Journal of Hazardous Materials 421 (2022) 126779

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Journal of Hazardous Materials

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Research Paper

Are your shoes safe for the environment? – Toxicity screening of leachates
from microplastic fragments of shoe soles using freshwater organisms
Lia Kim, Dokyung Kim, Sang A Kim, Haemi Kim, Tae-Yang Lee, Youn-Joo An *
Department of Environmental Health Science, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Republic of Korea

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

Editor: Dr. R Teresa This study investigated the toxic effects of leachates from microplastic fragments of soles from four different
types of shoes (slippers, trekking shoes, running shoes, and sneakers) on three aquatic organisms (Chlamydo­
Keywords: monas reinhardtii, Daphnia magna, and Danio rerio). The chemical components in each leachate were identified;
Microplastic furthermore, chlorophyll a contents of C. reinhardtii were measured, and immobilization of D. magna and de­
Aquatic toxicity
formities in D. rerio were observed. The abnormalities observed in the test species exposed to the leachates were
Principal component analysis
compared and chemical compounds majorly influencing the species were determined by principal component
Plastic pollution
analysis (PCA). Sneaker leachate showed growth inhibitions in C. reinhardtii, immobility and mortality in
D. magna, and severe abnormalities in D. rerio. Consequently, aquatic toxicity was majorly associated with
benzothiazole, carbon disulfide, ethyl acetate, and p-xylene. The results showed that toxic chemicals could leach
from load-originated microplastics when exposed to aquatic media, and consequently, induce significant nega­
tive effects on aquatic organisms. Since microplastics from shoe soles discharge the above-mentioned toxic
chemicals, regulating the chemical use during plastic production is critical to prevent severe effects of micro­
plastic toxicity in aquatic organisms, and to maintain the health of aquatic environments.

1. Introduction
Global plastic consumption has increased exponentially and conse­
quently, increased the proportion of plastic wastes (Geyer et al., 2017;
PlasticsEurope, 2020). Additionally, environmental research has
revealed increasing micro and nanoplastic effects on ecosystems, ranging
from molecular and individual levels to community levels (Huang et al.,
2021; Hwang et al., 2019; Seeley et al., 2020; Suman et al., 2020). Small
plastic particles originating from different sources are found in varying
environmental media (Awasthi et al., 2020; Enfrin et al., 2019; Wu et al.,
2019). In terrestrial environments, tires, asphalt, shoe soles, plastic bags,
and other polymer-based products are major sources of plastic wastes
(Hanik et al., 2019; Vogelsang et al., 2019). Furthermore, small plastic
fragments, such as expanded polystyrene (EPS) floats, and plastic wastes
originating from land have been observed in aquatic environments (Chae
et al., 2015; Statista, 2021).
Micro and nanoplastic research has majorly focused on plastic
pollution, toxicity effects, trophic transfer, and modelling of plastic
fragment movement, especially in marine environments (Browne et al.,
2011; Chae et al., 2019; Jung et al., 2021; Wang et al., 2021). However,
microplastics from the terrestrial environment have not been considered
although plastic fragments in freshwater environments could have
terrestrial origins. Urban sewage sludge is known to contain tire or shoe
sole fragments, which have been disrupted on roads (Rutgersson et al.,
2020; Vogelsang et al., 2019).
Previous studies include the application of spherical micro and
nanoplastics in artificial manufacturing or experimental designs. How­
ever, plastic fragments that pose a concern for the environment are
majorly irregular or fiber-shaped than spherical (Park et al., 2020; Jung
et al., 2021). Moreover, chemicals added during manufacturing of
plastic products can leach from these fragments (Chen et al., 2019;
Hahladakis et al., 2018; Rummel et al., 2019; Schrank et al., 2019).
Thus, the actual negative effects of microplastics in the environment are
related to the eluted materials from plastics and plastic fragment
toxicity. Tire leachates include toxic chemicals, such as benzothiazole,
benzo[a]pyrene, pyrene, and zinc (Zn), which have also been detected in
shoe sole leachates (Capolupo et al., 2020; Ingre-Khans et al., 2010;
Kolomijeca et al., 2020).
According to previous studies, microplastic particles originating
from terrestrial environments could leach toxicants when released in

* Corresponding author.
E-mail address: anyjoo@konkuk.ac.kr (Y.-J. An).

https://doi.org/10.1016/j.jhazmat.2021.126779
Received 3 May 2021; Received in revised form 27 July 2021; Accepted 27 July 2021
Available online 29 July 2021
0304-3894/© 2021 Elsevier B.V. All rights reserved.
L. Kim et al. Journal of Hazardous Materials 421 (2022) 126779

aquatic environments (Canepari et al., 2008; Ingre-Khans et al., 2010;
Wagner et al., 2018; Shruti et al., 2021). The present study aimed to
examine the effects of leachates of irregular-shaped plastic fragments,
which were acquired from manual fragmentation of shoe soles, on three
aquatic model species (algae, water fleas, and fish embryos). Bioassays
were conducted on these aquatic organisms belonging to different tro­
phic levels, while the leachates were chemically analyzed. We hypoth­
esized that specific chemical components of the leachate would be
associated with high risks. The results of the bioassay and chemical
analyses were compared and analyzed to determine the chemicals
responsible for inducing adverse effects on the test aquatic organisms by
principal component analysis (PCA).
2. Material and methods
2.1. Test materials
Four types of commonly used footwear (slippers, trekking shoes,
running shoes, and sneakers) were purchased from a commercial mar­
ket. As slippers and sneakers are used more frequently in daily life, there
are high chances of exposure of the microplastic fragments from their
soles to occur in run-off surface water and groundwater in the town. In
contrast, trekking shoes and running shoes are rarely used by general
people, except during physical workout. The soles get damaged due to
the irregular surface of the ground where the shoes are commonly used.
As a result, many tears could develop every time they are worn.
The slipper sole comprises ethylene vinyl acetate, while the soles of
the other three types of shoes is mainly composed of synthetic rubber
(Table 1) per the information from the retailer. Soles of each shoe were
scrubbed using a foot file and the resultant fragments were sieved
through a 1 mm pore size mesh. The sole fragments of each shoe type
were examined with an optical microscope (Olympus BX51 optical mi­
croscope; Olympus, Tokyo, Japan). Feret’s diameter, particle area,
circularity, and roundness of each fragment were measured using free
software ImageJ (da Costa Araújo et al., 2020). The composition of each
particle was identified by obtaining a Fourier-transform infrared (FT/IR)
Table 1
Characteristics of selected shoe models (Slippers, trekking shoes, running shoes,
and sneakers) used to develop leachates of shoe sole fragments. Fragment
characteristics (Feret’s diameter, fragment area, circularity, and roundness)
have been shown as mean values including standard deviations.
Characteristics Slippers Trekking Running Sneakers
shoes shoes
spectrum using FT/IR 4100 spectrometer (Jasco Co. Ltd., Tokyo, Japan),
and the results were compared with the details provided by the retailer.
Four types of shoe sole leachate were prepared as per the leaching
method described by Ingre-Khans et al. (2010) with some modifications.
The original method used shoe sole fragment with size less than 1 mm
were used to produce leachates as a liquid/solid (L/S) ratio of 1000. One
gram of sole fragments was added in 1 L of 2 mM NaHCO3 solution and
placed on a shaker at 21 ◦ C and 45 rpm under dark conditions for 29 d.
The toxicity assays were then conducted using 20 µm filtrates. In the
present study, four types of shoe sole leachates were prepared as L/S
ratio of 500. It was prepared by adding 2 g of shoe sole fragment in 1 L of
2 mM NaHCO3 solution under the same leaching condition. The resul­
tant solutions were filtered using a 0.45 µm cellulose-acetate filter;
subsequently, the acquired filtrates were stored at 4 ◦ C for one week
before conducting the bioassays. This experiment was designed with
extremely high concentration of microplastics compared to the actual
freshwater environment (Stanton et al., 2020). The possibility of over­
estimation of ecotoxicity was considered by the higher concentration of
shoe sole leachates.
To analyze the chemical composition of the leachates, 1% solution
of the shoe sole leachate was prepared and measured using inductively
coupled plasma-mass spectrometry (ICP MS) and gas chromatography-
mass spectroscopy (GC-MS). Eight metals (Arsenic (As), Barium (Ba),
Cadmium (Cd), Chromium (Cr), Copper (Cu), Nickel (Ni), Lead (Pb),
and Zn) in the test solution were analyzed using three replicates via
ICP-MS (Agilent 7700; Agilent Technologies, Santa Clara, CA, USA),
with a sample depth of 8 mm injected into the chamber at 2 ◦ C at a flow
rate of 0.82 L/min using argon (Ar) as a carrier gas. The metals were
analyzed using helium (He) mode at 3.5 mL/min of cell gas flow. A
MicroMist concentric nebulizer was used, and the detection limit was
0.1 μg/L. The limit of detection level of each heavy metal component is
described in Table S1(A).
Organic compounds in each leachate were measured in duplicates
through GC MS (Agilent 7890B; Agilent Technologies, Santa Clara, CA,
USA) having a DB-5MS column (30 m × 0.25 mm ID; 0.25 µm thickness).
Samples (1 μL) were injected at 230 ◦ C in an electron impact mode, at 1
mL/min of flow rate of He carrier gas. The organic compounds were
identified and matched with the records in the reference library (NIST
mainlib EI MS Library) and compounds with a similarity of more than
800 were selected. In this study, organic compound analysis was con­
ducted at a non-targeted screening level and the analysis of the signal-to-
noise ratios (S/N ratio) is described in Table S1(B).
2.2. Test species

Shoe sole Black soles Black and Grey waffle Light brown Bioassays for assessing the four leachate types was conducted one
description made of grey waffle shoe made of sole made of
ethylene sole made of synthetic synthetic
week after leachate preparation. Model aquatic organisms belonging to
vinyl synthetic rubber rubber tropic levels were selected to conduct the bioassays. Similar to this
acetate rubber study, previous studies have assessed the ecotoxicity of waste by
(EVA) exposing the leachates to test species, which belonged to different tro­
Country of China China China China
phic levels (Pandard and Römbke, 2013; Wuttke and Walther, 2013).
manufacture
Retailer eMart eMart (Seoul, eMart (Seoul, eMart The selected test species were Chlamydomonas reinhardtii, Daphnia
(Seoul, Korea) Korea) (Seoul, magna, and Danio rerio. Green algae are sensitive to environmental
Korea) Korea) changes in aquatic environment and water fleas are usually used to
Fragment characteristics assess the toxicity of wastewater (Lee et al., 2015). Fish exposed to
Description of Black A mixture of Grey Light brown
leachates since the embryonic stage have demonstrated developmental
shoe sole powder with grey and fragments particles
fragments size < 1 mm black with size < 1 with a size sub-chronic effects and acute effects (Kwak et al., 2016).
particles with mm < 1 mm Double-concentrated test mediums were prepared for each bioassay
a size < 1 mm to supplement each test species with same nutrients during their expo­
Feret’s 56.61 ± 204.38 ± 228.61 ± 155.72 ±
sure to the shoe sole leachates. These mediums were mixed with 100%
diameter 45.80 131.14 107.82 143.68
(μm) concentration of the four shoe sole leachate types. In the control group,
Fragment area 1468 ± 20253 ± 21134 ± 12243 ± double-concentrated test medium was mixed with 2 mM NaHCO3, which
(μm2) 1993 20465 17240 16446 was used as a base solution in the four types of shoe sole leachates. All
Circularity 0.68 ± 0.25 0.62 ± 0.21 0.56 ± 0.16 0.67 ± 0.26 test mediums commonly included 2 mM NaHCO3 and since it did not
Roundness 0.57 ± 0.18 0.61 ± 0.16 0.60 ± 0.16 0.61 ± 0.18
exhibit adverse effects in any of the test species, 2 mM NaHCO3 was
assumed to not contribute to toxicity.

2
L. Kim et al.
Toxicity tests involving sneaker leachate used only 50% concentra­
tion of the leachate as a stock solution that was diluted with the test
medium and mixed with 2 mM NaHCO3. Different solutions of low
concentration sneaker leachate were applied to test organisms; specif­
ically, algae (belonging to low trophic levels) and fish were exposed to
the leachate at the embryonic stages. Since these organisms are highly
sensitive to contaminants, low concentration of sneaker leachate
(1–25%) was expected to demonstrate a high risk. However, bioassays
using water fleas required a high concentration of sneaker leachate with
dilution applied to 3.125%. The compositions of the test media are
mentioned in Table S2.
2.2.1. Algal assays
Green algal species, C. reinhardtii, acquired from the algal culture in
the University of Texas (Texas, Austin, USA) was selected for the algal
toxicity assay. C. reinhardtii was pre cultured for 7 days in Tris-Acetate
Phosphate (TAP) medium. Test solution with TAP medium was sterilized
in an autoclave at 120 ◦ C and 0.15 MPa for 15 min. The four shoe sole
leachates were diluted with the TAP medium to achieve a final con­
centration of 50% for C. reinhardtii; subsequently, 0.9 mL of the solution
was added per well in a 48-well microplate. Later, 0.1 mL of C. reinhardtii
culture was inoculated with an initial cell density of 105 cell/well and
placed on a shaker at 24 ◦ C and 100 rpm under a 16:8 light: dark con­
dition for 3 days. To measure the algal growth, chlorophyll a contents
were extracted for 3 h using ethanol under dark conditions, and were
measured at excitation (420 nm) and emission (670 nm) wavelengths
using a microplate reader Varioskan LUX (Thermo Fisher Scientific,
Waltham, MA, USA). The entire test was conducted in four replicates.
The pH was measured before and after the experiment. Microalgae
subjected to low sneaker leachate concentration were fixed using 2%
glutaraldehyde, and 20 μL of the microalgae solution was placed on a
slide glass and observed under an optical microscope—Olympus BX51
(Olympus, Tokyo, Japan).
2.2.2. Cladoceran (water flea) assay
Water flea acute toxicity test was conducted according to the Orga­
nization for Economic Co-operation and Development (OECD) test
guideline no. 202 using Daphnia magna (OECD, 2004). Shoe sole
leachates (50% concentration) were prepared by diluting the solution
with moderate hard water. Five neonates born within 24 h were directly
exposed to 10 mL of the 50% leachate in 20 mL vials and kept in an
incubator at 20 ◦ C with a 16:8 light: dark condition for 2 days. Subse­
quently, D. magna immobilization and mortality at 24 and 48 h was
observed. The additional toxicity test was conducted in low concentra­
tions at 3.125%, 6.25%, 12.5%, and 25% for the sneaker leachate. Each
test was conducted in four replicates for each concentration of the four
shoe sole leachates, and the pH was measured before and after the
experiment. No food was provided to D. magna neonates nor was the
medium changed during the test.
2.2.3. Fish embryo assay
Fish embryo assay was conducted according to the OECD standard
guideline no. 36 using wild zebrafish (D. rerio) with some modifications
(OECD, 2013). Adult fish were cultured in the dechlorinated water for
three months (pH 7.0 ± 0.1) under 16:8 light:dark condition at 26 ◦ C in
the laboratory fish tank (30 cm × 30 cm × 45 cm). A spawning box,
which could separate fertilized embryos from adult fish, was installed in
the fish tank, and male and female D. rerio individuals were mated at a
ratio of 1:2.5 to induce spawning under the dark condition. Newly
fertilized embryos were collected in the morning of the day when the
exposure study was conducted, and they were washed using embryonic
rearing solutions. The embryos at the blastula stage were directly
exposed to the four types of shoe sole leachates (50% concentration),
200 μL of each leachate per well in a 96-well microplate. The embryos
were individually exposed to the leachates for 7 days with static con­
dition. Danio rerio were placed in an incubator at 26 ◦ C with a 16:8 light:
3
Journal of Hazardous Materials 421 (2022) 126779
dark condition for 7 days and observed every 24 h using an optical
microscope. The endpoints for D. rerio embryo and larvae included
mortality, hatching and developmental delays, tail malformation, bent
spine, peritoneum edema, and abnormal eyes. Developmental delays
were compared with the growth stages described in a previous study
(Kimmel et al., 1995). Negative effects and hatching rate were observed
daily, and the results were shown as the accumulative abnormality of 7
d post fertilized (dpf) D. rerio. At the end of the exposure, each organism
was placed on a glass slide and its images were obtained. Total body
length of the surviving individuals was measured with Olympus cellSens
Entry software (Version 1.16; Olympus, Tokyo, Japan). Each test was
conducted thrice with 10 replicates for each shoe sole leachate. The
toxicity of the sneaker leachate was investigated at low concentrations
ranging from 0% to 25%. Additionally, the degree of spine incurvation
was measured for survivors using the angle measurement function in
ImageJ program. The incurvation was calculated as the angle of incli­
nation from the straight spine that was calculated at the center of the
two eyeballs center for each organism. The pH was measured before and
after the experiment. Neither food was supplied to the individuals nor
the medium was changed. The fish embryo tests were conducted ac­
cording to the regulations and ethics of the experimental plan autho­
rized by Institutional Animal Care and Use Committee in Konkuk
University (KU 20089).
2.3. Principal component analysis
To determine the major leachate chemical factor which caused
toxicity in the test aquatic organisms, principal component analysis
(PCA) was used. PCA is a type of selection analysis used in data mining
to minimize the number of factors. PCA has been included in previous
studies for identifying suitable factors to understand the toxicity in or­
ganisms (Chae et al., 2017; Kim et al., 2018). Principal components
(PCs) with an eigenvalue > 1 were considered to select the chemicals
causing toxicity; subsequently, two principal components (PC1 and
PC2), each with a maximum eigenvalue of two, were selected.
In this study, metals and organic compounds detected in the four
leachate types were used for PCA of leachate chemical compound in­
dicators. Four metals (As, Cr, Ni, and Zn), and few organic compounds
were used for the PCA of leachate chemical compound indicators. To
compare the aquatic toxicity, the endpoints of all the test species were
considered at the end of the leachate exposure. Degree of decreased
amount of Chlorophyll a contents of C. reinhardtii compared with those
of the control group was used for PCA. Immobilization rate of D. magna
at 48 h, and cumulative abnormalities of D. rerio at 7 d were also used as
aquatic toxicity indicators. Among the different chemical compounds,
the indicators responsible for the most significant negative effects on
C. reinhardtii, D. magna, and D. rerio were determined.
2.4. Statistical analyses
Chlamydomonas reinhardtii data were reported as relative cell
growth rate, whereas the D. magna and D. rerio embryo tests were
indicated graphically as the sum of negative effects. Before performing
the analysis of variance (ANOVA), the normality and variance homo­
geneity of data were checked using the Shapiro Wilk test and Levene
test, respectively. Statistically significant differences between each type
of leachate and the low concentration of sneaker fragments (p < 0.05)
were determined by one-way ANOVA along with Tukey’s post-hoc test.
In D. magna, two factors (leachate type and exposure time) were
analyzed against immobilization using two-way ANOVA along with DD
test, but since the exposure time was divided only in two groups (24 and
48 h), post-hoc test was not conducted. Additionally, the chemical
components in the leachate of shoe sole fragments were compared with
the results of adverse effects on C. reinhardtii, D. magna, and D. rerio.
Pearson’s correlation coefficient was used to estimate the correlation
between each chemical and test species along with a two-tailed test.
L. Kim et al.
Statistical analyses were conducted using SPSS software (Version 24.0;
IBM Corp., Armonk, NY, USA).
3. Results
3.1. Shoe sole fragment characterization
Four types of shoe sole fragments were observed via microscopy
(Fig. 1), and their corresponding physical characteristics were measured
including the Feret’s diameters, mean fragment areas
fragment areas, circularity, and roundness of the fragments (Table 1).
The slipper shoe sole fragments were relatively smaller than other types
of shoe sole fragments. Size distributions of four parameters are described
in Fig. S1. According to FT/IR spectra (Fig. S2(A)), the slipper particles is
composed of ethyl vinyl acetate (EVA), as described in Table 1. However,
the chemical composition of other shoes did not match the details pro­
vided by the retailer. As shown in Fig. S2(B–D), the FT/IR spectra pat­
terns of trekking shoes, running shoes and sneakers depart from the FT/
IR spectrum of synthetic rubbers (Gorassini et al., 2016). The running
shoes and sneakers showed the transmittance peak at 1050–1085 cm− 1.
3.2. Chemical analyses
The four shoe sole leachates were screened for metals and organic
compounds, and the differences in the chemical composition of each
leachate were compared. According to the ICP–MS results, seven metals
out of the total eight were detected, with the exclusion of Cd, in which
the detection limit was 0.1 μg/L. Furthermore, except for As and Zn, the
concentration of the other metals was not significantly different between
the control and the four shoe sole leachates. Overall, the As and Zn
concentrations were high. However, only the Zn concentration in the
sneaker leachate reached a maximum value of 69.46 μg/L, while the As
concentration was 283.21 μg/L, which was lower than that in the
trekking shoe sole leachate (323.08 μg/L). Moreover, chemicals, such as
benzothiazole, carbon disulfide, 1-(4-propan-2-ylphenyl)ethanone, and
2-(3-isopropylphenyl)propan-1-ol were detected only in the sneaker
leachate. The average peak area in the GC–MS results were in accor­
dance with the retention time. The results of the chemical analyses for
metals and organic compounds are shown in Fig. S3. The results of
organic compounds were enhanced by labelling the organic compounds
alphabetically.
Fig. 1. Fragments of shoe soles after sieving with 1 mm mesh to develop
leachates of shoe sole fragments. Fragment images of (A) slippers, (B) trekking
shoes, (C) running shoes, and (D) sneakers. Scale bar = 250 µm (blue) and
500 µm (black). (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
4
Journal of Hazardous Materials 421 (2022) 126779
3.3. Algal assay
Fig. 2 shows the growth rate of C. reinhardtii exposed to the four shoe
sole leachates. The growth of C. reinhardtii was inhibited only in the
sneaker leachate and was normal in the slipper, trekking shoe, and
running shoe leachates compared with the control group. The chloro­
phyll contents of C. reinhardtii exposed to 50% of the trekking shoe
leachate increased significantly compared with other exposure groups
(slippers, running shoes, and sneakers). Furthermore, pH before and
after the 3 D-exposure increased in all the leachates, except sneaker
leachate. The leachate pH value of control, slippers, trekking shoes,
running shoes, and sneakers with 50% concentration before the test was
7.39, 7.75, 7.72, 7.81, and 7.79, respectively; it increased to 8.05, 8.28,
8.25, 8.20, and 7.73, respectively, after the 3 D-exposure (Table S4).
Furthermore, toxicity tests at low concentrations of sneakers
leachate were conducted, but no significant decrease in the cell growth
was observed, except at 12.5% concentration (Fig. 2(B)). Each algal cell
in the different sneaker leachate concentrations was observed through
optical microscopy. Consequently, the delay in cell division was found at
6.25% concentration of the sneaker leachate (Fig. S4) with decrease in
yield, and cell growth inhibition observed at 50% concentration; how­
ever, images could not be acquired because of the low cell density.
3.4. Cladoceran (water flea) assay
Fig. 3 shows the cumulative effects (immobilization and mortality) of
D. magna exposed to the four leachates. As seen in the Fig. 3(A),
immobilization rate in D. magna significantly increased at 48 h in or­
ganisms exposed to sneaker leachate and the mortality rapidly increased
between 24 and 48 h. There was no significant increase in the cumula­
tive effects in other types of shoe sole leachates. The leachate pH value of
control, slippers, trekking shoes, running shoes, and sneakers at 50%
concentration before the test was 8.20, 8.25, 8.25, 8.41, and 8.30,
respectively; it increased to 8.33, 8.44, 8.50, 8.30, and 8.11, respec­
tively, after the 48 h-exposure (Table S4). Organisms were immobilized
at 12.5% concentration of the sneaker leachate, whereas no significant
effects were observed at exposures ranging from 3.125% to 6.25%.
3.5. Fish embryo assay
Fig. 4(A–D) indicates the total negative effects on 7 dpf D. rerio
exposed to the four leachates. There was no significant difference in the
50% leachate exposure group in slipper, trekking shoe, and running shoe
fragments. Conversely, only sneaker leachate significantly affected fish
and other test species in the early stages. Embryos exposed to sneaker
leachate demonstrated developmental delay, hatching delay, pigmenta­
tion loss, and eventually hatching failure by the end of leachate exposure
(Fig. S5). The pH values of 50% leachate decreased after the 7-d exposure;
the detailed pH values are stated in Table S4. Significant abnormalities,
including bent spine and body edema in the 7 dpf larvae were observed
after exposure to 3.125% of the sneaker leachate. The intensity of the
negative effects increased with increasing sneaker leachate concentration
and the corresponding abnormalities in D. rerio are shown in Fig. 5. As the
concentration of sneaker leachate increased, the degree of spine incur­
vation intensified (Fig. S6(C)); furthermore, eye edema was observed,
and pigmentation decreased. Hemostasis and hatching delay were
accompanied by the developmental delay in the 25% of sneaker leachate
concentration. Total body length of the survivals was measured and was
observed to decrease in the groups exposed to 12.5% and 25% of the
sneaker leachate concentration (Fig. S6).
3.6. Results of PCA
PCA was employed to characterize the four leachates and identify the
chemical compounds, which negatively affected the three test organ­
isms. Chemicals detected with a higher concentration than the control
L. Kim et al.
(including test medium and 2 mM NaHCO3) were used as the input data
since the negative effects in the organisms were observed only in in­
dividuals exposed to the sneaker leachate.
Furthermore, only the sneaker leachate showed a positive PC1
value among the other shoe sole leachates (slippers, trekking, and
running shoes), while the control solution showed a highly negative
value. As shown in Table 2, 1-(4-propan-2-ylphenyl)ethenone, 2-(3-
isopropylphenyl)propan-1-ol, benzothiazole, carbon disulfide, hex­
amethylcyclotrisiloxane, methyl salicylate, p-xylene, and Zn were
observed in PC1, representing 61.23% of the total variance percentage.
Furthermore, 2-ethylhexanol, dichloromethane and methyl (Z)-N-
hydroxybenzenecarboximidate were observed in PC2, representing
17.51% of the total variance percentage.
The chemical composition of sneakers differed from that of other
leachates and was separated based on PC1. The loading spots of negative
effects on the three test species were positively correlated (r = 0.91)
with the chemicals shown in PC1 (Fig. 6 and Table S5).
Additionally, the toxicity of the sneaker leachate was highly associ­
ated with the chemicals in PC1. Benzothiazole, carbon disulfide, ethyl
5
Journal of Hazardous Materials 421 (2022) 126779
Fig. 2. Chlorophyll a contents (% control) in Chlamydo­
monas reinhardtii exposed to (A) four types of shoe sole
leachates (slippers, trekking shoes, running shoes, and
sneakers) at 50% concentration and (B) low concentration
(3.125%, 6.25%, 12.5%, and 25%) of the sneaker leachate
for 3 days. Standard deviations of each mean are shown as
error bars. (A) Letters a, b, c, d, and e represent significant
differences compared with control, slippers, trekking shoes,
running shoes, and sneakers, respectively (p < 0.05). (B)
Asterisks represent significant differences between control
and exposure groups (p < 0.05).
acetate, methyl salicylate, p-xylene, and Zn in the sneaker leachate
induced severe effects on the test species. The findings were similar to
those of the PCA for individual analysis of metals or organic compounds;
the subsequent results are presented in Tables S6 and S7 and Fig. S7.
4. Discussions
Plastic products need various types of additives or chemicals during
their production process (Hahladakis et al., 2018). According to the FT/IR
peak, only slipper fragments showed the typical EVA spectrum (Adelnia
et al., 2015; Jaunich et al., 2016). Compared to previous studies, the soles
of trekking shoes showed a similar trend as styrene-butadiene-styrene
rubber (Baeta et al., 2009; Iancu et al., 2013; Luna et al., 2020). The
sole fragments of running shoes and sneakers showed FT/IR spectra that
were similar, not entirely same, to those of nitrile butadiene rubbers.
(Alhareb et al., 2017; Samantarai et al., 2017, 2019). Nevertheless, the
specific transmittance peak in their FT/IR spectra was observed at
1050–1085 cm− 1; hence, it is possible to have a single bond between
carbon and oxygen in their structure (Mecozzi et al., 2012).
L. Kim et al.
Fig. 3. Immobilization of Daphnia magna exposed to (A) four types of shoe sole
leachates (slippers, trekking shoes, running shoes, and sneakers) at 50% con­
centration and (B) low concentration (3.125%, 6.25%, 12.5%, and 25%) of the
sneaker leachate for 48 h. Standard deviations of each mean are shown as error
bars. (A) Letters a, b, c, d, and e represent significant differences compared with
control, slippers, trekking shoes, running shoes, and sneakers at 48 h, respec­
tively (p < 0.05). (B) Asterisks represent significant differences between control
and exposure groups and compounds represent significant differences in the
values between 24 and 48 h (p < 0.05).
Previous studies have observed that the plastic particles, such as tire
and shoe sole fragments, originating from terrestrial environments were
found in the aquatic environment (Hanik et al., 2019; Vogelsang et al.,
2019). High proportion of tire particles were detected in land, sewage
sludge, pond water, and sediment (Ingre-Khans et al., 2010; Rutgersson
et al., 2020; Vogelsang et al., 2019). Long exposure duration in the
aquatic environment could leach toxicants and consequently, Zn and
benzothiazole were commonly detected in their leachates (Capolupo
et al., 2020; Ingre-Khans et al., 2010; Khoshgoftarmanesh et al., 2013;
Kolomijeca et al., 2020; Sullivan, 2006). These chemicals generally
induce toxicity on human lung cells, bloodworms, and gut microbiota of
soil fauna (Ding et al., 2020; Gualtieri et al., 2005; Panko et al., 2013).
In this study, the negative effects of different shoe sole leachates on
three aquatic organisms were observed. The most sensitive trophic level
organism was the early-stage zebrafish. At the low concentration of
sneakers leachate, only fish larvae at 7-dpf showed adverse effects. In
some cases of bioassays for complex compound, the toxicity has been
shown against the trophic levels and their trends have been shown as
well as in this study (Kim et al., 2021; Kwak et al., 2021).
According to the PCA results, specific chemicals in the sneaker
leachate induced significant negative effects on algae (C. reinhardtii),
crustacean (D. magna), and fish larvae (D. rerio). The negative effects
6
Journal of Hazardous Materials 421 (2022) 126779
were highly associated with benzothiazole, carbon disulfide, ethyl ace­
tate, methyl salicylate, and p-xylene. Furthermore, Zn concentration had
significant negative effects on the organisms.
The overall concentrations of As and Zn were comparatively higher
than those of other heavy metal species in the four shoe sole leachates.
During rubber production, zinc oxide is used as a vulcanization catalyst
and As is used as a color-dye and preservative (Bridson et al., 2021;
Edwin and Pillai, 2004; Khoshgoftarmanesh et al., 2013). According to
the PCA results of this study, Zn was highly related to the aquatic
toxicity. According to the ecotoxicity database of the United States
Environmental Protection Agency (US EPA; US EPA, 2021), 1.01 μg/L to
8.8 μg/L of Zn affects green algae depending on the species sensitivity.
Furthermore, Zn exposure to C. reinhardtii for 6 h inhibited 50% of the
algal cells because of photoelectric effects at 248 μg/L (Govorunova
et al., 2000). Chronic toxicity in D. magna in the form of reduced
fertilization was observed at 59.2 μg/L of Zn (de Schamphelaere et al.,
2005). However, D. rerio embryo exposed to Zn did not exhibit any
abnormal effects up to 0.1 mg/L (Choi et al., 2016). However, the Zn
concentration detected in the sneaker leachate was 69.46 μg/L and each
test species was exposed to 50% of the leachate, having a Zn concen­
tration of 34.75 μg/L. The detected Zn concentration was significantly
lower than the concentration at which toxicity was observed and thus,
organic compounds were determined as major factors involved in
toxicity in aquatic organisms.
Organic compounds are used as additives for manufacturing plastic
products. Benzothiazole, which is used as a vulcanization additive for
rubber (Wik and Dave, 2006), has been detected in the leachate of tire
wear fragments (Wik and Dave, 2006; Wik, 2007). Mussels (Mytilus
galloprovincialis) exhibited a decrease in the lysosomal membrane sta­
bility and gamete fertilization rate (Capolupo et al., 2020). Furthermore,
rubber leachate with high benzothiazole concentration inhibited the
growth in marine algae, Skeletonema costatum (Capolupo et al., 2020).
Gill functions in fish exposed to benzothiazole decreased, while necrosis
and apoptosis was observed in zebrafish (Chen et al., 2017; Ma et al.,
2019), and sheepshead minnow larvae demonstrated reduced mobility
(Evans et al., 2000; Liao et al., 2018).
Carbon disulfide is a by-product obtained during the processing of
materials containing petroleum components or is used as a solvent for
resins, rubbers, and sulfur monochloride (Newhook et al., 2002).
However, it includes toxic sulfur, and is generated by the chemical
degradation of ecotoxic dithiocarbamate pesticide (Haendel et al.,
2004). Carbon disulfide caused notochord distortion during the early
developmental stages of zebrafish (Tilton et al., 2006, 2008; Van Leeu­
wen et al., 1985), and eventually delayed development and hatching.
Similarly, the embryos exposed to sneaker leachate in this study showed
a delay in development and hatching. Additionally, the degree of bent
spine increased with increasing concentration of the shoe sole leachates.
At exposures to 25% of sneaker leachate, unhatched individuals
exhibited a bent spine tail.
Furthermore, ethyl acetate is an additive during polyurethane and
rubber production and extensive research has been conducted for
assessing its toxicity. The toxicity of ethyl acetate is high for various
species of algae, crustacea, and fish and its relative toxicity is lower than
other materials (US Environmental Protection Agency, 2020). For
example, its 50% effective concentration (EC50) on Scenedesmus sub­
spicatus and 50% lethal concentration (LC50) on D. magna and Oryzias
latipes at an exposure of 48 h were 5.6 g/L, 590 mg/L, and 125 mg/L,
respectively (Kühn and Pattard, 1990; Slooff et al., 1983). Fish exposed
to high concentrations of ethyl acetate inhibit carbohydrate metabo­
lisms and induce low glycogen concentrations in liver (Gupta and Sri­
vastava, 1982). However, this study did not conduct biochemical
analysis and did not expose the test organisms to individual chemicals,
but to leachate chemicals as a whole; however, the ethyl acetate con­
centration exposed to the organisms is expected to be minimal.
Xylene is a conventional toxic solvent used for manufacturing rubber
products (Le Floch et al., 2012) and is marginally toxic to algae and
L. Kim et al. Journal of Hazardous Materials 421 (2022) 126779

Fig. 4. Cumulative negative effects and hatching rates of Danio rerio at 7 day post fertilization (dpf) exposed to (A and B) four types of shoe sole leachate (slippers,
trekking shoes, running shoes, and sneakers) at 50% concentration, and (C and D) low concentration (3.125%, 6.25%, 12.5%, and 25%) of the sneaker leachate for 7
days. (A) Letters a, b, c, d, and e represent significant differences compared with control, slippers, trekking shoes, running shoes, and sneakers at 48 h, respectively
(p < 0.05). (C) Asterisks represent significant differences between control and exposure groups (p < 0.05).

crustacea, and is comparatively less toxic to fish (Duan et al., 2017);
however, few abnormalities were observed in the early developmental
stage of fish. Xylene exhibits adverse harmful effects, such as mortality,
and development and hatching delays in amphibians and zebrafish
(Rajan and Malathi, 2014). Furthermore, p-xylene inhibited growth and
increased mortality in Xenopus laevis; additionally, xylene modified
histones that cause apoptosis in organisms (Gao et al., 2016).
The chlorophyll contents of C. reinhardtii exposed to trekking shoe
leachate increased marginally and trekking shoes were highly related to
2-ethylhexanol and dichloromethane according to PCA results. These
organic compounds could be used as a carbon source for algae, and
exhibit relatively low risk on aquatic organisms because of their high
degradation rates (Staples, 2001).
Each person consumes one pair of shoes every year with approxi­
mately 2 g of shoe sole fragments abraded during their life cycle
(Ingre-Khans et al., 2010). Ingre-Khans et al. (2010) conducted a
leaching test under extreme storm conditions. In natural environments,
water flow naturally dilutes leachates and thus, our findings may have
overestimated the toxicity associated with shoe sole leachate. Although
environmentally relevant particles abrade from terrestrial environ­
ments, fractions and contaminants adhere to their fragment surfaces as
carriers of toxicants (Issac and Kandasubramanian, 2021; Stoney et al.,
2016). Such contaminated particles are taken up by fish larvae or water
flea by chemical adsorption; thus, the toxicity of plastic fragments
originating from terrestrial environments is unpredictable (Canniff and
Hoang, 2018; Roch et al., 2020).
This study investigated the toxicity of chemicals and their relative
abundances along with organic compounds using GC-MS. Accurate
analysis was not conducted to detect the exact concentration of organic
chemicals. Additionally, sufficient research on the aquatic toxicity of
chemicals, such as (1-(4-propan-2-ylphenyl) ethenone and 2-(3-iso­
propylphenyl) propan-1-ol does not exist. Furthermore, the exact
toxicity of these chemicals is assumed to be unknown and differs from
the actual toxicity. Thus, the results of this study described the chemical
toxicity effects of shoe sole fragments at a screening level.
The results indicated that specific chemicals, which induced risks on
three aquatic organisms in this study, are commonly used as additives,
solvents, and generated as by-products during manufacturing of plastic
products (Bridson et al., 2021; Hahladakis et al., 2018). European
countries have been regulating the consumption of plasticizers, and risk
assessment of additives in plastic products is being evaluated in the in­
dustrial field by the European Chemicals Agency (European Chemicals
Agency, 2020; Wagner and Schlummer, 2020). Since the products used
could induce toxicity during their lifetime, regulation on additives used
during plastic manufacturing cannot be avoided. However, using plas­
ticizers cannot be regulated and restricted immediately since they need
to be pre-examined or screened through PCA, as conducted in this study,
to prioritize additives.

7
L. Kim et al. Journal of Hazardous Materials 421 (2022) 126779

Fig. 5. Negative effects of Danio rerio after exposure to low concentrations of the sneaker leachate for 7 days. Multiple abnormalities (e.g., bent spine, eye edema,
hemostasis, peritoneum edema, and lack of retina pigment) significantly increased in the exposure group with concentration increasing from 3.125%. Scale
bar = 1 mm.

Table 2
Results of principal component analysis (PCA) of metals, organic compounds,
and abnormalities in the test organisms (Chlamydomonas reinhardtii, Daphnia
magna, and Danio rerio) that were used to determine significant differences
among the different types of shoe sole leachates (slippers, trekking, running, and
sneakers) and control. The table presents the maximum two principal compo­
nents (PC) with eigenvalue > 1.
PCA PC1 PC2

Eigenvalue 14.08 4.03

Percentage of variance (%) 61.23 17.51
Cumulative percentage (%) 61.23 78.74
Eigenvector
1-(4-propan-2-ylphenyl)ethanone 0.983 0.119
2-(3-isopropylphenyl)propan-1-ol 0.983 0.119
2-Ethylhexanol -0.457 0.868
4,6-Dimethylheptan-2-one 0.407 -0.576
Acetophenone -0.336 0.430
Benzothiazole 0.983 0.119
Carbon disulfide 0.983 0.119
Decamethylcyclopentasiloxane 0.825 0.488
Dichloromethane -0.239 0.906 Fig. 6. Loading plots of the chemicals, which were detected in the four types of
Dodecamethylcyclohexasiloxane 0.805 -0.459 shoe sole leachates (slippers, trekking shoes, running shoes, and sneakers) after PCA
Ethyl Acetate 0.921 0.167 to predict the chemicals affecting the abnormalities on the three aquatic organisms
Hexamethylcyclotrisiloxane 0.951 -0.257 (Chlamydomonas reinhardtii, Daphnia magna, and Danio rerio). The x and y axes
methyl (Z)-N-hydroxybenzenecarboximidate -0.121 0.882 indicate the principal component 1 (PC1) and PC2, respectively. Loading factors
Methyl salicylate 0.991 0.005
related to the chemicals are represented as a black line. Relative negative
Octamethylcyclotetrasiloxane 0.796 0.507
effects of C. reinhardtii, D. magna, and D. rerio are shown as green, blue, and
p-Xylene 0.978 -0.041
Arsenic (As) 0.217 0.155 dark yellow lines, respectively. OC1 = 1-(4-propan-2-ylphenyl)etha
Chrome (Cr) 0.487 0.286 none, OC2 = 2-(3-isopropylphenyl)propan-1-ol, OC3 = 2-Ethylhexanol, OC4
Nickel (Ni) 0.127 -0.407 = 4,6-Dimethylheptan-2-one, OC5 = Acetophenone, OC6 = Benzothiazole,
Zinc (Zn) 0.917 0.034 OC7 = Carbon disulfide, OC8 = Decamethylcyclopentasiloxane, OC9 = Dic
C. reinhardtii growth inhibition (%) 0.990 -0.040 hloromethane, OC10 = Dodecamethylcyclohexasiloxane, OC11 = Ethylacetate,
D. magna abnormality (%) 0.981 0.126 OC12 = Hexamethylcyclotrisiloxane, OC13 = methyl(Z)-N-hydroxybenzenecarbox­
D. rerio abnormality (%) 0.962 0.087 imidate, OC14 = Methyl salicylate, OC15 = Octamethylcyclotetrasiloxane, and
OC16 = p-Xylene. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)

8
L. Kim et al.
5. Conclusions
In this study, the four types of shoe soles were fragmented, and the
leachates from the fragments were exposed to aquatic model organisms,
including green algae, water flea, and fish embryo. Consequently, all test
organisms exposed to sneaker leachate demonstrated severe negative
effects even though each organism belonged to different trophic levels.
The relative toxicity of fragment leachates at 50% concentration in test
species, and the results of chemical analyses of the four types of shoe sole
leachates were used as factors for PCA. Subsequently, the chemicals
detected only in the sneaker leachate were highly associated with the
abnormalities in the three test species. Benzothiazole, carbon disulfide,
and p-xylene, which are commonly used as additives and solvents and
are by-products of manufacturing of plastic products, were the most
significant components. According to PCA used for screening the
chemicals toxic to the environment, non-target analysis data can be
applied effectively. Future studies can include PCA to select the major
toxicants among various additives. Furthermore, PCA could assist in
prioritizing the regulation of chemicals that are commonly used as ad­
ditives during manufacturing of plastic products.
CRediT authorship contribution statement
L. Kim performed the experiments and drafted the first manuscript.
D. Kim, S.A. Kim, H. Kim, and T.-Y. Lee performed the experiments. The
project was planned and the manuscript was edited by Y.-J. An. The
project was supervised by Y.-J. An.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
This research was supported by the Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded by the
Ministry of Science, ICT and future planning (2020R1A2B5B02001734).
The authors thank the Korean Basic Science Institute for ICP-MS and GC-
MS analyses. The authors also thank S. An, Y. Kim, H. Lee, and S. Han for
their initial assistance.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.jhazmat.2021.126779.
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