Unaccounted Microplastics in the Outlet of Wastewater Treatment Plants—Challenges and Opportunities

Abstract

Since the 1950s, plastic production has skyrocketed. Various environmental and human activities are leading to the formation and accumulation of microplastics (MPs) in aquatic and terrestrial ecosystems, causing detrimental effects on water, soil, plants, and living creatures. Wastewater treatment plants (WWTPs) are one of the primary MP management centers meant to check their entry into the natural systems. However, there are considerable limitations in effectively capturing, detecting, and characterizing these MPs in the inlet and outlet of WWTPs leading to “unaccounted MPs” that are eventually discharged into our ecosystems. In order to assess the holistic picture of the MPs’ distribution in the ecosystems, prevent the release of these omitted MPs into the environment, and formulate regulatory policies, it is vital to develop protocols that can be standardized across the globe to accurately detect and account for MPs in different sample types. This review will cover the details of current WWTP adoption procedures for MP management. Specifically, the following aspects are discussed: (i) several processes involved in the workflow of estimating MPs in the outlet of WWTPs; (ii) key limitations or challenges in each process that would increase the uncertainty in accurately estimating MPs; (iii) favorable recommendations that would lead to the standardization of protocols in the workflow and facilitate more accurate analysis of MPs; (iv) research opportunities to tackle the problem of ‘missing MPs’; and (v) future research directions for the efficient management of MPs. Considering the burgeoning research interest in the area of MPs, this work would help early scientists in understanding the current status in the field of MP analysis in the outlet of WWTPs.

1. Introduction

Since the start of the large-scale production of plastics in the 1950s, their global use has surged to gigantic proportions (from 2 million metric tons (MT), 1950 to 380 MT, 2015) due to their superiority over metal and paper in various parameters including lightness, durability, economic production, low maintenance, diverse design opportunities, high strength, inertness, resistance to water, etc. [1,2,3]. In the US, total municipal solid waste generation increased by 188% between 1960 and 2013, whereas total plastic waste generation increased by 8238% over the same period [4]. Unfortunately, around 79% of plastic generated is dumped into terrestrial (13–25 MT) and aquatic (9–23 MT) environments resulting in long-lasting detrimental effects on the habitats of our planet [1,5].

Of global plastic pollution, the most widely studied contaminant is microplastics (MPs), which are tiny fragments (size < 5 mm) of plastic generated by various environmental and human activities (Figure 1). Human activities that release MPs include the use of products that already contain MPs, or long-lasting physical and mechanical force on some of the plastic tools used for domestic or recreational purposes. Environmental factors that degrade plastics into MPs are UV irradiation, heat, mechanical stress due to water and wind forces, and enzymatic/bio disintegration. Based on the origin, MPs are classified as primary MPs, which are originally manufactured as micro-size plastics (fibers, pellets, films, and spheres) in industries such as toothpaste, resin pellets, cosmetics, and fishing nets, and secondary MPs which are produced by the breakdown of larger plastic items discarded in the environment due to physical, chemical, and biological processes [6,7,8]. Only recently (2015) was the existence of nanoplastics (NPs) of sizes < 1 µm brought to light, and a lot more needs to be explored about their source, abundance, and health impacts [6,7,8].

MPs enter our ecosystems through various sources. Their distribution and abundance are chiefly determined by environmental and anthropogenic factors [9]. Primary MPs enter the environment mainly through the domestic and industrial drainage system, while secondary MPs can form at any place on earth and relocate based on the combination of several environmental factors such as sunlight, heat, physical abrasion by winds, wave currents, tides, cyclones, river dynamics, land dynamics, and wind directions [10,11,12].

Irrespective of the site of origin, most of the generated MPs can end up in surrounding water bodies through sewage, incorrect waste disposal, runoff from urban areas, environmental factors, littering, or other anthropogenic activities, and eventually reach the ocean [12,13]. Hence, wastewater of domestic, industrial, commercial, and surface run-off origin is one of the major sources to release MPs in our ecosystems and inflicts harmful effects due to the toxicity of the polymers, additives, and adsorbents associated with MPs [12,13]. Wastewater treatment plants (WWTPs) thus play a pivotal role in checking the release of MP-contaminated water into environmental resources.

In a typical WWTP, the wastewater (influent) is passed through primary, secondary, and tertiary stages of treatment to remove contaminants that include MPs as semi-solid sludge and produce cleaner water (effluent) suitable for release into the environment. However, the total number of MPs detected in the outlet of a WWTP (effluent plus sludge) is significantly lower than the number of MPs entering the WWTP via influent [14]. This mismatch results in the underestimation of MPs in the sludge and effluent that are released into the environment. Therefore, sludge and effluent were identified as primary carriers of ‘unaccounted’ MPs in our ecosystems [13,14,15,16,17,18,19,20].

The disparity can be attributed to key limitations such as (i) lack of accurate, sensitive, and easy methods to isolate, identify, and characterize MPs of all sizes and shapes, especially in the outlet of WWTPs; (ii) lack of standard operating procedures for the workflow involved in the analysis of MPs in environmental samples; and (iii) conventional designs of WWTPs are not specifically tailored to remove MPs [14,21]. Due to these deficiencies, ~90% of captured MPs in the sludge remain undetected. This results in a serious underrating of both the extent of contamination of agricultural soil with the MPs loaded through sludge, and the scale of associated exposure and risk to the ecosystems [14,22]. Some ways to address this problem are to achieve the standardization of protocols for MP analysis, integration of multi-disciplinary approach, and application of advanced technologies such as Artificial Intelligence (AI) in the field of MP analysis [23,24,25,26,27]. In the current review, we discuss the following key aspects: (i) several processes involved in the workflow of estimating MPs in the outlet of WWTPs; (ii) key limitations or challenges in each process that would increase the uncertainty in accurately estimating MPs; (iii) favorable recommendations that would lead to the standardization of protocols in the workflow and facilitate more accurate analysis of MPs; (iv) research opportunities to tackle the problem of ‘missing MPs’; and (iv) future research directions for the efficient management of MPs.

2. Methods

The relevant scientific literature reviewed in this manuscript was collected using web search engines for scholarly literature. The articles from the past few years were retrieved using the keywords “microplastics”, “unaccounted” and “wastewater treatment plants” after filtering for English as a language. Further, all the articles were manually analyzed based on their title and abstract to exclude duplicates, and those articles which are not directly relevant to the central theme of this review. One of the primary inclusion criteria was to choose those articles which have reported MP concentrations in influent, effluent, and sludge.

3. Impact of MPs

MPs are inert in nature and may not produce direct fatal effects on living organisms. However, they can exert harmful effects through any of the following ways: (i) the polymer material of MP itself can cause inflammation, reproductive disorders, and other toxic effects on certain organisms [12,28]; (ii) additives added to plastics to improve the plasticity (for example phthalates, diphenyl ethers, etc.) are well-known endocrine disruptors [12]; (iii) MPs, due to their large surface area, adsorb and carry other organic and inorganic toxic pollutants to the environment [12,29].

While in soil, MPs can be ingested by soil organisms and can affect the reproductive system of earthworms, which are an essential part of soil ecosystems [30]. However, there are limited studies regarding the effects of MPs on other terrestrial systems, including soil health and plant development [31]. Due to the size similarity between MPs and prey, marine and aquatic species can inadvertently ingest MPs, resulting in exposure to toxic substances through chemical leaching and the release of adsorbed pollutants [12]. For example, the synergistic toxic effect of MPs and organic pollutants has resulted in damage to the liver cells of the Japanese medaka (Oryzias latipes) and has affected the gene expression of medaka fish [21]. The exposure to MPs can affect growth, reproductive functions, neurological development, and mortality, depending on the concentration of chemicals ingested by the organisms [32].

The penetration of MPs into the food chain is possible primarily through tiny living creatures and aquatic species. Aquatic organisms can consume MPs by filter feeding, direct engulfment, the ingestion of suspension materials, and water intake. These lower trophic species are engulfed by fish and can ultimately reach humans via the food chain [33]. The possibility of the direct consumption of MPs by humans cannot be ruled out, as there are multiple channels such as salt contamination with MPs [34], bottled water and beer [35], toothpaste, and toothbrushes [36]. Multiple reports have shown that polystyrene MPs can have pro-inflammatory activities in human lung cells and liver [37,38,39]. Other toxic substances, such as di (2-ethylhexyl) phthalate (DEHP) that comes adsorbed onto MPs, can have detrimental effects on humans, causing cancer, birth defects, and immune system issues [12]. Overall, the rapidly growing research efforts have been constantly revealing new health effects of MPs, thus highlighting the dire need to design effective strategies to mitigate the contamination of MPs in natural resources.

4. Distribution and Fate of MPs in Soil and Water

The predominant route of the contamination of the food chain with MPs is through soil and water. It is therefore essential to assess the distribution of MPs in these natural resources in order to understand the natural and anthropogenic causes behind plastic pollution, and thereby formulate necessary management and preventive actions. Sebille et al. reported that only 1% of all plastic waste is released directly into the marine ecosystem, with the remainder being discarded into the terrestrial land and water system [11].

Soil is the central part of terrestrial ecosystem and mediates nutrient cycle for food production. MPs have the tendency of penetrating deep into the terrestrial land and accumulating through deposition processes [40]. The degradation of plastics in soil through different pathways, mainly by UV radiation and physical abrasion, has been investigated. A recent study report indicates a 0.4% weight loss of polypropylene after one year’s incubation of plastic material in the soil [41]. However, the degradation could be very slow in soil, and thus the soil ecosystem is becoming a large and long-term reservoir of MPs. There are reports that compost and sewage sludge would contain significant plastics/MPs, and their use in agriculture can lead to the accumulation of plastic material in the soil [42,43]. Agricultural plastic mulching is a practice widely used by farmers that can also contribute to the MP pollution in soil [44,45]. In addition, irrigation and aerial deposition can introduce plastic materials into the soil system [12,46]. There is a high possibility that MPs can migrate and eventually impact soil health and function, as well as infiltrate groundwater [47,48].

Apart from the land accumulation, a large portion of MPs get settled into the freshwater and marine ecosystems. Marine MP contamination has long been a subject of interest to researchers and is well-documented [47,49,50,51]. Fresh water being an essential commodity for humans, MP pollution in these systems has garnered significant attention from the scientific community [52,53,54]. In the US, the presence of MPs has been detected in the freshwater continuum in the Milwaukee River to Lake Michigan, including the Mississippi River and other lakes [55,56]. Additionally, MP accumulation was found in the surface waters of the Laurentian Great Lakes of the USA [57]. A detailed study to understand the incidence and to quantify the MPs ingested by fishes in several freshwater drainages and the estuary of the Gulf of Mexico indicated that ingestions in urbanized streams are predominant compared to non-urbanized ones, suggesting an increased spread of plastic pollution in urban areas [58]. In Illinois, studies conducted on fish collected from the two hypereutrophic drinking water reservoirs dominated by row crop agriculture revealed the presence of MPs inside the fish, suggesting the possibility of high levels of MP contamination linked to agricultural activity in these watersheds [59]. Overall, fresh, surface, and marine waters, municipal WWTPs, and agriculture soil have shown the presence of millions to trillions of MPs [6,10,15,60,61,62,63,64,65,66]. These MPs were reported to have entered the food chain through various animals such as echinoderms, mollusks, mammals, birds, fish, reptiles, etc., and via plants such as algae, thereby inflicting undesirable consequences on organisms at all of the trophic levels [40,67].

5. MPs Removal Strategies in WWTPs

The current treatment procedures adopted in WWTPs in different countries worldwide depends upon the wastewater origins, such as domestic, industrial, commercial, or surface water runoff [13]. Currently, conventional wastewater treatment procedures are used, while modifications to adapt for MP removal are continuously being explored and implemented. In general, removal of MPs in WWTPs is carried out in primary, secondary, and tertiary treatments, using one or a combination of the following procedures: skimming and sedimentation [68], dissolved air flotation filtration [69], sand filtration [70], biofiltration [71], membrane filtration [72,73], coagulation [74], ozonation [75], adsorption [76], magnetic extraction [77], membrane bioreactor (MBR) [78], conventional activated sludge [16], and degradation using microorganisms [79,80].

Although MP removal has been demonstrated using different techniques in WWTPs, smaller-size particles still evade the treatment procedures, and some MPs are lost due to their bio or enzymatic degradation followed by infiltration and adsorption. These particles remain in either the effluent or the sludge [13]. Though only <5% of MPs entering the WWTPs are present in the effluent, it is a significant source of contamination considering the huge volumes of effluents that are released daily into water bodies [19,20,81]. For example, the primary source of MP contamination in the US is from WWTP effluents causing a release of over 4 million MPs per facility per day [15,60,82,83,84,85,86,87,88,89]. On the other hand, the sludge that captures 90% of MPs present in the influent is another major source of MP contamination when used for agricultural purposes. However, several studies suggest that of the 90% of MPs captured in the sludge, only 4% of them were isolated and detected, while the remainder ~96% remain unaccounted [14,22,87,90]. These unaccounted MPs are the root cause of ambiguity in understanding of the magnitude of the contamination of soil, water, and subsequently the food chain.

6. Unaccounted MPs—A Mass Balance Error

Recent reports suggested that the mass balance of MPs in WWTPs is not accurate [18,19,20,82]. The typical life cycle of MPs is illustrated in Figure 2. The primary and secondary MPs most often enter the water resources by disposals from domestic, industrial, and commercial sources or surface water runoff. The contaminated water (influent) is treated in WWTPs to remove the MPs, and the cleaner water (effluent) is released back into the water bodies. The solid waste of the WWTPs (sludge) is often used for agricultural purposes. Since both effluent and sludge are reintroduced into the environment, it is critical to estimate the exact load of MPs in them in order to assess the magnitude of risks from the exposure of MPs to species at all trophic levels. However, it has been a challenge to estimate and quantify the amount of MPs in the outlet of WWTPs. The number of MPs in the influent of any WWTP should equal the number of MPs in the effluent plus sludge. However, there are many reports that indicate significant discrepancies between the amount of MPs entering the WWTP and the amount detected in the outlet causing an error in mass balance (EMB) [19,20,82,91].

EMB: Number of MPs in Influent > Number of MPs in Effluent + Number of MPs in Sludge.

This discrepancy can be primarily attributed to the fact that 22 to 89% of MPs in sludge remain unaccounted [14]. A summary of studies that report MPs in the influent, effluent, and sludge of WWTPs in different countries and the percentage of unaccounted MPs and EMB due to this discrepancy is listed in

Table 1. Summary of error in mass balance and % of unaccounted MPs in WWTP in several studies across the globe. Adopted with permission from Koutnik, V et al. [14]. unaccounted microplastics in wastewater sludge: where do they go? ACS EST Water 2021, 1, 1086–1097, Copyright © 2023, American Chemical Society.

Study	MPs in Influent (p/day)	MPs in Effluent (p/day)	MPs in Sludge (p/day)	Error in Mass Balance	% unaccounted	Ref.
WWTP, Los Angeles, CA, USA.	1,510,000,000	930,000	1,090,000,000	419,070,000	28%	[82]
WWTP, Vancouver, British Columbia, Canada.	14,040,000,000	230,000,000	3,506,849,315	10,303,150,685	73%	[19]
WWTP, Mikkeli, South Savo, Finland.	626,428,761	3,328,421	460,000,000	163,100,340	26%	[91]
WWTP, Mikkeli, South Savo, Finland.	645,483,628	3,328,421	460,000,000	182,155,207	28%	[91]
WWTP, Mikkeli, South Savo, Finland.	676,447,788	4,740,478	460,000,000	211,707,309	31%	[91]
WWTP, Mikkeli, South Savo, Finland.	1,343,368,142	31,620,000	460,000,000	851,748,142	63%	[91]
WWTP, Republic of Korea.	234,821,918	3,945,205	149,712,329	90,164,384	38%	[20]
WWTP, Republic of Korea.	399,315,069	18,904,110	143,315,069	237,095,890	59%	[20]
WWTP, Republic of Korea.	123,780,822	2,000,000	32,301,370	89,479,452	72%	[20]
WWTP, Republic of Korea.	1,097,260,274	10,493,151	589,315,069	497,452,055	45%	[20]
WWTP, Republic of Korea.	1,074,575,342	7,095,890	444,602,740	622,876,712	58%	[20]
WWTP, Republic of Korea.	481,945,206	6,328,767	266,547,945	209,068,493	43%	[20]
WWTP, Wuxi, Jiangsu, China.	33,600,000	3,500,000	290,000	29,810,000	89%	[18]
WWTP, Wuxi, Jiangsu, China.	33,600,000	6,500,000	1,650,000	25,450,000	76%	[18]
WWTP, Hunter Region, NSW, Australia.	566,400,000	111,724,800	12,165,580	442,509,620	78%	[93]
WWTP, Zhengzhou, Henan, China.	4,800,000,000	870,000,000	315,000,000	3,615,000,000	75%	[81]
WWTP, Helsinki, Uusimaa, Finland.	193,649,400,000	197,000,000	151,000,000,000	42,452,400,000	22%	[94]

. Carr et al. analyzed the concentration of MPs in the influent, effluent, and sludge of seven tertiary and one secondary WWTPs in Southern California [82]. While 99.91% of MPs in the influent were removed by the WWTPs, all the captured MPs were not detected in the outlet of WWTPs, resulting in 28% of unaccounted MPs. This number was much higher (73% unaccounted MPs) in the study conducted by Gies et al. in a major secondary WWTP near Vancouver, Canada [19]. Lares et al. studied the MP removal efficiency of WWTPs in Finland by a conventional activated sludge process and a pilot-scale advanced membrane bioreactor [91]. The concentration of MPs in the inlet and outlet were estimated at different stages of treatment using visual, FTIR, and Raman microscopy methods. Though 98.3% of MPs were removed by the treatment, the percentage of unaccounted MPs in the outlet of WWTP ranged from 26% to 63%. The variability is attributed to inadequate sampling events, variations in sample collection times and locations, and the inability to accurately distinguish non-plastics from plastics. In a similar study conducted by Lee et al. in Korea, the authors compared the MP removal efficiency of sewage treatment facility operated by three different processes: anaerobic-anoxic-aerobic (A2O), sequence batch reactor (SBR), and the Media [20]. While all the three processes showed >98% treatment efficiency, all the captured MPs were not detected in the outlet of the treatment facility resulting in a percentage of unaccounted MPs in the range of 38% to 72%. Surprisingly, some studies have reported an overestimation of MPs due to the misinterpretation of non-plastics as plastics [92]. In two separate studies conducted in China and Australia, the percentage of unaccounted MPs in the outlet of WWTPs are 89% and 78% respectively [18,93]. Overall, a consistent trend of an inability to account for MPs in the outlet of WWTPs in different countries across the globe highlights the challenges being faced in the analysis of MPs and the importance of addressing them.

7. Challenges in Accounting for the MPs

The “unaccounted MPs” that are present either in the effluent or the sludge are the covert contaminants of soil and water, and it is vital to improve techniques to isolate and characterize them. The trend of uneven mass balance clearly indicates the problem in the analysis of MPs, which can be attributed to inherent limitations in the steps involved in the process flow of MP examination, i.e., sample preparation, purification, extraction, separation, filtration, oxidation, detection, and quantification. The danger of human exposure to several types of toxics carried by MPs thereby highlighting the important of accounting for MPs in the outlet of WWTPs is schematically represented in Figure 3. The workflow involved in the estimation of MPs in the outlet of WWTPs can be broadly divided into three stages: sampling, pre-treatment and separation, and characterization. Sampling deals with tools, techniques, and protocols to collect samples containing MPs, while pretreatment and separation encompasses several steps involved in the isolation of MPs from the samples collected. During the last stage, the isolated MPs are characterized physically and chemically and quantified using various analytical techniques. Below, we have briefly discussed the challenges in the steps involved in all these stages. Additionally, a summary and brief description of each of the steps involved MP analysis, factors that influence their performance, and best practices recommended is outlined in

Table 2. Summary of various steps involved in the process flow of MPs analysis, and recommended practices based on research evidence.

Process	Goal	Methods	Influencing Factors & Recommendations	Ref
Sampling	Collect samples to analyze their MP content.	Net, trawl, pump, tweezer, spoon, shovel, box corer, grab sampler.	Sampling technique: A composite or pooled sampling of several collections at different times, locations, and depths yield a more accurate analysis of MPs than individual sampling. Sample volume: Analysis of larger sample volumes decreases the error in MPs estimation. Material: Stainless steel tools are preferred over plastic to avoid possible contamination. Sample composition: Apart from MPs, the property and composition of the soil sample needs to be characterized to enable interstudy comparisons, since these factors would influence MP recovery. Drying: In case of soil sample, drying the sample is recommended for easy and reliable analysis of MPs. However, drying >40 ℃ for longer durations is not recommended as it can fragment the MPs and change their chemical composition, both of which can affect MP detection.	[14,21,95,96,97],
Sieving	Isolate the bigger (>1 mm) /easily accessible MPs from the sample before further processing for smaller MPs.	Sieves of different pore sizes.	Pore size: Use sieves of various pore sizes starting from a larger size (≈500 µm) and gradually going to a smaller size (≈1 µm); Material: Stainless steel tools are preferred over plastic to avoid possible contamination.	[14,96,97,98]
Pre-oxidation or Purification	Break down the organic debris in the sample to increase the efficiency of extraction and detection of MPs.	Treatment with hydrogen peroxide, Fenton’s reagent, acid, alkali, enzymes.	Choice of the method: Fenton’s reagent can facilitate more efficient degradation of organics and estimation of MPs than traditional peroxide treatment; acid/alkali digestion is very aggressive, and enzymatic digestion is efficient but expensive and complex. Reaction parameters: Reagent concentration, treatment time, and temperature need to be optimized to achieve maximum digestion of organic debris with the least effect on the physical and chemical properties of MPs. Sample composition: In case of soil samples, due to the complexity and heterogeneity of organic matter, the treatment methodology should be adapted and standardized following the examination of the composition and properties of soil.	[21,96,98,99,100]
Separation	Extract the MPs from the sample matrix into a salt solution.	Density separation (separation of low-density MPs using high-density solutions such as NaCl, NaI, ZnCl2, sodium polytungstate.	Choice of the salt solution: NaI or ZnCl2 result in superior extraction efficiency of MPs than NaCl. Pre-oxidation step: Performing a pre-oxidation step before the density separation is recommended as it releases tightly-bound small MPs from organic matter which would otherwise not be extracted.	[21,98,100]
Filtration and Sieving	Filter out the extracted MPs in the salt solution.	Vacuum filtration using membrane filters made of quartz, glass fiber, PTFE, or nylon.	Choice of the membrane filter: Use of quartz and glass membranes needs to be scrutinized, as they tend to leak inherent fibers and cause interference in MPs detection. Nylon is superior to hydrophobic PTFE in terms of ease of filtration. Pore size: Membranes with small pore size improve the efficiency of MP estimation, as most of the unaccounted MPs in sludge are <50 µm. Smaller pore size can lead to organic/mineral clogging of membranes. To mitigate this, solutions can be passed through stainless steel sieves first to remove organic matter, followed by membrane filtration.	[21,98,100]
Post-digestion oxidation	Remove any residual organic matter from the pre-oxidation and filtration steps to further reduce their interference in MPs detection.	A mixture of peroxide and acid	Choice of reagents: Post-digestion oxidation, being a second round of oxidation, can lead to chemical and physical changes to MPs, thereby interfering with their estimation. Though it is not universally followed, it would help the MPs’ analysis if the reagents and their concentration are optimized to positively influence MP estimation.	[98]
Detection	Identify and characterize the MPs physically and chemically.	Visual identification (naked eye or microscope), GC/MS, FTIR, Raman Spectroscopy.	Choice of technique: Visual identification alone can lead to inaccurate estimation due to inter-person variability and false classification, especially for smaller MPs. Hence, it needs to be supported with characterization by analytical instruments.	[14,21]
Quantification	To measure the concentration of MPs in the sample to understand the extent of pollution in a particular environment	Manual counting, image analysis softwares.	Choice of method: Image analysis capable of high throughput and automatic quantification are rapid, more accurate, and convenient than manual analysis. Choice of Units: Reporting in units of the number of particles rather than weight can result in over- or under-estimation due to possible fragmentation or aggregation of particles along the process flow. This can lead to data being inconsistent within and between studies.	[14,21]

. This will provide the reader with an overview of the current status of the field of MP analysis.

7.1. Sample Collection

Water, sediments, and biota are the three main sampling sites for characterizing MPs, the first two specifically being appropriate for WWTPs. In water samples, MPs can usually float because of their physical and chemical properties and are mostly collected using different mesh size trawls, whereas in sediments samples, MPs are collected using containers, tweezers, and metal spoons [21]. One of the main reasons for “unaccounted MPs” is the sample collection techniques that are used for the characterization of MPs. The variation in time, duration, and location of the sample collection can lead to an inaccurate number of MPs in the samples [82,91,101,102,103]. Specifically, variation in the depth of the sample collecting point can result in false estimations for sludge samples as the MP distribution is not homogenous throughout the various depths of WWTP sludge [97]. Additionally, due to the difference in the physical state between liquid and solid samples, sampling techniques such as tools, location, depth, and volumes are recommended to be unique for each sample type [95,96]. Such inconsistencies would directly influence the sampling efficiency of MPs. Hence, standardization of every detail involved in the sampling techniques is one of the critical requirements to account for missing MPs.

7.2. Pretreatment and Separation

The samples from WWTPs undergo a pretreatment step to remove high organic and inorganic contents from the samples. Peroxidation [104], enzymatic degradation [105], and acid/alkali treatment [106] are the most widely used pretreatment techniques to remove organic contents. Though effective in recovering MPs, some of these protocols are reported to change the chemical composition of MPs and affects detection accuracy [21]. In addition, the effect of these harsh chemicals on biodegradable plastics is poorly understood [96]. Further, most studies were limited to agricultural soils. Hence, the effect of these reagents on soils with different compositions such as chernozems or kastanozems requires further investigation [96]. The density separation technique using a salt solution is commonly employed to remove inorganic contents from the samples [107]. Sodium chloride is generally used to separate MPs due to it being an inexpensive, easily available, and non-toxic salt; however, its density is not high enough to separate MPs in samples with higher inorganic content and those particles with densities > 1.20 g/cm³ [21,108]. Instead, sodium iodide can separate even high-density MPs with an overall recovery rate of 99%; however, other limitations include high cost, toxicity, and challenges in visual inspection as it turns cellulose filters black [109].

7.3. Filtration

After the pretreatment, samples are separated by sieving and filtering. One of the reasons for a getting lower number of MPs in effluent and sludge in comparison to influent could be the missing out of smaller MPs in sludge due to the limitations in the pore/mesh size of the filters/sieves. As the wastewater goes through the primary, secondary, and tertiary treatment in WWTPs, the process further breaks down the MPs into smaller pieces which can be difficult to capture by the filters and sieves being used. Most often, filters with a size >50 µm are used to avoid clogging during filtering. However, this would lead to adequate loss of MPs as most of them are <50 µm [20,98,100].

7.4. Detection

MPs are generally characterized physically by size distribution, shape, and color, as well as chemically by composition of MPs. The most used technique to physically detect MPs is using an optical or stereomicroscope primarily for counting larger MPs in samples because of its accessibility, simple technique and inexpensiveness. This, however, has limitations in detecting smaller MPs due to the lower magnification capacity [110]. In addition to this, the visual identification of MPs using microscopes is prone to user bias leading to a higher susceptibility of error [111]. Moreover, it is difficult to differentiate the tiny MP particles from other non-plastic fabrics. The fibrous particles with the color in them are relatively easier to detect in comparison to other shapes with white or transparent particles. The usage of visual identification by microscope has, because of its convenience, raised the risk of error in many studies. Nearly 70% of fabrics visually identified as MPs by microscopes are characterized as non-plastics by another commonly employed technique—Fourier-transform infrared (FTIR) spectroscopy [17].

The chemical characterization of MPs using techniques such as GC-MS, FTIR, Raman spectroscopy, and others can enhance the accuracy of characterization [112]. The use of scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS) has been found to be effective as a technique, coupled with the physical identification of MPs by SEM and the chemical composition of MPs by EDS. SEM can provide high-magnification and resolution images of the particle surfaces, and EDS can distinguish between the types of MPs, organics, and inorganics. However, these techniques have inherent limitations such as cost and the inability to distinguish between opaque and thick MPs [113].

Raman spectroscopy uses the scattered light detected upon irradiating MPs with a laser to characterize the chemical and molecular structure of the MPs. This is one of the reliable techniques to identify MPs mostly up to 1 µm. However, the background and overlapping spectra from other impurities such as additives or organic contents can be challenging to eliminate. Neat sample preparation is required in order to remove organic and inorganic impurities, and an appropriate wavelength selection is required to reduce the fluorescence emitted by the sample [114]. In FTIR characterization, samples are introduced to infrared radiation and the spectra obtained is compared with known libraries of spectra to determine the functional groups of MP polymers [115,116]. This can detect particles as small as 20 µm, and fabrics are clearly identified as non-plastics by this procedure. Chromatographic techniques such as liquid chromatography (LC) and gas chromatography coupled with mass spectrometry (GC-MS) are improved analytical tools that have been utilized when MPs were properly separated from complex environmental samples. However, morphological characterization cannot be achieved with these techniques [117].

The practical application of all the chemical characterization techniques mentioned above is limited due to laborious and time-consuming methodology, the need for expensive instrumentation and well-trained operators, and an inability to detect smaller particles [118]. Lv et al. has compared the 4 different detection techniques (visual observation, Raman spectroscopy, FTIR, GC-MS) and concluded that the MP detection methods are still challenging for certain environmental samples [21].

7.5. Other Potential Challenges

Apart from the drawbacks discussed above, there are other possible challenges that need attention in order to tackle the problem of the unaccountability of MPs. In general, the sizes and volumes of MPs present in the sample source can lead to detection error. The detection technique employed in the study is usually optimized for standard sizes and volumes of MPs that are expected in the samples. Researchers often develop detection strategies based on the assumption that the size and volume of the MPs found in influent, effluent, and sludge are similar. However, depending on the treatment types employed in the WWTPs, the MPs in the effluent and sludge can be significantly smaller and go undetected by the technique optimized for the study. This argument is further supported by the fact that MPs undergo biodegradation and fragmentation during the treatment process that leads to relatively smaller MPs in effluent in comparison to influent [7]. Moreover, the use or exposure of foreign plastic materials during the extraction and characterization procedures introduce possibilities of cross contamination [96]. Therefore, it is vital to include blank samples to account for contamination due to tools and other external factors.

8. Opportunities to Improve the Outcome of Analytical Methods of MPs

Since the discovery of the existence of MPs by Professor Richard Thompson in 2004, research efforts in the field of MPs have grown exponentially in order to understand the source, abundance, distribution, fate, and impact of MPs [21]. In the past two decades, a lot has been understood about MPs while simultaneously raising many questions that needs to be answered. Considering the growing research evidence on the health implications of MPs, a collaborative and multi-disciplinary research effort is the need of the hour to address the knowledge gaps in the field of MP analysis. We have briefly described some of the many opportunities available for the scientific community to tackle the existing challenges.

8.1. Account for Transport of MPs from Water to Air

A potential contributing factor for ‘unaccounted MPs’ which is often overlooked is the relocation of MPs from the surface of water to the surrounding air. This is particularly true in the case of very small MPs (<1 µm) which often stay on the upper layers of water due to the Brownian motion, rather than the relatively bigger particles which are likely to sediment down [119,120]. Moreover, MPs composed of polyethylene and polypropylene, the most commonly used polymers for fabricating plastics, float in water due to their buoyancy [121]. MPs on the water surface are likely to escape to air by mechanisms such as formation and bursting of air bubbles at the water-air interface. Indeed, Masry et al. have shown that MPs less than 1 µm are transported from water to air by the bursting of air bubbles at the water’s surface, with the transfer rate increasing with the decrease in the bubble size [122].

It is known that MPs present in the influent undergo further fragmentation to smaller particles during several stages of treatment in WWTPs [7]. These smaller MPs are more likely to be trapped in air bubbles. In fact, the dissolved air floatation technique used in the WWTPs will result in the formation of bubbles that carry MPs to the surface of water [123]. Further, operation factors such as the aeration, dynamic movement of water, and use of surfactants in the WWTPs would also favor the formation of bubbles. Combining these factors, there is a high probability that much of the smaller MPs in the influent are trapped in the air bubbles formed during the wastewater treatment process. Further, the mechanical forces due to the physical processes in the WWTPs can cause these bubbles to burst, thereby releasing the MPs into the air. Thus, some of the MPs present in the influent would have been transported to the air even before they reach the effluent phase. A similar scenario can occur in the pre-treatment and isolation steps during the analysis of MPs in effluent and sludge. Taken together, the transport of MPs from water to air in the WWTP as well as during the analysis of effluent and sludge results in mass balance error between inlet and outlet of WWTPs.

However, adequate research attention has not been devoted to understanding the loss of MPs due to their transport from water to air. Hence, there is an urgent need to address this gap. Some of the ways would be to: (i) simulate the conditions of WWTPs in the laboratory and study the release of MPs from water to air, (ii) understand the factors that promote this transport, (iii) design easy and accurate analytical methods to detect MPs in aerosol; (iv) standardize the protocols to measure the MPs’ concentration at every stage of the WWTPs, rather than the end, to determine the loss caused due to water-to-air transport at each of the treatment stages.

8.2. Optimization of Techniques for Characterizing Ultra-Small MPs

The disparity in the equation of the mass balance of MPs between the inlet and outlet is primarily attributed to the inability of analytical techniques to accurately detect smaller plastics such as ultra-small MPs and NPs [14,21]. The ideal analytical method should be sensitive enough to detect all MPs and NPs at very low concentrations (ng/L) irrespective of the size, shape, composition and nature of environmental matrices. In this context, some of the common techniques used for the characterization of nanoparticles in laboratories such as the dynamic light scattering (DLS) technique, Nanosight (nanoparticle tracking analysis), and scanning/transmission electron microscopy, would be also deemed suitable for the detection of NPs.

However, the direct application of these techniques for characterizing smaller MPs or NPs is limited by two factors: The first is non-specificity: the environmental sample matrices of MPs or NPs would contain many interfering non-plastic components in the similar size ranges [124]. The above-mentioned techniques are not designed to delineate between the MPs or NPs and interfering particles resulting in false positive analysis. Therefore, new sample preparations that will eliminate interfering substances should be designed. The second factor is a lack of reference standards for MPs or NPs: The techniques such as DLS and Nanosight detect nanoparticles based on the reference standards which are spherical polymeric nanobeads of particular size, concentration, and composition [124]. These standards cannot be used for MPs or NPs because these plastic fragments are highly heterogenous in shape, size, and composition. Hence, there is a need to develop a reference standard that resembles all the properties of MPs or NPs. This provides an opportunity for the research community to address these two limitations to be able to expand the repertoire of tools that can characterize ultra-small MPs and reduce the error in mass balance.

8.3. Application of AI for Sorting of Plastics

The interference from the highly heterogenous and complex sample matrix housing the MPs is one of the major issues responsible for the inaccurate analysis of MPs [125]. Hence, an additional step of pretreatment of samples in harsh environments is generally carried out to reduce the interference of the matrix, though it cannot be completely eliminated. In addition, the MPs are formed from different polymers and vary in size and shape, further adding to the heterogeneity. Hence, there is a genuine need to develop new technologies that can sort the MPs from non-plastics as well as categorize per the shape, size, and composition in complex matrixes with minimum pretreatment.

AI-based algorithms have been successfully implemented in such sorting applications in other fields. For example, in the field of biotechnology, Thinkcyte is a recently developed AI-powered device that can accomplish high throughput sorting on a single cell level and isolate target cells based on the intended purpose [126]. Similarly, TrashBot is an AI and machine learning-based recycling solution develop by CleanRobotics that sorts trash at the time of disposal and diverts the components into their respective bins with more accuracy than humans [127]. Clearly, such AI-powered solutions can be integrated in the field of MP analysis to improve the accuracy of detection in environmental samples in a high throughput manner. It is evident that it would require significant efforts in developing and training the AI model, but considering the rapid increase in the global prevalence of MPs, it is a critical requirement for our safety and a good opportunity for AI scientists.

8.4. Mimicking Laboratory Testing to Natural Conditions

Most of the research related to development or improvement of analytical methods for MPs is conducted in laboratory settings. However, the natural environment of MPs and the morphology of MPs in reality would be considerably different from laboratory conditions [128]. This would lead to inconsistency in the outcomes between laboratory conditions and natural environments even when the same analytical method is used. Some of the noticeable differences between laboratory and natural environments that would have an influence on the analytical outcome are as follows: (i) most laboratory studies on MPs were performed at sample concentrations higher than those observed in real samples [128]. In such cases, the sensitivity of analytical methods is not challenged to realistic limits. (ii) Laboratory studies on MPs often deal with particles of one polymer type, and a particular size and shape [128]. However, the efficiency of analytical methods might be affected on naturally existing MPs which are highly heterogenous in terms of composition, size and shape; (iii) MPs existing in natural environments adsorb several biotic and abiotic factors over a period of time [128]. The sample preparation and workflow for analyzing MPs in such a complex milieu should not be the same as the ones used for plain MPs used in laboratories. Overall, it is important for scientists to closely mimic the natural conditions of MPs and conduct environmentally representative studies that can be relied upon with greater confidence.

8.5. Models to Understand the Fragmentation of MPs

Fragmentation of MPs to even smaller particles such as NPs during the wastewater treatment process further increases the error in mass balance due to the inefficiency of analytical methods to detect smaller particles. Hence, it is vital to understand the factors that would promote the fragmentation of MPs and the kinetics involved in it. Ter Halle et al. demonstrated that mathematical models can be designed to understand the pattern of fragmentation [129]. For example, cubic-shaped MPs were expected to cause faster fragmentation than parallelepipeds. Similarly, models based on the maximum entropy method were used to highlight mechanistic details behind several processes that would cause fragmentation [130]. Development of such mathematical models that would predict the Influence of Intrinsic properties of MPs as well as operational factors of WWTPs on the process of fragmentation is important to account for the missing particles. In this context, researchers should adapt the six principles of good modelling practice recommended by Buser et al. [131]. The notable ones include specifying the input and output data, recognizing the input parameters that have maximum influence on the key outcomes, and specifying the limits of the application of the model.

9. Conclusions and Future Directions

The evolving designs and technologies of WWTPs have achieved significant removal (≈90%) of MPs from water bodies. However, most of the captured MPs remain undetected, resulting in serious underestimation of the amount of loading of MPs into our ecosystems through the WWTP outlets. This is primarily attributed to the lack of standardization in the methods used for sampling, purification, extraction, filtering/sieving, and detection of MPs causing an error in the mass balance of MPs. Increasing research efforts toward addressing this discrepancy have provided evidence that supports recommendations in each of these processes involved. For example, composite sampling, the use of Fenton’s reagent for sample digestion, NaI-mediated density separation, and visual identification based on microscopic and spectroscopic techniques are some of the recent improvements that can aid in accurate MP analysis [14,21]. In addition, standardization on units (number or mass) while reporting the MPs’ quantity in the samples is vital, as it leads to consistency and comparability between research studies [95].

The progress so far is partial and future research efforts must be directed toward existing challenges responsible for the ‘unaccountability’ of MPs. Some of these include the development of efficient filters and highly sensitive techniques for the capture and detection of ultra-small MPs and nano plastics (NPs) which invariably remain unnoticed and reintroduced into the environment.

The current techniques utilized in WWTPs for MP removal were originally designed to purge general impurities, such as floating and suspended matter, dissolved organic matter, and organic and inorganic impurities that include synthetic organic compounds, heavy metal ions, fluorides, and microorganisms. Direct adoption and utilization of these conventional procedures are not sufficient to remove MPs. Smaller MPs still evade the treatment procedures, and most of these unaccounted MPs are presumably lost due to their bio or enzymatic degradation. Exclusive procedures for MP detection and removal are currently being designed and are still in their infancy. There is a pressing need to reevaluate the retention efficiency of WWTPs of smaller MPs by developing more sensitive methods of detection, assessing the variations in concentrations of MPs over time, and determining the contribution of WWTPs and sludge as sources of MPs. Additionally, improvements must be made in all steps such as sample collection, filtration, and detection to accurately quantify MPs. Fragmentation of MPs due to physical, chemical, and biological processes involved in wastewater treatment and extraction steps is one of the causes of the unaccountability of MPs, as fragments below 50 µm often remain undetected or not captured by the filters/sieves in the separation step. Therefore, the percentage breakdown of MPs into smaller fragments in WWTPs during the extraction/identification processes needs to be assessed, and the mechanism behind the fragmentation needs to be comprehensively understood in order to account for the mass imbalance between the inlet and outlet of WWTPs. A comprehensive knowledge of the mechanism behind the fragmentation process would further contribute toward the standardization of methodologies involved in MPs analysis.

The development of portable, hand-held, easy-to-use, and accurate analytical tools for rapid and high-throughput identification of MPs in environmental samples is a necessity that demands immediate attention. Ongoing development of advanced and highly efficient filters could only capture MPs in sizes up to 20 µm, thus highlighting the need for novel materials and designs to isolate nano and fibrous plastics [23].

The problem of MPs can be better managed by initiating and supporting collaborative and interdisciplinary research among varied disciplines of science. Analytical chemists and information scientists could partner to establish a comprehensive database of spectra of all the original polymers used in the manufacture of plastics, including the spectral changes that would occur on the interaction of these polymers with the environment for varying periods of time. This would help clearly distinguish the MPs from other interfering materials with similar morphological characteristics. The use of evolving Artificial Intelligence (AI) technology can be explored to develop methods and procedures for characterization, control the treatment processes involved in MP management, and enhance their efficiencies. Based on the encouraging recent reports, the adoption of AI technology to accurately quantify MPs in the effluent and sludges of WWTPs would potentially reduce contamination issues due to “unaccounted plastics” [24,25,26]. Additionally, machine learning (ML) algorithms were proven to improve the spectral resolution of conventional characterization tools of MPs, thereby improving detection efficiencies [27]. However, additional research is warranted on the application of AI technology and ML models for developing analytical tools for the identification and characterization of MPs. In conclusion, given the steady rise in the research studies establishing the harmful impact of MPs on organisms at all tropic levels, it is imperative that governmental, industrial, and research organizations devote serious attention toward the efficient management and analysis of MPs, before it becomes too late.