117, 118]. It has been reported that about 35% of MPs of sizes <20 μm are underestimated by μFTIR compared to μRaman [119]. However, μFTIR has an extensive polymer library that makes it easier for polymer identification. However, with the increase in use of μRaman spectroscopy, it is expected that its polymer library resources will also increase that will provide polymer identification opportunities equivalent to μFTIR in near future.
Chromatography based analytical methods such as GC-MS and LC-MS are used for rapid MPs identification in wastewater samples. In GC-MS, sample of MPs are heat-treated, and the gas obtained is analysed [120]. GC-MS can be of different types such as Pyrolysis-GC-MS (Pyr/GC-MS), Thermal Desorption-GC-MS (TD/GC-MS) and Thermal Extraction Desorption-GC-MS (TED-GC-MS). In these methods, quantities of the MPs polymers are evaluated by the number of ions released by pyrolysis or thermal desorption [121]. Due to very high sensitivity of GC-MS, it can even analyse very small MPs (<10 μg) both quantitatively and qualitatively, and the volume of sample required is very small [122]. In addition, the analysis is not affected by the physical characteristics of MPs (colour, size shape etc.) or by the additives in the MPs. However, the drawbacks of this method are, it can only analyse small number of samples with only one sample at a time thereby limiting its wider use and the destructive nature of analysis, where there is total loss of sample, and any subsequent analysis of the sample is impossible [123].
SEM based techniques such as, SEM-Energy Dispersive X-ray spectroscopy (SEM-EDX) and Environmental SEM-EDS (ESEMEDS) are also used for MPs analysis. In this, first surface morphology of the MPs is characterized followed by determination of elemental composition of the MP polymer by diffraction and reflection of emitted radiation from the MPs surface [68, 85]. Due to micro-scale and nano-scale features with magnification up to 500,000X It can determine particle sizes, and elemental composition of microplastics in the sample [85]. Both SEM variations can investigate biodegradation, weathering as well as composition of the MP. EDS is an extra module that is connected to SEM, to measure the X-ray radiations emitted from the surface during the interactions between beam and MPs surface [124]. This emitted X-ray beam is used to obtain a spectrum that gives the information about the characteristics of the MP. The spectrum is then compared against a database to identify the elemental composition of the MP. By calibrating the instrument for specific elements, relative quantification can also be obtained.
There are a few studies that have used Size Exclusion Chromatography (SEC) for characterization of MPs. This method is based on high-performance liquid chromatography (HPLC). In this method, porous column material containing cross-linked polymers of different pore sizes are used in series for separation. In the dissolved polymer chain, larger polymer chains can only pass-through larger pores whereas smaller polymer chains can pass through any available pore, thereby separating the molecules based on size. Only sizes between the smallest and largest pores can be characterized. Following separation, a set of detectors that detect the refractive index (RI) are used to determine the concentration of the chains. For qualitative evaluation, Infrared (IR), ultraviolet-visible (UV-Vis), and fluorescence detectors are used to determine the functional groups in the molecules. This method is usually used to understand the mechanism of weathering of MPs. SEC was used for the identification of polystyrene (PS) and polyethylene (PE) by using fluorescence detector, it has also been used for dissolved polymers quantification [112, 125]. In Table 2 a summary of different analytical methods used for analysis of MPs is presented along with advantages and disadvantages of each method.

7. Conclusions and Recommendation for Future Research

Many studies have identified WWTPs as a point source for release of MPs into the aquatic environment. Even though studies report that most of the existing WWTPs are capable of removing more than 90% of the influent MPs, the quantity of wastewater effluents discharged daily is very high, small percent of MPs can lead to release of hundreds of millions/billions of MPs particles into natural aquatic environment. Quantity of MPs in the wastewater depends on several factors including the location of the WWTP. It has been identified that, WWTPs in the cities or highly populated areas have higher MPs than those at lower populated areas.
Wastewater is known to contain different toxic chemicals such as pesticides, pharmaceutical chemicals, metal ions etc. In addition, harmful biological organisms such as the bacteria, viruses, fungi, protozoa etc. are also present in the wastewater. MPs by themselves can adsorb many toxic organic/inorganic chemicals, however, after the adsorption, a biofilm forms around the MP particle that consists of microorganisms like bacteria, protozoa, fungi etc. The formation of biofilm further increases the adsorption capacity of the MPs. These toxicities can potentially spread into the natural aquatic bodies and lead to distinct toxicological hazards.
A standard method for sampling and analysis of MPs from WWTPs is not yet available and therefore, the results obtained in different studies vary greatly and therefore comparing results between different studies is very difficult. Among the different sampling methods, Separate pumping and filtration method is identified as the best option. For analysis and characterization of MPs, spectroscopic techniques such as FT-IR or Raman spectroscopy are identified as best available options as they provide complete information about, number, size, and chemical compositions of the MP particles.
The review has identified that there are still research gaps in this field of research, such as:
  1. MPs from atmospheric fallout as a source of into WWTPs is not quantified and must be considered.

  2. Investigate the mechanism of removal of MPs in the secondary treatment stage.

  3. Study the MPs removal in different advanced treatment technologies like DAF and MBR to develop technologies for higher MP removal.

  4. Standardized procedures for sampling, extraction, characterization of MPs in distinct environmental compartments is urgently needed for more clarity.

  5. Interactions of MPs with toxic chemicals and biofilm formation should be studied further. Does the biofilm formation increase the biodegradability of the MPs?


The authors gratefully thank the anonymous reviewers for corrections and comments.


Author Contributions

T.H.N (PhD student): Conceptualization, Investigation, Data curation, Writing – original draft, Writing – review & editing. A.J.A (senior environmental engineer): Data analysis, Writing – review & editing, correction. I.M.A (Lecturer Environmental Engineering): Critical review of manuscript, editing, language improvement.


The authors declare that they have no conflict of interest.


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Fig. 1
Schematic Diagram of estimated microplastics removal at each stage of a typical wastewater treatment plant.
Fig. 2
Schematic diagram of general procedure for sampling, processing and analysis of MPs from wastewater samples. Modified from [62].
Fig. 3
Suspected MPs as observed under Light microscopy. Items shown are (a) pellets, (b) fibres, and (c) fragments [62].
Table 1
Microplastics removal at different stages of WWTP
Sl. No Location Microplastics Concentration (Particles L-1) Secondary treatment Tertiary Treatment Removal Efficiency (%) Study

Influent Primary effluent Secondary Effluent Tertiary Effluent
1 Helsinki (Finland) 636.7(±38.8) 14.2(±4.0) 1.0(±0.6) N/A ASP N/A 99.8(±0.18) [21]

2 Mikkeli (Finland) 115.2(±24.7) 1.1 (±0.4) 0.7(±0.3) N/A MBR N/A 99.4(±0.14) [42]

3 Helsinki (Finland) 610 304(±28.9) 82.4(±7.9) N/A ASP N/A 86.5(±1.3) [27]

4 Glasgow (Scotland) 15.7(±5.23) 8.70(±1.56) 0.25(±0.28) N/A ASP N/A 98.4(1.27) [46]

5 Detroit (USA) 133.0(±35.6) 21.15(±5.4) 8.24(±2.14) N/A ASP N/A 93.8(3.7) [51]

6 Northfield (USA) 289.0(±69.5) 33.52(±8.0) 29.48(±7.0) 8.09(±1.94) ASP Tertiary granular sand filter 97.2(1.4) [51]

7 Lysekil (Sweden) 15.1 __ 0.00825 N/A ASP N/A 99.9 [59]

8 Paris (France) 260–390 50–120 14–50 N/A Biological treatment (Biofilters) N/A 88.1((±6.8) [9]

9 Netherlands 68–910 __ 55–81 N/A ASP and MBR N/A 11 to 94 [52]

10 Germany __ __ 0.08–7.52 N/A __ N/A __ [48]

11 Australia 12–117 __ 0.21–1.5 0.28–0.21 Secondary Biological Chlorination, UF, RO __ [50]

12 Denmark 7216 __ __ 54 Secondary Biological Retention Soil Filters 99.3 [22]

13 South Korea __ 0.09 N/A N/A 98–99% [52]
A2O 29.8 0.435 A2O 98.54
SBR 16.45 0.14 SBR 99.14
Media 13.865 0.28 Media Process 97.98

14 Italy [56]
ASP 3.64 1.9 0.76 0.52 ASP Disinfection 85.7
UASB+MBR 3.64 N/A 1.72 0.2 UASB AnMBR 94.5

15 Iran 12.67 3.514 0.423 N/A Anoxic tank and clarifier N/A 96.7 [60]

16 South Korea Bioreactor [55]
WWTP-1 4200 1568 710 33 Ozone 99.2
WWTP-2 31400 12580 7863 297 MDF 99.0
WWTP-3 5840 2080 433 66 RSF 98.9

17 China [61]
Process 1 N/A N/A N/A 0.168±0.02 (mg/l) Oxidation ditch UV 97
Process 2 5.6 ± 0.09 (mg/l) N/A N/A 0.028±0.01 (mg/l) A2O MBR 99.5

18 UK 3–10 1–5 1–4 <1–3 Biological aeration Nitrification tank >96 [62]

19 Thailand 12.2 12.2 2.0 N/A Aeration N/A 83.6 [63]

20 Australia 92 2.25 0.18 N/A Biological treatment N/A 99.8 [64]
80 9.65 0.96 98.8
55 10.8 0.91 98.3

21 Spain N/A 171±43 10.7±5.2 N/A A2O N/A 93.7 [65]

22 Turkey N/A Anaerobic digestion [66]
Karaduvar 2.8 N/A 1.6 Biological 42.8
Tarsus 3.1 0.7 N/A Aeration N/A 77.4
Silifke 1.5 0.6 N/A N/A 60.0

ASP: Activated Sludge Process; N/A: Not available; A2O: Anaerobic, Anoxic, and aerobic process; SBR: Sequence batch reactor; MBR: Membrane Bioreactor; MDF: Membrane Disk Filter; RO: Reverse Osmosis; RSF: Rapid sand filter; UF: Ultrafiltration; UV: Ultraviolet; UASB: Up flow Anaerobic Sludge Blanket.

Table 2
Comparison of different MPs analysis techniques
Sl. No Method of Analysis Method Details Advantages Limitations Study
1 Fourier Transform Infrared Spectroscopy (FT-IR) (FT-NIR) MPs exposed to IR radiations and spectra obtained is compared with the reference spectra
  1. Preparation of sample is not required.

  2. Dust free and water vapour free environment for microscope due to fully purge able IR beam.

  3. Software based, therefore easy sample visualization, spectra collection and sample mapping.

  4. FT-NIR can be used for faster detection

  1. Need to have reference spectrum for all types of MP particles present.

  2. Reference spectra produced is usually for clean MP materials.

  3. Takes longer time for analysing MPs <20 μm size

  4. FT-NIR requires minimum mass content of 1%.

[25,48, 126]
2 Raman Spectroscopy A laser is used to excite the molecular vibrations. It gives information of molecular bonding and structure.
  1. Non-destructive analysis

  2. Can be performed on different types of materials.

  3. MPs smaller than 10μm can be analysed.

  1. Coloured MPs particles can interfere with the analysis and can lead to errors in identification.

  2. Thorough sample purification is required.

3 GC-MS with thermo analytical techniques (Pyr-GC/MS and TED-GC/MS) Mass spectroscopy of MP by analysis of their degradation products.
  1. Faster analysis.

  2. Polymer type and organic plastic additives in one go, without solvent use.

  3. No background contamination.

  4. TED-GC/MS a precise can precisely quantify PE, PET, PP, and PS in 20 to 100 mg samples within 3 h.

  5. It can give polymer concentration in terms of mass per litre.

  1. Destructive analysis.

  2. Environmental Hazard due to plastic burning.

  3. Pigments and impurities on MPs can interfere with the results.

[94, 126]
4 Scanning Electron Microscopy (SEM) Interaction of electron beam with the sample to produce an image
  1. High image resolution

  2. Non-destructive analysis.

  3. MPs can be detected down to nanometre range.

  1. Time consuming.

  2. Expensive set-up

  3. During scanning, amorphous carbon can deposit on the sample from residual organic contamination in the chamber or within the sample, which can cause error in analysis.

5 SEM-EDS/ESEM-EDS Diffraction and reflection of emitted radiations from MP surface.
  1. Non-destructive method

  2. No sample preparation

  3. Elemental composition and surface morphology can be determined.

  1. Expensive equipment

  2. Time consuming

  3. Light elements are not detected by EDS

[85, 128]
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