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

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.

Graphical abstract

Keywords

Plastic particles / Fluorescence dyes / Identification / Concentration quantification

References

[1] Alimi O S, Farner Budarz J, Hernandez L M, Tufenkji N (2018). Microplastics and nanoplastics in aquatic environments: Aggregation, deposition, and enhanced contaminant transport. Environmental Science & Technology, 52(4): 1704–1724

[2] Avio C G, Gorbi S, Regoli F (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

[3] Barnes D K A, Galgani F, Thompson R C, Barlaz M (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

[4] Besley A, Vijver M G, Behrens P, Bosker T (2017). A standardized method for sampling and extraction methods for quantifying microplastics in beach sand. Marine Pollution Bulletin, 114(1): 77–83

[5] Bradney L, Wijesekara H, Palansooriya K N, Obadamudalige N, Bolan N S, Ok Y S, Rinklebe J, Kim K H, Kirkham M B (2019). Particulate plastics as a vector for toxic trace-element uptake by aquatic and terrestrial organisms and human health risk. Environment International, 131: 104937

[6] Browne M A, Crump P, Niven S J, Teuten E, Tonkin A, Galloway T, Thompson R (2011). Accumulation of microplastic on shorelines woldwide: Sources and sinks. Environmental Science & Technology, 45(21): 9175–9179

[7] Carbery M, O’Connor W, Palanisami T (2018). Trophic transfer of microplastics and mixed contaminants in the marine food web and implications for human health. Environment International, 115: 400–409

[8] Catarino A I, Macchia V, Sanderson W G, Thompson R C, Henry T B (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

[9] Chen Y, Leng Y, Liu X, Wang J (2020). Microplastic pollution in vegetable farmlands of suburb Wuhan, Central China. Environmental Pollution, 257: 113449

[10] Claessens M, Van Cauwenberghe L, Vandegehuchte M B, Janssen C R (2013). New techniques for the detection of microplastics in sediments and field collected organisms. Marine Pollution Bulletin, 70(1-2): 227–233

[11] Cole M, Lindeque P, Halsband C, Galloway T S (2011). Microplastics as contaminants in the marine environment: A review. Marine Pollution Bulletin, 62(12): 2588–2597

[12] Cook S, Chan H L, Abolfathi S, Bending G D, Schäfer H, Pearson J M (2020). Longitudinal dispersion of microplastics in aquatic flows using fluorometric techniques. Water Research, 170: 115337

[13] Ding J, Zhang S, Razanajatovo R M, Zou H, Zhu W (2018). Accumulation, tissue distribution, and biochemical effects of polystyrene microplastics in the freshwater fish red tilapia (Oreochromis niloticus). Environmental Pollution, 238: 1–9

[14] Dowarah K, Patchaiyappan A, Thirunavukkarasu C, Jayakumar S, Devipriya S P (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

[15] Duan J, Bolan N, Li Y, Ding S, Atugoda T, Vithanage M, Sarkar B, Tsang D C W, Kirkham M B (2021). Weathering of microplastics and interaction with other coexisting constituents in terrestrial and aquatic environments. Water Research, 196: 117011

[16] Erni-Cassola G, Gibson M I, Thompson R C, Christie-Oleza J A (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

[17] Fu W, Min J, Jiang W, Li Y, Zhang W (2020). Separation, characterization and identification of microplastics and nanoplastics in the environment. Science of the Total Environment, 721: 137561

[18] Gagné F, Auclair J, Quinn B (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

[19] Greenspan P, Fowler S D (1985). Spectrofluorometric studies of the lipid probe, nile red. Journal of Lipid Research, 26(7): 781–789

[20] Guo B, Meng J, Wang X, Yin C, Hao W, Ma B, Tao Z (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] Hanke G, Galgani F, Werner S, Oosterbaan L, Nilsson P, Fleet D, Kinsey S, Thompson R, Van Franeker J A, Vlachogianni T (2013). Guidance on monitoring of marine litter in European seas. Luxembourg. doi, 10: 99475

[22] Hidalgo-Ruz V, Gutow L, Thompson R C, Thiel M (2012). Microplastics in the marine environment: A review of the methods used for identification and quantification. Environmental Science & Technology, 46(6): 3060–3075

[23] Jee A Y, Park S, Kwon H, Lee M (2009). Excited state dynamics of Nile Red in polymers. Chemical Physics Letters, 477(1–3): 112–115

[24] Karakolis E G, Nguyen B, You J B, Rochman C M, Sinton D (2019). Fluorescent dyes for visualizing microplastic particles and fibers in laboratory-based studies. Environmental Science & Technology Letters, 6(6): 334–340

[25] Klein M, Fischer E K (2019). Microplastic abundance in atmospheric deposition within the Metropolitan area of Hamburg, Germany. Science of the Total Environment, 685: 96–103

[26] Konde S, Ornik J, Prume J A, Taiber J, Koch M (2020). Exploring the potential of photoluminescence spectroscopy in combination with Nile Red staining for microplastic detection. Marine Pollution Bulletin, 159: 111475

[27] Kumar M, Xiong X, He M, Tsang D C W, Gupta J, Khan E, Harrad S, Hou D, Ok Y S, Bolan N S (2020). Microplastics as pollutants in agricultural soils. Environmental Pollution, 265(Pt A): 114980

[28] Lares M, Ncibi M C, Sillanpää M, Sillanpää M (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

[29] Li Y, Wang X, Fu W, Xia X, Liu C, Min J, Zhang W, Crittenden J C (2019). Interactions between nano/micro plastics and suspended sediment in water: Implications on aggregation and settling. Water Research, 161: 486–495

[30] Liu X, Wang J (2020). Algae (Raphidocelis subcapitata) mitigate combined toxicity of microplastic and lead on Ceriodaphnia dubia. Frontiers of Environmental Science & Engineering, 14(6): 97

[31] Liu Y, Shao H, Liu J, Cao R, Shang E, Liu S, Li Y (2021). Transport and transformation of microplastics and nanoplastics in the soil environment: A critical review. Soil Use and Management, 37(2): 224–242

[32] Lv L, Qu J, Yu Z, Chen D, Zhou C, Hong P, Sun S, Li C (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

[33] Maes T, Jessop R, Wellner N, Haupt K, Mayes A G (2017). A rapid-screening approach to detect and quantify microplastics based on fluorescent tagging with Nile Red. Scientific Reports, 7: 44501

[34] Mai L, Bao L J, Shi L, Wong C S, Zeng E Y (2018). A review of methods for measuring microplastics in aquatic environments. Environmental Science and Pollution Research International, 25(12): 11319–11332

[35] Maxwell S H, Melinda K F, Matthew G (2020). Counterstaining to separate Nile Red-stained microplastic particles from terrestrial invertebrate biomass. Environmental Science & Technology, 54(9): 5580–5588

[36] McCormick A, Hoellein T J, Mason S A, Schluep J, Kelly J J (2014). Microplastic is an abundant and distinct microbial habitat in an urban river. Environmental Science & Technology, 48(20): 11863–11871

[37] Nor N H, Obbard J P (2014). Microplastics in Singapore’s coastal mangrove ecosystems. Marine Pollution Bulletin, 79(1–2): 278–283

[38] Nuelle M T, Dekiff J H, Remy D, Fries E (2014). A new analytical approach for monitoring microplastics in marine sediments. Environmental Pollution, 184: 161–169

[39] Patchaiyappan A, Ahmed S Z, Dowarah K, Jayakumar S, Devipriya S P (2020). Occurrence, distribution and composition of microplastics in the sediments of South Andaman beaches. Marine Pollution Bulletin, 156: 111227

[40] Prata J C, Alves J R, da Costa J P, Duarte A C, Rocha-Santos T (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

[41] Prata J C, Da Costa J P, Duarte A C, Rocha-Santos T (2019a). Methods for sampling and detection of microplastics in water and sediment: A critical review. Trends in Analytical Chemistry, 110: 150–159

[42] Prata J C, Reis V, Matos J T V, da Costa J P, Duarte A C, Rocha-Santos T (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

[43] Rocha-Santos T, Duarte A C (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] Rumin J, Bonnefond H, Saint-Jean B, Rouxel C, Sciandra A, Bernard O, Cadoret J P, Bougaran G (2015). The use of fluorescent Nile red and BODIPY for lipid measurement in microalgae. Biotechnology for Biofuels, 8: 42

[45] Scircle A, Cizdziel J V, Tisinger L, Anumol T, Robey D (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

[46] Sfriso A A, Tomio Y, Rosso B, Gambaro A, Sfriso A, Corami F, Rastelli E, Corinaldesi C, Mistri M, Munari C (2020). Microplastic accumulation in benthic invertebrates in Terra Nova Bay (Ross Sea, Antarctica). Environment International, 137: 105587

[47] Shahul Hamid F, Bhatti M S, Anuar N, Anuar N, Mohan P, Periathamby A (2018). Worldwide distribution and abundance of microplastic: How dire is the situation? Waste Management and Research, 36(10): 873–897

[48] Shim W J, Hong S H, Eo S E (2017). Identification methods in microplastic analysis: A review. Analytical Methods, 9(9): 1384–1391

[49] Shim W J, Song Y K, Hong S H, Jang M (2016). Identification and quantification of microplastics using Nile Red staining. Marine Pollution Bulletin, 113(1–2): 469–476

[50] Simmerman C B, Wasik J K C (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] Sobhani Z, Al Amin M, Naidu R, Megharaj M, Fang C (2019). Identification and visualisation of microplastics by Raman mapping. Analytica Chimica Acta, 1077: 191–199

[52] Sobhani Z, Zhang X, Gibson C, Naidu R, Megharaj M, Fang C (2020). Identification and visualisation of microplastics/nanoplastics by Raman imaging (i): Down to 100 nm. Water Research, 174: 115658

[53] Stanton T, Johnson M, Nathanail P, Gomes R L, Needham T, Burson A (2019). Exploring the efficacy of Nile Red in microplastic quantification: A costaining approach. Environmental Science & Technology Letters, 6(10): 606–611

[54] Sun L, Sun N, Bai L, An X, Liu B, Sun C, Fan L, Wei C, Han Y, Yu M, Lin J, Lu D, Wang N, Xie L, Shen K, Zhang X, Xu Y, Cabanillas-Gonzaleze J, Huang W (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] Tamminga M (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] Thompson R C, Olsen Y, Mitchell R P, Davis A, Rowland S J, John A W G, McGonigle D, Russell A E (2004). Lost at sea: where is all the plastic? Science, 304(5672): 838

[57] Tiwari M, Rathod T D, Ajmal P Y, Bhangare R C, Sahu S K (2019). Distribution and characterization of microplastics in beach sand from three different Indian coastal environments. Marine Pollution Bulletin, 140: 262–273

[58] Valine A E, Peterson A E, Horn D A, Scully-Engelmeyer K M, Granek E F (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

[59] Vermaire J C, Pomeroy C, Herczegh S M, Haggart O, Murphy M, Schindler D E (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] Wang X, Bolan N, Tsang D C W, Sarkar B, Bradney L, Li Y (2021). A review of microplastics aggregation in aquatic environment: Influence factors, analytical methods, and environmental implications. Journal of Hazardous Materials, 402: 123496

[61] Wiggin K J, Holland E B (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

[62] Wu W M, Yang J, Criddle C S (2017). Microplastics pollution and reduction strategies. Frontiers of Environmental Science & Engineering, 11(1): 4

[63] Zhang G S, Liu Y F (2018). The distribution of microplastics in soil aggregate fractions in southwestern China. Science of the Total Environment, 642: 12–20

[64] Zhang S, Liu X, Hao X, Wang J, Zhang Y (2020). Distribution of low-density microplastics in the mollisol farmlands of northeast China. Science of the Total Environment, 708: 135091

[65] Ziajahromi S, Neale P A, Rintoul L, Leusch F D (2017). Wastewater treatment plants as a pathway for microplastics: Development of a new approach to sample wastewater-based microplastics. Water Research, 112: 93–99

RIGHTS & PERMISSIONS

The Author(s) 2022. This article is published with open access at link.springer.com and journal.hep. com.cn