© 2023 The Authors Water Practice & Technology Vol 18 No 12, 3124 doi: 10.2166/wpt.2023.

215

Identification and quantification of microplastics in wastewater treatment

plants by spectroscopic and microscopic techniques in Johannesburg East,
South Africa

Tendani Mphaga a, *, T. N. Mhlongo b, Sithembele Zikalalac and James Topkin d

a
Department of Environmental Sciences, Faculty of Agriculture and Environmental Sciences, University of South Africa, UNISA Science Campus,
P.O. Box 392 UNISA 003, Florida 1709, Johannesburg, South Africa
b
Centre for Postgraduate Studies, Cape Peninsula University of Technology, Bellville South Industrial, Cape Town 7530, South Africa
c
Department of Sciences Engineering and Technology, Institute for Nanotechnology and Water Sustainability
d
Research Department, Scientific Services, Ekurhuleni Water Care, P.O. Box 13108 Norkem Park, 1631, Kempton Park, South Africa
*Corresponding author. E-mail: tendanimphaga@gmail.com

TM, 0000-0003-4721-2556

ABSTRACT

The occurrence of microplastics in water, their chemistry, physical characteristics, and the efficiency of public wastewater
treatment work (WWTW) processes in the removal of microplastics are investigated. Samples were collected from the period
2021 December to 2022 September from two WWTWs in Johannesburg East using 24-h autosamplers. The microplastics
were imaged using polarised optical microscopy (POM) and the images were processed using image J 1.53 K to determine
the particle counts. The total concentration of microplastics at WWTW A was 3,098 MP/L while WWTW B had 3,561 MP/L.
The microplastics identified across the seasons were dominated by angular, fibres, fragments, and films. Fourier transforms
infrared (FTIR) spectroscopy identified the polymers such as polyethylene, acrylic, polyethylene terephthalate, and polystyrene
in WWTW A and B influent while identifying the polymers such as polystyrene, polyacrylamide, polypropylene, polycarbonate,
acrylonitrile butadiene styrene, poly(ethyl cyanoacrylate), carboxyl, poly(ethylene terephthalate), polyethylene, poly(methyl
methacrylate), and cellulose in the final effluent. Scanning electron microscopy (SEM) with energy-dispersive X-ray spec-
troscopy (EDX) identified Cr, Ca, Fe, Al, Na, Mg, Zn, Cl, P, S, and silicon as additives to microplastics with high-intensity
peaks of oxygen and carbon. It is recommended to monitor and regulate microplastics in discharged effluents from WWTWs
to minimise environmental pollution.

Key words: chemical composition, microplastics, microscopy, polymers, spectroscopy

HIGHLIGHTS

• Quantity, polymer functionality, and physical morphology of microplastics are studied.

• Variations of microplastic concentration across seasons are studied.
• Different polymers are composed of a variety of functional groups.
• Microplastics are carriers of metals.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY 4.0), which permits copying,
adaptation and redistribution, provided the original work is properly cited (http://creativecommons.org/licenses/by/4.0/).

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 Water Practice & Technology Vol 18 No 12, 3125

GRAPHICAL ABSTRACT

1. INTRODUCTION
Across the world, it is estimated that a total of 381 million tons of plastics is produced annually with an increase
of 4% (Nhon et al. 2022). Due to limited recycling, plastics end up in the environment. The larger plastics (pri-
mary plastics .5 mm) undergo physical processes such as weathering, disintegration by waves in wastewater
conveyance systems, and microbial processes to form smaller particles referred to as microplastics (particles
,5 mm) (Li et al. 2022). Microplastics that end up in the wastewater treatment works (WWTWs) are not com-
pletely removed and, therefore, regarded as the pathway to enter surface water sources (Habib et al. 2020).
Microplastics have been identified in the final effluent of WWTWs in Europe, Asia, Africa, and the United
States of America (USA) that regulates microplastics in drinking water (Wickham et al. 2019). On average, it
is estimated that 4% of global microplastics reach surface water (Rubio-Armendariz et al. 2022).
Although microplastics are known to originate from weathering of larger particles, it is well known that the
production of pharmaceutical and personal care products involves the addition of microplastics, e.g. scrubbers,
soaps, toothpaste, and cosmetics products (Bashir et al. 2021). During the production processes of primary and
secondary microplastics, additives such as plasticisers, flame retardants, antioxidants, acid scavengers, light
and heat stabilisers, lubricants, pigments, antistatic agents, slip compounds, and thermal stabilisers are added
(Hahladakis et al. 2018). The most common chemical additives of microplastics include bisphenol A (BPA),
phthalates, and polybrominated diphenyl ethers (Campanale et al. 2020). The microplastics detected in the
final effluent end up in the rivers and, therefore, may act as pathways of toxic additives (Rubio-Armendariz
et al. 2022). The additives released from microplastics alter water quality, cause toxicity to aquatic species and
may cause endocrine disruption and cancer, and accumulate in organs such as the liver and heart resulting in
their failures (Campanale et al. 2020). Traces of microplastics have been found in the digestive tracts of fish
and sea birds and that adversely affects their health and increases mortality (Katyal et al. 2020).
The influent of WWTWs originates from diverse sources such as industries, domestic sewage, and storm water
(Reddy & Nair 2022). The different sources of wastewater result in the occurrence of a wide array of substances in
the wastewater ranging from metallic species including toxic metals to persistent organic compounds (Hejabi
et al. 2021). Microplastics’ chemical composition is associated with the release of contaminants into the environ-
ment, and the hydrophobic characteristics of microplastics play an important role in contaminate distribution
(Joo et al. 2021). Seidensticker et al. (2018) confirmed the ability of microplastic polymers such as polyethylene
(PE) and polystyrene (PS) to sorption of different contaminants exposed to different pH levels. The results from
Seidensticker et al. (2018) showed that PE and PS highly adsorb chemical compounds that do not attract water
molecules such as alkanes, hydroxyl compounds and hydrocarbons at neutral pH. Similarly, Wang et al. (2022)

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studied the ability of PE in the sorption of different pharmaceutical compounds. The study by Wang et al. (2022)
was comparable to the study by Seidensticker et al. (2018), where the PE showed high adsorption capacity on
hydrophobic pharmaceutical compounds such as sulfamethoxazole, propranolol and sertraline at pH value of
7. This study seeks to identify and quantify microplastics in the influent and final effluent of WWTWs A and
B in the Johannesburg East in South Africa.

2. MATERIALS AND METHODS

2.1. Study location and description
The city of Johannesburg is located at 26.2044° S, 28.0456° E in Gauteng Province, South Africa, covering an area
of 1,645 km2 and with a population of about 4, 434, 827 million. The wastewater treatment plants are in the East
of Johannesburg. Figure 1 shows the location map of wastewater treatment plants A and B.

Figure 1 | Map showing the location of sampling points.

2.2. Site and process description

2.2.1. WWTW A description
The WWTW A was designed to treat 63 ML/d and in the year 2021/2022, the work operated at 100% of its
capacity (WWTW A Water Quality Report 2021). Although the work operated at its design capacity, it is at a
risk of exceeding its design capacity. The treatment works receive its influent from 51 industries scattered
around Kempton Park and 53,777 households with a population of 171 575 in Kempton Park. The influent is
received in a proportion of 60% domestic and 40% industrial wastewater (WWTW A Manual 2015). There is
a potential source of microplastics from industrial and domestic influent which is received in the WWTW.
The WWTW A utilises a conventional treatment technology. It comprises inlet works equipped with three
mechanical fine screens and degritting chambers for the screen, sand and grit removals as a preliminary treat-
ment process (WWTW A Manual. 2015). Preliminary treatment is followed by four primary settling tanks
(PST) aimed at solid and COD reduction to reduce the loading into the activated sludge processes (Archer
2018). The supernatant from PST overflows into activated sludge processes which are configured as University
of Cape Town (UCT configuration) in treatment sections 1–3 and Modified Ludzac Etinger processes in treatment
section 4 (WWTW A Manual 2015). Although treatment configurations are different in the sections, the

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secondary treatment of the BNR process remains the same across the treatment units. In the secondary treatment,
microorganisms are used to oxidise nutrients and organic matter. Overflow from the BNR is subjected to tertiary
treatment using final settling tanks (FSTs). This is the final polishing step for the removal of SS before the effluent
is disinfected using chlorine gas (WWTW A Water Quality Report 2021). The wastewater residue such as waste-
activated sludge (WAS) and primary sludge from PSTs is treated in cold open digesters equipped with rotor
mixers. However, the anaerobic digestion process is not heated (Eustina et al. 2018).
The final effluent and sludge treated at WWTW A are expected to comply with the Water Use License (WUL)
granted in terms of the National Water Act (Act 36 of 1998) under Section 21. Although there is WUL in place,
microplastics are not regulated.

2.2.2. WWTW B description

WWTW B is designed to treat a maximum of 155 ML/d (6458,33 m3/h) from primarily domestic households with
an average population of 111,612 and 38,046 households (WWTW B Water Quality Report 2021). Only
5.41 ML/d (4%) of wastewater is contributed by industries. The wastewater treated is from domestic households
in Everton and surrounding communities. The plant is comprised of four treatment modules with three modules
treating 35 ML/d and one treating 50 ML/d. The conventional treatment process employed in the plant with pri-
mary, secondary, and tertiary treatment mainly as the final stage (WWTW B Water Quality Report 2021). Primary
treatment consists of six mechanical screens and six pre-screen grits for screen and debris removal. It is also
equipped with six grit chambers for sand and grit removal (WWTW B Process Audit 2019). Four modules com-
prised of four primary settling tanks for further removal of SS and sand which escaped from grit chambers. Solids
settle as raw sludge and are further treated in an anaerobic hydrolysis process, filter belt pressers, and paddies.
The activated sludge process is used for secondary treatment for oxidation of nutrients in the presence of micro-
organisms (Eustina et al. 2018). It also entails secondary clarification for further removal of SS and polishing of
final effluent. The last treatment process is disinfection for the removal of microorganisms using chlorine gas.
The WWTW B is also regulated by the WUL. The final effluent discharged into Klip River must comply with
the conditions of WUL (WWTW B WUL audit report 2021).

2.3. Study design and sampling

Samples were collected from December 2021 to September 2022 to give representative samples over four sea-
sons. A total of 16 samples were collected from the wastewater treatment plant; from the influent before the
inlet works fine screens and final effluent sampling points after disinfection. Microplastic sampling was done
using the MS 10 composite autosampler, which is equipped with a 3-L sampling bottle. The MS 10 composite
autosamplers equipped with a vacuum pump, which allowed the suction of water at 10–20 min intervals were
placed in the inlet and effluent of the WWTWs. The sampler timer was set to collect an equivalent of 2.5 L of
the representative sample over 24 h. A sample volume equivalent to 1 L of composite sample was collected by
opening the autosampler dispenser pipe to fill up the glass bottle to its maximum capacity. The samples were pre-
served in a cooler box and transported to the Council for Scientific and Industrial Research (CSIR) laboratories
for further analysis.

2.4. Microplastic pre-treatment

The microplastics were pre-treated prior to analysis by filtering using a Millipore xx2004708 stainless steel sieve
with 5-mm pores purchased at Merck group. Materials retained from a 5-mm stainless steel sieve were discarded.
The filtering of samples was done to remove particles greater than 5 mm which were not considered
microplastics.
The wet peroxide oxidation procedure for removal of organic contaminants to isolate microplastics in waste-
water samples was followed to reduce interferences that might cause errors during microplastic identification.
100 mL of wastewater sample was taken from the filtered sample and catalysed by adding 20 mL of iron sulphate
at (0.05 M of Fe (II)), which was allowed to stand for 5 min at room temperature prior to heating at 75 °C on a
hotplate and later allowed to stand for 30 min at 23 °C. The organic compounds were degraded and only micro-
plastics remained due to their resistance to peroxide oxidation. The solution of wet H2O2 was then transferred
into a density separator and allowed to settle over 24 h at room temperature of 23 °C. The settled organics
were discarded, and the clear sample was transferred into the 250-mL glass beaker for drying that was placed
in the Memmert vacuum oven-PM400 at 90 °C for 72 h to ensure proper drying. Microplastics remained on

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the surface of the glass beaker. In order to avoid contamination from ambient air and cross-contamination, the
glass beaker was covered with an aluminium foil.

2.5. Identification of microplastics

The identification of microplastics included the physical and chemical properties of microplastics as well as their
quantities in wastewater samples. Physical identification was done using scanning electron microscopy (SEM)
and the polarised optical microscope (POM). To do an elemental analysis of microplastics, Fourier transform
infrared (FTIR) spectroscopy was used to identify polymers and the SEM coupled with energy-dispersive X-ray
(EDX) was used to perform an elemental analysis of microplastics. The identification and classification of micro-
plastics was carried out using a POM and SEM.

2.5.1. Physical identification using POM

For the physical identification of the microplastics, a POM was used. The sample preparation was done by sub-
merging the microplastic particles in the refractive index oil, this was done to allow clear visibility of the sample.
The magnification of the POM was adjusted to 40
using the top light. Microplastics were then placed on the
POM stage micrometre with a rectangular size of 25 mm
75 mm. The dried microplastics on the micrometre
were observed and images were taken to categorise particles based on the colour and shapes of fragments,
fibre, film, micro-beads, and foam.

2.5.2. Physical identification using SEM/EDX

For the physical identification of shapes and sizes of microplastics, a SEM was used. The first procedure was to
open the specimen chamber on the SEM to allow the specimen stage to be visible. Microplastic particles were
transferred from the petridish into the specimen stage of the SEM coated with carbon at 50 nm to avoid conflict-
ing with peaks of other compounds. The chamber on the SEM containing the specimen was closed and the
pressure equalisation was maintained by switching on the vacuum pumps for 5–10 s at 103 Torr. The SEM
coupled with EDX was operated at 15 kV voltage. To produce primary electron, the magnification was set
between 400 and 6,400
and the sample was scanned with a beam at high energy with a map resolution of
512
384. The SEM coupled with energy-dispersive X-ray was operated at backscattered imaging mode where
the primary electron produced was allowed to interact with the samples to produce secondary electrons, back-
scattered electrons, and X-rays. SEM and X-ray detectors were allowed to collect signals from electrons and
X-rays to create images at a pixel size of 0.06 μm and a maximum magnification of 5,200 which were displayed
on the computer monitor.

2.5.3. Chemical identification of microplastics

To identify the spectra of samples, a Perkin Elmer Spectrum 100 spectrometer was used. To achieve the required
signal-to-noise ratio, FTIR spectroscopy spectra of all microplastic samples were measured in the range of 550–
4,000 cm1 and 32 scans were made per spectra, which were co-added and averaged. In all the samples, the spec-
tra resolution was maintained at 16 cm1. A systematic procedure was followed in the sample preparation and
observation to minimise contamination and optimise the identification of the polymer. First, the detection unit
was cleaned with alcohol and a clean cloth free from fibre to avoid contamination. The microplastics were
placed on a rectangular glass micrometre with dimensions 25 mm
75 mm. The control joystick was used to pos-
ition the specimen and microplastics were scanned on the sample table. An optical photo of the specimen was
taken and the location where the sample was characterised was marked. The spectrum of the measured particle
was matched to the database of spectra in the library to identify the closest match and, therefore, the polymer
types on the microplastics. FTIR spectroscopy was composed of various reference spectra of polymer which
included PE, polyethylene terephthalate (PET), PS, poly(methyl methacrylate) (PMM), cellulose, polypropylene
(PP), polyvinylchloride (PVC), polycarbonate carboxylic acid amine salt film and other spectra not identified
on the samples.
The elemental analysis was done using the SEM equipped with energy-dispersive X-ray. This was done to ident-
ify elements such as heavy metals that are linked to the presence of microplastics in water. The presence of
elements such as chloride, aluminium, manganese, zinc, iron, silicon and heavy metals in microplastics present
the potential of microplastics to distribute contaminants. The step-by-step systematic procedure for SEM and
EDX has been explained above on morphological characterisation. The identification of elements was based

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on the reaction of primary electrons that interacted with samples to create secondary electrons, scattered elec-
trons and X-rays. The X-ray detector was used to detect elements.

2.5.4. Quantification of microplastics using ImageJ 1.53K

The ImageJ 1.53 K java-based application by the National Institute of Health (NIH) of the USA was used to per-
form particle counts to determine the microplastic concentration in the influent and final effluent of WWTW.
First, the sequence images were imported and uploaded to ImageJ 1.53 K and then processed to accentuate
the image edges for ease of automated particle count and analysis. The image was edited by setting the threshold
colour to white and saturation ranges of 120–255. The image size was interpolated as bilinear at a pixel width of
481 and height of 332 with a constrained aspect ratio and average when downsizing. The image contrast and
colour balance were adjusted to 50%. The particles were processed by setting the image to smooth and sharpened
mode. The edges on the image particles were determined automatically on the ImageJ application. The particles
were analysed by setting the pixel from 0 to infinity and circularity ranging between 0.001 and 1. The application
analysed the particles automatically and the results summary table was displayed with particle counts of micro-
plastics in MPs/L. The edited image was compared with the original image from the POM and SEM.

3. RESULTS AND DISCUSSIONS

3.1. Microplastics concentrations
The concentrations of microplastics were obtained by adjusting and processing POM images to determine the
particle counts on image J 1.53 K. The seasonal concentrations of microplastics for influent and effluent at
WWTW A and B are presented in Figure 2. Detailed descriptions of microplastic shapes and their concentrations
are also presented in Tables 1 and 2 for both WWTWs.

Figure 2 | Seasonal microplastic concentrations for influent and effluent at WWTW A and WWTW B.

Table 1 | Seasonal microplastic concentrations based on shapes for WWTW A

Shapes

Sample ID Seasons Particle size (mm) Angular Fragments Fibres Film

Influent Summer 0.01 0 148 145 0

Autumn 0.01 0 399 0 0
Winter 0.05 250 0 203 0
Spring 0.01 402 271 0 0
Effluent Summer 0.002 0 138 54 0
Autumn 0.01 0 258 0 0
Winter 0.01 134 0 189 0
Spring 0.05 296 0 0 211

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Table 2 | Seasonal microplastic concentrations based on shapes for WWTW B

Shapes

Sample ID Seasons Particle size (mm) Angular Fragments Fibres

Influent Summer 0.01 0 0 180

Autumn 0.01 0 401 0
Winter 0.01 354 0 798
Spring 0.01 287 420 0
Effluent Summer 0.002 0 78 21
Autumn 0.01 0 210 0
Winter 0.02 119 316 0
Spring 0.01 152 225 0

The concentrations of microplastics vary seasonally across the sampling points. According to Figure 2 and
Table 1, during the summer season, the influent concentrations at WWTW A contained 145 MPs/L fibres and
148 MPs/L fragments while the effluent contained 54 MPs/L fibres and 138 MPs/L fragments. The composition
of WWTW B did not vary in the influent during summer; the influent concentrations ranged between 79 and
180 MPs/L and a total of 180 MPs/L were fibres (see Figure 2 and Table 2). The effluent at WWTW B during
summer contained 78 MPs/L fragments and 21 MPs/L fibres. During summer both WWTWs influent and efflu-
ent were composed of fragments and fibres. The concentrations of microplastics are generally higher in the
influent and lower in the effluent across all sampling points with WWTW A receiving higher concentrations of
microplastics during the summer season.
The concentrations of microplastics during autumn slightly increased in the influent and effluent of both
WWTW A and B. With reference to Figure 2 and Tables 1 and 2, the influent concentrations of microplastics
at WWTW A during the autumn season ranged between 219 and 180 MPs/L, in which a total of 399 MPs/l
was composed of fragments. The effluent concentration ranged between 129 and 138 MPs/L and a total of
298 MPs/L were observed as fragments.
The WWTW B influent microplastics concentrations ranged between 170 and 231 MPs/L, with a total of
401 MPs/L composed of fragments. The effluent concentration also consisted of 210 MPs/L predominantly frag-
ments. During autumn, the concentrations of microplastics were only composed of fragments in both WWTWs.
The influent microplastic concentrations for WWTW B were higher than WWTW A.
During winter, the concentrations of microplastics continued to increase on the influent and effluent of
WWTW A and B, with the highest peaks being 250 MPs/L and 798 MPs/L in the influent and 189 MPs/L
and 316 MPs/L in the effluent. A morphology analysis showed that the influent at WWTW A contained
250 MPs/L angular and 203 MPs/L fibres-shaped particles. Although the effluent concentrations dropped, the
angular and fibre-shaped particles were identified with concentrations of 134 MPs/L and 189 MPs/L respectively.
The WWTW B influent microplastics concentrations were 354 MPs/L angular and 798 MPs/L fibrous particles.
Of these concentrations, the effluent microplastics were also composed of angular and fibre-shaped particles with
concentrations of 119 MPs/L and 316 MPs/L respectively. During the winter season, WWTW B received the
highest influent concentrations of microplastics resulting in high effluent concentrations. This is likely due to acti-
vated sludge processes being unable to handle higher microplastic loading.
The spring season was accompanied by a slight drop in the influent concentrations. However, the effluent
concentrations remained high in both WWTWs. The influent microplastics concentrations at WWTW A com-
prises of 271 MPs/L fragments and 402 MPs/L angular-shaped particles while the final effluent comprises
211 MPs/L films and 296 MPs/L angular particles. The WWTW B influent in spring comprises 287 MPs/L angu-
lar and 420 MPs/L fragmented particles. The concentrations dropped in the effluent with 152 MPs/L angular and
225 MPs/L fragmented particles. Both WWTWs concentrations of microplastics consisted of angular and fibres
at the influent and effluent. Across the four seasons, films were only noticeable during the spring season
at WWTW A. The concentrations of microplastics at WWTW A influent were higher than at WWTW B. How-
ever, WWTW A recorded the highest concentrations in the final effluent indicating insufficient treatment of
microplastics.

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Microplastics concentrations were quantified based on the particle count of different shapes such as angular,
fragments, fibres and film. According to the literature, various shapes of microplastic concentrations have been
detected in the influent and effluent of WWTWs (Kang et al. 2018; Conley et al. 2019). Although various studies
used similar techniques, the concentrations of microplastics observed were different. In a study done by Conley
et al. (2019) the microplastic concentrations in the influent had a difference factor of 2.5 count/L while the efflu-
ent factor was 4.8 count/L. There were no seasonal variations observed. The findings in the current study show
variations in the microplastic influent and effluent concentrations. However, lower concentrations were observed
in summer and autumn while high concentrations were observed in winter and spring. Conley et al. (2019) did
not quantify microplastics based on their shapes while the current study quantified the number of angular, frag-
ments, fibres and films identified as seasonal as shown in Tables 1 and 2.
Long et al. (2019) reported that microplastic concentrations ranging from 1.57–13.69 items/L and 0.2–1.73
items/L in the influent and final effluent respectively. Long et al. (2019) further quantified the concentrations
based on shapes such as pellets, fragments, fibres and granules. The study done by Long et al. (2019) is compar-
able to the findings of the current study in that the concentration of microplastics was high in the influent and
lower in the final effluent with different shapes identified. Although the two studies are comparable, the micro-
plastic samples in the study done by Long et al. (2019) were collected over 2 days in September, therefore,
seasonal differences in the concentrations could not be quantified. The reported high concentrations in the influ-
ent and low concentrations in the effluent suggest that microplastics are reduced in other treatment units of the
activated sludge process. In the current study, the microplastic concentrations varied across the seasons. The vari-
ations of microplastics across the seasons are associated with the changes in polymer compositions and changes
in human behaviours during specific seasons ( Jiang et al. 2022). Microplastics discharged in wastewater end up in
the rivers, where seasonal variations may determine the abundance and accumulation of different types of micro-
plastics. According to Xia et al. (2021) the distribution of microplastics during the dry season is higher than in the
rainy season, wet weather conditions result in the distribution of smaller microplastics along river banks. There-
fore the toxicity of microplastics may differ across seasons based on the polymers present attributed to changes in
flow patterns, water quality and changes in human habits that lead to diverse and complex microplastic charac-
teristics across the seasons ( Jiang et al. 2022).

3.2. Physical characteristics of microplastics

The size of microplastics ranged between 0.01 and 0.02 mm in the influent and 0.01 mm to 0.005 mm in the efflu-
ent. Angular, fragments and fibres were dominant across all four seasons. Rosal (2021) stated that fragments and
fibres are amongst the most reported shapes of microplastics in most studies. Most angular and fibrous-shaped
microplastics occurred in white colour with some showing fouling in the recent study. Some of the fragmented
materials appeared blue and reddish in colour similar to the current study. Angular and fibrous-shaped particles
were observed across all four seasons in the influent and effluent of both WWTWs, except for film-shaped par-
ticles, which were only observed in spring. The shapes of microplastics for WWTW A were comprised of
34.9% angular, 39.2% fragments, 19.1% fibres and 6.8% film-shaped particles (see Figure 3) for reference.

Figure 3 | Microplastic shapes for WWTW A.

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The WWTW B had particle shape dominated by fragments at 46.3%, fibres at 28.1% and angular-shaped par-
ticles at 25.6% with sizes ranging between 0.01 and 0.02 mm in the influent and 0.01–0.05 mm in the effluent (see
Figure 4) for reference. WWTW B had more fibres and fragments as compared to WWTW A, which was domi-
nant with angular types of microplastics. WWTW A is abundant in fibres because it receives 90% influent from
domestic sources, fibres originate from human activities such as the washing of clothes and disposing of waste-
water into the sewage systems, acting as pathways of fibres into wastewater treatment.

Figure 4 | Microplastic shapes for WWTW B.

The results of the current study were comparable with the findings of Rosal (2021) where dominant microplas-
tics identified were fragments and fibres with various colours ranging from white, blue and black, however, the
differences were observed in the sizes of microplastics which ranged between 0.0016 and 5 mm.

3.3. Polymers and functional groups

According to Figure 5(a) WWTW A influent was composed of 7% PE, 11.8% acrylic, 22.4% PET and 33% PS. The
final effluent for WWTW A was composed of 6.3% PS, 16.2% polyacrylamide (PAM), 16.2% PP, 24.7% polycar-
bonate and 36.6% acrylonitrile butadiene styrene (see Figure 5(b)) for reference. There was a difference in the
polymers identified in the influent and effluent of WWTW A, the variation of polymers in the influent and effluent
could be due to the representativeness of samples and the time of sampling for the influent and the effluent.

Figure 5 | Influent and effluent polymers identified for WWTW A: (a) influent polymers and (b) effluent polymers identified for
WWTW A.

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According to Figure 6(a) WWTW B influent was composed of 11.8% PAM, 16.4% poly(ethyl cyanoacrylate),
17.2% polycarbonyl, 21.9% PET and 32.7% PS. The final effluent for WWTW B was composed of 1.8% polycar-
bonate, 6.6% PE, 17.8% poly(methyl methacrylate), 22.9% cellulose, 24.1% polycarbonyl and 26.8% PP (see
Figure 5(b)) for reference. The polymer composition for WWTW B influent and effluent is also different, however,
PS and acrylonitrile butadiene styrene were present in both sampling points.

Figure 6 | Influent and effluent polymer identified for WWTW B: (a) influent polymers and (b) effluent polymer identified for
WWTW B.

Williams et al. (2020) conducted a study on microplastics quantification comparing two WWTWs. The findings
of the study revealed that the two studied WWTWs contained polymers such as PP, PE, PET and silicon in the
influent and effluent over 10 months of monitoring. The current study showed a complex range of polymers ident-
ified in the influent and effluent of WWTW A and B. The influent and effluent of two WWTWs in the current
study were comprised of polymers such as PE, acrylonitrile butadiene styrene, PAM, polymethyl methacrylate,
PS, cellulose, acrylic, PP carbonate, thermoplastic vulcanisates, poly(methyl methacrylate) and poly(ethyl cya-
noacrylate). Contrary to Williams et al. (2020), the current study showed that the microplastic polymer types
observed in the influent and effluent tend to vary seasonally. Studies such as Olesen et al. (2017) and Stockin
et al. (2021) showed similar findings to the current study.
According to FTIR spectroscopy spectra in Figures 7 and 8, the functional groups identified linked to polymers
identified include alcohol, hydroxy compound, methylene (.CH2), methyl (CH3), carbonyl compound, olefinic

Figure 7 | FTIR spectra for polymer functional group identification.

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Figure 8 | FTIR spectra for polymer functional group identification.

(alkene), silicon-oxy compound, aromatic ring (Aryl), nitrogen-oxy compounds, inorganic ion and ether and oxy
compound. The presence of alkenes also indicates the compounds with hydrophobic characteristics, which are
likely to be adsorbed and carried by microplastics discharged into the rivers (Zhang et al. 2022).
In the current study, different polymers were identified from the FTIR spectroscopy library while the spectra
were used to identify the functional group region. According to Bayo et al. (2021) there are two regions of
FTIR spectroscopy spectra that are the fingerprint region (between 600 and 1500 cm1) and the functional
group region. However, the functional group is composed of single bond region (2500–4000 cm1), triple
bonds (2,000–2,500 cm1), and double bond region (1,500–2,000 cm1) wavelength. The fingerprint region is
complex and is composed of a large number of peaks, therefore, it was not used as opposed to the functional
group region (Aurelio Ramírez-Hernández & Universidad 2019). In this study the functional group regions
were between 1,500 and 3,400 cm1 as in the results obtained by FTIR spectroscopy.
The functional groups identified represented various compounds such as ethylene (.CH2), methyl (CH3),
methyne (.CH) carbonyl compounds, olefinic (alkene), alcohol and hydroxy compounds identified between
1,500 and 4,000 cm1 wavelength. According to Shekoohiyan & Akbarzadeh (2022) microplastic particles can
be consumed by fauna and may end up in human bodies through the hierarchy of the food web and this is
likely to affect the health of human beings. The identified saturated aliphatic, alcohol and hydroxyl compounds
as well as alkene in the current study form part of polymer synthetic and are regarded as microplastic additives
substances. The presence of hydroxyl compounds and alkenes indicates the presence of hydrophobic compounds,
therefore, the likelihood of adsorption on polymer surfaces is high, exposing the aquatic species to a more toxic
environment and likely to change the water quality regime (Seidensticker et al. 2018; Wang et al. 2022).
It is also documented that different polymers have sorption capacity on various compounds, for example, a
study conducted by Sheng et al. (2021) investigated the ability of different polymers such as PE, PP and polyvinyl
chloride on the ability to adsorb triclosan (TCS) and health impacts on the Zebrafish. The results of the study by
Sheng et al. (2021) indicated that all polymers adsorbed TCS, however, PP microplastics highly adsorbed TCS as
compared to other polymers increasing the distribution and concentrations of TCS in the tissues and livers of
Zebra fish, followed by polyvinyl chloride then PE.

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Kang et al. (2018) observed that the occurrence of polymer types such as PS in wastewater may indicate dom-
estic sewage as the main source since it is largely fibres that disintegrate during washing in laundries. When
comparing the influents and effluents of both WWTW, we observe similar types of polymers with close % (PS
33% for A and 32.7% for B; PET at 22.4% for A and 21.9% for B) and other different polymers. The main poly-
mers identified in both WWTW influents are PE, PET, and PS while the effluent components are PC and PP.

3.4. Elemental analysis of microplastics particles

Elemental analysis of particles was done to determine the composition of microplastics using a SEM coupled
with EDX (SEM/EDX). Figures 9 and 10 present the SEM images and graphs for elemental analysis for influent
and effluent samples collected at WWTW A and B. Tables 3–6 summarise the results obtained for elements ident-
ified. SEM coupled with EDX analysis was performed for samples that were composed of PE, PAM, and PET.

Figure 9 | SEM/EDX analysis for WWTW A and B: (a) HART-A Influent SEM image, (b) HART-A influent elemental analysis graph,
(c) WART-B effluent SEM image, and (d) WART-B effluent elemental analysis graph.

Figure 10 | SEM/EDX analysis for influent and effluent at WWTW A and B: (a) HART-A influent SEM image, (b) HART-A influent
elemental analysis graph, (c) WART-B effluent SEM image, and (d) WART-B effluent elemental analysis graph.

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Table 3 | Details of microplastic particle composition from EDX analysis for Figure 9(a)

Sampling point Atoms Weight % from EDX analysis Error, % Reference spectrum

Raw influent sample 1 HART C 73.46 +0.35 PE

O 15.90 +0.26
Cr 0.19 +0.01
Fe 0.41 +0.03
Mg 0.61 +0.03
Al 0.86 +0.03
Si 0.37 +0.03
P 1.23 +0.03
S 0.41 +0.01
Cl 1.47 +0.02
K 0.50 +0.02
Ca 0.55 +0.02
Na 4.02 +0.05
Total 100

Table 4 | Details of microplastic particle composition from EDX analysis for Figure 9(b)

Sampling point Atoms Weight % from EDX analysis Error, % Reference spectrum

Final effluent sample 2 HART C 58 0.42 PAM

O 26.9 0.22
Fe 0.09 0.02
Mg 1.06 0.02
Si 2.26 0.03
P 0.77 0.01
S 1.66 0.03
Cl 8.73 0.03
K 4.41 0.03
Ca 0.40 0.02
Na 5.19 0.05
Total 100

Table 5 | Details of microplastics particle composition from EDX analysis for Figure 9(c)

Sampling point Atoms Weight % from EDX analysis Error, % Reference spectrum

Raw influent sample 3 WART C 52.26 +1.34 PET

O 19.92 +0.51
Fe 1.23 +0.09
Mg 1.21 +0.04
Al 0.18 +0.03
Si 0.52 +0.03
P 3.2 +0.08
S 0.49 +0.04
Cl 8.73 +0.12
K 1.39 +0.08
Ca 2.21 +0.10
Na 8.66 +0.05
Total 100

Figure 9(a) shows the SEM images for influent at WWTW A with particle sizes at 0.005 mm (5 μm). The sample
was composed of PE showing fibrous-shaped microplastics. Figure 9(b) shows the SEM image for effluent at
WWTW A with particle sizes at 0.05 mm (50 μm). The shapes of particles are long translucent fibres and fragmen-
ted materials containing PAM polymer.

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Table 6 | Details of microplastic particle composition from EDX analysis for Figure 9(d)

Sampling point Atoms Weight % from EDX analysis Error, % Reference spectrum

Final effluent sample 4 WART C 58.31 +0.43 PE

O 22.01 +0.19
Mg 0.63 +0.03
Al 0.21 +0.01
Si 2.31 +0.02
P 0.48 +0.01
S 0.64 +0.01
Cl 6.38 +0.04
K 2.09 +0.02
Total 100

Figure 9(c) shows the SEM images for influent at WWTW B with particle sizes averaging 0.025 mm (25 μm).
The microplastics appear to be thick fibres of PET. SEM image for effluent at WWTW B confirms the presence of
fibrous particles, but the fibres appear to be bio fouled, this could be attributed to microplastics having passed
through different treatment stages in the activated sludge processes such as anaerobic zone, anoxic zone, and
aeration. The polymer composition of the WWTW B effluent sample was PE (see Figure 9(d)) for reference.
Figure 10(a) presents the elemental analysis of PE particles on the influent of WWTW A. The elemental analy-
sis shows highly intense peaks between 0 and 3 KeV which are indexed to elements such as Carbon (C) and
Oxygen (O). Other peaks noticeable such as Calcium (Ca), Chromium (Cr), Magnesium (Mg), Chloride (Cl),
Iron (Fe), Aluminium (Al), Silicone (Si), Phosphorous (P), Sodium (Na) and Sulphur (S) were between the
KeV ranges 0.5–4.
Figure 10(b) shows elemental analysis peaks of high intensity between 0 and 4 KeV which are indexed to the
elements C, O, P, Na, Mg, and Cl. The most intense peak occurs in the range 2.5–3.0 KeV peak and is caused by
Cl followed by the peaks attributed to Na and P between 1 and 2 KeV. The O and C peaks were noticeable
between 0 and 1 KeV. The polymer linked to the elements detectable in the effluent of WWTW B is PAM.
The influent and effluent at WWTW A are composed of elements that can be linked to the presence of microplas-
tics, with heavy metals prevailing in the analysed samples which contain PE and PAM polymers.
Figure 10(c) presents the elemental analysis of PET particles identified in the influent of WWTW B. The most
intense peaks were identified in the range 0–4 KeV indicating the abundance of C, O, Na, Si, S, Cl, and P in the
microplastics. Other elements identified at lower peaks include Fe, Mg, and Ca. The presence of C, O, and Cl
provides evidence of polymer presence in the samples and associated metal (see Figure 10(c)) for reference.
Figure 10(d) presents the elemental peaks in the effluent sample for WWTW B, with the highest peaks occur-
ring between 0 and 4 KeV. The highest peaks recorded are Cl between 2.8 and 3 KeV, Na between 1 and 1.5 KeV,
Si between 1 and 2 KeV and O and C between 0 and 1 KeV. Other elemental peaks recorded include Mg, Al, P,
and S. The elemental composition is associated with PE particles observed in the effluent of WWTW B.
SEM imaging was used to generate high-resolution micrographs which were used to differentiate the microplas-
tics from organic materials. EDX was used to determine elements present in the microplastics. Woo et al. (2021)
further explained that elements such as Al, Ca, Mg, Na and Si are regarded as components of colourants, plas-
ticisers and flame retardants in which microplastics are manufactured. In the current study, heavy metals
related to microplastic additives were observed on polymers such as PAM, PET and PE. The analysis of samples
containing PE, PAM, and PET showed a high ratio of C and O which can be linked to fibres and fragments
observed in the current study. The presence of heavy metals in microplastics is associated with toxic effects on
aquatic species. In a study by Banaee et al. (2019) cyprinus carpio species were exposed to Cd and Cl leading
to immune disorders and blood flow malfunctioning, indicating the toxic effects of the presence of toxic
metals and microplastics in water.
The summary of elemental composition at WWTW A influent is presented in Table 3 showed that the particles
of PE were dominated by C (73.46%) followed by O (15.9%). Other elements constitute 11%, mostly sodium was
abundant at 4% as compared to the other remaining elements such as Fe, Mg, Si, P, S, Cl, K, and Ca.
The elemental composition for effluent is presented in Table 4 for PAM particles identified on the effluent of
WWTW A. The elements dominating the effluent sample at WWTW A were C (58%), O (26.9%), Cl (8.73%) and

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Na (8.66%) (see Table 4) for reference. Other elements such as Fe, Mg, Si, P, S, Cl, K, Ca and Na were detectable
in smaller percentages between ranges of 0.04 and 5%. Although some metal elements were detectable in smaller
amounts, microplastic particles of PAM may contain contaminants that can be distributed into surface water.
Table 5 presents the elemental analysis for the influent sample for WWTW B, with PET particles dominating
the samples as identified from the reference spectrum. The elemental analysis of the particles indicated high per-
centages of C and O at 52.26% and 19.92% respectively. The influent for WWTW B also contained elements such
as Cl (8.73%) and Na (8.6%) in smaller percentages as compared to C and O. Other elements identified ranged
between 0.18 and 2.21%, with Al in the smallest abundance.
According to Table 6, the effluent at WWTW B was predominantly composed of elements such as C (58.31%),
O (22.01%), and Cl (6.38%). Other elements such as Mg, Al, Si, P, and S were prevalent in smaller percentages.
The particles were dominated by PE as identified from the reference spectrum. The influent and effluent of
WWTW B both contained high percentages of C and O elements, indicating that the particles comprised similar
elemental compositions but different polymers identified, i.e. PE, PAM and PET.
According to Zhang et al. (2022) when plastic waste is transformed, aromatic oxygenates are formed, this is
attributed to the fact that during plastic manufacturing, oxygenated additives are added. The high percentages
of oxygen obtained in the elemental analysis of particles conducted in the current study point out that polymers
such as PE, PAM and PET are made out of additives rich in oxygen. For example, the transformation of 0.5 g of
PS resulted in 0.36 g of aromatic oxygenates produced as stated by Zhang et al. (2022). Therefore the presence of
carbon in higher percentages in samples containing polymers in Tables 3–6 indicates polymers made with carbon
linkages. Other compounds identified were metals, which form bases in the manufacturing and production of
microplastics.

4. CONCLUSIONS
The identification and quantification of microplastics in wastewater treatment plants by spectroscopic and micro-
scopic techniques were carried out in Johannesburg East, South Africa. The study considered the physical and
chemical characteristics of microplastics and briefly quantification in terms of concentrations. The concen-
trations of microplastics in WWTW A and B varied across seasons. The seasonal changes showed that from
summer to autumn concentrations are slightly low as compared to winter and spring.
The shapes, colours, and sizes of microplastics are not different across WWTWs, the physical properties of
microplastics in WWTW A and B were similar across four seasons. The polymers identified include acrylic, acry-
lonitrile butadiene styrene, cellulose, polycarbonyl, PAM, PE, PET, poly(ethyl cyanoacrylate), PS, poly(methyl
methacrylate) and polycarbonate. The chemical composition of polymers was dominated by trace metals and
organic compounds such as hydroxyl compounds, Alcohol, saturated aliphatic, polymeric –OH stretches
which confirms the presence of plastic particles in the samples. The elemental composition was dominated by
non-metallic elements such as S, Si, O, C and Cl occurring together with metallic elements such as Mg, Al, P,
and non-metals such as S and Si.
Due to high concentrations of microplastics escaping from WWTW A and B, the receiving water bodies are
affected and the water quality regime is changed by the chemical compositions of microplastics. The seasonal
variations in microplastic concentrations point out that there are changes in the patterns of human habits
which play a role in the abundance of microplastics in wastewater. The abundance of fibres indicates domestic
sources such as laundry machines, therefore, is important to equip them with filters to avoid high accumulation in
wastewater. Due to the amount of microplastics that pass through the wastewater treatment works, there is an
urgent need to replace the existing wastewater treatment technologies with the new emerging technologies
that are tailored for the efficient removal of microplastics from wastewater.

DATA AVAILABILITY STATEMENT

All relevant data are included in the paper or its Supplementary Information.

CONFLICT OF INTEREST
The authors declare there is no conflict.

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First received 11 June 2023; accepted in revised form 21 November 2023. Available online 4 December 2023

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