Chemosphere 315 (2023) 137757

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Microplastics in road dust: A practical guide for identification

and characterisation
Rebecca Myszka, Marie Enfrin, Filippo Giustozzi *
Royal Melbourne Institute of Technology, Civil and Infrastructure Engineering, Melbourne 3001, Victoria, Australia

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• A fluorescence staining method was

optimised to identify microplastic in
road dust.
• Microplastic shape, amount and size
identification was achieved to 1 μm.
• The optimised Nile red solution was
used to assess 33 road dust materials.
• Autofluorescence can be used to deter­
mine microplastic origin in road dust.

A R T I C L E I N F O A B S T R A C T

Handling Editor: Michael Bank The contamination of the environment by microplastics (MPs) in road dust poses a serious ecological and health
concern. MPs have been detected in road dust worldwide and their presence has been mainly attributed to plastic
Keywords: litter fragmentation and vehicle tyre abrasion. Although current technologies such as Raman and Fourier
Microplastic Transform InfraRed spectroscopy as well as Scanning Electron Microscopy are capable of detecting MPs in road
Fluorescence
dust, the analysis of MPs shape and MPs smaller than 20 μm is limited and often labour demanding. More ac­
Recycled plastic
curate, cost-effective and rapid techniques have now become necessary to analyse MPs in road dust, particularly
Road dust
Asphalt since the development of large infrastructure projects that incorporate recycled plastic into road assets and
Nile red roadside furniture. Nile red (NR) staining is a promising technique to identify MPs in environmental samples;
however, it has not yet been applied to road dust. This study investigates the use of NR fluorescence microscopy
to detect MPs in road dust and provides information about MP amount, shape and size distribution. The staining
duration and temperature, solvent selection and NR concentration were optimised considering 33 different road
dust materials, including 13 types of plastic. The NR staining procedure developed in this work is capable of
successfully differentiating between MPs down to 1 μm and other non-plastic road dust materials. Future ap­
plications include assessing the contribution of plastic-modified roads to MP pollution, comparing the level of MP
pollution in urban and rural areas and providing a rapid, simple, inexpensive and reliable monitoring approach
for further studies to compare MP using a singular optimised methodology.

* Corresponding author.
E-mail address: [email protected] (F. Giustozzi).

https://doi.org/10.1016/j.chemosphere.2023.137757
Received 17 April 2022; Received in revised form 8 December 2022; Accepted 3 January 2023
Available online 4 January 2023
0045-6535/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-
nc/4.0/).
R. Myszka et al. Chemosphere 315 (2023) 137757

1. Introduction period (24–168 h) after staining to obtain optimal visibility for MPs
under a fluorescent microscope (Prata et al., 2019b). Direct comparisons
The abundance of plastic released into the environment is an between MP research in road dust or urban wastewater/air quality are
increasing global concern (Rillig et al., 2021). Annual plastic emissions limited as studies fail to provide sufficient and comparable details on MP
in 2030 are expected to reach up to 53 million metric tons/year due to size. Some identified limitations include time and labour constraints
increases in global plastic production and usage (Borrelle et al., 2020). It (Browne et al., 2011; Magnusson and Norén, 2014; Carr et al., 2016;
is estimated that tyre and road wear particles constitute up to 42% of the Fang et al., 2022), while other studies have tried to solve this issue by
total microplastics (MPs) transported to the ocean, including vehicle categorising MP groups by size with MPs <1 mm commonly being re­
tyres, polymers used for bitumen modification and road marking con­ ported as the minimum size range (Hidalgo-Ruz et al., 2012; Doyle et al.,
stituents (Siegfried et al., 2017; Vogelsang et al., 2019; Dehghani et al., 2011; Monira et al., 2022a). Although promising for facilitating MP
2017). Road dust generation occurs due to the continuous passage of identification, fluorescence staining is limited by road dust composition
vehicles along urban and regional road pavements, resulting in the since MPs may be mixed with non-plastic particles that could also
fragmentation of large pieces of roadside debris, including plastic food fluoresce, such as leaves and soil (Nalbone et al., 2021). The main road
containers and packaging as well as asphalt and other construction constituents, aggregates and bitumen, could also impact the detection of
materials, soils and organics (Monira et al., 2021). Road dust consists of MPs as well as metals and inorganic materials originating from nearby
a combination of organic and inorganic particles, among which MPs construction sites. The extraction of MPs from contaminants via organics
were found to contribute up to 0.06% of dry mass within an urban city removal, density separation and filtration is commonly performed for
environment (O’Brien et al., 2021). However, the incorporation of environmental samples, however, such procedures are time-consuming
recycled plastic in roads raised awareness about the environmental and can lead to particle loss due to the multiple steps involved (Shim
impact of MPs found in road dust since plastic-modified roads have the et al., 2016a).
potential to release and increase the number of MPs in the environment Recent studies relating to MPs analysis in road dust involved labo­
due to weathering as well as surface abrasion from vehicle tyres (Zhang rious methods such as Scanning Electron Microscopy, electrochemical
et al., 2017; Boom and Guistozzi, 2021; Giustozzi et al., 2021). detection including the use of boron-doped diamond electrodes or
MPs are widely defined as plastic particles smaller than 5 mm; μ-Raman and μ-FTIR spectroscopy, which may provide an inaccurate
however, several studies have indicated a preferable upper particle size estimation of MP release due to the low volume of particles that can be
limit of 1 mm due to increased toxicity concerns for particles with a analysed as well as the manual handling of plastics and individual
higher surface area to volume ratio (ISO, 2020; Thiele et al., 2021; Van counting (Martić et al., 2022; Narmadha et al., 2020; Kang et al., 2022;
Cauwenberghe Lisbeth et al., 2012). MPs can occur in the environment Khan and Strand, 2018). Although effective in identifying plastic, NR
either as a direct input or through the degradation and fragmentation of staining procedures can be biased by the background fluorescence of
large plastic waste fragments and constitute an environmental concern filters used for pre-treatment to extract MPs, which may interfere with
due to their toxicity (Rillig et al., 2021; Julienne et al., 2019; Song et al., MP fluorescence and therefore prevent proper identification (Shim et al.,
2017). Although MPs have been found to only contribute 0.06% of dry 2016a; Shruti et al., 2022). The presence of non-plastic materials in road
mass in road dust, MPs along with rubber are still considered hazardous dust, such as asphalt residues, metals and other organic materials such
for human health and the environment (O’Brien et al., 2021; Zhang as leaves soil and bark also limits the applicability of these techniques
et al., 2017; Boom and Guistozzi, 2021; Giustozzi et al., 2021). MP (Donaldson, 2020). Hence, this study has been attuned to identifying,
toxicity is enhanced with decreasing particle size due to severe effects characterising and quantifying MPs in road dust mixtures considering
from the ingestion of small MPs by organisms, including the subsequent time constraints, technical constraints related to sample preparation and
uptake and accumulation of contaminants such as persistent organic the presence of non-plastic materials in road dust.
pollutants (POPs) present on the surface of MP particles (Lehner et al., The aim of this study was to develop a rapid, accurate and cost-
2019; Andrady, 2011). Hence, the contribution to MP pollution in road effective technique to detect MPs in road dust based on NR fluores­
dust from plastic roads constitutes a threat to the environment that has cence microscopy. Within this paper, the authors present ideal staining
yet to be investigated. conditions and parameters required to quantify MPs within road dust.
The identification of MPs in environmental samples is complex; This procedure was developed to address limitations that exist in current
current analytical methods vary greatly within the literature due to research, including minimal investigations into the fluorescence of road
variance in sampling techniques as well as extraction and analysis pro­ dust constituents such as metals, soils, foliage, and road-marking frag­
tocols (Stanton et al., 2019; Dean et al., 2010). Spectroscopic techniques ments (Siegfried et al., 2017; Monira et al., 2021). The staining pro­
may be used to identify MPs, such as Raman or Fourier Transform cedure parameters were optimised for 33 different road dust materials,
Infrared (FTIR) spectroscopy, as well as chromatographic techniques, including 13 types of plastic. The shape and size of MPs down to 1 μm
including pyrolysis-gas chromatography (Maes et al., 2017a; Tamminga were successfully analysed, opening doors for road authorities to assess
et al., 2017; Prata et al., 2021). However, MP particle size and shape the contribution of urban/rural areas and plastic-modified roads to MP
cannot be assessed using these techniques; hence, complementary pollution. Additionally, although bitumen has been modified with
microscopic analyses are commonly performed to visually examine MPs. plastics for decades, there have been no investigations targeting the
The successful removal of organic matter from road dust may be used to presence of MPs in road dust from these plastic-modified roads; hence,
improve MP identification (Prata et al., 2019a; Enfrin et al., 2022). this study may be adapted for this focus throughout future applications.
Fluorescence staining can be used following sample pre-treatment to
characterise MPs particle shape and size (Andrady, 2011; Prata et al., 2. Materials and methods
2019a; Erni-Cassola et al., 2017). Alongside providing a rapid method
for MP detection, the use of fluorescence staining is a cost-effective 2.1. Materials
technique for MPs analysis with compared to expensive instruments
such as FPA-FTIR. Acetone (>99%), ethanol (>99%), n-heptane (>95%), chloroform
Nile red (NR) is typically used to stain hydrophobic plastic particles (>99%), n-hexane (>95%) and Nile red (9-diethylamino5H-benzo[a]
that cannot be detected by common instruments such as FTIR, which can phenoxazine-5-one) (>97%) were provided by Merck Pty Ltd.
detect MPs down to 20 μm, or μ-FTIR, which has a minimum size Virgin low-density polyethylene (LDPE) was obtained from Good­
detection limit of 3 μm (Giustozzi et al., 2021; Stanton et al., 2019; Vinay fellow Corporation Pty Ltd. in the form of pellets and powder. Sonali Pvt
Kumar et al., 2021). NR is a lipophilic solvatochromic dye which fluo­ Ltd. provided virgin linear low-density polyethylene (LLDPE) and
resces in hydrophobic-rich environments over a suitable incubation polypropylene (PP) pellets. Qenos Pty Ltd. supplied virgin high-density

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polyethylene (HDPE) pellets. Virgin polyethylene terephthalate (PET) Table 1

pellets, styrene-ethylene-butylene-styrene (SEBS), polylactide acid Database of road dust materials investigated by Nile Red staining.
(PLA) and styrene-butadiene-styrene (SBS) were provided by Merck Pty Plastic Non-plastic
Ltd. Virgin ethylene-vinyl acetate (EVA) (D2836 m, 28% vinyl acetate)
Recycled ABS Asphalt Aggregate
and virgin acrylonitrile butadiene styrene (ABS) pellets were provided Plastics constituents
by Repsol S. A. and Plasgroup Pty Ltd., respectively. Crumb rubber was HDPE Bitumen
provided by a local tyre recycler. Recycled plastic samples consisting of LDPE Sand
recycled LDPE, PP, PET and ABS were sourced from 3 plastic recyclers LLDPE Organics and Soils Calcium
Carbonate
within Australia (Giustozzi et al., 2021). Asphalt dust and microplastics PET Cardboard
extracted after abrasion of asphalt modified by recycled LDPE, PP, ABS PP Clay
and PET were sourced from a previous work of the authors (Enfrin et al., Virgin Plastics ABS Foliage
2022). In brief, a recycled plastic-modified asphalt sample was abraded Crumb rubber Paper
HDPE Starch
under controlled laboratory conditions to simulate traffic, and the res­
LDPE Sulphur
idues abraded from the asphalt were collected. MPs were extracted from LLDPE Talc
the residues and subsequently used in this work to represent real MPs PET Timber
that could be released from plastic-modified roads and end-up in road PP Metals Aluminium
dust (Enfrin et al., 2022). Copper
Iron
Aluminium (>99%), iron (>98%), lead (>99%), tin (>99%), silver Lead
(>99%), copper (>99%), tungsten (>99%), clay (>99%), calcium car­ Silver
bonate (>99%), sulphur (>98%), starch (>98%) and talcum powder Steel
(>99%) were purchased from Sigma–Aldrich Pty Ltd. Organic material, Tin
Tungsten
including bark, sand, cardboard, paper, foliage and leaves were
collected in the field.
100 ◦ C, where the samples were left to dry in a refrigerator at 4 ◦ C, a
2.2. Staining methodology well-ventilated room at 25 ◦ C or an oven at 100 ◦ C. Temperature testing
was designed to account for the temperature-selective staining of NR
This study focuses on a new method to quantify MPs extracted from and ensure selective plastic fluorescence within road dust (Tamminga
road dust residues after minimal sample pre-treatment and therefore et al., 2017). NR was found to be stable and exhibited fluorescence after
potentially mixed with other road dust contaminants. The well-known staining at all tested temperatures.
organics removal and density separation steps commonly used for MPs Each plastic particle sample was evenly stained using 0.1 ml of NR
extraction are therefore not considered in this study. This investigation solution in the selected solvent and up to 1 ml for large filter pieces. The
focuses on 33 materials commonly found in road dust, including or­ sample was stained on a microscope slide prior to being enclosed with
ganics, plastic, asphalt material, metals and MPs artificially released another slide to ensure secure and flat coverage of the sample. The slide
from recycled plastic-modified asphalt upon abrasion. All materials was then covered with aluminium foil to prevent light exposure for
were analysed individually to determine their fluorescence properties consistent staining and imaging (Escorcia et al., 2018). The slides were
after staining. Real road dust samples were not considered in this study. transferred to a fume hood where the duration between staining and
Glass particles in road pavement markings and paints were also omitted microscope examination varied between 24, 48, and 168 h for procedure
due to the high polarity of glass particles preventing NR staining. Note optimisation.
that all cover slides were made of glass and hence glass was assumed not
to impact the staining methodology. 2.2.2. Visual identification methodology
A Nikon N-storm inverted stage Ti-E Nikon confocal microscope was
2.2.1. Fluorescence staining used to image the stained plastics and road contaminants. The instru­
The staining procedure was developed to ensure that only MPs ment was equipped with ×10 and ×20 objectives and lasers Coherent
fluoresced in a road dust mixture of plastic and other non-plastic ma­ OBIS L×647–120, 1,185,049 Coherent OBIS LX 405–100, 1,165,327
terials. 13 different types of plastics and 20 non-plastic materials Coherent Sapphire 488–100 and 1,137,504 Coherent Sapphire 561–100,
commonly found in road dust were investigated individually to generate fitted with a digital camera for image capture. The laser intensity was
a comprehensive database presented in Table 1. Two different types of standardised at 1 and the pinhole was varied between 1, 5, and 10.
filters were also considered since filtration is commonly used to remove Details are provided in Table 2.
large contaminants from samples collected in the field. Although filters
are useful to prevent the overlapping of particles and facilitate the
identification of MPs, fluorescence from the filters can interfere with
that of MPs and result in overestimations.
The staining procedure was initially optimised using LDPE particles,
as polyethylene (PE) is a dominant MP in stormwater as well as road Table 2
dust (Pramanik et al., 2020; Monira et al., 2022b). Approximately 30 Nile Red staining optimisation.
pieces of recycled LDPE particles (>1 mm) were transferred onto a mi­ Parameter Initial Variation prior to Optimisation of parameters
croscope slide, covered by NR staining solution and left to dry for 24 h at solvent selection following solvent selection
25 ◦ C in the dark to select the most suitable solvent to provide optimal Staining Parameters
fluorescence. A series of solutions containing 1 μg/ml NR were prepared Solvent Acetone, ethanol, n-heptane, Acetone and ethanol
in acetone, ethanol, n-hexane, chloroform, n-heptane and a mixture chloroform, and n-hexane
Concentration 1 μg 0.2, 0.5, 1 and 2 μg
containing 50% acetone and 50% n-heptane; the initial concentration
Duration 24 h 24, 48, and 168 h
was selected based on previous work using NR staining (Maes et al., Temperature 25 ◦ C 4, 25, and 100 ◦ C
2017b). The two NR solutions providing the maximum fluorescence Imaging Parameters
were further tested at varying concentrations between 0.2, 0.5, and 2 Laser 405, 488 and 561 nm 488 nm
μg/ml and examined over periods of 24, 48, and 168 h at 25 ◦ C. The Wavelength
Pinhole 5 1, 5 and 10
solutions were also tested at all 3 concentrations for 24 h at 4, 25, and

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3. Results and discussion improved dissolution of NR crystals, ensuring a more even stain around
the plastic particle (Appendix A.1). A concentration of 0.2 μg/ml was
The NR staining procedure was optimised to identify MPs in road identified as the optimal concentration for MP analysis. The high solu­
dust mixtures containing non-plastic materials such as metals, soil/or­ bility of NR in acetone and ethanol contrasted with the low solubility of
ganics and asphalt residues. The staining protocol was also applied to NR in chloroform, N-heptane, N-hexane and a combination of these
filters that are commonly used to extract MPs from environmental solvents, which left undissolved NR residues onto the plastics after
samples to ensure that they do not prevent the identification of MPs. staining (Appendix A.2.). Acetone and ethanol were therefore selected
to ensure the homogeneity and reproducibility of the plastic staining
procedure.
3.1. Staining optimisation
Increasing the time between particle staining and imaging from 24 to
168 h did not impact the fluorescence intensity of the stained recycled
3.1.1. Impact of laser wavelength, Nile red concentration, solvent and
LDPE particles, showing that samples can be prepared in advance pro­
staining duration on material fluorescence
vided that they are kept in a dark and well-ventilated space (Appendix
Recycled and virgin LDPE exhibited particle fluorescence in 488 nm
A.3) (Shim et al., 2016a). The ability for NR to remain active for up to
(green) laser light after being stained with an NR solution of 0.2 μg/ml in
168 h is a great advantage for field-based collection of samples and
acetone (Fig. 1). The incident light emission was found to be consistent
consequent testing. However, one must ensure that the collection of road
with literature studies indicating optimum fluorescence for LDPE, PP,
dust does not make the particles overlap before staining, which could
ABS and PET in blue/green light (Stanton et al., 2019; Tamminga et al.,
prevent the accurate identification of plastic particles (Shim et al.,
2017). Aggregates and bitumen comprise the highest fraction of road
2016a). There is a lack of information regarding sample weight re­
dust, with studies suggesting up to 97% of road dust would originate
quirements for MP identification within road dust; however for MP
from asphalt (Tervahattu et al., 2006; Kupiainen et al., 2003). Hence,
quantification in marine sediment, the National Oceanic and Atmo­
this study was optimised using bitumen and aggregates to prevent er­
spheric Administration (NOAA) recommends the use of 400 g per
rors. Compared to the 405 nm (blue) and 561 nm (red) laser lights, the
replicate (Masura et al., 2015). Filtration is a frequently used method for
488 nm (green) laser light did not render NR-stained bitumen and
particle separation in water and sediment; recent studies recommend
aggregate residues fluorescent. As a result, the 488 nm (green) laser light
that the system have a pore or mesh size between 0.3 and 200 μm (Prata
was selected to assess the presence of MPs in road dust. Acetone was
et al., 2019b).
found to degrade ABS, hence ethanol was used instead to prevent the
Literature studies have examined the success of using NR on vari­
loss of particle size, shape and count information during staining.
ously shaped particles, including microspheres, irregularly shaped MPs
Decreasing the NR concentration from 2 μg/ml to 0.2 μg/ml
and microfibers (Cole, 2016; Karakolis et al., 2019; Lv et al., 2019).
increased the fluorescence intensity of the stained particles due to

Fig. 1. Fluorescence images of a) recycled LDPE, b) virgin LDPE, c) bitumen and d) aggregate stained with NR in acetone at 0.2 μg/ml for 24 h at 25 ◦ C. A pinhole of
1 was used. The first column refers to a laser light of 647 nm, the second column refers to 405 nm, the third refers to 488 nm (green) and the final column refers to a
laser light of 561 nm (red). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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R. Myszka et al. Chemosphere 315 (2023) 137757

However, these features have never been applied to plastics within road with acetone NR solutions.
dust. The fluorescence intensity of stained plastic particles was found to In addition to providing a rapid count of particles on the filters,
depend on the polymer type as well as the particle surface morphology fluorescence-based automated MP detection was able to provide mea­
(Appendix A.4). Road dust particles are irregularly shaped since they are surements of particle size, area and shape. Although NR ethanol solution
constantly fragmented due to their prolonged exposure to vehicle tyres prevented ABS particles from degrading, this solvent reduced the fluo­
and weathering events, which increases the accumulation and NR rescence intensity of the stained ABS particles and a pinhole of 5.0 was
retention onto plastic particles (Appendix A.4) (Shim et al., 2016a). therefore required to reach a suitable image brightness detectable by the
In this study, the use of a 10× magnification objective allowed the software (Appendix A.5) (Tamminga et al., 2017).
successful assessment of the size and shape of MPs from 1 to 8000 μm,
which is one order of magnitude lower than the detection limit of
standard IR microscopy (Cole, 2016). This procedure may also allow for 3.2. Plastics commonly found in road dust
the detection of plastic particles down to 0.1 μm, assuming maximum
microscopic resolution at 100× magnification, which is one order or MP pollution was identified to be prominent in metropolitan areas
magnitude lower than advanced IR microscopic techniques such as (Patchaiyappan et al., 2021; Yukioka et al., 2020), hence the efficiency
μ-FTIR (Cole, 2016). The elevated fluorescence intensity associated with of the staining procedure to detect and quantify a wide range of plastics
a smaller MP size is a favourable characteristic of the optimised NR potentially found in road dust was investigated (Roychand and Prama­
staining method, particularly for particles below the <3 μm (detection nik, 2020). The most common plastics used for household and industrial
limit of μ-FTIR). purposes include PP, LLDPE, HDPE, LDPE, PET and synthetic rubber
(Andrady, 2011; Boucher and Billard, 2019; Kole et al., 2017). Plastics
3.1.2. Automated MP quantification and background interference commonly used as asphalt modifiers, such as EVA, SBS and ABS, were
The identification and quantification of the stained plastic particles also considered since they could potentially be released from
were automated using Nikon Instruments Software (NIS-elements) par­ plastic-modified road materials into road dust (Monira et al., 2021;
ticle detection software based on the contrast between the fluorescent Giustozzi et al., 2021).
particles and the background at a standard pinhole brightness of 1. As All stained plastics were fluorescent under 488 nm (green) laser
mentioned in Section 3.1.1, prefiltration is recommended for large light, however, the fluorescence of recycled plastics was higher than that
sample volumes (400 g recommendation by NOAA). Hence, common of their virgin counterparts (Fig. 3) (Maes et al., 2017a). NR was re­
filter materials including paper and steel were examined to ensure no ported to preferentially interact with non-polar materials (Shim et al.,
background fluorescence at the optimised pinhole (Fig. 2a and b). The 2016a), hence the variation in polarity during the recycling of plastic
phenomenon of background fluorescence must be avoided to prevent can impact their interaction with NR and therefore their detection by
underestimation of the particle count and overestimation of the particle fluorescence microscopy. The impact of MPs weathering on the particle
size, hence a pinhole of 1 was preferred. Increasing the pinhole to 5, 10 fluorescence after NR staining is still in debate among the scientific
and 15 enhanced the brightness of the particles. However, at the expense community, as the oxidation of the MPs surface may inhibit the chemical
of their resolution (Fig. 2c), PP was assumed to be representative of all interactions between the MPs and NR stain resulting in a lower fluo­
plastic types. Hence, a pinhole of 1 was selected for all plastics stained rescence intensity (Shruti et al., 2022; Shim et al., 2016b). For example,
the presence of varying additives and fillers could provide different

Fig. 2. Fluorescence images of a) paper, b) steel and c) recycled PP stained with NR in acetone at 0.2 μg/ml for 24 h at 25 ◦ C. The first column refers to a laser light of
647 nm with no pinhole. The subsequent columns used a 488 nm (green) laser and a pinhole of 1 for the second column, a pinhole of 5 for the third column, a pinhole
of 10 for the fourth column and a pinhole of 15 for the final column. (For interpretation of the references to colour in this figure legend, the reader is referred to the
Web version of this article.)

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R. Myszka et al. Chemosphere 315 (2023) 137757

Fig. 3. Fluorescence images of a) virgin LDPE, b) recycled LDPE, c) virgin HDPE, d) recycled HDPE, e) virgin LLDPE, f) recycled LLDPE, g) virgin PP, h) recycled PP,
i) virgin PET, j) recycled PET, k) virgin ABS* l) recycled ABS m) rubber stained with NR in acetone at 0.2 μg/ml for 24 h at 25 ◦ C. The first column for each subset
(virgin and recycled) refers to a laser light of 647 nm with no pinhole and the second column used a 488 nm (green) laser and a pinhole of 1. *Note: ABS was stained
in ethanol under the same parameters. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

surface chemistry to the plastic particles and therefore impact their household or commercial waste, hence containing a variety of coloured
sensitivity to NR staining due to MP size, polarity and presence of ad­ pigments and colour stabilisers (Appendix A.6). In this study, the anal­
ditives and fillers (Shruti et al., 2022). The plastic used in this study was ysis of coloured recycled PP and ABS plastic demonstrated that NR
made of either virgin plastic or recycled plastic originating from staining was not impacted by the presence of pigments.

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R. Myszka et al. Chemosphere 315 (2023) 137757

The presence of other non-plastic contaminants may be identified if any fluorescence at 488 nm or any of the lasers studied (Appendix A.8).
samples show an unusual fluorescence pattern. However, additional This finding has been confirmed within recent research regarding rubber
non-invasive techniques such as FTIR are still recommended to deter­ fluorescence (Stanton et al., 2019; Hitchcock and Mitrovic, 2019; Araujo
mine the exact polymer type assuming a particle size distribution above et al., 2018). Recommended additional methods, such as density sepa­
the 20 μm range required for standard spectroscopic analysis (Prata ration or FTIR, may be required to reduce this limitation of MP associ­
et al., 2021; Shim et al., 2016a). Interestingly, incorporating plastic into ated with vehicle tyres (Appendix B) (Fu et al., 2020; Halle et al., 2020).
bitumen resulted in auto-fluorescing plastic particles where unstained
plastic extracted from the bitumen was naturally fluorescent (Appendix 3.3. External soil and organic contamination
A.7). Part of the bitumen maltene molecules were likely adsorbed by the
polymer, which became naturally fluorescent due to the aromatic Soil and soil modifiers, as well as organic materials such as paper,
functional groups of the maltenes (Handle et al., 2016). Although the timber and foliage, were examined to determine whether such materials
fluorescence of the unstained particles was less intense than particles - naturally occurring in road dust - could be fluorescent and prevent the
stained with NR, this phenomenon could be used as an assessment of identification of plastic particles (Fig. 4). Soil (clay soil) did not exhibit
whether the MPs found in road dust originated from plastic-modified any fluorescence, nor did calcium carbonate which is a common filler in
roads rather than from other sources. A comparison between stained construction applications (Wilford et al., 2015). Organic materials
and unstained samples of road dust would allow the identification of including paper, cardboard and foliage were found to partially fluoresce.
naturally fluorescing particles and the determination of the fraction This may pose issues for plastic analysis since these organic materials
originating from the road material itself. could be mistaken for plastic (Shruti et al., 2022; Donaldson, 2013,
Within road dust, rubber particles would be inevitable due to the 2020; Donaldson et al., 2015). Therefore, preliminary treatment of the
continual abrasion of vehicle tyres. Tyre rubber was found to not show sample such as chemical digestion with hydrogen peroxide (Mikutta

Fig. 4. Fluorescence images of a) clay, b) calcium carbonate c) talc, d) folliage, e) timber, f) paper g) sulphur, h) cardboard and i) starch stained with NR
in acetone at 0.2 μg/ml for 24 h at 25 ◦ C. The first column refers to a laser light of 647 nm with no pinhole, the second column used a 488 nm (green) laser
and a pinhole of 1. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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R. Myszka et al. Chemosphere 315 (2023) 137757

et al., 2005; Prata et al., 2019c) is required to remove these organic pose difficulties for the detection of MPs in road dust (Maeaba et al.,
materials, particularly in rural environments where foliage surrounds 2019; Pagotto et al., 2001; Aryal et al., 2016). Metal contamination from
the road, with no adverse effects on the fluoresce of NR reported in field-based testing is not expected to be a major concern on rural roads,
literature (Sturm et al., 2021; More et al., 2021). However, samples however roads with heavy traffic are expected to contain a high pres­
generated under controlled laboratory conditions may not require any ence of metals (Tang et al., 2017; Lin et al., 2017). Metal presence is
pre-treatment, such as during the analysis of MPs abraded from expected to be elevated around modern industrial plants and areas
plastic-modified roads (Enfrin et al., 2022). On rural roads, starch has where past exploitation of ores and processing of the metals occurred
been used as a dust suppressant and may remain present within the road (Logiewa et al., 2020). The metals may also originate from a variety of
dust collected. Due to a lack of fluorescence, however, misidentification sources, including emissions from the local industry as well as traffic
as plastic is not an identifiable concern (Parsakhoo et al., 2020). Addi­ emissions directly related to vehicles, such as crashes, exhaust emis­
tionally, talc did not fluoresce and hence would not pose misidentifi­ sions, or leakages (Logiewa et al., 2020). In the urban environment,
cation risks, particularly in urban environments, as it is a common however, there would be additional sources of residential and industrial
constituent in paint products or roofing (Talc, 2022). Sulphur is present pollution with greater potential for contaminating road dust samples
in road dust due to vehicle and boiler-oil combustion, as well as with substances including lead, tin, aluminium, iron, gypsum, copper, or
manufacturing, however it does not fluoresce under optimal conditions precious metal cargo such as silver and tungsten (Logiewa et al., 2020;
(Wang et al., 2005). Adachi and Tainosho, 2004).
Additionally, organics such as wood and paper have also been found Although density separation was previously used to remove metals
to autofluorescence under blue/green light (~488 nm) due to lignin from road dust samples (Kang et al., 2022), such separation may not be
(Donaldson, 2020). An additional pre-treatment would therefore be required when using fluorescence microscopy provided that metals do
required prior to analysis to remove organics from road dust and not fluoresce. The staining procedure was applied to 8 different types of
ascertain the presence of MPs (Appendix B). Lignin fluorescence is metals, such as aluminium, iron, lead, steel, tin, silver, copper and
environmentally sensitive, hence treatments such as acidic pH, the usage tungsten. None was fluorescent after staining (Fig. 5), showing that
of quenching agents and heat may reduce lignin autofluorescence and metal residues cannot be misidentified with MPs when using the fluo­
allow for MP isolation within road dust (Donaldson, 2013; Donaldson rescence procedure. Hence, only a minimal pre-treatment of the road
et al., 2015; Kim et al., 2015; Thygesen et al., 2010). dust is required to prevent particle overlap on the filter, ensuring
maximum particle retention with rapid and accurate results. (Khan and
Strand, 2018).
3.4. Metal contamination

Metal residues have been found in road dust worldwide, which could

Fig. 5. Fluorescence images of a) aluminium, b) iron, c) lead, d) tin, e) steel, f) silver, g) copper and h) tungsten stained with NR in acetone at 0.2 μg/ml for 24 h at
25 ◦ C. The first column refers to a laser light of 647 nm with no pinhole, the second column used a 488 nm (green) laser and a pinhole of 1.

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R. Myszka et al. Chemosphere 315 (2023) 137757

3.5. External asphalt residue contamination adjacent to the road. The procedure developed allows the determination
of the number of plastics coming from the roads compared to the sur­
The main constituents of road dust are expected to be asphalt con­ rounding areas, with 1 μm being the minimum particle size detected in
stituents removed through abrasion with vehicle tyres and weathering, this study. The resolution may be improved to 0.1 μm by utilising a
namely aggregates and bitumen (Giustozzi et al., 2021). Stained ag­ higher magnification with the size limitation being due to instrument
gregates did not fluoresce regardless of their mineralogy, hence aggre­ capability. This research may be complemented further by a detailed
gates are not a concern for the analysis of MPs in road dust and no study of the fate of MPs within road dust under different conditions that
additional pre-treatment is suggested (Fig. 1). The aromatic molecules of need to be explored, including a representation of samples at different
bitumen did show some fluorescence under 488 nm with a pinhole value areas, seasons and weather conditions. The future work expands the
of 5 and higher. Hence, a careful selection of the pinhole is required to potential application of this method to road dust collected from various
remove this interference and ensure that bitumen residues do not fluo­ areas and frequencies. The application of this procedure on field samples
resce (Fig. 6) (Handle et al., 2016). allows for a rapid, simple, inexpensive and reliable monitoring approach
taken to determine the presence of MP particles within road dust,
4. Conclusion and prospects particularly in light of the recent interest in plastic-modified asphalt
roads. In addition, this optimised procedure for field sampling, including
A novel optimisation of the existing NR MP staining identification ideal staining conditions and parameters required to isolate MPs within
method was adapted to quantify the amount and determine the size and road dust, can allow for further MP analysis, including the number,
shape properties of MPs in road dust. The optimisation procedure was shape and size of particles in road dust. Furthermore, MPs released from
designed to enhance MP visibility, allowing road dust samples to be plastic-modified roads would exhibit autofluorescence without utilising
analysed with minimal pre-treatment. The application of staining on NR staining. This phenomenon may provide a novel assessment of road
field-based samples was determined through a comprehensive contam­ dust to determine whether the MPs found in road dust originated from
inant analysis including metals, soils and organics commonly located plastic-modified roads.

Fig. 6. Acetone staining of asphalt constituents a) bitumen, b) aggregate and c) sand at a concentration of 0.2 μg/ml after 24 h. The first column refers to a laser light
of 647 nm with no pinhole, the second column used a 488 nm (green) laser and a pinhole of 1.

9
R. Myszka et al. Chemosphere 315 (2023) 137757

Credit author statement the work reported in this paper.

All authors: Conceptualization; All authors: Methodology; R. Data availability

Myszka: Investigation and testing; R. Myszka and M. Enfrin: Data
analysis; R. Myszka and M. Enfrin: Writing - original draft; All authors: Data will be made available on request.
Writing - review & editing. F. Giustozzi: Funding, project administration
and supervision. Acknowledgement

Declaration of competing interest The authors would like to acknowledge Austroads Ltd. for their
financial contribution to this research through the project APT6305 –
The authors declare that they have no known competing financial Road-grade plastics for sustainable asphalt roads.
interests or personal relationships that could have appeared to influence

Appendix A

Fig. A.1. Fluorescence images of recycled LDPE stained with NR in acetone at concentrations of a) 0.2, b) 0.5 and c) 2 μg/ml for 24 h at 25 ◦ C. The first column refers
to a laser light of 647 nm with no pinhole, the second column used a 488 nm (green) laser and a pinhole of 1.

Fig. A.2. Fluorescence images of recycled LDPE stained with NR in chloroform at a concentration of 1 μg/ml for 24 h at 25 ◦ C. The first column refers to a laser light
of 647 nm with no pinhole, the second column used a 488 nm (green) laser and a pinhole of 1.

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R. Myszka et al. Chemosphere 315 (2023) 137757

Fig. A.3. Fluorescence images of recycled LDPE stained with NR in acetone at concentrations of 0.2 μg/ml for a) 24 h b) 48 h and c) 168 h post-staining at 25 ◦ C. The
first column refers to a laser light of 647 nm with no pinhole, the second column used a 488 nm (green) laser and a pinhole of 1.

Fig. A.4. Fluorescence images of recycled LDPE with a) rough and b) smooth surfaces stained with NR in acetone at concentrations of 0.2 μg/ml for 24 h at 25 ◦ C.
The first column refers to a laser light of 647 nm with no pinhole, the second column used a 488 nm (green) laser and a pinhole of 1.

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R. Myszka et al. Chemosphere 315 (2023) 137757

Fig. A.5. Fluorescence images of a) recycled ABS b) bitumen and c) aggregate stained with NR in ethanol at concentrations of 0.2 μg/ml for 24 h at 25 ◦ C. The first
column refers to a laser light of 647 nm with no pinhole, the second column used a 488 nm (green) laser and a pinhole of 1, the third column used a 488 nm (green)
laser and a pinhole of 5.

Fig. A.6. Recycled plastics LDPE, PE/PP, PET and ABS used in this study pictured prior to staining depicting a wide variety of coloured additives included due to the
variety of original uses and recycling practices.

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R. Myszka et al. Chemosphere 315 (2023) 137757

Fig. A.7. Autofluorescence images of recycled LDPE extracted from bitumen after 24 h. A laser of a) 405 nm b) 488 nm and c) 561 nm and a pinhole of 1 were used.

Fig. A.8. Fluorescence images of vehicle tyres rubber stained with NR in acetone at a concentration of 0.2 μg/ml for 24 h at 25 ◦ C. A laser of a) 647 b) 405 nm c) 488
nm and d) 561 nm and a pinhole of 1 were use.

Appendix B

Pre-treatment recommendations for road dust prior to NR staining

Prefiltration of particles on paper or steel filters is recommended to reduce loading of road dust, allowing for size separation of particles to prevent
overlap of large particles hindering the detection of finer particles prior to NR staining. Due to the lack of research encompassing MPs in road dust, a
maximum sample weight of 400 g per replicate is recommended following guidelines by NOAA (Masura et al., 2015). Similarly, MP studies within
water and sediment recommend filtration for particle separation to have a pore or mesh size between 0.3 and 200 μm (Prata et al., 2019b). Hence, a
filtration series for road dust is recommended to collect particles >0.3, >3, >30 and >200 μm to prevent particle overlap. Additionally, pre-treatment
may be used to quantify a mass of MP associated with vehicle tyres by density separation. The removal of non-fluorescent rubber would result in
improved detection of fluorescent MP due to mitigating particle overlap.
The size of MPs following this procedure has been found to encompass particles down to 1 μm assuming 10× magnification. This may be improved

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R. Myszka et al. Chemosphere 315 (2023) 137757

by the use of a 100× magnification to achieve MP identification of 0.1 μm on the Nikon N-storm inverted stage Ti-E Nikon confocal microscope.
Additional pre-treatment involving heat, pH alteration or chemical digestion with hydrogen peroxide may be recommended; these are further dis­
cussed in sections 3.2 and 3.3 of the manuscript.

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