Introduction

Plastic products are widely manufactured, many of which are applied only once. Global plastic production has continued to rise, but recycling has lagged behind. It is estimated that between 4.8 and 12.7 million tons of plastic waste end up in oceans annually, via river inputs (Jambeck et al. 2015). Different effects of various types of pollutants have been widely studied (Mirzajani et al. 2015, 2016; Padash Barmchi et al. 2015; Rezaei Kalvani et al. 2019) and recently, there has been more research conducted on emerging pollutants (Jafari Ozumchelouei et al. 2020), such as microplastics. Microplastics are synthetic particles with regular or irregular shape and with size ranging from 1 to 5 mm, which are insoluble in water and have primary (manufactured in micro-sized dimensions) or secondary (large plastics broken down via degradation forces) origin (Frias and Nash 2019; Razeghi et al. 2021a; Zhang et al. 2021). While, there are huge efforts on removal of pollutants from wastewater (Mojoudi et al. 2018, 2019), including biological methods (Alavian et al. 2018; Mansouri et al. 2013; Mirzajani et al. 2017), some evidences suggest that key sources of microplastic pollution in freshwater sources are wastewater effluent and terrestrial run (Hamidian et al. 2021; Lasee et al. 2017). The potential harm to humans҆ health and organisms associated with microplastics can be categorized into three forms, including physical harms, chemicals, and microbial pathogens of biofilms (Campanale et al. 2020a; Naqash et al., 2020; Prinz and Korez 2020). Removal of microplastics by adsorption, filtration, chemical methods to treat microplastics, biological removal, and ingestion methods has been also reported (Othman et al. 2021; Padervand et al. 2020; Tofa et al. 2019). However, this important topic needs more attention from the scientific community.

Prevention or minimization of plastic production, identifying the current state of pollution and filtration are three important strategies toward removal of small-sized plastic particles in environment. After selecting appropriate sampling methods and tools for microplastic detection in environmental samples (Razeghi et al. 2021b), microplastic isolating procedure is the next important stage in microplastic studies, before identifying physical and chemical characteristics of plastic particles. Lack of standard methods in sampling, isolating, and instrumental analysis of microplastic particles from environmental samples leads us to review the literature on microplastic contamination in freshwater environments. Several separation techniques using numerous density separation and digestion solutions have been developed to isolate microplastics from water, sediment, and biological tissues. As a result, a full report on laboratory isolation methods/materials of microplastics and their frequency of use in freshwater studies is presented in this review study. Advantages and limitations of each method are then discussed throughout the paper. As an attempt, the following questions would be answered:

  1. a.

    What are microplastic isolating procedure steps in freshwater studies?

  2. b.

    Which are the most prevalent chemicals utilized in digestion and separation steps?

  3. c.

    What are scientific advantages and disadvantages of water and sediment isolating methods and chemicals?

Data acquisition

Relevant scientific studies were gathered through online search in the databases of ISI Web of Knowledge, Science Direct, and Google Scholar. Keywords, including “microplastic” OR AND “freshwater,” OR AND “plastic particle,” OR AND “plastic fragment,” OR AND “pellets” OR AND “river” OR AND “estuary” OR AND “lake” were considered. Then, the retrieved papers were screened with respect to types of freshwater, including rivers, estuaries, reservoirs, lakes, etc. It should be noted that microplastic research focusing only on microplastics in freshwater species was excluded. However, a combination of water or sediment studies with biota or three of them simultaneously were included. A total of 150 published studies during 2010–2020 were selected and evaluated. Initial data were extracted and recorded in an EXCEL spreadsheet for subsequent analysis.

Isolating procedures of microplastics

After collecting water, sediment, or biological samples from environment, plastic particles in the samples should be separated from organic and inorganic materials. Separation makes it possible to calculate quantity and quality of plastic particles. Pretreatment is done to improve accuracy of subsequent processes for plastic particles, such as isolation, material identification, and counting/weighing (Michida et al. 2019). Challenges in detection of microplastics in environment comprise of three main aspects: (1) the ability to capture plastic particles from water, sediment, and biota samples; (2) separation of plastic particles from other matter (organic and inorganic); and (3) the exact identification of plastic types (Li et al. 2020).

In general, the collected samples from environment go through all or some steps, including size selection, digestion, density separation, and filtration (Fig. 1). After that, the prepared samples are used for further examination and identification. Although, there is not a unique standard method for analysis of microplastics, currently, the main basis of studies includes the guidelines developed by the national oceanic and atmospheric administration (NOAA) (Masura et al. 2015) with slight modifications. A combination of chemical, thermal, physical, and mechanical processes can be used to prepare samples before instrumental analysis (Yonkos et al. 2014).

Fig. 1
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Microplastic main separation steps in freshwater studies. The collected samples from environment go through all or some steps

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Order of the steps may be different in some studies or may be omitted in the others regarding complexity of the sample matrix, organic matter load, and plastic particles҆ size distribution (Vaughan et al. 2017). Zbyszewski and colleagues cleaned visible particles in an ultrasonic bath with deionized water in order to remove sand and other potential surface residues and plastics were carefully separated by hand (Zbyszewski and Corcoran 2011; Zbyszewski et al. 2014).

Sieving

Sieving is an important step to separate large natural materials like sticks, leaves, grass, shells, or human-made waste material. Regarding the accepted defined large size for microplastics, which is equal to 5 mm, most of the studies have used one or a few sieves with different sizes at the beginning of detection of micro-sized plastic particles. In some papers, the collected samples have been subjected to a size selection step using sieves or filters with various pore sizes and other steps have been omitted. For example, water samples from the “Three Gorges Dam,” China, were prepared only by passing the sampled water through the 1.6 mm sieve. Then, liquid was transferred into a separating funnel. Materials retained on the sieve were assessed by the naked eye to pick the suspected plastic debris. Samples were allowed to settle for a week. The floating debris on surface was transferred to petri dishes for identification step (Zhang et al. 2015). Sadri and Thompson (2014) filtered water sample through a set of sieves with varying mesh sizes and then transferred particles onto petri dishes for detection (Sadri and Thompson 2014). Although sieving and visual identification are important and essential steps in microplastic identification, they may not be sufficient, especially for complex sediment samples. Sieving may result in size distribution artifacts, with different particle morphologies, but given nature of the collected material, it is important to remove larger materials as much as possible (Vaughan et al. 2017). It seems that there is a consensus on moving toward a combination of size and density separation using sieves and density separation techniques, which will be discussed in the following.

Digestion

Digestion is a commonly used method to remove non-plastic organic materials that otherwise may negatively interfere with isolation and identification of microplastics. Oxidation, enzymatic digestions, and acid–alkaline digestion are used for organic digestion. Each digestion method is discussed further.

Oxidation

Hydrogen peroxide (H2O2) is a chemical compound with oxidizing ability. In microplastic studies, hydrogen peroxide could be used alone or in combination with a catalyst to increase speed of chemical reaction. Fenton’s reagent or wet peroxide oxidation method is a common digestion procedure in the microplastic studies. In this method, a combination of hydrogen peroxide as oxidizing agent and Fe (ΙΙ) as catalyst are used to digest organic matter. It has been noted that plastic is resistant to the wet peroxide oxidation method (Baldwin et al. 2016; McCormick et al. 2014). Prata et al. (2019) suggested that H2O2 + Fe is appropriate for removal of plant material and KOH (potassium hydroxide) for animal tissues and virgin and weathered plastics did not change in the presence of these oxidizing agents, except for cellulose acetate (Prata et al. 2019). Some studies have reported that this method may alter or potentially digest some of materials in samples (especially nylon and low-density polyethylene). Certain low-density polymers, such as nylon and low-density polyethylene are known to be reactive in exposure to 30% H2O2 (Anderson et al. 2017). Most of the studies have used 30% or 35% peroxide to digest organic matter. However, wet digestion with 50% H2O2 has been used in the previous research as well (Liu et al. 2019). Oxidative digestion is inexpensive but temperature needs to be controlled.

Enzymatic digestion

Enzymatic digestion is a less damaging process that could be a suitable alternative for the wet peroxide oxidation method but it also could be very time-consuming especially for samples containing several different types of organic material (e.g., cellulose, chitin, proteins, and lipids) (Lusher et al. 2018; Michida et al. 2019). Sometimes, a combination of digestion and enzymatic methods is used. Materials of biological origin have been degraded with lipase, protease, amylase, chitinase, and cellulose, in combination with peroxide oxidation (Mani et al. 2015). In a study, protease, amylase, and lipase with hydrogen peroxide were used in microplastic assessment of Saigon River, in Vietnam (Lahens et al. 2018). Samples from urban and highway stormwater retention ponds from Denmark were wet-oxidized on the filters for 2 days by adding 50% H2O2. Subsequent digestion was performed by enzymatic digestion (enzymes of Cellubrix, Viscozyme, and Alcalase) (Liu et al. 2019). Depending on degree of biogenic or silicate debris, some sediment samples from Warnow Estuary, Germany, underwent enzymatic treatment (Enders et al. 2019). Extraction protocol for stormwater pond samples from Viborg City, Denmark, consisted of enzymatic digestion with cellulase followed by oxidation with Fenton’s reagent (Olesen et al. 2019).

Acid–alkaline digestion

Acid digestion is rapid but can degrade some polymers. A combination of nitric acid (HNO3) and hydrochloric acid (HCl) has been employed to digest biogenic matter (Noik and Tuah 2015). A mixture (1:3, v:v) of hydrogen peroxide solution (30%) and concentrated sulfuric acid (H2SO4) was used to destroy natural debris in study of microplastics in river shores҆ sediments of the Rhine-Main area in Germany (Klein et al. 2015). Dubaish and Liebezeit treated the retained inorganic particles with hydrofluoric acid (HF). However, it was noted that polystyrene and polycarbonate may be lost due to their susceptibility toward this acid (Dubaish and Liebezeit 2013). Sediment samples from Lake Bolsena and Lake Chiusi, Italy, were treated with hydrochloric acid for 48 h at room temperature and were additionally digested under heat in order to destroy or at least leach lipid contents of organic material (Fischer et al. 2016). The sediment samples from beach of Lake Garda, Italy, were treated with 100–200 mL of peroxymonosulfuric acid (H2SO5) in order to remove organic residue (Imhof et al. 2016, 2018).

Alkaline digestion causes minimal damages to most of polymers in comparison with acid digestion but it damages cellulose acetates (Michida et al. 2019). Potassium hydroxide is a commonly used chemical compound, especially for digesting biological tissues. Alkaline hydrolysis has been employed for hydrolyzing protein compounds with sodium hydroxide (NaOH) and has been verified as a good tool for separating animals҆ soft tissue (Nan et al. 2020).

The use of a combination of different methods has been reported in research papers. The samples from urban and highway stormwater retention ponds underwent oxidation with Fenton’s reagent and 0.1 M NaOH to further remove organic matter (Liu et al. 2019). Removing organic material via alkaline digestion using KOH/NaClO (Hitchcock and Mitrovic 2019) or two-step protocol using H2O2 followed by sodium hypochlorite solution (NaClO) (Tamminga et al. 2019) has been done in the reviewed studies. Campbell et al. (2017) utilized Fenton’s reagent, 10% NaClO solution and subsequently HNO3/NaClO solution to digest fish’s gastrointestinal tracts (Campbell et al. 2017). Some sediment samples from Warnow Estuary, Germany, were treated with enzymes, both acid and base ones, including HCl and NaOH depending on degree of biogenic or silicate debris (Enders et al. 2019). Treatment of biological tissue with HNO3, NaOH, and H2O2 led to loss of particle fluorescence as well as a strong agglomeration of particles. Tetramethylammonium hydroxide caused a slight decrease in particle fluorescence, resulting in an incomplete dissolution of tissues (Rist et al. 2017).

Density separation

Density separations are conducted by subjecting environmental samples to the concentrated or saturated salt solutions, followed by filtration or other separation techniques to decrease sample mass and mineral matter. Density separation has been adopted for most of the reviewed papers. Various methods and materials have been used in different studies to separate microplastics from water, sediment, and biota samples. Some authors have reported density separation using water for polystyrene, polyethylene, and polypropylene (Vaughan et al. 2017). However, this procedure seems to be insufficient for separation of polymers with higher density. Chemicals used for density separation of microplastics in freshwater studies are listed in Table 1. These solutions represent different density separation limits, and with respect to their densities they can separate polymers smaller than this density. For example, sodium chloride (NaCl), sodium polytungstate (SPT), sodium iodide (NaI), and zinc chloride (ZnCl2) solutions have densities of 1.2 (Kataoka et al. 2019), 1.4 (Hidalgo-Ruz et al. 2012), 1.6 (Claessens et al. 2013), and 1.7 g cm−3 (Imhof et al. 2012), respectively. It has been suggested that for sediment samples, microplastic density separation can be performed with sodium chloride, sodium bromide (NaBr), sodium iodide, or zinc bromide (ZnBr2) (Hu et al. 2018). Sodium chloride is suitable for separation of many microplastics, such as polyethylene, some blends of polypropylene, and foamed polystyrene, which are typically the most common types of plastic found in aquatic environment (Crawford and Quinn 2016). Sodium chloride is commonly used in density separation techniques for microplastic particles and has advantages over other salts, because it is inexpensive, readily available, and has less potential for negative environmental effects (Hendrickson et al. 2018). However, the use of sodium chloride solutions for density separation has been found to be inefficient for separating more dense plastic polymers like polyvinyl chloride (density = 1.30–1.70 g cm−3) and polyethylene terephthalate (density = 1.40–1.50 g cm−3) from environmental matrices (Crawford and Quinn 2016).

Table 1 Chemicals used for microplastic density separation step in freshwater studies
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Other salts, such as zinc chloride, sodium iodide, and sodium polytungstate solution are less commonly used due to their high cost and substances interfering with sediments that might be extracted as well (Wang et al. 2017a). However, they have the advantage of sufficient density for polymers with higher densities, e.g., polyvinylchloride and polyethylene terephthalate and all of them have been used successfully (Crawford and Quinn 2016). Each of these salts has different densities. Most sediment grains have a density of approximately 2.6 g cm−3, which is higher than densities of salty solution; therefore, they sink to the bottom when standing still (Lusher et al. 2018).

Two-step density separation methods have been applied in some studies. The basic idea in this method is using a combination of different density saturated salts. In this method, fluidization of particles occurs in a lower density salt (NaCl) followed by flotation of microplastics in a higher density salt (NaI) (Di et al. 2019; Di and Wang 2018; Hurley et al. 2018b). However, two-step extraction is more time-consuming than flotation using only one type of salt. Air-induced overflow method is sometimes used in two-step separation to force specific lighter particles to move more quickly and frequently to the top layer of the solution (Nuelle et al. 2014).

Separation solution has been prepared in some studies by dissolving potassium formate (KHCO2) in deionized water to a density of 1.5 g cm−3 (Xiong et al. 2018; Zhang et al. 2016, 2017,2019) and calcium chloride (CaCl2) to a density of 1.4 g cm−3 (Grbić et al. 2020). Lithium metatungstate solution (LMT) with the density of 1.6 g cm−3 was used to separate microplastics from denser inorganic particles. It was noted that the original density of lithium metatungstate is 2.95 g cm−3 but it was diluted with water to the specific density (Eo et al. 2019; Watkins et al. 2019a). This is also true for other salts.

Recently, microplastic separation by means of hydrophobic interactions and using oils (e.g., silicone oils, paraffin oils, and corn oil) has been reported as well. In this technique, lipophilic microplastics are extracted from their environmental matrix by attracting the microplastics to an oil layer and non-microplastic particles are segregated in a separation funnel. However, no generally valid recommendations can be given in this method (Dong et al. 2020; Mani and Burkhardt-Holm 2020).

In a study, for efficiently extracting the microplastics from sediment and checking performance of each salt, sodium chloride, sodium iodide, zinc chloride, and potassium formate were tested as separation solutions. Potassium formate was finally chosen due to its relatively better recovery for samples from Pearl River, in China (Fan et al. 2019).

The use of high-density solutions increases extraction efficiency, but very dense solutions cause floating of other wastes in the sample or even sediments, thus reducing efficiency of separation process. The extraction process must be repeated at least three times to achieve the maximum efficiency.

Filtration

Filtration step is the last stage in all the research activities on microplastics and is done before visual/instrumental polymer identification. In this stage, the micro-sized plastic particles in solution or water samples are retained on the top of filter paper or sieve, with specific mesh size. Vacuum pump filtration is used to accelerate and to facilitate this process. Gridded filter paper can make it easier to search and count particles under a microscope.

Other methods for isolating microplastics from environmental samples

In a research, elutriation step using a 1-m long tube fitted with 63 μm mesh at the bottom was performed to reduce amount of sediment to be further treated for assessing 18 streams in and around Auckland City, New Zealand (Dikareva and Simon 2019). Froth flotation is a process that uses affinity for hydrophilicity or hydrophobicity of water to separate materials and is used for separation of plastics (Alter 2005). Centrifugation is a good method to collect supernatants from residue after flotation. Sometimes more than one extraction stage is utilized for better particle separation (Han et al. 2020; Phillips 2020). Sodium hexametaphosphate has been applied in some studies as a dispersant to disperse any microscopic aggregates in sediment samples and then, samples have been subjected to ultrasonic dispersion or sieving (Browne et al. 2010; Egessa et al. 2020; Firdaus et al. 2020; Vermaire et al. 2017). In some papers, extraction protocol and purification step have been started using sodium dodecyl sulfate (SDS) as an anionic surfactant, which is supplemented to avoid agglomeration and to ensure stability of microplastic suspensions (Enders et al. 2019; Lahens et al. 2018; Liu et al. 2019; Mintenig et al. 2020; Olesen et al. 2019). For disaggregation of sediment particles, Rodrigues et al. (2018) employed sodium polyphosphate before sieving sediment sample and stirred the sample for a specific time (Rodrigues et al. 2018). Diluted hydrochloric acid was used for washing the microplastics to remove metal attached to them (Wang et al. 2020b).

Imhof et al. (2012, 2018) extracted plastic particles using the developed semi-automated device called as Munich plastic sediment separator, which was constructed for sediment samples with recovery rates of 100% for large microplastic particles (large microplastics, in the size range of 1–5 mm) and 95.5% for small microplastics. Zinc chloride is used as a separation fluid (Imhof et al. 2012, 2018). This device has advantages like high recovery and reduced time, but it is a highly specialized piece of equipment that is not widely available. Elutriation is a process, in which particles are separated based on their shape, size, and density using a stream of gas or liquid flowing in a direction opposite to that of sedimentation. The technique was first used to separate microplastics from sediment by directing an upward flow of water through a column (Claessens et al. 2013).

For separating microplastic particles lower than 0.5 mm, Wessel et al. (2016) designed a separation process with a series of PVC pipes and connectors that used density differences to mechanically separate sand and plastic particles (Wessel et al. 2016). Plastic debris that escaped the 1 mm sieve with the bulk of sand was separated using a low-cost fluidized density separation system, which used air pump (Noik and Tuah 2015).

Horton et al. (2017) processed sediment samples in three steps in order to determine efficiency of these steps in removing microplastics. The steps included visual inspection of the whole sample, flotation technique, and post-flotation visual inspection. The most effective method of particle removal was flotation, which extracted between 51 and 82% from the total particles. The final post-flotation visual inspection extracted less than 3% of the total particles recovered for three sites of this study (Horton et al. 2017).

Wang et al. (2020) demonstrated that hybrid biochar sand filter is able to remove 60–80% of microspheres presented and has strong potential for removal of microplastic spheres with a size of 10 µm. Plastic particles are immobilized through stuck, trap, and entanglement in biochar porous media (Wang et al. 2020c). The techniques used for separating microplastics in freshwater studies are summarized in Tables 2, 3, 4, 5.

Table 2 Microplastic separation and digestion in freshwater media
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Table 3 Microplastic separation and digestion in freshwater media
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Table 4 Microplastic separation and digestion in freshwater media
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Table 5 Microplastic separation and digestion in freshwater media
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Discussion

There are numerous brine solutions used for density separation of microplastics from environmental samples. Despite lower efficiency of the sodium chloride solution in separating all types of polymer, sodium chloride is the most prevalent salt used in separating microplastics from freshwater studies. The advantages of this salt over other options include ease of access, cheapness, and less potential for negative environmental effects. It has been suggested that the saturated sodium chloride solution commonly used in studies could float up the materials with a density lower than 1.2 g cm−3; therefore it might underestimate microplastic concentration (Wang et al. 2017a). For ensuring about appropriate estimation of the total microplastics, especially in sediment samples, it is recommended to use alternative methods and salts. Solutions with the required density can be prepared by adding distilled water to heavy solutions (Crawford and Quinn 2016). ZnCl2 is the second option among different materials (Fig. 2). It has been revealed that ZnCl2 and NaI solutions are not commonly used due to their high cost and more interfering substances in the sediments that might be extracted as well (Wang et al. 2017a). Zinc chloride has offered adequate density for separation of most polymer types and aids in effective extraction of microplastics. Also, lower cost of ZnCl2 makes it suitable for large-volume samples compared to other high-density separators, such as sodium polytungstate and sodium iodide (Shruti et al. 2019). Advantages and disadvantages of three salts with the highest frequency of use in density separation solution in freshwater studies are listed in Table 6.

Fig. 2
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Frequency of chemicals used for microplastics separation in freshwater studies

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Table 6 Advantages and disadvantages of some commonly used salts for microplastic isolation using density separation method
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The use of oil, CaCl2, and KF has been recently reported. Although liquids with higher density help to better recover microplastic particles, they may be expensive and toxic to environment. Some researchers have used methods, such as elutriation and froth flotation to reduce sample size and consequently, reducing costs (Crawford and Quinn 2016).

Hydrogen peroxide and Fenton’s reagent are the most frequently used chemicals in digestion step and more than 73% of the freshwater studies have reported the use of these chemicals (Fig. 3). Some researchers have reported that this method may potentially digest some polymers (Anderson et al. 2017), while other researchers have addressed the lack or minimal effect of it on micro-sized plastic particles (Hurley et al. 2018a; Tagg et al. 2017).

Fig. 3
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Frequency of digestion methods used in freshwater studies

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Acid–base digestion using different chemical compounds like H2SO4, HNO3, H2SO5, KOH, NaClO, and NaOH has been also reported. Acid digestion can degrade some polymers. For example, polystyrene (PS) particles are pH-sensitive polymers (Erni-Cassola et al. 2017). Base digestion seems to have less effect in altering microplastics, but it may be more time-consuming. Advantages and disadvantages of different digestion methods/materials in microplastic studies are discussed in Table 7.

Table 7 Advantages and disadvantages of different digestion methods/materials in microplastic studies
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Generally, the smaller the size (particularly particles lower than 1 mm) of plastic particle, the more difficult it is to separate them from sample matrix. It seems that the current experimental methods are still insufficient to deal with this fact. It is often challenging to quantify microplastics in complex matrices, such as fine-grained organic-rich samples and biological tissues. Currently employed density and size separation techniques to isolate plastic particles from aquatic environmental samples are not well suited and information cannot easily be compared. Quality control procedures, including blanks and spike recovery should be employed and related results should be reported in experimental studies. Another important issue is the use of high amounts of salts to achieve appropriate density in the density separation step. Therefore, new methods should move toward less chemical use and reusing of solutions. The current methods need to be optimized to increase efficiency, to reduce contamination potential, and to avoid color and structure changes in plastic particles during sample processing.

Conclusion

Size selection, digestion, density separation, and filtration are the main steps in microplastic separation from environmental samples. Among the papers with clearly determined experimental methods, sodium chloride was the most commonly used salt in separating microplastics in freshwater studies. Though, it seems that, sodium chloride solution might not fully isolate high-density microplastic polymers and may lead to false negative errors. Hydrogen peroxide and Fenton’s reagent were the most prevalent chemicals used in digestion step. The preliminary results call for more research efforts to better characterize the microplastics in inland waters. Continuous research is needed to develop efficient methods for separating particles from different substrates. For this purpose, it is necessary to develop standard protocols for better comparison of data and results at different times and places. New methodologies are still emerging and their ability to separate out a wide range of micro-sized polymers with appropriate shapes and sizes found in environment needs to be investigated. Exploring new techniques like centrifugation and anticoagulant use along with common methods is suggested for achieving better results.