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What have we known so far for fluorescence staining and quantification of microplastics: A tutorial review

What have we known so far for fluorescence staining and quantification of microplastics: A... Front. Environ. Sci. Eng. 2022, 16(1): 8 https://doi.org/10.1007/s11783-021-1442-2 REVIEW ARTICLE What have we known so far for fluorescence staining and quantification of microplastics: A tutorial review 1 2 1 1 3,4,5 Shengdong Liu , Enxiang Shang (✉) , Jingnan Liu , Yining Wang , Nanthi Bolan , 6 1 M.B. Kirkham , Yang Li (✉) 1 Key Laboratory of Water and Sediment Sciences of Ministry of Education, State Key Laboratory of Water Environment Simulation, School of Environment, Beijing Normal University, Beijing 100875, China 2 College of Science and Technology, Hebei Agricultural University, Huanghua 061100, China 3 School of Agriculture and Environment, The University of Western Australia, Perth, WA 6001, Australia 4 The UWA Institute of Agriculture, The University of Western Australia, Perth, WA 6001, Australia 5 Global Innovative Centre for Advanced Nanomaterials, College of Engineering, Science and Environment, University of Newcastle, Callaghan, NSW 2308, Australia 6 Department of Agronomy, Throckmorton Plant Sciences Center, Kansas State University, Manhattan, KS 66506, USA HIGH LIGHTS GRAPHIC A BSTRA C T � Fluorescence staining provides a fast and easy method to quantify microplastics. � Factors that influence staining are summarized to obtain an optimum staining effect. � Natural organic matter can be stained by dye and interfere with quantification. � Fluorescence staining is applied in both field and laboratory studies. � Future work involves developing new dyes and automated image-analysis methods. AR TICL E IN F O ABSTRA CT Article history: Understanding the fate and toxicity of microplastics (MPs,< 5 mm plastic particles) is limited by quantification methods. This paper summarizes the methods in use and presents new ones. First, sampling Received 23 July 2021 and pretreatment processes of MPs, including sample collection, digestion, density separation, and quality Revised 22 September 2021 control are reviewed. Then the promising and convenient staining procedures and quantification methods Accepted 12 October 2021 for MPs using fluorescence dyes are reviewed. The factors that influence the staining of MPs, including Available online 15 November 2021 their physicochemical properties, are summarized to provide an optimal operation procedure. In general, the digestion step is crucial to eliminate natural organic matter (NOM) to avoid interference in quantification. Chloroform was reported to be the most appropriate solvent, and 10–20 μg/mL are Keywords: recommended as optimal dye concentrations. In addition, a heating and cooling procedure is Plastic particles recommended to maintain the fluorescence intensity of MPs for two months. After staining, a fluorescence microscope is usually used to characterize the morphology, mass, or number of MPs, but Fluorescence dyes compositional analysis cannot be determined with it. These fluorescence staining methods have been Identification implemented to study MP abundance, transport, and toxicity and have been combined with other chemical Concentration quantification characterization techniques, such as Fourier transform infrared spectroscopy and Raman spectroscopy. More studies are needed to focus on the synthesis of novel dyes to avoid NOM’s interference. They need to be combined with other spectroscopic techniques to characterize plastic composition and to develop image-analysis methods. The stability of stained MPs needs to be improved. © The Author(s) 2021. This article is published with open access at link.springer.com and journal.hep. com.cn 2021 ✉ Corresponding author E-mail: shangenx@163.com (E. Shang), liyang_bnu@bnu.edu.cn (Y. Li) Special Issue—Microplastic and Nanoplastic Pollution: Characterization, Transport, Fate, and Remediation Strategies (Responsible Editors: Wen Zhang, Melissa Pasquinelli & Yang Li) 2 Front. Environ. Sci. Eng. 2022, 16(1): 8 to develop a convenient and cheap quantification method 1 Introduction for MPs present in the environment. Over the past decades, the production and consumption of The fluorescence staining and quantification method plastic products has risen rapidly (Thompson et al., 2004). provides a fast, convenient, and cheap way to quantify Due to the continued discharge of plastic products and their MPs (Maes et al., 2017). This technique uses a dye called low degradation rates, they accumulate in environmental Nile Red (NR, 9-diethylamino-5-benzo[α]-phenoxazi- matrixes, including water, sediments, soils, and the none), a lipid soluble fluorescence dye, to stain neutral atmosphere (Barnes et al., 2009; Liu et al., 2021). Once lipids in biological samples (Greenspan and Fowler, 1985). Afterwards, NR is used to stain synthetic polymers (Jee the plastic products are discharged into the natural et al., 2009). Since 2016, the staining ability of MPs by NR environment, large particles may be fractured, weathered, has drawn the attention of researchers (Shim et al., 2016; or degraded into microplastics (MPs, 0.1 μm–5 mm) and Erni-Cassola et al., 2017; Maes et al., 2017). Maes et al. nanoplastics (NPs, £0.1 μm) via biodegradation and proposed the identification and quantification method for physical and chemical weathering processes (Browne MPs using NR staining and fluorescence microscopy, and et al., 2011; Cole et al., 2011; Duan et al., 2021). they got an average recovery rate of 96.6% for marine Commercial products, such as personal care products and industrial beads, contain ultrafine plastic particles, which MPs, which was cross-validated by FTIR (Maes et al., are regarded as the primary source of MPs and NPs (Alimi 2017). Recently, heating protocols have been developed to et al., 2018). The loads of MPs in freshwater, seawater, enhance the staining effect, and programmed-image- –5 –6 soil, and sediment are 10 –10 items/L, 10 –10 items/L, analysis software has been applied to quantify fluorescence 4 3 1–10 items/kg, and 1–10 items/kg, respectively (McCor- of MPs automatically (Shim et al., 2016; Erni-Cassola mick et al., 2014; Shahul Hamid et al., 2018; Zhang and et al., 2017; Karakolis et al., 2019). The MP fluorescence Liu, 2018; Chen et al., 2020; Zhang et al., 2020). Studies staining and quantification method has achieved a recovery have shown that MPs pose a threat to human health and rate of over 90%, which has been validated by FTIR using ecosystems, because MPs can be transported via food the same batch of laboratory samples (Shim et al., 2016; chains (Carbery et al., 2018; Sun et al., 2019; Liu and Erni-Cassola et al., 2017; Cook et al., 2020; Karakolis Wang, 2020). The increasing load of MPs has drawn the et al., 2019). Therefore, the fluorescence staining and attention of researchers (Wu et al., 2017; Guo et al., 2020; quantification method for MPs provides a promising and convenient technique for researchers. Wang et al., 2021). Thus, studying their abundance is To summarize research on MP characterization and important to assess the impact of MPs on health and to quantification, a literature review of papers published ensure the security of ecosystems. between January 2016 and March 2021 was conducted. A A fundamental step to understand the distribution, fate, total of 1411 articles were investigated with the following and toxicity of MPs is the identification and quantification keywords: microplastics, quantification, characterization, of MPs. A variety of approaches have been developed to detection, sampling, fluorescence, and NR. As shown in detect and characterize MPs (Shim et al., 2017; Fu et al., Fig. 1, 32% of the research focused on quantification of 2020). However, the advantages and limitations of these MPs, which was more than research in any other area. methods vary. Manual counting of MPs by optical microscopy is one of the most widely used methods to Currently, most reviews have concentrated on character- quantify MPs, which has the advantage of convenience but ization of physicochemical properties, transport, and is limited by the operator’s subjectivity (Fu et al., 2020). transformation of MPs, as well as sampling, separation, Scanning electron microscopy (SEM), transmission elec- and digestion procedures of MPs (Hidalgo-Ruz et al., tron microscopy (TEM), and atomic force microscopy 2012; Fu et al., 2020), but the reviews have ignored the (AFM) can provide clear and high-resolution images of fluorescence-quantification method for MPs. MPs, but these methods need to be combined with Thus, we focus on providing a comprehensive overview spectroscopy methods to characterize the chemical com- of the fluorescence staining method to quantify MPs in position of MPs (Patchaiyappan et al., 2020). Analysis by aquatic environments. The objective of this review is to Fourier transform infrared spectroscopy (FTIR) and identify the optimum protocols and best operating Raman spectroscopy can characterize the specific chemical conditions for fluorescence staining and quantification of bonds of MPs, but they are difficult to use to quantify MPs MPs. First, procedures dealing with sample collection, (Fu et al., 2020). Gas chromatography-mass spectrometry separation, digestion, identification, and quantification are (GC-MS) is also an alternative method for measuring the summarized. Next, the advantages and limitations of dye concentrations of monomers or additives in MPs, but it is a staining and the main factors that influence the dying destructive method (Shim et al., 2017). All the above effect, including the physicochemical properties of MPs methods require expensive instruments, experienced and environmental conditions, are summarized. Then, the operators, time-consuming pretreatment processes, and method itself for the identification and quantification of complex data analysis, except for the manual counting stained MPs is reviewed. After discussing the application method via optical microscopy. Therefore, it is necessary of fluorescence staining and quantification methods for Shengdong Liu et al. Fluorescence staining for characterization of microplastics 3 MPs being done in current studies, knowledge gaps and shorelines and bottoms of rivers, lakes, or seafloors, the top future perspective regarding standardized protocols for MP 0–5 cm layer of the surface of beaches and sediments is quantification are proposed. most commonly collected (Patchaiyappan et al., 2020). Sediment cores are also collected to study the occurrence and transport of MPs in aquatic environments. For biota 2 Fluorescence staining methods for MPs samples, fish, invertebrates, and bivalves are most frequently collected. Biota samples are generally collected 2.1 Sampling and pretreatment processes of MPs from wild environments, but some are sampled from commercial operations (Mai et al., 2018). Processes used Sampling and pretreatment processes of MPs include the to separate the MPs from collected samples include collection of samples from the environment and the sieving, filtration, and drying, as shown in Table 1. Sieving extraction of MPs from the samples. Approaches for is generally considered as the first step in sample sampling and extracting MPs from various environmental processing for water and sand or sediment samples, and matrixes, such as freshwater and seawater (Hidalgo-Ruz a mesh of 3 mm or 5 mm is used. et al., 2012; Karakolis et al., 2019), sand and sediments (Nuelle et al., 2014; Besley et al., 2017), and organisms 2.1.2 Digestion and density separation processes of MPs and tissues (Claessens et al., 2013; Avio et al., 2015) have been critically reviewed (Hidalgo-Ruz et al., 2012; Mai The widely distributed NOM in the environment can lead et al., 2018; Prata et al., 2019a). Therefore, we summarize to overestimation of the particle number and environment just the basic steps for the sampling and pretreatment concentration of MPs. Thus, the digestion step is usually processes of MPs. We emphasize the importance of followed by sieving to eliminate NOM (Mai et al., 2018). digestion protocols in fluorescence staining for MPs, The most commonly applied digestion protocol is adding which directly influence the staining effect and the H O (30%) and Fe(II) solution to samples (Prata et al., 2 2 accuracy of detection. 2019a). Then the mixture was heated at 75°C for 0.5–4h. This digestion method is recommended by US National 2.1.1 Sample collection processes Oceanic and Atmospheric Administration (NOAA) (Hanke et al., 2013). Moreover, the procedures of digestion In field studies, different methods and equipment are depend on the source of environmental samples, which applied to collect samples in specific environmental contain different concentrations of NOM. For instance, the matrixes, including water, sand, sediment, the atmosphere, digestion for water samples with low content of NOM by and biota (Table 1) (Dowarah et al., 2020; Scircle et al., H O (30%) is enough, while for sediment or sand samples 2 2 2020; Valine et al., 2020). For water samples, manta nets or containing high concentration of NOM requires H O 2 2 trawls are commonly used to collect large-sized MPs (30%) with Fe(II) solution (0.05 M) and to be heated at (>100 μm) in surface waters, whereas pumping with filters 75°C (Fu et al., 2020). When dealing with biota samples, (100 or 300 μm) is used as a complemental sampling the digestion protocol is extremely important because of method for smaller sized MPs (< 100 μm). For sediment the high biomass content, and an enzyme (such as and sand samples, which are generally sampled from proteinase K, chitinase, and cellulase) is commonly Fig. 1 The proportion of research papers investigating the quantification and characterization of microplastic. 4 Front. Environ. Sci. Eng. 2022, 16(1): 8 Table 1 Sampling and pretreatment processes of MPs for fluorescence staining Sample collection process Sample pretreatment process Sample origin Ref. Sampling Sampling Sampling Density Extraction Sieving Digestion Filtration location equipment details separation recovery Fresh water Kinnickinnic Glass jar NA NA NA 0.05 M FeSO Polycarbonate NA Simmerman River, USA 1L H SO filter & Wasik, 2 4 30% H O 0.4 μm 2020 2 2 Fresh water Four rivers, Plankton Below NA 84.2 mg/L 10% KOH Strainer NA Valine USA tow net river NaCl 200 µm et al., 200-μm surface 2020 0.3-1 m Sea water Mississippi Glass jar Below 25-μm NA 0.05 M Fe (II) Polycarbonate NA Scircle Sound, USA 946 mL surface mesh 30% H O filter et al., 2 2 water 10 µm 2020 Beach sand Three beaches, NA Top of 5-mm CaCl H O Mesh 89.5%- Tiwari 2 2 2 India beach sand mesh 1.34 g/cm 38 μm 97.5% et al., 3–4cm 2019 Sediment South Metal Top layer 3-mm NaCl 0.05 M Fe (II) Vacuum NA Patchaiyappan Andaman spoon of beach 5-mm 1.2 g/cm 30% H O filtration et al., 2020 2 2 beaches, 1cm mesh India Biota Kinnickinnic D-shaped NA NA NA 0.05 M FeSO Steel sieve NA Simmerman (Macroinvertebrates) River, USA kick net 3mL H SO 20 μm & Wasik, 2 4 600-μm 30% H O 2020 2 2 Biota Puducherry Bought NA NA NA 10% KOH Vacuum NA Dowarah (bivalve) coastline, in fish filtration et al., 2020 India market 11 μm Biota Forth Stainless- NA NA Super- Enzyme Vacuum NA Catarino (mussels) River, steel saturated mixture filtration et al., 2018 UK wired NaCl (Corolase 0.8 μm scrubber 7089) Atmosphere Hamburg PE-funnel Above NA NA 15% v/v Vacuum NA Klein & metropolitan PE bottle ground NaClO filtration Fischer, area, level 5–13 μm 2019 Germany 100 cm NA means it is not available in the references. applied to eliminate the tissue (Prata et al., 2019a). In samples are retained on the filter and the pour size of filter addition, there are other digestion methods, such as acid determines the retention particle size. The filters with pour digestion, alkali digestion, oxidizing digestion, and enzy- sizes in the range between 0.2 μm and 55 μm have been matic digestion (Prata et al., 2019a). Detailed digestion used in different studies based on their purpose. The procedures for each method have been discussed in smaller pour size of filter they used, the smaller sized published reviews (Prata et al., 2019a). plastic particles can be retained on the filter for further After the digestion step, density-separation procedure is detection. In addition, considering the visual observation conducted to separate specific MPs from water, soil, or of MPs, the filter should not exhibit a fluorescence signal sediment samples. Based on the different densities of and interfere the detection result. For example, a study 3 3 plastics (0.8–1.6 g/cm ) and sediment (2.7 g/cm ), density- tested 6 types of filters and found that only glass fiber filter separation methods have been developed to separate MPs (1.2 μm) and black polycarbonate filters (0.2 μm) are and sediment or sand by mixing the samples in salt- appropriate for the detection of NR-stained MPs without saturated solutions and afterwards collecting the super- introduction of fluorescence intensity from the filters (Prata natant, which contains MPs (Rocha-Santos and Duarte, et al., 2019b). Some plastic particles directly identified by 2015). In the density-separation step, the most frequently visual observation cannot use the special filter membrane. used salt is NaCl, while other salts such as CaCl , NaI, and After filtration, drying procedures (generally at 60°C) are ZnBr are also available (Prata et al., 2019a). Filtration is carried out, and they are indispensable as the final steps for necessary after the digestion protocol, where all the sample processing. Shengdong Liu et al. Fluorescence staining for characterization of microplastics 5 2.1.3 Quality assurance and quality control in the sampling important to get accurate and reliable data and high process recovery rates during the entire sampling processes. Guaranteeing quality assurance and quality control (QA/ 2.2 Methods for fluorescence staining of MPs QC) in the sampling process is essential to get reliable findings concerning the abundance of MPs. For example, Fluorescence methods are distinguished from regular, procedural blanks (only containing water) and spiked optical approaches to detect and quantify MPs, because blanks (containing water with known composition and they require a staining process during pretreatment. The number of MPs) should be analyzed during sample fluorescence staining methods for MPs can be categorized collection and processing of samples (Hanke et al., 2013; into staining MPs on filter paper or in solution. The first Catarino et al., 2018). In addition, the recovery rate of MPs dying method for MPs on filter paper includes collecting throughout the whole sampling process should be reported the environmental samples, separating and extracting the to reveal the actual level of MPs in the environment. MPs from the environmental samples, placing the MPs on Determining the recovery rate is also beneficial, because it filter paper (usually a polycarbonate filter paper), and allows comparison of the abundance with other studies adding the dye solution on the filter paper to stain the MPs (Wiggin and Holland, 2019). Currently, the recovery rates for a period of time (2–4 h) (Shim et al., 2016; Erni- of generally used sampling processes (sieving, digestion, Cassola et al., 2017). After the staining procedure, the density separation, and filtering) for MPs can reach number or concentrations of the stained MPs are detected 83.3%–96.6% (Maes et al., 2017; Tamminga, 2017). In by a fluorescence microscope or spectrophotometer. field sampling, non-plastic collection tools and storage The other method is staining MPs in solution (Cook containers should be used to avoid cross contamination. et al., 2020; Karakolis et al., 2019), as shown in Fig. 2. The During laboratory sampling, it is necessary to wear latex first step is preparation of the staining solution (Fig. 2a), gloves and cotton clothes to avoid contamination from which is an organic solvent that dissolves the NR. The airborne fibers, which are widely detected in the environ- most commonly used solvents include methanol, chloro- ment via atmospheric fallout (Ziajahromi et al., 2017). form, acetone, and n-hexane (Tamminga, 2017). The Overall, quality assurance and quality control protocols are second step is suspending MPs in the staining solution Fig. 2 MP staining process in solution. (a) adding Nile Red in solvent, (b) staining in solution while heating and cooling, (c) vacuum filtering, (d) Nile Red stained MPs. 6 Front. Environ. Sci. Eng. 2022, 16(1): 8 (Fig. 2b). The heating and cooling procedures may fibers are especially difficult to be stained compared with enhance the intensity of the fluorescence signal and inhibit particles and fragments due to their irregular shapes leaching of the dye (Lv et al., 2019). The third step is (Tamminga, 2017). Moreover, the color of MPs may filtering the staining solution to get the fluorescence- disturb the staining effect and quantification. Most stained MPs and washing them with deionized water to get commonly used plastics are white, which plays a minor rid of the adsorbed dye (Fig. 2c). Then, the fluorescence- role in detection after staining. But certain polymer types, stained MPs are obtained, and they usually show red or like PVC in black color, are difficult to be stained and purple color when NR is used as dye (Fig. 2d). detected (Shim et al., 2016). The composition of MPs determines the affinity between In early studies, the staining method on filter paper was MPs and dyes. As a hydrophobic dye, NR preferentially commonly applied and was used for most types of combines with polymer materials such as PP and PE that polymers (Shim et al., 2016; Erni-Cassola et al., 2017). In recent years, staining in solution is an improved method have a low polarity. Fluorescence staining using lipophilic due to stirring and the addition of heating or cooling dyes, such as NR, has proven to be effective in procedures. Therefore, recent studies tend to stain MPs in quantification of small particles of PE, PP, PS, PC, PUR, solution (Stanton et al., 2019; Konde et al., 2020). This and PEVA, because of their high hydrophobicity, whereas staining method requires MPs to be distributed in a PVC, PA, and PES could not be detected after staining due homogeneous solution. Thus, it may be difficult to use with to their low hydrophobicity (Shim et al., 2016). Among the certain types of MPs such as PE, which tends to float on the physicochemical properties of MPs, the chemical compo- surface of water due to its low density. To solve this sition of the polymer plays a primary role in the staining problem, researchers have suspended a powder of PE in a effect, and it determines the affinity between MPs and mixture of ultrapure water and dimethyl sulfoxide (v = 1:1) dyes. To note, the fluorescence staining has minor effect on for better distribution in solution (Cook et al., 2020). In the aggregation of MPs. On the one hand, Raman spectrum conclusion, to get a better staining effect, the staining and FTIR spectra measurements prove that the stained method for MPs in solution is recommended, which has MPs have almost no alteration on composition compared the advantages of flexible heating or stirring procedures. to pristine MPs, because only a few dye molecules are added into MPs surface by diffusion (Lv et al., 2019). On 2.3 Factors influencing the staining effect the other hand, the Zeta potentials of pristine PET MPs and NR stained PET MPs have been measured, their Zeta The staining effect of MPs depends on many factors, such potentials are – 6.000.78 mV and – 7.640.81 mV, as dye concentration, solvent type, and temperature respectively. This proves that staining process has a minor (Wiggin and Holland, 2019). In this section, we compre- effect on the surface charge of MPs. Similarly, the Zeta hensively discuss how these factors influence the staining potentials of stained PE, PP, and PVC MPs were also effect to develop an optimum MP staining protocol. measured and no significant alteration of Zeta potentials has been observed. 2.3.1 Physicochemical properties of MPs 2.3.2 Experimental parameters The physicochemical properties of MPs, such as particle size, shape, and composition, affect the staining process 1) NR concentration (Wiggin and Holland, 2019). The recovery rate of fluorescent-stained MPs decreases as the size of particle Fluorescence intensity of stained MPs is influenced by the decreases. For example, studies have found that recovery concentration of NR, which has been applied in the range rates of NR-stained MPs decreased from 82% to 49% of 0.1 μg/mL and 500 μg/mL in current studies (Rumin when the size decreased from 500 to 1000 μmto20–63 et al., 2015; Lv et al., 2019). For example, Lv et al. found μm, respectively (Wiggin and Holland, 2019), which may that fluorescence intensity first increased (0.1–25 μg/mL be because the smaller particles are more difficult to be NR concentration) and then decreased (25–100 μg/mL NR detected under a microscope. Moreover, approximately concentration) with NR concentration (Lv et al., 2019), 95% of particles with sizes larger than 1 mm could be which was attributed to NR aggregation at high dye detected after staining by NR, while only 71.7% of concentration that decreased fluorescence intensity (Rumin particles smaller than 1 mm could be detected (Tamminga, et al., 2015). Similarly, the fluorescence intensity increased 2017), which was primarily because the small particles and then decreased with rising dye concentration (0.1– were stained less strongly and exhibited a weaker 100 μg/mL) when selecting Fluorescein isophosphate fluorescence intensity; therefore, they were more difficult (FITC) to stain MPs. The fluorescence intensity increased to be detected than larger particles. with Safranine T dye concentration increasing from In addition, the shapes of the MPs can influence the 0.1 μg/mL to 100 μg/mL, while it changed slightly until staining effect, which are generally categorized as the dye concentration reached saturation at 100 μg/mL (Lv particles, fragments, or fibers. Studies have indicated that et al., 2019). Moreover, a high NR concentration, such as Shengdong Liu et al. Fluorescence staining for characterization of microplastics 7 500 μg/mL, may lead to saturation of spectrometer surface, or the entrance of dye inside polymer molecules, intensity. An obvious red shift in fluorescence spectra of depends on temperature. Fluorescence is exhibited only for PVC could interfere with the fluorescence measurement a short period when MPs are stained at room temperature and analysis (Konde et al., 2020). Thus, attention should be (25°C) without heating (Prata et al., 2020). For example, paid to avoid using too high a NR concentration for after 2 months, 73.5% of NR-stained MPs without heating staining. Conversely, low NR concentration (< 1 μg/mL) were found to have lost fluorescence (Prata et al., 2020), which was attributed to the desorption of dye molecules exhibits too weak fluorescence signals to detect (Konde from the MP surface because the dyes were just adsorbed et al., 2020). Therefore, an appropriate NR concentration onto the surface. Studies have found that the heating between 10 μg/mL and 20 μg/mL is suggested in NR protocol promotes the staining effect resulting in both staining protocols, which gets both adequate fluorescence higher fluorescence intensity and a more stable florescence intensity and minimum interference (Tamminga, 2017). signal for a long period (over 2 months) (Lv et al., 2019). 2) Solvent Fluorescence intensity of MPs has shown an increasing trend with increasing temperatures from 20°C to 50°C NR is a hydrophobic dye with poor solubility and weak (Konde et al., 2020). Most types of MPs have their fluorescence in water (Greenspan and Fowler, 1985). It strongest fluorescence intensity at 50°C, except for PVC first needs to be dissolved in a solvent for staining. The which has its strongest fluorescence at 75°C (Lv et al., solvent has a significant impact on the NR staining effect. 2019). Furthermore, heating protocols have enabled On the one hand, the solvent directly affects the recovery stained MPs to maintain a stable fluorescence intensity rate of the fluorescent-stained MPs. On the other hand, NR over 2 months (Lv et al., 2019). Heating protocols have contains a polar carboxyl function (-COOH) on its been applied in several studies and to obtain high recovery aromatic rings; therefore, NR fluorescence spectra exhibit rates (> 95%) (Cook et al., 2020; Karakolis et al., 2019; Lv dependency on the polarity of solvents due to the et al., 2019). The heating temperature of staining solutions solvatochromism of NR (Rumin et al., 2015). Because of in previous studies have generally been set from 50°C to the relatively polar nature of NR molecules when 75°C (Cook et al., 2020; Karakolis et al., 2019; Lv et al., compared to plastics, the partitioning of NR molecules 2019; Konde et al., 2020). The temperature should be from the solvent to plastics can be made more feasible in lower than the melting point of some polymer materials non-polar solvents, such as n-hexane, than in polar such as LDPE (85°C). PVC and PET are less hydrophobic solvents (Shim et al., 2016). plastics and, thus, are difficult to be stained by NR. The use The use of various solvents, such as chloroform, of a heating protocol enables PVC and PET to exhibit acetone, n-hexane, and methanol, has been summarized strong fluorescence intensity (Lv et al., 2019). Overall, to in the literature (Tamminga, 2017; Konde et al., 2020). obtain stronger fluorescence intensity and a more stable Tamminga et al. investigated the influence of three staining effect for MPs, the protocol of heating staining solvents, which were acetone, chloroform, and n-hexane, solutions is recommended. When the melting point of on the staining effect of MPs by NR. Chloroform was the polymer materials is considered, 50°C–60°C would be an most appropriate solvent for HDPE, LDPE, PP, and PVC, appropriate temperature range to use for NR staining and the lowest recovery rate was 83.3% (Tamminga, protocols. 2017). Nevertheless, they found that some polymer materials, such as cellulose acetone and PS, had the 2.4 Advantages of fluorescence staining for MPs tendency to dissolve in chloroform or acetone (Tamminga, 2017). Konde et al. proposed a mixed solvent consisting of Fluorescence staining for quantification of MPs provides a acetone and ethanol with v/v = 1:1 for the investigation of straightforward, quick, cheap, and convenient technique to MP photoluminescence spectra, and the mixed solvent detect mass concentration of MPs and to investigate their exhibited strong fluorescence intensity without PVC distribution in environmental samples. Spectral methods or deformation (Konde et al., 2020). Overall, it is necessary chromatographic methods can cost up to hundreds of to select an appropriate solvent to avoid leaching of the dollars for each batch of samples, while the cost of NR is additive or monomer from MPs; to develop specific only $ 8.36 USD/g plastic with the additional need for fluorescence spectra for each solvent; and to get reliable fluorescence microscopy (Karakolis et al., 2019). detection results of fluorescence-stained MPs. A mixture of Fluorescence-staining methods also have been repre- solvents, like acetone and ethanol, appears to offer a good sented as atimeefficient and convenient way for choice for staining. quantification of MPs. The staining protocol of MPs could be as short as 30 min for one batch of samples, while 3) Temperature spectral methods take a longer time for detection of MPs. For example, FTIR requires at least nine hours to scan one Temperature has a significant impact on the staining effect, filter paper (Shim et al., 2017) including the requirement of because the adsorption of the dye onto the NP or MP sample pretreatment time. The work experience required 8 Front. Environ. Sci. Eng. 2022, 16(1): 8 for quantification of MPs via fluorescence techniques is After confirming the fluorescent particles as MPs, these much less than that needed for spectral (i.e. FTIR) or mass particles are counted either by manual work or automated spectrometry methods (Rocha-Santos and Duarte, 2015). software (such as ImageJ) to obtain the number of Quantification of MPs via fluorescence can be operated by particles. To be specific, the total number of low abundance automated photo-analysis software. In contrast, the mass of MPs can be counted directly on the entire filter. While spectrometry method, like GC-MS, requires well-trained counting the high abundance of MP samples, it is operators as well as time-consuming pretreatment pro- commonly to count the total particles from 3 random cesses (Rocha-Santos and Duarte, 2015). Overall, the fields of view and averaging these counts, then comparing major advantages of fluorescence-staining methods are that the viewed area with the entire filter area and normalizing the observed numbers to the whole filter area (Simmerman they are time efficient and easy to use, and they are and Wasik, 2020). appropriate for the detection of the abundance of MPs in bulk environmental samples. The influence of the excitation wavelength on the In addition, NR staining protocols improve the count detection of stained MPs has been explored. Prata et al. efficiencies of smaller-sized MPs compared with those tested NR-stained mixtures of polymers (PE, PP, PS, PVC, taken with regular visual quantification methods. A study EPS, nylon) and organic matter and excited them under showed that NR staining increased the detection number of light with multiple wavelengths (254, 365, 470, 495, 530, MPs in every sample, and the greatest increase of the 625 nm) (Prata et al., 2020). The results showed that numbers observed was in the smaller sized fraction (< 124 excitation light at 254 nm had the advantage of a high μm) (Wiggin and Holland, 2019). Furthermore, this study contrast with the background signals without interference showed that the reported levels of MPs determined via the with organic matter. But PS, PVC, nylon, virgin HDPE, NR staining and counting approach were higher than those and weathered PE could hardly be detected under the determined by other methods worldwide, and this was 254 nm excitation wavelength. Most polymers (PE, PP, likely due to the inclusion of smaller sized MPs after HDPE, PS, EPS) and NOM could be excited under 470 nm dyeing samples from a highly urbanized aquatic environ- at the same time. Therefore, application of the 470 nm ment (Los Angeles, California) (Wiggin and Holland, excitation wavelength requires a digestion step to remove 2019). Overall, smaller-sized MPs are more likely to be NOM (Prata et al., 2019b). In conclusion, the 254 nm detected by the NR staining method, and it avoids excitation wavelength is suitable for limited types of underestimation of MP abundance in the environment. polymers (PE, PP) with the advantage of less interference However, there are challenges for detecting the small-sized by NOM. The excitation wavelength at 470 nm can excite MPs, and the detection limit by the current fluorescence most types of polymers but it can excite NOM as well, which requires digestion procedures to avoid interference staining method for the size of MPs was down to 3 μm (Wiggin and Holland, 2019). Meanwhile, the detection with the NOM’s fluorescence signal. limit of MPs mass concentration by fluorescence staining During the identification procedures, the fluorescence methods lies in the range between 1 mg/L and 100 mg/L signals from MPs have different colors (Fu et al., 2020). (Li et al., 2019). The NR-stained plastics appear orange color in a chloro- form solvent when excited by blue or UV light (Maes et al., 2017; Tamminga, 2017). In addition, MPs can fluoresce in 3 Fluorescence identification and quantifi- varied light ranges when stained by different dyes. For cation methods for MPs example, iDye pink (pink dye), iDye blue (blue dye), and Rit DyeMore Kentucky Sky (Kentucky dye) can fluoresce 3.1 Identification in the red range, the far red range, and the green and red (both) ranges, respectively (Karakolis et al., 2019). Basically, identifying MPs with fluorescence microscopes With the help of image-analysis techniques, the hydro- includes the following steps: 1) the samples are placed phobic and hydrophilic characteristics of fluorescence under the fluorescence microscope and are excited with the particles can be identified. Using this method, a simple proper excitation wavelength; 2) the samples are observed “fluorescence index” can be calculated as (R + G)/R (‘R’ or photographed under the proper emission wavelength; and ‘G’ are the 8-bit color intensity values of red and 3) the fluorescence signal or images are detected and green, respectively). This index represents the “polarity” of analyzed for identification. In detail, the particles were the polymer surface, and the larger the value of the index identified as MPs by the following criteria: 1) no cellular or is, the higher hydrophobicity of the polymer particles is organic structure are observed; 2) fiber particles are (Maes et al., 2017; Tamminga, 2017; Wiggin and Holland, uniform in thickness throughout their whole length and 2019). Recently, an automated counting software (MP- have no three-dimensional bending; 3) the colored VAT, Microplastics Visual Analysis Toll) has been particles should present clear and homogeneous colors; developed, which can be applied to detect the sizes and 4) fiber particles have no segment; 5) particles do not shine shapes of stained MPs through their emitted light (Prata (Nor and Obbard, 2014; Klein and Fischer, 2019). et al., 2019b). Shengdong Liu et al. Fluorescence staining for characterization of microplastics 9 3.2 Quantification investigate their aggregation and settling mechanisms in sandy water. In this study, a fluorescence spectrophot- The identification of MPs is commonly followed by a ometer was used to measure the fluorescence intensity of quantification procedure. Identification can provide infor- MPs, and then the MP mass concentration was further mation about the morphology and surface properties of calculated using a standard curve (Li et al., 2019). In MPs, while quantification can show the number and mass conclusion, fluorescence MPs provide a fast method for concentrations of MPs in samples, in order to study quantification of MP concentration, and the particles can abundance and occurrence of MPs (Mai et al., 2018). easily be detected using their fluorescence intensity. Procedures for quantification with fluorescence micro- The quantification methods used for field samples and scopes involve the following steps: 1) the MPs are excited laboratory samples are different. In one study, field under an excitation wavelength; 2) their fluorescence samples were first photographed under a certain excitation signal is observed; and 3) the fluorescence particles are wavelength (Klein and Fischer, 2019). Then, images were counted or selected MPs are picked up for further mass analyzed by software to count the number of fluorescence weighing and quantifying (Prata et al., 2020). plastic particles. The size and particle circularity could also In addition, using a fluorescence spectrophotometer to be determined by image-analysis software (Sfriso et al., determine the mass concentration of MPs in a water matrix 2020). As for laboratory samples, they can be detected is also feasible, based on a standard curve that is developed easily and accurately by using a fluorescence spectro- to show the relationship between fluorescence intensity photometer, because they have a known polymer type and and MP concentration (Li et al., 2019). An example for few impurities (Li et al., 2019). Recently, fully- or semi- quantification of stained PET using a fluorescence spectro- automated analytical methods have been applied to photometer is shown in Fig. 3. This method is especially quantify MPs, which offer a promising way of MP appropriate for simulated samples done under laboratory quantification in the future. Confocal microscopy also conditions, which consist of pure polymers and little NOM has provided a straightforward way to observe stained (Shim et al., 2016). For example, a study was done MPs. A study demonstrated that artificially generated MPs with fluorescence polystyrene nanoplastics (PSNPs, are more recognizable under confocal microscopy than 100 nm) and polyethylene MPs (PEMPs, 1.0–1.2 mm) to field samples (Maxwell et al., 2020). Therefore, confocal Fig. 3 Quantification of MPs using fluorescence spectrophotometer (a) fluorescence stained PET microplastic when being excited, (b) fluorescence stained PET microplastic under bright-field, Scale bar = 650 μm, (c) correlation curve between fluorescence intensity and mass concentration of PET microplastic. 10 Front. Environ. Sci. Eng. 2022, 16(1): 8 microscopy could be more appropriate in laboratory tions of MPs were 45–220 items/kg of dry sand (South studies (Maxwell et al., 2020). The use of Laser Confocal Andaman beach) and in the other study they were 161.7– Raman Spectroscopy in identifying and quantifying MPs 973.3 items/kg of dry sand (Girgaon Mumbai, Tuticorin can increase the sensitivity and accuracy of analysis. This beach, Dhanushkodi beach) (Tiwari et al., 2019; Patch- advanced technology can identify the physical properties aiyappan et al., 2020). Simmerman et al. examined the MP and chemical compositions of MPs, with the advantages of levels in water and organisms in a cold-water stream in high spectral coverage, high lateral resolution, specific western Wisconsin, USA. They found that the concentra- fingerprint spectrum and low interference from organic tions of MPs stained by NR in water ranged from 545 to matter/water/fluorescence background signals (Sobhani 3622 items/L and increased significantly from upstream to et al., 2019). Thus, Laser Confocal Raman Spectroscopy downstream. The mean MP concentrations downstream of an urban area were 2–3 times those found upstream has a promising future especially in the study of transport and transformation of nanostructured materials in natural (Simmerman and Wasik, 2020), which may be because waters. MPs move down stream with the water flow. Klein et al. examined the MP abundance in atmospheric disposition via fluorescence MP quantification. They determined that 4 Application of information from studies of the mean MP abundance was 275 items/m /d. To fluorescence-stained MPs characterize the chemical composition of MPs, Raman spectroscopy was combined with fluorescence quantifica- 4.1 Quantification of MPs input in the environment tion, and polyethylene/ethylvinyl acetate copolymers were found to dominate within the metropolitan area of Microplastics can enter the terrestrial, aquatic, and atmo- Hamburg, Germany (Klein and Fischer, 2019). In conclu- spheric environments directly through indiscriminate sion, dye stained MPs and the fluorescence quantification disposal of plastic wastes and indirectly through applica- approach have been applied in field samples to examine the tion of waste resources containing plastics such as abundance of MPs, which are generally coupled with biosolids and composts (Bradney et al., 2019; Kumar spectroscopic methods to characterize the composition of et al., 2020). The fluorescence-quantification method of MPs (Vermaire et al., 2017; Klein and Fischer, 2019; MPs has been developed in recent years, and it has been Patchaiyappan et al., 2020). validated to be a straightforward and cost-effective way to quantify the input of MPs into environments through 4.3 Investigation of distribution of MPs in organisms various waste sources (Lares et al., 2019). For example, Lares et al. compared six different methods to detect MPs Assessment of the toxicity of MPs in organisms requires in municipal wastewater and digested sludge samples, characterization of their distribution in organisms. Fluor- spiked with seven different types of plastic particles and escence detection methods have been widely applied in fiber, and they suggested that a staining method using Rose distribution studies of MPs, which may facilitate toxicity Bengal could be useful in separating MPs from other assessments of marine organisms and evaluation of human materials (Lares et al., 2019). Another study examined the health risks (Catarino et al., 2018; Maxwell et al., 2020). occurrence of MPs in wild mussels via fluorescence Maxwell et al. reported a novel counterstaining method by staining using NR. The results showed that the mean NR, Evans blue, and Calcofluor white dyes to detect MPs concentration of MPs entering the body of Modiolus (a in terrestrial, invertebrate samples. The results could be kind of mussel) was 0.0860.031 items/g wet weight used for investigation of MP ingestion by soil animals (Catarino et al., 2018). The authors also reported that the (Maxwell et al., 2020). They studied the tissue distribution ingestion of airborne fibers by humans during a meal can of PS-MPs in red tilapia (O. niloticus) during a 14-d be up to 13731– 68415 items/y/person. In these studies, the exposure period. They found that the concentration of PS- 4 4 staining method was applied to separate MPs from other MPs in the fish gut was 171.1  10 3.5  10 μg/kg, and materials and to quantify the input of MPs into the the concentration of microplastics in organs were in the environment. order of gut>gills>livers≈brain (shown in Fig. 4) (Ding et al., 2018). Overall, fluorescence staining of MPs can be 4.2 Measurement of MPs abundance in the environment used to study the distribution and accumulation of MPs in tissues of organisms, and the results can be helpful for the The fluorescence-quantification method has been applied assessment of the toxicity of MPs to evaluate human health to measure the concentrations of MPs in samples of fresh risk. water, seawater, soil, beach sand, sediment, airborne MPs, and biota (Vermaire et al., 2017; Gagné et al., 2019; Klein 4.4 Investigation of the environmental fate and transport of and Fischer, 2019; Simmerman and Wasik, 2020). In two MPs studies in India, the abundance of MPs in beach sand was determined by NR-staining MPs. In one study, concentra- Fluorescence staining and quantification methods have Shengdong Liu et al. Fluorescence staining for characterization of microplastics 11 Fig. 4 Photographs of fish tissues under a bright-field microscope (top row) and representative fluorescence images of PS-MPs in different fish tissues after 14 d of the exposure to 100 μg/L (bottom row), Scale bar = 100 μm. Graph was adapted from ref (Ding et al., 2018) with permission. been applied to determine the environmental fate of MPs. 5.1 Strengthen research on the synthesis of novel dye to Results from simulated, laboratory experiments are avoid interference of organic matter extremely useful. In simulated experiments done under laboratory conditions, Li et al. studied the aggregation Organic matter is ubiquitous in natural aquatic, terrestrial, behavior of MPs in suspended sediment (SS) using or biotic environments. Dyes can co-stain it along with plastic particles, which interferes with the detection of MP fluorescence PSNPs and PEMPs to quantify the concen- fluorescence intensity and results in overestimation of the tration of MPs (Li et al., 2019). They found that PSNPs and abundance of plastic particles in samples, and, conse- SS formed heteroaggregates and settled in a water column quently, their accumulation and toxicity is overstated at a settling velocity of 0.010 m/s, which can affect the (Stanton et al., 2019). Therefore, pre-purification of distribution and fate of PSNPs in aquatic environments. In samples is required to remove organic matter. In a study contrast, the settling velocity for heteroaggregates of –5 PEMPs and SS was low (~10 with benthic invertebrate samples, it was found that the use m/s), which indicated that the effect of SS on the settling and distribution of of the NR staining method should take digestion protocols PEMPs is negligible. In addition, Cook et al. used NR- into consideration, because chitin cannot be removed by stained PEMPs to measure their longitudinal dispersion H O digestion. It can get stained by NR and exhibits a 2 2 coefficients in laboratory flumes (Cook et al., 2020). They strong fluorescence intensity leading to false positives calculated that the longitudinal dispersion coefficients (Sfriso et al., 2020). Thus, future studies should be done to ranged from 0.0030 to 0.0690 m /s for PE particles. Their develop counterstaining methods with novel dyes to results were used to establish a model concerning the distinguish the fluorescence intensity of MPs from that of transport of MPs in natural rivers. In summary, fluores- organic matter in order to avoid interfere in the quantifica- cence staining and quantification methods facilitate a tion of plastic particles. comprehensive understanding of the fate and transport of MPs in aquatic and terrestrial environments. 5.2 Combine fluorescence staining with other chemical methods to analyze MPs composition 5 Conclusions and future prospects The fluorescence-staining technique facilitates the quanti- fication of the number, concentration, or abundance of MPs in various environmental matrixes. However, the The novel fluorescence staining and quantification method is straightforward, quick, cheap, and reliable for quantifi- fluorescence-staining method cannot stain all types of cation of MP abundance and MP distribution in the polymers, because of the different affinity between dyes environment, including water, soil, sediments, and organ- and plastics (Stanton et al., 2019). For example, NR can isms (Duan et al., 2021; Liu et al., 2021; Wang et al., stain plastics like PP, PE, PS, PC, EPS, PU, and PEVA but 2021). The method is especially suitable for detection of cannot stain plastics like PVC, PA, and PES (Shim et al., MPs in large, bulk environmental samples and laboratory 2016). In addition, the NR-staining method does not samples. However, challenges regarding detection of MPs provide information about the chemical bonds of the and quantification of their concentrations remain, as detected MPs, and the method may be combined with described in the following sections. spectroscopic techniques to determine the chemical 12 Front. Environ. Sci. Eng. 2022, 16(1): 8 composition of plastic particles (Erni-Cassola et al., 2017). developed to provide the mapping image of the sample To better elucidate the transport, availability, and toxicity and high lateral resolution of optical images (Sobhani of MPs, combinational analysis, in which fluorescence et al., 2019). However, how to improve the resolution of staining is used with chemical identification methods like measurement when detecting nanoplastics and overcoming FTIR and IR, is a current trend in studies concerning the nano- scale effects are huge challenges. Future work is occurrence and abundance of MPs. suggested to develop detection methods of nanoplastics to improve superior analytical algorithms and enhance the 5.3 Develop image-analysis methods for quantification of weak signal from nanoplastics. stained MPs Acknowledgements This study was supported by the National Key R&D Program of China (Grant No. 2017YFA0605001), the National Natural Developing fully- or semi-automated image-analysis Science Foundation of China (Grant Nos. 52170024, 21677015 and protocols when using the fluorescence staining and 22006031), the Natural Science Foundation of Hebei Province (No. quantification method has a broad future. Currently, B2019204315), and the Sponsored Research Overhead Fund (Grant No. manual counting of the numbers of MPs under fluorescent 472120) from Kansas State University. or optical microscopy is the most applied of the methods Open Access This article is licensed under a Creative Commons used for the quantification of the abundance and Attribution 4.0 International License, which permits use, sharing, adaptation, concentration of MPs. It has a low efficiency and may distribution and reproduction in any medium or format, as long as you give cause error. Therefore, automated image analysis would be appropriate credit to the original author(s) and the source, provide a link to the helpful and provide researchers with both accuracy and Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative efficiency to save time and labor. The challenge for the Commons licence, unless indicated otherwise in a credit line to the material. future of image-analysis methods includes the counting of If material is not included in the article’s Creative Commons licence and your complex aggregates, which are widely distributed in intended use is not permitted by statutory regulation or exceeds the permitted aquatic and terrestrial environments. use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 5.4 Improve the stability of stained MPs The migration, transport, and transformation of MPs in References natural aquatic, terrestrial, or biotic systems is a long-term Alimi O S, Farner Budarz J, Hernandez L M, Tufenkji N (2018). process. 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What have we known so far for fluorescence staining and quantification of microplastics: A tutorial review

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Abstract

Front. Environ. Sci. Eng. 2022, 16(1): 8 https://doi.org/10.1007/s11783-021-1442-2 REVIEW ARTICLE What have we known so far for fluorescence staining and quantification of microplastics: A tutorial review 1 2 1 1 3,4,5 Shengdong Liu , Enxiang Shang (✉) , Jingnan Liu , Yining Wang , Nanthi Bolan , 6 1 M.B. Kirkham , Yang Li (✉) 1 Key Laboratory of Water and Sediment Sciences of Ministry of Education, State Key Laboratory of Water Environment Simulation, School of Environment, Beijing Normal University, Beijing 100875, China 2 College of Science and Technology, Hebei Agricultural University, Huanghua 061100, China 3 School of Agriculture and Environment, The University of Western Australia, Perth, WA 6001, Australia 4 The UWA Institute of Agriculture, The University of Western Australia, Perth, WA 6001, Australia 5 Global Innovative Centre for Advanced Nanomaterials, College of Engineering, Science and Environment, University of Newcastle, Callaghan, NSW 2308, Australia 6 Department of Agronomy, Throckmorton Plant Sciences Center, Kansas State University, Manhattan, KS 66506, USA HIGH LIGHTS GRAPHIC A BSTRA C T � Fluorescence staining provides a fast and easy method to quantify microplastics. � Factors that influence staining are summarized to obtain an optimum staining effect. � Natural organic matter can be stained by dye and interfere with quantification. � Fluorescence staining is applied in both field and laboratory studies. � Future work involves developing new dyes and automated image-analysis methods. AR TICL E IN F O ABSTRA CT Article history: Understanding the fate and toxicity of microplastics (MPs,< 5 mm plastic particles) is limited by quantification methods. This paper summarizes the methods in use and presents new ones. First, sampling Received 23 July 2021 and pretreatment processes of MPs, including sample collection, digestion, density separation, and quality Revised 22 September 2021 control are reviewed. Then the promising and convenient staining procedures and quantification methods Accepted 12 October 2021 for MPs using fluorescence dyes are reviewed. The factors that influence the staining of MPs, including Available online 15 November 2021 their physicochemical properties, are summarized to provide an optimal operation procedure. In general, the digestion step is crucial to eliminate natural organic matter (NOM) to avoid interference in quantification. Chloroform was reported to be the most appropriate solvent, and 10–20 μg/mL are Keywords: recommended as optimal dye concentrations. In addition, a heating and cooling procedure is Plastic particles recommended to maintain the fluorescence intensity of MPs for two months. After staining, a fluorescence microscope is usually used to characterize the morphology, mass, or number of MPs, but Fluorescence dyes compositional analysis cannot be determined with it. These fluorescence staining methods have been Identification implemented to study MP abundance, transport, and toxicity and have been combined with other chemical Concentration quantification characterization techniques, such as Fourier transform infrared spectroscopy and Raman spectroscopy. More studies are needed to focus on the synthesis of novel dyes to avoid NOM’s interference. They need to be combined with other spectroscopic techniques to characterize plastic composition and to develop image-analysis methods. The stability of stained MPs needs to be improved. © The Author(s) 2021. This article is published with open access at link.springer.com and journal.hep. com.cn 2021 ✉ Corresponding author E-mail: shangenx@163.com (E. Shang), liyang_bnu@bnu.edu.cn (Y. Li) Special Issue—Microplastic and Nanoplastic Pollution: Characterization, Transport, Fate, and Remediation Strategies (Responsible Editors: Wen Zhang, Melissa Pasquinelli & Yang Li) 2 Front. Environ. Sci. Eng. 2022, 16(1): 8 to develop a convenient and cheap quantification method 1 Introduction for MPs present in the environment. Over the past decades, the production and consumption of The fluorescence staining and quantification method plastic products has risen rapidly (Thompson et al., 2004). provides a fast, convenient, and cheap way to quantify Due to the continued discharge of plastic products and their MPs (Maes et al., 2017). This technique uses a dye called low degradation rates, they accumulate in environmental Nile Red (NR, 9-diethylamino-5-benzo[α]-phenoxazi- matrixes, including water, sediments, soils, and the none), a lipid soluble fluorescence dye, to stain neutral atmosphere (Barnes et al., 2009; Liu et al., 2021). Once lipids in biological samples (Greenspan and Fowler, 1985). Afterwards, NR is used to stain synthetic polymers (Jee the plastic products are discharged into the natural et al., 2009). Since 2016, the staining ability of MPs by NR environment, large particles may be fractured, weathered, has drawn the attention of researchers (Shim et al., 2016; or degraded into microplastics (MPs, 0.1 μm–5 mm) and Erni-Cassola et al., 2017; Maes et al., 2017). Maes et al. nanoplastics (NPs, £0.1 μm) via biodegradation and proposed the identification and quantification method for physical and chemical weathering processes (Browne MPs using NR staining and fluorescence microscopy, and et al., 2011; Cole et al., 2011; Duan et al., 2021). they got an average recovery rate of 96.6% for marine Commercial products, such as personal care products and industrial beads, contain ultrafine plastic particles, which MPs, which was cross-validated by FTIR (Maes et al., are regarded as the primary source of MPs and NPs (Alimi 2017). Recently, heating protocols have been developed to et al., 2018). The loads of MPs in freshwater, seawater, enhance the staining effect, and programmed-image- –5 –6 soil, and sediment are 10 –10 items/L, 10 –10 items/L, analysis software has been applied to quantify fluorescence 4 3 1–10 items/kg, and 1–10 items/kg, respectively (McCor- of MPs automatically (Shim et al., 2016; Erni-Cassola mick et al., 2014; Shahul Hamid et al., 2018; Zhang and et al., 2017; Karakolis et al., 2019). The MP fluorescence Liu, 2018; Chen et al., 2020; Zhang et al., 2020). Studies staining and quantification method has achieved a recovery have shown that MPs pose a threat to human health and rate of over 90%, which has been validated by FTIR using ecosystems, because MPs can be transported via food the same batch of laboratory samples (Shim et al., 2016; chains (Carbery et al., 2018; Sun et al., 2019; Liu and Erni-Cassola et al., 2017; Cook et al., 2020; Karakolis Wang, 2020). The increasing load of MPs has drawn the et al., 2019). Therefore, the fluorescence staining and attention of researchers (Wu et al., 2017; Guo et al., 2020; quantification method for MPs provides a promising and convenient technique for researchers. Wang et al., 2021). Thus, studying their abundance is To summarize research on MP characterization and important to assess the impact of MPs on health and to quantification, a literature review of papers published ensure the security of ecosystems. between January 2016 and March 2021 was conducted. A A fundamental step to understand the distribution, fate, total of 1411 articles were investigated with the following and toxicity of MPs is the identification and quantification keywords: microplastics, quantification, characterization, of MPs. A variety of approaches have been developed to detection, sampling, fluorescence, and NR. As shown in detect and characterize MPs (Shim et al., 2017; Fu et al., Fig. 1, 32% of the research focused on quantification of 2020). However, the advantages and limitations of these MPs, which was more than research in any other area. methods vary. Manual counting of MPs by optical microscopy is one of the most widely used methods to Currently, most reviews have concentrated on character- quantify MPs, which has the advantage of convenience but ization of physicochemical properties, transport, and is limited by the operator’s subjectivity (Fu et al., 2020). transformation of MPs, as well as sampling, separation, Scanning electron microscopy (SEM), transmission elec- and digestion procedures of MPs (Hidalgo-Ruz et al., tron microscopy (TEM), and atomic force microscopy 2012; Fu et al., 2020), but the reviews have ignored the (AFM) can provide clear and high-resolution images of fluorescence-quantification method for MPs. MPs, but these methods need to be combined with Thus, we focus on providing a comprehensive overview spectroscopy methods to characterize the chemical com- of the fluorescence staining method to quantify MPs in position of MPs (Patchaiyappan et al., 2020). Analysis by aquatic environments. The objective of this review is to Fourier transform infrared spectroscopy (FTIR) and identify the optimum protocols and best operating Raman spectroscopy can characterize the specific chemical conditions for fluorescence staining and quantification of bonds of MPs, but they are difficult to use to quantify MPs MPs. First, procedures dealing with sample collection, (Fu et al., 2020). Gas chromatography-mass spectrometry separation, digestion, identification, and quantification are (GC-MS) is also an alternative method for measuring the summarized. Next, the advantages and limitations of dye concentrations of monomers or additives in MPs, but it is a staining and the main factors that influence the dying destructive method (Shim et al., 2017). All the above effect, including the physicochemical properties of MPs methods require expensive instruments, experienced and environmental conditions, are summarized. Then, the operators, time-consuming pretreatment processes, and method itself for the identification and quantification of complex data analysis, except for the manual counting stained MPs is reviewed. After discussing the application method via optical microscopy. Therefore, it is necessary of fluorescence staining and quantification methods for Shengdong Liu et al. Fluorescence staining for characterization of microplastics 3 MPs being done in current studies, knowledge gaps and shorelines and bottoms of rivers, lakes, or seafloors, the top future perspective regarding standardized protocols for MP 0–5 cm layer of the surface of beaches and sediments is quantification are proposed. most commonly collected (Patchaiyappan et al., 2020). Sediment cores are also collected to study the occurrence and transport of MPs in aquatic environments. For biota 2 Fluorescence staining methods for MPs samples, fish, invertebrates, and bivalves are most frequently collected. Biota samples are generally collected 2.1 Sampling and pretreatment processes of MPs from wild environments, but some are sampled from commercial operations (Mai et al., 2018). Processes used Sampling and pretreatment processes of MPs include the to separate the MPs from collected samples include collection of samples from the environment and the sieving, filtration, and drying, as shown in Table 1. Sieving extraction of MPs from the samples. Approaches for is generally considered as the first step in sample sampling and extracting MPs from various environmental processing for water and sand or sediment samples, and matrixes, such as freshwater and seawater (Hidalgo-Ruz a mesh of 3 mm or 5 mm is used. et al., 2012; Karakolis et al., 2019), sand and sediments (Nuelle et al., 2014; Besley et al., 2017), and organisms 2.1.2 Digestion and density separation processes of MPs and tissues (Claessens et al., 2013; Avio et al., 2015) have been critically reviewed (Hidalgo-Ruz et al., 2012; Mai The widely distributed NOM in the environment can lead et al., 2018; Prata et al., 2019a). Therefore, we summarize to overestimation of the particle number and environment just the basic steps for the sampling and pretreatment concentration of MPs. Thus, the digestion step is usually processes of MPs. We emphasize the importance of followed by sieving to eliminate NOM (Mai et al., 2018). digestion protocols in fluorescence staining for MPs, The most commonly applied digestion protocol is adding which directly influence the staining effect and the H O (30%) and Fe(II) solution to samples (Prata et al., 2 2 accuracy of detection. 2019a). Then the mixture was heated at 75°C for 0.5–4h. This digestion method is recommended by US National 2.1.1 Sample collection processes Oceanic and Atmospheric Administration (NOAA) (Hanke et al., 2013). Moreover, the procedures of digestion In field studies, different methods and equipment are depend on the source of environmental samples, which applied to collect samples in specific environmental contain different concentrations of NOM. For instance, the matrixes, including water, sand, sediment, the atmosphere, digestion for water samples with low content of NOM by and biota (Table 1) (Dowarah et al., 2020; Scircle et al., H O (30%) is enough, while for sediment or sand samples 2 2 2020; Valine et al., 2020). For water samples, manta nets or containing high concentration of NOM requires H O 2 2 trawls are commonly used to collect large-sized MPs (30%) with Fe(II) solution (0.05 M) and to be heated at (>100 μm) in surface waters, whereas pumping with filters 75°C (Fu et al., 2020). When dealing with biota samples, (100 or 300 μm) is used as a complemental sampling the digestion protocol is extremely important because of method for smaller sized MPs (< 100 μm). For sediment the high biomass content, and an enzyme (such as and sand samples, which are generally sampled from proteinase K, chitinase, and cellulase) is commonly Fig. 1 The proportion of research papers investigating the quantification and characterization of microplastic. 4 Front. Environ. Sci. Eng. 2022, 16(1): 8 Table 1 Sampling and pretreatment processes of MPs for fluorescence staining Sample collection process Sample pretreatment process Sample origin Ref. Sampling Sampling Sampling Density Extraction Sieving Digestion Filtration location equipment details separation recovery Fresh water Kinnickinnic Glass jar NA NA NA 0.05 M FeSO Polycarbonate NA Simmerman River, USA 1L H SO filter & Wasik, 2 4 30% H O 0.4 μm 2020 2 2 Fresh water Four rivers, Plankton Below NA 84.2 mg/L 10% KOH Strainer NA Valine USA tow net river NaCl 200 µm et al., 200-μm surface 2020 0.3-1 m Sea water Mississippi Glass jar Below 25-μm NA 0.05 M Fe (II) Polycarbonate NA Scircle Sound, USA 946 mL surface mesh 30% H O filter et al., 2 2 water 10 µm 2020 Beach sand Three beaches, NA Top of 5-mm CaCl H O Mesh 89.5%- Tiwari 2 2 2 India beach sand mesh 1.34 g/cm 38 μm 97.5% et al., 3–4cm 2019 Sediment South Metal Top layer 3-mm NaCl 0.05 M Fe (II) Vacuum NA Patchaiyappan Andaman spoon of beach 5-mm 1.2 g/cm 30% H O filtration et al., 2020 2 2 beaches, 1cm mesh India Biota Kinnickinnic D-shaped NA NA NA 0.05 M FeSO Steel sieve NA Simmerman (Macroinvertebrates) River, USA kick net 3mL H SO 20 μm & Wasik, 2 4 600-μm 30% H O 2020 2 2 Biota Puducherry Bought NA NA NA 10% KOH Vacuum NA Dowarah (bivalve) coastline, in fish filtration et al., 2020 India market 11 μm Biota Forth Stainless- NA NA Super- Enzyme Vacuum NA Catarino (mussels) River, steel saturated mixture filtration et al., 2018 UK wired NaCl (Corolase 0.8 μm scrubber 7089) Atmosphere Hamburg PE-funnel Above NA NA 15% v/v Vacuum NA Klein & metropolitan PE bottle ground NaClO filtration Fischer, area, level 5–13 μm 2019 Germany 100 cm NA means it is not available in the references. applied to eliminate the tissue (Prata et al., 2019a). In samples are retained on the filter and the pour size of filter addition, there are other digestion methods, such as acid determines the retention particle size. The filters with pour digestion, alkali digestion, oxidizing digestion, and enzy- sizes in the range between 0.2 μm and 55 μm have been matic digestion (Prata et al., 2019a). Detailed digestion used in different studies based on their purpose. The procedures for each method have been discussed in smaller pour size of filter they used, the smaller sized published reviews (Prata et al., 2019a). plastic particles can be retained on the filter for further After the digestion step, density-separation procedure is detection. In addition, considering the visual observation conducted to separate specific MPs from water, soil, or of MPs, the filter should not exhibit a fluorescence signal sediment samples. Based on the different densities of and interfere the detection result. For example, a study 3 3 plastics (0.8–1.6 g/cm ) and sediment (2.7 g/cm ), density- tested 6 types of filters and found that only glass fiber filter separation methods have been developed to separate MPs (1.2 μm) and black polycarbonate filters (0.2 μm) are and sediment or sand by mixing the samples in salt- appropriate for the detection of NR-stained MPs without saturated solutions and afterwards collecting the super- introduction of fluorescence intensity from the filters (Prata natant, which contains MPs (Rocha-Santos and Duarte, et al., 2019b). Some plastic particles directly identified by 2015). In the density-separation step, the most frequently visual observation cannot use the special filter membrane. used salt is NaCl, while other salts such as CaCl , NaI, and After filtration, drying procedures (generally at 60°C) are ZnBr are also available (Prata et al., 2019a). Filtration is carried out, and they are indispensable as the final steps for necessary after the digestion protocol, where all the sample processing. Shengdong Liu et al. Fluorescence staining for characterization of microplastics 5 2.1.3 Quality assurance and quality control in the sampling important to get accurate and reliable data and high process recovery rates during the entire sampling processes. Guaranteeing quality assurance and quality control (QA/ 2.2 Methods for fluorescence staining of MPs QC) in the sampling process is essential to get reliable findings concerning the abundance of MPs. For example, Fluorescence methods are distinguished from regular, procedural blanks (only containing water) and spiked optical approaches to detect and quantify MPs, because blanks (containing water with known composition and they require a staining process during pretreatment. The number of MPs) should be analyzed during sample fluorescence staining methods for MPs can be categorized collection and processing of samples (Hanke et al., 2013; into staining MPs on filter paper or in solution. The first Catarino et al., 2018). In addition, the recovery rate of MPs dying method for MPs on filter paper includes collecting throughout the whole sampling process should be reported the environmental samples, separating and extracting the to reveal the actual level of MPs in the environment. MPs from the environmental samples, placing the MPs on Determining the recovery rate is also beneficial, because it filter paper (usually a polycarbonate filter paper), and allows comparison of the abundance with other studies adding the dye solution on the filter paper to stain the MPs (Wiggin and Holland, 2019). Currently, the recovery rates for a period of time (2–4 h) (Shim et al., 2016; Erni- of generally used sampling processes (sieving, digestion, Cassola et al., 2017). After the staining procedure, the density separation, and filtering) for MPs can reach number or concentrations of the stained MPs are detected 83.3%–96.6% (Maes et al., 2017; Tamminga, 2017). In by a fluorescence microscope or spectrophotometer. field sampling, non-plastic collection tools and storage The other method is staining MPs in solution (Cook containers should be used to avoid cross contamination. et al., 2020; Karakolis et al., 2019), as shown in Fig. 2. The During laboratory sampling, it is necessary to wear latex first step is preparation of the staining solution (Fig. 2a), gloves and cotton clothes to avoid contamination from which is an organic solvent that dissolves the NR. The airborne fibers, which are widely detected in the environ- most commonly used solvents include methanol, chloro- ment via atmospheric fallout (Ziajahromi et al., 2017). form, acetone, and n-hexane (Tamminga, 2017). The Overall, quality assurance and quality control protocols are second step is suspending MPs in the staining solution Fig. 2 MP staining process in solution. (a) adding Nile Red in solvent, (b) staining in solution while heating and cooling, (c) vacuum filtering, (d) Nile Red stained MPs. 6 Front. Environ. Sci. Eng. 2022, 16(1): 8 (Fig. 2b). The heating and cooling procedures may fibers are especially difficult to be stained compared with enhance the intensity of the fluorescence signal and inhibit particles and fragments due to their irregular shapes leaching of the dye (Lv et al., 2019). The third step is (Tamminga, 2017). Moreover, the color of MPs may filtering the staining solution to get the fluorescence- disturb the staining effect and quantification. Most stained MPs and washing them with deionized water to get commonly used plastics are white, which plays a minor rid of the adsorbed dye (Fig. 2c). Then, the fluorescence- role in detection after staining. But certain polymer types, stained MPs are obtained, and they usually show red or like PVC in black color, are difficult to be stained and purple color when NR is used as dye (Fig. 2d). detected (Shim et al., 2016). The composition of MPs determines the affinity between In early studies, the staining method on filter paper was MPs and dyes. As a hydrophobic dye, NR preferentially commonly applied and was used for most types of combines with polymer materials such as PP and PE that polymers (Shim et al., 2016; Erni-Cassola et al., 2017). In recent years, staining in solution is an improved method have a low polarity. Fluorescence staining using lipophilic due to stirring and the addition of heating or cooling dyes, such as NR, has proven to be effective in procedures. Therefore, recent studies tend to stain MPs in quantification of small particles of PE, PP, PS, PC, PUR, solution (Stanton et al., 2019; Konde et al., 2020). This and PEVA, because of their high hydrophobicity, whereas staining method requires MPs to be distributed in a PVC, PA, and PES could not be detected after staining due homogeneous solution. Thus, it may be difficult to use with to their low hydrophobicity (Shim et al., 2016). Among the certain types of MPs such as PE, which tends to float on the physicochemical properties of MPs, the chemical compo- surface of water due to its low density. To solve this sition of the polymer plays a primary role in the staining problem, researchers have suspended a powder of PE in a effect, and it determines the affinity between MPs and mixture of ultrapure water and dimethyl sulfoxide (v = 1:1) dyes. To note, the fluorescence staining has minor effect on for better distribution in solution (Cook et al., 2020). In the aggregation of MPs. On the one hand, Raman spectrum conclusion, to get a better staining effect, the staining and FTIR spectra measurements prove that the stained method for MPs in solution is recommended, which has MPs have almost no alteration on composition compared the advantages of flexible heating or stirring procedures. to pristine MPs, because only a few dye molecules are added into MPs surface by diffusion (Lv et al., 2019). On 2.3 Factors influencing the staining effect the other hand, the Zeta potentials of pristine PET MPs and NR stained PET MPs have been measured, their Zeta The staining effect of MPs depends on many factors, such potentials are – 6.000.78 mV and – 7.640.81 mV, as dye concentration, solvent type, and temperature respectively. This proves that staining process has a minor (Wiggin and Holland, 2019). In this section, we compre- effect on the surface charge of MPs. Similarly, the Zeta hensively discuss how these factors influence the staining potentials of stained PE, PP, and PVC MPs were also effect to develop an optimum MP staining protocol. measured and no significant alteration of Zeta potentials has been observed. 2.3.1 Physicochemical properties of MPs 2.3.2 Experimental parameters The physicochemical properties of MPs, such as particle size, shape, and composition, affect the staining process 1) NR concentration (Wiggin and Holland, 2019). The recovery rate of fluorescent-stained MPs decreases as the size of particle Fluorescence intensity of stained MPs is influenced by the decreases. For example, studies have found that recovery concentration of NR, which has been applied in the range rates of NR-stained MPs decreased from 82% to 49% of 0.1 μg/mL and 500 μg/mL in current studies (Rumin when the size decreased from 500 to 1000 μmto20–63 et al., 2015; Lv et al., 2019). For example, Lv et al. found μm, respectively (Wiggin and Holland, 2019), which may that fluorescence intensity first increased (0.1–25 μg/mL be because the smaller particles are more difficult to be NR concentration) and then decreased (25–100 μg/mL NR detected under a microscope. Moreover, approximately concentration) with NR concentration (Lv et al., 2019), 95% of particles with sizes larger than 1 mm could be which was attributed to NR aggregation at high dye detected after staining by NR, while only 71.7% of concentration that decreased fluorescence intensity (Rumin particles smaller than 1 mm could be detected (Tamminga, et al., 2015). Similarly, the fluorescence intensity increased 2017), which was primarily because the small particles and then decreased with rising dye concentration (0.1– were stained less strongly and exhibited a weaker 100 μg/mL) when selecting Fluorescein isophosphate fluorescence intensity; therefore, they were more difficult (FITC) to stain MPs. The fluorescence intensity increased to be detected than larger particles. with Safranine T dye concentration increasing from In addition, the shapes of the MPs can influence the 0.1 μg/mL to 100 μg/mL, while it changed slightly until staining effect, which are generally categorized as the dye concentration reached saturation at 100 μg/mL (Lv particles, fragments, or fibers. Studies have indicated that et al., 2019). Moreover, a high NR concentration, such as Shengdong Liu et al. Fluorescence staining for characterization of microplastics 7 500 μg/mL, may lead to saturation of spectrometer surface, or the entrance of dye inside polymer molecules, intensity. An obvious red shift in fluorescence spectra of depends on temperature. Fluorescence is exhibited only for PVC could interfere with the fluorescence measurement a short period when MPs are stained at room temperature and analysis (Konde et al., 2020). Thus, attention should be (25°C) without heating (Prata et al., 2020). For example, paid to avoid using too high a NR concentration for after 2 months, 73.5% of NR-stained MPs without heating staining. Conversely, low NR concentration (< 1 μg/mL) were found to have lost fluorescence (Prata et al., 2020), which was attributed to the desorption of dye molecules exhibits too weak fluorescence signals to detect (Konde from the MP surface because the dyes were just adsorbed et al., 2020). Therefore, an appropriate NR concentration onto the surface. Studies have found that the heating between 10 μg/mL and 20 μg/mL is suggested in NR protocol promotes the staining effect resulting in both staining protocols, which gets both adequate fluorescence higher fluorescence intensity and a more stable florescence intensity and minimum interference (Tamminga, 2017). signal for a long period (over 2 months) (Lv et al., 2019). 2) Solvent Fluorescence intensity of MPs has shown an increasing trend with increasing temperatures from 20°C to 50°C NR is a hydrophobic dye with poor solubility and weak (Konde et al., 2020). Most types of MPs have their fluorescence in water (Greenspan and Fowler, 1985). It strongest fluorescence intensity at 50°C, except for PVC first needs to be dissolved in a solvent for staining. The which has its strongest fluorescence at 75°C (Lv et al., solvent has a significant impact on the NR staining effect. 2019). Furthermore, heating protocols have enabled On the one hand, the solvent directly affects the recovery stained MPs to maintain a stable fluorescence intensity rate of the fluorescent-stained MPs. On the other hand, NR over 2 months (Lv et al., 2019). Heating protocols have contains a polar carboxyl function (-COOH) on its been applied in several studies and to obtain high recovery aromatic rings; therefore, NR fluorescence spectra exhibit rates (> 95%) (Cook et al., 2020; Karakolis et al., 2019; Lv dependency on the polarity of solvents due to the et al., 2019). The heating temperature of staining solutions solvatochromism of NR (Rumin et al., 2015). Because of in previous studies have generally been set from 50°C to the relatively polar nature of NR molecules when 75°C (Cook et al., 2020; Karakolis et al., 2019; Lv et al., compared to plastics, the partitioning of NR molecules 2019; Konde et al., 2020). The temperature should be from the solvent to plastics can be made more feasible in lower than the melting point of some polymer materials non-polar solvents, such as n-hexane, than in polar such as LDPE (85°C). PVC and PET are less hydrophobic solvents (Shim et al., 2016). plastics and, thus, are difficult to be stained by NR. The use The use of various solvents, such as chloroform, of a heating protocol enables PVC and PET to exhibit acetone, n-hexane, and methanol, has been summarized strong fluorescence intensity (Lv et al., 2019). Overall, to in the literature (Tamminga, 2017; Konde et al., 2020). obtain stronger fluorescence intensity and a more stable Tamminga et al. investigated the influence of three staining effect for MPs, the protocol of heating staining solvents, which were acetone, chloroform, and n-hexane, solutions is recommended. When the melting point of on the staining effect of MPs by NR. Chloroform was the polymer materials is considered, 50°C–60°C would be an most appropriate solvent for HDPE, LDPE, PP, and PVC, appropriate temperature range to use for NR staining and the lowest recovery rate was 83.3% (Tamminga, protocols. 2017). Nevertheless, they found that some polymer materials, such as cellulose acetone and PS, had the 2.4 Advantages of fluorescence staining for MPs tendency to dissolve in chloroform or acetone (Tamminga, 2017). Konde et al. proposed a mixed solvent consisting of Fluorescence staining for quantification of MPs provides a acetone and ethanol with v/v = 1:1 for the investigation of straightforward, quick, cheap, and convenient technique to MP photoluminescence spectra, and the mixed solvent detect mass concentration of MPs and to investigate their exhibited strong fluorescence intensity without PVC distribution in environmental samples. Spectral methods or deformation (Konde et al., 2020). Overall, it is necessary chromatographic methods can cost up to hundreds of to select an appropriate solvent to avoid leaching of the dollars for each batch of samples, while the cost of NR is additive or monomer from MPs; to develop specific only $ 8.36 USD/g plastic with the additional need for fluorescence spectra for each solvent; and to get reliable fluorescence microscopy (Karakolis et al., 2019). detection results of fluorescence-stained MPs. A mixture of Fluorescence-staining methods also have been repre- solvents, like acetone and ethanol, appears to offer a good sented as atimeefficient and convenient way for choice for staining. quantification of MPs. The staining protocol of MPs could be as short as 30 min for one batch of samples, while 3) Temperature spectral methods take a longer time for detection of MPs. For example, FTIR requires at least nine hours to scan one Temperature has a significant impact on the staining effect, filter paper (Shim et al., 2017) including the requirement of because the adsorption of the dye onto the NP or MP sample pretreatment time. The work experience required 8 Front. Environ. Sci. Eng. 2022, 16(1): 8 for quantification of MPs via fluorescence techniques is After confirming the fluorescent particles as MPs, these much less than that needed for spectral (i.e. FTIR) or mass particles are counted either by manual work or automated spectrometry methods (Rocha-Santos and Duarte, 2015). software (such as ImageJ) to obtain the number of Quantification of MPs via fluorescence can be operated by particles. To be specific, the total number of low abundance automated photo-analysis software. In contrast, the mass of MPs can be counted directly on the entire filter. While spectrometry method, like GC-MS, requires well-trained counting the high abundance of MP samples, it is operators as well as time-consuming pretreatment pro- commonly to count the total particles from 3 random cesses (Rocha-Santos and Duarte, 2015). Overall, the fields of view and averaging these counts, then comparing major advantages of fluorescence-staining methods are that the viewed area with the entire filter area and normalizing the observed numbers to the whole filter area (Simmerman they are time efficient and easy to use, and they are and Wasik, 2020). appropriate for the detection of the abundance of MPs in bulk environmental samples. The influence of the excitation wavelength on the In addition, NR staining protocols improve the count detection of stained MPs has been explored. Prata et al. efficiencies of smaller-sized MPs compared with those tested NR-stained mixtures of polymers (PE, PP, PS, PVC, taken with regular visual quantification methods. A study EPS, nylon) and organic matter and excited them under showed that NR staining increased the detection number of light with multiple wavelengths (254, 365, 470, 495, 530, MPs in every sample, and the greatest increase of the 625 nm) (Prata et al., 2020). The results showed that numbers observed was in the smaller sized fraction (< 124 excitation light at 254 nm had the advantage of a high μm) (Wiggin and Holland, 2019). Furthermore, this study contrast with the background signals without interference showed that the reported levels of MPs determined via the with organic matter. But PS, PVC, nylon, virgin HDPE, NR staining and counting approach were higher than those and weathered PE could hardly be detected under the determined by other methods worldwide, and this was 254 nm excitation wavelength. Most polymers (PE, PP, likely due to the inclusion of smaller sized MPs after HDPE, PS, EPS) and NOM could be excited under 470 nm dyeing samples from a highly urbanized aquatic environ- at the same time. Therefore, application of the 470 nm ment (Los Angeles, California) (Wiggin and Holland, excitation wavelength requires a digestion step to remove 2019). Overall, smaller-sized MPs are more likely to be NOM (Prata et al., 2019b). In conclusion, the 254 nm detected by the NR staining method, and it avoids excitation wavelength is suitable for limited types of underestimation of MP abundance in the environment. polymers (PE, PP) with the advantage of less interference However, there are challenges for detecting the small-sized by NOM. The excitation wavelength at 470 nm can excite MPs, and the detection limit by the current fluorescence most types of polymers but it can excite NOM as well, which requires digestion procedures to avoid interference staining method for the size of MPs was down to 3 μm (Wiggin and Holland, 2019). Meanwhile, the detection with the NOM’s fluorescence signal. limit of MPs mass concentration by fluorescence staining During the identification procedures, the fluorescence methods lies in the range between 1 mg/L and 100 mg/L signals from MPs have different colors (Fu et al., 2020). (Li et al., 2019). The NR-stained plastics appear orange color in a chloro- form solvent when excited by blue or UV light (Maes et al., 2017; Tamminga, 2017). In addition, MPs can fluoresce in 3 Fluorescence identification and quantifi- varied light ranges when stained by different dyes. For cation methods for MPs example, iDye pink (pink dye), iDye blue (blue dye), and Rit DyeMore Kentucky Sky (Kentucky dye) can fluoresce 3.1 Identification in the red range, the far red range, and the green and red (both) ranges, respectively (Karakolis et al., 2019). Basically, identifying MPs with fluorescence microscopes With the help of image-analysis techniques, the hydro- includes the following steps: 1) the samples are placed phobic and hydrophilic characteristics of fluorescence under the fluorescence microscope and are excited with the particles can be identified. Using this method, a simple proper excitation wavelength; 2) the samples are observed “fluorescence index” can be calculated as (R + G)/R (‘R’ or photographed under the proper emission wavelength; and ‘G’ are the 8-bit color intensity values of red and 3) the fluorescence signal or images are detected and green, respectively). This index represents the “polarity” of analyzed for identification. In detail, the particles were the polymer surface, and the larger the value of the index identified as MPs by the following criteria: 1) no cellular or is, the higher hydrophobicity of the polymer particles is organic structure are observed; 2) fiber particles are (Maes et al., 2017; Tamminga, 2017; Wiggin and Holland, uniform in thickness throughout their whole length and 2019). Recently, an automated counting software (MP- have no three-dimensional bending; 3) the colored VAT, Microplastics Visual Analysis Toll) has been particles should present clear and homogeneous colors; developed, which can be applied to detect the sizes and 4) fiber particles have no segment; 5) particles do not shine shapes of stained MPs through their emitted light (Prata (Nor and Obbard, 2014; Klein and Fischer, 2019). et al., 2019b). Shengdong Liu et al. Fluorescence staining for characterization of microplastics 9 3.2 Quantification investigate their aggregation and settling mechanisms in sandy water. In this study, a fluorescence spectrophot- The identification of MPs is commonly followed by a ometer was used to measure the fluorescence intensity of quantification procedure. Identification can provide infor- MPs, and then the MP mass concentration was further mation about the morphology and surface properties of calculated using a standard curve (Li et al., 2019). In MPs, while quantification can show the number and mass conclusion, fluorescence MPs provide a fast method for concentrations of MPs in samples, in order to study quantification of MP concentration, and the particles can abundance and occurrence of MPs (Mai et al., 2018). easily be detected using their fluorescence intensity. Procedures for quantification with fluorescence micro- The quantification methods used for field samples and scopes involve the following steps: 1) the MPs are excited laboratory samples are different. In one study, field under an excitation wavelength; 2) their fluorescence samples were first photographed under a certain excitation signal is observed; and 3) the fluorescence particles are wavelength (Klein and Fischer, 2019). Then, images were counted or selected MPs are picked up for further mass analyzed by software to count the number of fluorescence weighing and quantifying (Prata et al., 2020). plastic particles. The size and particle circularity could also In addition, using a fluorescence spectrophotometer to be determined by image-analysis software (Sfriso et al., determine the mass concentration of MPs in a water matrix 2020). As for laboratory samples, they can be detected is also feasible, based on a standard curve that is developed easily and accurately by using a fluorescence spectro- to show the relationship between fluorescence intensity photometer, because they have a known polymer type and and MP concentration (Li et al., 2019). An example for few impurities (Li et al., 2019). Recently, fully- or semi- quantification of stained PET using a fluorescence spectro- automated analytical methods have been applied to photometer is shown in Fig. 3. This method is especially quantify MPs, which offer a promising way of MP appropriate for simulated samples done under laboratory quantification in the future. Confocal microscopy also conditions, which consist of pure polymers and little NOM has provided a straightforward way to observe stained (Shim et al., 2016). For example, a study was done MPs. A study demonstrated that artificially generated MPs with fluorescence polystyrene nanoplastics (PSNPs, are more recognizable under confocal microscopy than 100 nm) and polyethylene MPs (PEMPs, 1.0–1.2 mm) to field samples (Maxwell et al., 2020). Therefore, confocal Fig. 3 Quantification of MPs using fluorescence spectrophotometer (a) fluorescence stained PET microplastic when being excited, (b) fluorescence stained PET microplastic under bright-field, Scale bar = 650 μm, (c) correlation curve between fluorescence intensity and mass concentration of PET microplastic. 10 Front. Environ. Sci. Eng. 2022, 16(1): 8 microscopy could be more appropriate in laboratory tions of MPs were 45–220 items/kg of dry sand (South studies (Maxwell et al., 2020). The use of Laser Confocal Andaman beach) and in the other study they were 161.7– Raman Spectroscopy in identifying and quantifying MPs 973.3 items/kg of dry sand (Girgaon Mumbai, Tuticorin can increase the sensitivity and accuracy of analysis. This beach, Dhanushkodi beach) (Tiwari et al., 2019; Patch- advanced technology can identify the physical properties aiyappan et al., 2020). Simmerman et al. examined the MP and chemical compositions of MPs, with the advantages of levels in water and organisms in a cold-water stream in high spectral coverage, high lateral resolution, specific western Wisconsin, USA. They found that the concentra- fingerprint spectrum and low interference from organic tions of MPs stained by NR in water ranged from 545 to matter/water/fluorescence background signals (Sobhani 3622 items/L and increased significantly from upstream to et al., 2019). Thus, Laser Confocal Raman Spectroscopy downstream. The mean MP concentrations downstream of an urban area were 2–3 times those found upstream has a promising future especially in the study of transport and transformation of nanostructured materials in natural (Simmerman and Wasik, 2020), which may be because waters. MPs move down stream with the water flow. Klein et al. examined the MP abundance in atmospheric disposition via fluorescence MP quantification. They determined that 4 Application of information from studies of the mean MP abundance was 275 items/m /d. To fluorescence-stained MPs characterize the chemical composition of MPs, Raman spectroscopy was combined with fluorescence quantifica- 4.1 Quantification of MPs input in the environment tion, and polyethylene/ethylvinyl acetate copolymers were found to dominate within the metropolitan area of Microplastics can enter the terrestrial, aquatic, and atmo- Hamburg, Germany (Klein and Fischer, 2019). In conclu- spheric environments directly through indiscriminate sion, dye stained MPs and the fluorescence quantification disposal of plastic wastes and indirectly through applica- approach have been applied in field samples to examine the tion of waste resources containing plastics such as abundance of MPs, which are generally coupled with biosolids and composts (Bradney et al., 2019; Kumar spectroscopic methods to characterize the composition of et al., 2020). The fluorescence-quantification method of MPs (Vermaire et al., 2017; Klein and Fischer, 2019; MPs has been developed in recent years, and it has been Patchaiyappan et al., 2020). validated to be a straightforward and cost-effective way to quantify the input of MPs into environments through 4.3 Investigation of distribution of MPs in organisms various waste sources (Lares et al., 2019). For example, Lares et al. compared six different methods to detect MPs Assessment of the toxicity of MPs in organisms requires in municipal wastewater and digested sludge samples, characterization of their distribution in organisms. Fluor- spiked with seven different types of plastic particles and escence detection methods have been widely applied in fiber, and they suggested that a staining method using Rose distribution studies of MPs, which may facilitate toxicity Bengal could be useful in separating MPs from other assessments of marine organisms and evaluation of human materials (Lares et al., 2019). Another study examined the health risks (Catarino et al., 2018; Maxwell et al., 2020). occurrence of MPs in wild mussels via fluorescence Maxwell et al. reported a novel counterstaining method by staining using NR. The results showed that the mean NR, Evans blue, and Calcofluor white dyes to detect MPs concentration of MPs entering the body of Modiolus (a in terrestrial, invertebrate samples. The results could be kind of mussel) was 0.0860.031 items/g wet weight used for investigation of MP ingestion by soil animals (Catarino et al., 2018). The authors also reported that the (Maxwell et al., 2020). They studied the tissue distribution ingestion of airborne fibers by humans during a meal can of PS-MPs in red tilapia (O. niloticus) during a 14-d be up to 13731– 68415 items/y/person. In these studies, the exposure period. They found that the concentration of PS- 4 4 staining method was applied to separate MPs from other MPs in the fish gut was 171.1  10 3.5  10 μg/kg, and materials and to quantify the input of MPs into the the concentration of microplastics in organs were in the environment. order of gut>gills>livers≈brain (shown in Fig. 4) (Ding et al., 2018). Overall, fluorescence staining of MPs can be 4.2 Measurement of MPs abundance in the environment used to study the distribution and accumulation of MPs in tissues of organisms, and the results can be helpful for the The fluorescence-quantification method has been applied assessment of the toxicity of MPs to evaluate human health to measure the concentrations of MPs in samples of fresh risk. water, seawater, soil, beach sand, sediment, airborne MPs, and biota (Vermaire et al., 2017; Gagné et al., 2019; Klein 4.4 Investigation of the environmental fate and transport of and Fischer, 2019; Simmerman and Wasik, 2020). In two MPs studies in India, the abundance of MPs in beach sand was determined by NR-staining MPs. In one study, concentra- Fluorescence staining and quantification methods have Shengdong Liu et al. Fluorescence staining for characterization of microplastics 11 Fig. 4 Photographs of fish tissues under a bright-field microscope (top row) and representative fluorescence images of PS-MPs in different fish tissues after 14 d of the exposure to 100 μg/L (bottom row), Scale bar = 100 μm. Graph was adapted from ref (Ding et al., 2018) with permission. been applied to determine the environmental fate of MPs. 5.1 Strengthen research on the synthesis of novel dye to Results from simulated, laboratory experiments are avoid interference of organic matter extremely useful. In simulated experiments done under laboratory conditions, Li et al. studied the aggregation Organic matter is ubiquitous in natural aquatic, terrestrial, behavior of MPs in suspended sediment (SS) using or biotic environments. Dyes can co-stain it along with plastic particles, which interferes with the detection of MP fluorescence PSNPs and PEMPs to quantify the concen- fluorescence intensity and results in overestimation of the tration of MPs (Li et al., 2019). They found that PSNPs and abundance of plastic particles in samples, and, conse- SS formed heteroaggregates and settled in a water column quently, their accumulation and toxicity is overstated at a settling velocity of 0.010 m/s, which can affect the (Stanton et al., 2019). Therefore, pre-purification of distribution and fate of PSNPs in aquatic environments. In samples is required to remove organic matter. In a study contrast, the settling velocity for heteroaggregates of –5 PEMPs and SS was low (~10 with benthic invertebrate samples, it was found that the use m/s), which indicated that the effect of SS on the settling and distribution of of the NR staining method should take digestion protocols PEMPs is negligible. In addition, Cook et al. used NR- into consideration, because chitin cannot be removed by stained PEMPs to measure their longitudinal dispersion H O digestion. It can get stained by NR and exhibits a 2 2 coefficients in laboratory flumes (Cook et al., 2020). They strong fluorescence intensity leading to false positives calculated that the longitudinal dispersion coefficients (Sfriso et al., 2020). Thus, future studies should be done to ranged from 0.0030 to 0.0690 m /s for PE particles. Their develop counterstaining methods with novel dyes to results were used to establish a model concerning the distinguish the fluorescence intensity of MPs from that of transport of MPs in natural rivers. In summary, fluores- organic matter in order to avoid interfere in the quantifica- cence staining and quantification methods facilitate a tion of plastic particles. comprehensive understanding of the fate and transport of MPs in aquatic and terrestrial environments. 5.2 Combine fluorescence staining with other chemical methods to analyze MPs composition 5 Conclusions and future prospects The fluorescence-staining technique facilitates the quanti- fication of the number, concentration, or abundance of MPs in various environmental matrixes. However, the The novel fluorescence staining and quantification method is straightforward, quick, cheap, and reliable for quantifi- fluorescence-staining method cannot stain all types of cation of MP abundance and MP distribution in the polymers, because of the different affinity between dyes environment, including water, soil, sediments, and organ- and plastics (Stanton et al., 2019). For example, NR can isms (Duan et al., 2021; Liu et al., 2021; Wang et al., stain plastics like PP, PE, PS, PC, EPS, PU, and PEVA but 2021). The method is especially suitable for detection of cannot stain plastics like PVC, PA, and PES (Shim et al., MPs in large, bulk environmental samples and laboratory 2016). In addition, the NR-staining method does not samples. However, challenges regarding detection of MPs provide information about the chemical bonds of the and quantification of their concentrations remain, as detected MPs, and the method may be combined with described in the following sections. spectroscopic techniques to determine the chemical 12 Front. Environ. Sci. Eng. 2022, 16(1): 8 composition of plastic particles (Erni-Cassola et al., 2017). developed to provide the mapping image of the sample To better elucidate the transport, availability, and toxicity and high lateral resolution of optical images (Sobhani of MPs, combinational analysis, in which fluorescence et al., 2019). However, how to improve the resolution of staining is used with chemical identification methods like measurement when detecting nanoplastics and overcoming FTIR and IR, is a current trend in studies concerning the nano- scale effects are huge challenges. Future work is occurrence and abundance of MPs. suggested to develop detection methods of nanoplastics to improve superior analytical algorithms and enhance the 5.3 Develop image-analysis methods for quantification of weak signal from nanoplastics. stained MPs Acknowledgements This study was supported by the National Key R&D Program of China (Grant No. 2017YFA0605001), the National Natural Developing fully- or semi-automated image-analysis Science Foundation of China (Grant Nos. 52170024, 21677015 and protocols when using the fluorescence staining and 22006031), the Natural Science Foundation of Hebei Province (No. quantification method has a broad future. Currently, B2019204315), and the Sponsored Research Overhead Fund (Grant No. manual counting of the numbers of MPs under fluorescent 472120) from Kansas State University. or optical microscopy is the most applied of the methods Open Access This article is licensed under a Creative Commons used for the quantification of the abundance and Attribution 4.0 International License, which permits use, sharing, adaptation, concentration of MPs. It has a low efficiency and may distribution and reproduction in any medium or format, as long as you give cause error. Therefore, automated image analysis would be appropriate credit to the original author(s) and the source, provide a link to the helpful and provide researchers with both accuracy and Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative efficiency to save time and labor. The challenge for the Commons licence, unless indicated otherwise in a credit line to the material. future of image-analysis methods includes the counting of If material is not included in the article’s Creative Commons licence and your complex aggregates, which are widely distributed in intended use is not permitted by statutory regulation or exceeds the permitted aquatic and terrestrial environments. use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 5.4 Improve the stability of stained MPs The migration, transport, and transformation of MPs in References natural aquatic, terrestrial, or biotic systems is a long-term Alimi O S, Farner Budarz J, Hernandez L M, Tufenkji N (2018). process. 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Journal

"Frontiers of Environmental Science & Engineering"Springer Journals

Published: Jan 1, 2022

Keywords: Plastic particles; Fluorescence dyes; Identification; Concentration quantification

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