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

doi:10.1007/s11783-021-1442-2

Front. Environ. Sci. Eng.

2022, Vol. 16

Issue (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

Shengdong Liu¹, Enxiang Shang²(

), Jingnan Liu¹, Yining Wang¹, Nanthi Bolan^3,^4,⁵, M.B. Kirkham⁶, Yang Li¹(

)

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

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Abstract

• 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.

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 and pretreatment processes of MPs, including sample collection, digestion, density separation, and quality control are reviewed. Then the promising and convenient staining procedures and quantification methods for MPs using fluorescence dyes are reviewed. The factors that influence the staining of MPs, including 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 recommended as optimal dye concentrations. In addition, a heating and cooling procedure is 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 compositional analysis cannot be determined with it. These fluorescence staining methods have been implemented to study MP abundance, transport, and toxicity and have been combined with other chemical 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.

Keywords Plastic particles Fluorescence dyes Identification Concentration quantification

Corresponding Author(s): Enxiang Shang,Yang Li

Issue Date: 18 November 2021

Cite this article:

Shengdong Liu,Enxiang Shang,Jingnan Liu, et al. What have we known so far for fluorescence staining and quantification of microplastics: A tutorial review[J]. Front. Environ. Sci. Eng., 2022, 16(1): 8.

URL:

https://academic.hep.com.cn/fese/EN/10.1007/s11783-021-1442-2
https://academic.hep.com.cn/fese/EN/Y2022/V16/I1/8

Fig.1 The proportion of research papers investigating the quantification and characterization of microplastic.

Tab.1 Sampling and pretreatment processes of MPs for fluorescence staining

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.

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.

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.

1	O S Alimi, J Farner Budarz, L M Hernandez, N Tufenkji (2018). Microplastics and nanoplastics in aquatic environments: Aggregation, deposition, and enhanced contaminant transport. Environmental Science & Technology, 52(4): 1704–1724 pmid: 29265806
2	C G Avio, S Gorbi, F Regoli (2015). Experimental development of a new protocol for extraction and characterization of microplastics in fish tissues: First observations in commercial species from Adriatic Sea. Marine Environmental Research, 111: 18–26 pmid: 26210759
3	D K A Barnes, F Galgani, R C Thompson, M Barlaz (2009). Accumulation and fragmentation of plastic debris in global environments. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences, 364(1526): 1985–1998 pmid: 19528051
4	A Besley, M G Vijver, P Behrens, T Bosker (2017). A standardized method for sampling and extraction methods for quantifying microplastics in beach sand. Marine Pollution Bulletin, 114(1): 77–83 pmid: 27614562
5	L Bradney, H Wijesekara, K N Palansooriya, N Obadamudalige, N S Bolan, Y S Ok, J Rinklebe, K H Kim, M B Kirkham (2019). Particulate plastics as a vector for toxic trace-element uptake by aquatic and terrestrial organisms and human health risk. Environment International, 131: 104937 pmid: 31284110
6	M A Browne, P Crump, S J Niven, E Teuten, A Tonkin, T Galloway, R Thompson (2011). Accumulation of microplastic on shorelines woldwide: Sources and sinks. Environmental Science & Technology, 45(21): 9175–9179 pmid: 21894925
7	M Carbery, W O’Connor, T Palanisami (2018). Trophic transfer of microplastics and mixed contaminants in the marine food web and implications for human health. Environment International, 115: 400–409 pmid: 29653694
8	A I Catarino, V Macchia, W G Sanderson, R C Thompson, T B Henry (2018). Low levels of microplastics (MP) in wild mussels indicate that MP ingestion by humans is minimal compared to exposure via household fibres fallout during a meal. Environmental Pollution, 237: 675–684 pmid: 29604577
9	Y Chen, Y Leng, X Liu, J Wang (2020). Microplastic pollution in vegetable farmlands of suburb Wuhan, Central China. Environmental Pollution, 257: 113449 pmid: 31706776
10	M Claessens, L Van Cauwenberghe, M B Vandegehuchte, C R Janssen (2013). New techniques for the detection of microplastics in sediments and field collected organisms. Marine Pollution Bulletin, 70(1-2): 227–233 pmid: 23601693
11	M Cole, P Lindeque, C Halsband, T S Galloway (2011). Microplastics as contaminants in the marine environment: A review. Marine Pollution Bulletin, 62(12): 2588–2597 pmid: 22001295
12	S Cook, H L Chan, S Abolfathi, G D Bending, H Schäfer, J M Pearson (2020). Longitudinal dispersion of microplastics in aquatic flows using fluorometric techniques. Water Research, 170: 115337 pmid: 31830655
13	J Ding, S Zhang, R M Razanajatovo, H Zou, W Zhu (2018). Accumulation, tissue distribution, and biochemical effects of polystyrene microplastics in the freshwater fish red tilapia (Oreochromis niloticus). Environmental Pollution, 238: 1–9 pmid: 29529477
14	K Dowarah, A Patchaiyappan, C Thirunavukkarasu, S Jayakumar, S P Devipriya (2020). Quantification of microplastics using Nile Red in two bivalve species Perna viridis and Meretrix meretrix from three estuaries in Pondicherry, India and microplastic uptake by local communities through bivalve diet. Marine Pollution Bulletin, 153: 110982 pmid: 32275539
15	J Duan, N Bolan, Y Li, S Ding, T Atugoda, M Vithanage, B Sarkar, D C W Tsang, M B Kirkham (2021). Weathering of microplastics and interaction with other coexisting constituents in terrestrial and aquatic environments. Water Research, 196: 117011 pmid: 33743325
16	G Erni-Cassola, M I Gibson, R C Thompson, J A Christie-Oleza (2017). Lost, but found with Nile Red: A novel method for detecting and quantifying small microplastics (1 mm to 20 μm) in environmental samples. Environmental Science & Technology, 51(23): 13641–13648 pmid: 29112813
17	W Fu, J Min, W Jiang, Y Li, W Zhang (2020). Separation, characterization and identification of microplastics and nanoplastics in the environment. Science of the Total Environment, 721: 137561 pmid: 32172100
18	F Gagné, J Auclair, B Quinn (2019). Detection of polystyrene nanoplastics in biological samples based on the solvatochromic properties of Nile red: Application in Hydra attenuata exposed to nanoplastics. Environmental Science and Pollution Research International, 26(32): 33524–33531 pmid: 31578681
19	P Greenspan, S D Fowler (1985). Spectrofluorometric studies of the lipid probe, nile red. Journal of Lipid Research, 26(7): 781–789 pmid: 4031658
20	B Guo, J Meng, X Wang, C Yin, W Hao, B Ma, Z Tao (2020). Quantification of pesticide residues on plastic mulching films in typical farmlands of the North China. Frontiers of Environmental Science & Engineering, 14(1): 2
21	G Hanke, F Galgani, S Werner, L Oosterbaan, P Nilsson, D Fleet, S Kinsey, R Thompson, J A Van Franeker, T Vlachogianni (2013). Guidance on monitoring of marine litter in European seas. Luxembourg. doi, 10: 99475
22	V Hidalgo-Ruz, L Gutow, R C Thompson, M Thiel (2012). Microplastics in the marine environment: A review of the methods used for identification and quantification. Environmental Science & Technology, 46(6): 3060–3075 pmid: 22321064
23	A Y Jee, S Park, H Kwon, M Lee (2009). Excited state dynamics of Nile Red in polymers. Chemical Physics Letters, 477(1–3): 112–115
24	E G Karakolis, B Nguyen, J B You, C M Rochman, D Sinton (2019). Fluorescent dyes for visualizing microplastic particles and fibers in laboratory-based studies. Environmental Science & Technology Letters, 6(6): 334–340
25	M Klein, E K Fischer (2019). Microplastic abundance in atmospheric deposition within the Metropolitan area of Hamburg, Germany. Science of the Total Environment, 685: 96–103 pmid: 31174127
26	S Konde, J Ornik, J A Prume, J Taiber, M Koch (2020). Exploring the potential of photoluminescence spectroscopy in combination with Nile Red staining for microplastic detection. Marine Pollution Bulletin, 159: 111475 pmid: 32692678
27	M Kumar, X Xiong, M He, D C W Tsang, J Gupta, E Khan, S Harrad, D Hou, Y S Ok, N S Bolan (2020). Microplastics as pollutants in agricultural soils. Environmental Pollution, 265(Pt A): 114980
28	M Lares, M C Ncibi, M Sillanpää, M Sillanpää (2019). Intercomparison study on commonly used methods to determine microplastics in wastewater and sludge samples. Environmental Science and Pollution Research International, 26(12): 12109–12122 pmid: 30827019
29	Y Li, X Wang, W Fu, X Xia, C Liu, J Min, W Zhang, J C Crittenden (2019). Interactions between nano/micro plastics and suspended sediment in water: Implications on aggregation and settling. Water Research, 161: 486–495 pmid: 31229729
30	X Liu, J Wang (2020). Algae (Raphidocelis subcapitata) mitigate combined toxicity of microplastic and lead on Ceriodaphnia dubia. Frontiers of Environmental Science & Engineering, 14(6): 97
31	Y Liu, H Shao, J Liu, R Cao, E Shang, S Liu, Y Li (2021). Transport and transformation of microplastics and nanoplastics in the soil environment: A critical review. Soil Use and Management, 37(2): 224–242
32	L Lv, J Qu, Z Yu, D Chen, C Zhou, P Hong, S Sun, C Li (2019). A simple method for detecting and quantifying microplastics utilizing fluorescent dyes: Safranine T, fluorescein isophosphate, Nile Red based on thermal expansion and contraction property. Environmental Pollution, 255(Pt 2): 113283 pmid: 31580990
33	T Maes, R Jessop, N Wellner, K Haupt, A G Mayes (2017). A rapid-screening approach to detect and quantify microplastics based on fluorescent tagging with Nile Red. Scientific Reports, 7: 44501 pmid: 28300146
34	L Mai, L J Bao, L Shi, C S Wong, E Y Zeng (2018). A review of methods for measuring microplastics in aquatic environments. Environmental Science and Pollution Research International, 25(12): 11319–11332 pmid: 29536421
35	H Maxwell S, F Melinda K, G Matthew (2020). Counterstaining to separate Nile Red-stained microplastic particles from terrestrial invertebrate biomass. Environmental Science & Technology, 54(9): 5580–5588 pmid: 32298090
36	A McCormick, T J Hoellein, S A Mason, J Schluep, J J Kelly (2014). Microplastic is an abundant and distinct microbial habitat in an urban river. Environmental Science & Technology, 48(20): 11863–11871 pmid: 25230146
37	N H Nor, J P Obbard (2014). Microplastics in Singapore’s coastal mangrove ecosystems. Marine Pollution Bulletin, 79(1–2): 278–283 pmid: 24365455
38	M T Nuelle, J H Dekiff, D Remy, E Fries (2014). A new analytical approach for monitoring microplastics in marine sediments. Environmental Pollution, 184: 161–169 pmid: 24051349
39	A Patchaiyappan, S Z Ahmed, K Dowarah, S Jayakumar, S P Devipriya (2020). Occurrence, distribution and composition of microplastics in the sediments of South Andaman beaches. Marine Pollution Bulletin, 156: 111227 pmid: 32510373
40	J C Prata, J R Alves, J P da Costa, A C Duarte, T Rocha-Santos (2020). Major factors influencing the quantification of Nile Red stained microplastics and improved automatic quantification (MP-VAT 2.0). Science of the Total Environment, 719: 137498 pmid: 32120106
41	J C Prata, J P Da Costa, A C Duarte, T Rocha-Santos (2019a). Methods for sampling and detection of microplastics in water and sediment: A critical review. Trends in Analytical Chemistry, 110: 150–159
42	J C Prata, V Reis, J T V Matos, J P da Costa, A C Duarte, T Rocha-Santos (2019b). A new approach for routine quantification of microplastics using Nile Red and automated software (MP-VAT). Science of the Total Environment, 690: 1277–1283 pmid: 31470490
43	T Rocha-Santos, A C Duarte (2015). A critical overview of the analytical approaches to the occurrence, the fate and the behavior of microplastics in the environment. Trends in Analytical Chemistry, 65: 47–53
44	J Rumin, H Bonnefond, B Saint-Jean, C Rouxel, A Sciandra, O Bernard, J P Cadoret, G Bougaran (2015). The use of fluorescent Nile red and BODIPY for lipid measurement in microalgae. Biotechnology for Biofuels, 8: 42 pmid: 25788982
45	A Scircle, J V Cizdziel, L Tisinger, T Anumol, D Robey (2020). Occurrence of microplastic pollution at Oyster Reefs and other coastal sites in the Mississippi Sound, USA: Impacts of freshwater inflows from flooding. Toxics, 8(2): 35 pmid: 32429184
46	A A Sfriso, Y Tomio, B Rosso, A Gambaro, A Sfriso, F Corami, E Rastelli, C Corinaldesi, M Mistri, C Munari (2020). Microplastic accumulation in benthic invertebrates in Terra Nova Bay (Ross Sea, Antarctica). Environment International, 137: 105587 pmid: 32097803
47	F Shahul Hamid, M S Bhatti, N Anuar, N Anuar, P Mohan, A Periathamby (2018). Worldwide distribution and abundance of microplastic: How dire is the situation? Waste Management and Research, 36(10): 873–897 pmid: 30103651
48	W J Shim, S H Hong, S E Eo (2017). Identification methods in microplastic analysis: A review. Analytical Methods, 9(9): 1384–1391
49	W J Shim, Y K Song, S H Hong, M Jang (2016). Identification and quantification of microplastics using Nile Red staining. Marine Pollution Bulletin, 113(1–2): 469–476 pmid: 28340965
50	C B Simmerman, J K C Wasik (2020). The effect of urban point source contamination on microplastic levels in water and organisms in a cold-water stream. Limnology and Oceanography Letters, 5(1): 137–146
51	Z Sobhani, M Al Amin, R Naidu, M Megharaj, C Fang (2019). Identification and visualisation of microplastics by Raman mapping. Analytica Chimica Acta, 1077: 191–199 pmid: 31307709
52	Z Sobhani, X Zhang, C Gibson, R Naidu, M Megharaj, C Fang (2020). Identification and visualisation of microplastics/nanoplastics by Raman imaging (i): Down to 100 nm. Water Research, 174: 115658 pmid: 32146170
53	T Stanton, M Johnson, P Nathanail, R L Gomes, T Needham, A Burson (2019). Exploring the efficacy of Nile Red in microplastic quantification: A costaining approach. Environmental Science & Technology Letters, 6(10): 606–611
54	L Sun, N Sun, L Bai, X An, B Liu, C Sun, L Fan, C Wei, Y Han, M Yu, J Lin, D Lu, N Wang, L Xie, K Shen, X Zhang, Y Xu, J Cabanillas-Gonzaleze, W Huang (2019). Alkyl-chain branched effect on the aggregation and photophysical behavior of polydiarylfluorenes toward stable deep-blue electroluminescence and efficient amplified spontaneous emission. Chinese Chemical Letters, 30(11): 1959–1964
55	M Tamminga (2017). Nile Red staining as a subsidiary method for microplastic quantification: A comparison of three solvents and factors influencing application reliability. SDRP Journal of Earth Sciences & Environmental Studies, 2(2): 165–168
56	R C Thompson, Y Olsen, R P Mitchell, A Davis, S J Rowland, A W G John, D McGonigle, A E Russell (2004). Lost at sea: where is all the plastic? Science, 304(5672): 838 pmid: 15131299
57	M Tiwari, T D Rathod, P Y Ajmal, R C Bhangare, S K Sahu (2019). Distribution and characterization of microplastics in beach sand from three different Indian coastal environments. Marine Pollution Bulletin, 140: 262–273 pmid: 30803642
58	A E Valine, A E Peterson, D A Horn, K M Scully-Engelmeyer, E F Granek (2020). Microplastic prevalence in 4 Oregon rivers along a rural to urban gradient applying a cost‐effective validation technique. Environmental Toxicology and Chemistry, 39(8): 1590–1598 pmid: 32430919
59	J C Vermaire, C Pomeroy, S M Herczegh, O Haggart, M Murphy, D E Schindler (2017). Microplastic abundance and distribution in the open water and sediment of the Ottawa River, Canada, and its tributaries. Facets, 2(1): 301–314
60	X Wang, N Bolan, D C W Tsang, B Sarkar, L Bradney, Y Li (2021). A review of microplastics aggregation in aquatic environment: Influence factors, analytical methods, and environmental implications. Journal of Hazardous Materials, 402: 123496 pmid: 32717542
61	K J Wiggin, E B Holland (2019). Validation and application of cost and time effective methods for the detection of 3–500 μm sized microplastics in the urban marine and estuarine environments surrounding Long Beach, California. Marine Pollution Bulletin, 143: 152–162 pmid: 31789151
62	W M Wu, J Yang, C S Criddle (2017). Microplastics pollution and reduction strategies. Frontiers of Environmental Science & Engineering, 11(1): 4
63	G S Zhang, Y F Liu (2018). The distribution of microplastics in soil aggregate fractions in southwestern China. Science of the Total Environment, 642: 12–20 pmid: 29894871
64	S Zhang, X Liu, X Hao, J Wang, Y Zhang (2020). Distribution of low-density microplastics in the mollisol farmlands of northeast China. Science of the Total Environment, 708: 135091 pmid: 31785906
65	S Ziajahromi, P A Neale, L Rintoul, F D Leusch (2017). Wastewater treatment plants as a pathway for microplastics: Development of a new approach to sample wastewater-based microplastics. Water Research, 112: 93–99 pmid: 28160700

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