The effects of tread rubber and road dust particles on stress, immunity and digestive biomarkers in the larvae of the mealworm Tenebrio molitor

Abstract

Airborne road and abrasive car parts particles penetrate into aquatic and soil environments, but also, settling on
vegetation along highways, enter trophic chains as a result of consumption by herbivorous invertebrates. The
effects of this exposure are poorly recognized. The study aimed to assess the toxicity of two traffic-connected
materials: tread rubber (TR) particles and environmentally relevant field-collected road dust (RD), to the Tenebrio molitor larvae under laboratory conditions using a set of protective (heat shock protein – HSP70, metallothionein – Mts levels), immunity (lysozyme – Lys, defensin – Def levels) and digestive (protease, amylase, and
celulase activities) biomarkers. ELISA assay was used for protein levels, while fluorimetric and spectrophotometric methods were used for enzymatic activity studies. RD and TR particles were characterized by SEM/EDS
techniques. The representative TR particle sizes were within the range of 31 µm and 274 µm. For the RD, the size
of the particles were 153–587 µm. Fat body HSP70 levels were, on average, twice lower in groups exposed to RD
particles. For fat body Mts, RD and TR caused the decrease while in the gut, the effect depended on the particle
type. Gut lysozyme levels increased for both particles while in fat body this effect was made by RD. Digestive
enzyme activity did not reflect exposure to TR and RD particles. RD induced changes in more experimental
groups than TR. This may be due to the greater complexity of their composition. Further studies focusing on
material type, concentration, exposure duration, and particle size are necessary to understand the effects of
traffic-connected material on terrestrial herbivores.

Full text

The effects of tread rubber and road dust particles on stress, immunity and
digestive biomarkers in the larvae of the mealworm Tenebrio molitor
Agnieszka Babczy´
nska
a,*
, Michalina Ba´
nska
a
, Katarzyna Mizera
a
, Monika Tarnawska
a
,
Maria Augustyniak
a
, Katarzyna Rozpędek
a
, Bartosz Łozowski
a
, Jolanta Bro˙
zek
a
,
Izabela Potocka
a
, Magdalena Kowalewska-Groszkowska
b
, Marta Sawadro
a
,
Agnieszka Czerwonka
a
, J¯
urat˙
e ˇ
Zaltauskait˙
e
c
, Gintar ˙
e Sujetovien ˙
e
c
, Piero Giulianini
d
,
Monia Renzi
d
, Anita Giglio
e
a
Institute of Biology, Biotechnology and Environmental Protection, Faculty of Natural Sciences, University of Silesia in Katowice Jagiello´
nska 28, Katowice 40-032,
Poland
b
Museum and Institute of Zoology, Polish Academy of Sciences, Wilcza 64, Warszawa 00-679, Poland
c
Department of Environmental Sciences, Vytautas Magnus University., Universiteto st. 10, Akademija, Kaunas LT-53361, Lithuania
d
Department of Life Sciences, University of Trieste, Via L. Giorgieri 5, Trieste 34127, Italy
e
Department of Biology, Ecology and Earth Sciences, Di.B.E.S.T., University of Calabria, Cosenza, Italy
ARTICLE INFO
Keywords:
Lysozyme
Defensin
HSP70
Metallothioneins
Digestive enzymes
Non-exhaust emission
ABSTRACT
Airborne road and abrasive car parts particles penetrate into aquatic and soil environments, but also, settling on
vegetation along highways, enter trophic chains as a result of consumption by herbivorous invertebrates. The
effects of this exposure are poorly recognized. The study aimed to assess the toxicity of two trafc-connected
materials: tread rubber (TR) particles and environmentally relevant eld-collected road dust (RD), to the Ten-
ebrio molitor larvae under laboratory conditions using a set of protective (heat shock protein – HSP70, metal-
lothionein – Mts levels), immunity (lysozyme – Lys, defensin – Def levels) and digestive (protease, amylase, and
celulase activities) biomarkers. ELISA assay was used for protein levels, while uorimetric and spectrophoto-
metric methods were used for enzymatic activity studies. RD and TR particles were characterized by SEM/EDS
techniques. The representative TR particle sizes were within the range of 31 µm and 274 µm. For the RD, the size
of the particles were 153–587 µm. Fat body HSP70 levels were, on average, twice lower in groups exposed to RD
particles. For fat body Mts, RD and TR caused the decrease while in the gut, the effect depended on the particle
type. Gut lysozyme levels increased for both particles while in fat body this effect was made by RD. Digestive
enzyme activity did not reect exposure to TR and RD particles. RD induced changes in more experimental
groups than TR. This may be due to the greater complexity of their composition. Further studies focusing on
material type, concentration, exposure duration, and particle size are necessary to understand the effects of
trafc-connected material on terrestrial herbivores.
1. Introduction
The unstoppable development of transport infrastructure on a global
scale has led to a spectacular number of cars being used, which in 2020
equaled 1.590.276.000 (International Organization of Motor Vehicle
Manufacturers, 2020). Based on these statistics, since the year 2015, the
number increased by approximately 4 %. It is followed by the same
growth rate regarding European motorway length calculated based on
available data within the same period (Eurostat, 2024). Both trends have
their environmental side effects. Exploiting the roads and cars generates
tire and road wear particles (TRWP), considered contaminants of
emerging concern (Garrard et al., 2022). Due to analogous properties
and origin, TRWP are often included in a broad conception of nano- and
microplastics, which signicantly contribute to the global load of plastic
pollution in the environment (Sommer et al., 2018; Mitrano et al., 2020;
Sun et al., 2022, Giechaskiel et al., 2024). On the European scale, this
source contributes 42 % out of over 14 kilotons to the total plastic
pollution that European rivers carry to the seas they ow into (Siegfried
* Corresponding author.
E-mail address: agnieszka.babczynska@us.edu.pl (A. Babczy´
nska).
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety
journal homepage: www.elsevier.com/locate/ecoenv
https://doi.org/10.1016/j.ecoenv.2025.118289
Received 7 February 2025; Received in revised form 6 May 2025; Accepted 6 May 2025
Ecotoxicology and Environmental Safety 298 (2025) 118289
Available online 8 May 2025
0147-6513/© 2025 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ).

et al., 2017). According to the analysis by Sommer et al. (2018), TRWP
results mainly from the abrasion of tires, road surfaces, brake parts, and
the concrete of road infrastructure, and it also contains particles from
the land along the highways. Thus, the dust contains particles of natural
and synthetic rubber (polymers of carbon and hydrogen), llers con-
taining silica and chalk, and softeners of various compositions.
Energy-dispersive X-ray spectroscopy (EDS) studies revealed heavy
metals, including Ba, Cu, Mo, and Zn (Sommer et al., 2018). Physical
properties such as particle size, shape, or density are also signicant for
TRWP environmental toxicity. The size ranges between 1 and 1000 µm,
and density is within the range of 1.3–2.2 g/cm
3
while the shape, highly
irregular, reects their aggregation, breakup, weathering, and other
interaction effects, including additional pollution with metals
(Kayhanian, 2012; ˇ
Zibret et al., 2013; Sommer et al., 2018;
Baensch-Baltruschat et al., 2020; Teran et al., 2020; Wagner et al.,
2022).
Airborne TRWP concentrations, according to the measurements by
Panko et al. (2013) conducted on three continents, ranged from 0.05 to
0.70
μ
g m
−3
. In the pool of airborne PM10 particles, TRWP constitutes
0.84 %, while Amato et al. (2014) identied the maximum level of
TRWP equal to 13.9 µg m
−3
, which constitutes 34 % of total PM10
particles. Concerning PM2.5, the values are 14.8 µg m
−3
and 40 %.
The TRWP airborne particles settle and are washed out, thus entering
the soil and aquatic environments. In aquatic environments these par-
ticles concentrations range from 0.5 and 1990 mg L
−1
(Wik and Dave,
2009; Rauert et al., 2022a,b), while in soil ecosystems TRWP concen-
tration may reach up to 1300 µg kg
−1
depending on the distance to the
road and sampling depth (Baensch-Baltruschat et al., 2020; Mengistu
et al., 2022; Müller et al., 2022; Thomas et al., 2023). Studies on TRWP
toxicity tests towards soil fauna reveal signicant side effects of expo-
sure to trafc particles at various biological levels, including survival,
enzymatic activity (Selonen et al., 2021), body length (Kim et al., 2022),
reproductive potential (Kim et al., 2021), immune cellular and molec-
ular parameters (Dolar, 2022a,b) or gut microbiota disturbance (Ding
et al., 2020). In response to tire rubber, the upregulation of defense and
immunity mechanisms, including genes coding for heat shock proteins,
decrease in protein and energy resources, and changes in immune de-
fense mechanisms was detected previously in various aquatic (midge
larvae) and soil (woodlice) invertebrates (Carrasco-Navarro et al., 2021;
Garrard et al., 2022; Dolar et al., 2022a). On the other hand, possible
downregulation of defensive parameters may result from inhibition of
enzymatic activity due to interaction with material particles or products
of their potential transformation by detoxication, neutralization or
digestion (Lackmann et al., 2022). Another reason for the down-
regulation of biomarkers could be changes at the molecular level, which
is not impossible given the results of Meland et al. (2019). The authors
found signicantly increased levels of DNA damage in dragon y larvae
exposed to pollution in highway sediment pond. The effects get even
more complex when considering concentration-dependent effects
(Zimmermann et al., 2020; Babczy´
nska et al., 2023) or when the parti-
cles interact with other chemicals, such as, e.g., pesticides (Dolar et al.,
2021; Selonen et al., 2023). The effects of TRWP consumption and the
biomarkers efciently describing TRWP effects in terrestrial in-
vertebrates inhabiting other than soil ecological niches have not been
studied sufciently. Harmful effects, similar to those described for soil
fauna, can be expected if studying the consequences of consumption of
plant leaves covered with airborne TRWP settled on the side road
vegetation. Herbivorous invertebrates, largely insects, often utilize this
food source, mainly in their larval stage. Insect larvae are usually
voracious, gathering energy resources for metamorphosis and/or sexual
maturation (Llandres et al., 2015). Optimal achievement of the mature
stage guarantees population stability and, in a broader sense, ecosystem
biodiversity. Energy deciencies at the individual level make develop-
ment suboptimal and prolonged, and may cause an end effect of
malnutrition demonstrating itself at the level of life history parameters
(Babczy´
nska et al., 2012; Augustyniak et al., 2020; Fujii et al., 2020).
This kind of effects, evoked by TRWP, was found by Garrard et al.,
(2022) in the clam Scrobicularia plana and the nereid Hediste diversicolor,
exposed to TRWP in the sediment. The individuals consuming the par-
ticles were characterized by signicantly lower protein concentration
(both species) and energy contents (H. diversicolor). Similar effect
described as the food assimilation rate was found for Gammarus fossarum
exposed to the polyamide microparticles in food (Blarer and
Burkhardt-Holm, 2016). Both examples may suggest ineffective uti-
lisation of food supplemented with various kinds of polymer
microparticles.
Tenebrio molitor, due to its features as a good model species (well-
recognized biology, convenience for laboratory breeding, high fecun-
dity, relatively short life cycle, and relatively big body size) is commonly
used for laboratory tests of various physicochemical and biological
stressors, including insecticides, heat, or pathogens (Adamski et al.,
2019; Pedersen et al., 2020; Plata-Rueda et al., 2021; Lozoya-P´
erez
et al., 2021; Herren et al., 2023). Of specic interest, from this study
perspective, are the reports on microplastic effects on T. molitor exposed
to environmentally relevant concentrations of the particles. In general,
survivorship and developmental parameters as well as energy-related
biomarkers seem to be slightly affected or unaffected in lower particle
concentration tests (Jemec Kokalj et al., 2022; 2024a,b). On the other
hand, T. molitor is recognized as a plastivorous insect, able to survive on
a sole synthetic polymer diet during 1-month exposure with some in-
dications of functional changes in intestine (Wu, 2019; Peng and Wang,
2024).
In the present project, we tested the toxicity of two trafc-connected
materials: tread rubber (TR) and road dust collected near high-trafc
viaduct (RD) to T. molitor larvae under laboratory conditions. The oral
exposure effects assessed using biomarkers connected with various
metabolic and cellular processes. These included stress (heat shock
proteins, HSP70; metallothioneines Mts), immunity (lysozyme, Lys;
defensins, Def) markers which are conservative across animal kingdom
(Bachali et al., 2002; Robert et al., 2004; Capdevila, Atrian, 2011; Zhu,
Gao, 2013). In addition, digestive enzyme (amylase, proteases, cellu-
lases) activities were tested. All biomarkers were related to the type of
the material and its concentrations considering tissue (fat body, gut)
specicity. The study aimed to test the following hypotheses:
H1.0. The exposure to trafc material does not induce any changes in
the levels of parameters of protective reactions (HSP70, Mts, Lys, Def)
that may suggest stress and/or potential threat, irrespectively of their
concentration in food.
H1.1. Exposure to TR or RD causes the initiation and/or intensi-
cation of the defensive process levels against environmental stressors.
Similar upregulation refers to digestive enzyme activity, assuming that
the nutritive value per unit of mass of food containing non-nutritive
ingredients (pollutants) may be lower than that of food with an
optimal composition.
H1.2. The effects of TR or RD exposure manifest themselves in the
form of reduced values of the biomarkers tested.
H1.3. TR and RD effects depend on the type of the material and
exposure intensity (here: material concentration). In this hypothesis the
possible hormetic effect cannot be neglected,
2. Materials and methods
2.1. Model insect
The yellow mealworm beetle, T. molitor L., from the laboratory
breeding population was used as a model species. The individuals tested
in the present study have never been pre-exposed or pre-selected to any
kind of stressor in the previous generations. Therefore their response
depends solely on the new factor (RD and TR particles) added to their
diet. This way the elimination of uncertainties that would apply to in-
sects collected in the eld for the experimental purposes was possible.
Under optimal laboratory conditions (25–30 ◦C), humidity 20–45 %,
A. Babczy´
nska et al.
Ecotoxicology and Environmental Safety 298 (2025) 118289
2

their life cycle lasts 75–90 days, with a larval period, consisting of 9–23
moltings, lasting, on average, 57 days, during which the larvae grow
from 1.25 mm to over 25 mm of the length. Then, after the pupal period
of 6–20 days, imagoes appear, and they start reproduction on the
approximately 3rd day from emerging. Imagoes die within, approxi-
mately, 30 days (Ribeiro et al., 2018). The insects in the basal laboratory
population were kept under constant controlled conditions (28 ±1 ◦C;
20 %–45 % RH photoperiod: L:D 12:12). The food consisted of crushed
oat akes up to 2 mm fragments, powdered yeast, powdered dry food for
dogs and cats, and water (93 g, 5 g, 2 g, and 30 mL, respectively (Gałęcki,
2021). The ingredients were mixed thoroughly and then dried. After
drying, the medium was crushed again to get food fragments no bigger
than 2 mm. The insects were kept in ca. 3 L insectaria (up to 200 in-
dividuals per container) containing the food mixture in excess (ad libi-
tum), and falcon tube clogged with cotton wool, containing tap water as
the source of humidity.
2.2. Control and experimental rearing conditions
Insects from the experimental groups were maintained under the
same microclimatic conditions as the basal population. The food for in
the control group, the food was the same as for the basal population, For
the experimental groups, RD or TR (random mixture of two brands of
tire, see below) dust was added to the medium prepared as for the
control group so that the materials constituted 1 %, 2 %, and 4 % of dry
weight (w/w) of the medium, respectively. Material concentrations
were selected based on Selonen et al. (2021) and Sheng et al. (2021).
Samples of TR supplemented food were subjected for SEM analyses.
Early instar larvae of the length of 5 mm were randomly divided into the
200 mL insectaria, lled with control and experimental medium and
equipped with cotton-clogged glass tubes with water, 20 individuals in
each, and left undisturbed until their last larval instar (on average,
43 ±4 days). The last instar individuals were recognized by size
(approximately 2.5 cm) and behavior signaling the approaching
pre-pupal stage (limited mobility and reactivity, lower foraging in-
tensity). During that time, the rearing conditions were monitored and
water was exchanged weekly. To verify the particle transfer through the
alimentary tract, randomly selected individuals were placed individu-
ally in empty Eppendorf tubes and after 24 h the faeces were collected
for SEM analyses and the individuals were moved to post-experimental
containers till trheir natural death. Ethical approval was not required for
this experiment.
2.3. RD and TR particles acquisition and description
RD was collected by sweeping the dust under the heavy trafc
viaduct where the accumulated dust was not blown away by the wind,
on the national road N◦86 in Tychy (Poland, 86, 50.165248,
19.016312). TR was obtained by the abrasion of the tread layers of tires
using a bench grinder in laboratory workshop conditions. Tires of two
anonymized leading brands, “X”, produced in 2003, and “Y”, produced
in 2015, were used. TR and r RD were described using SEM and EDS
techniques (Spanheimer and Katrakova-Krüger, 2022; Torreggiani et al.,
2024). TR as well as food and faeces samples were analyzed with
scanning electron microscope S-3400N by HITACHI using a BSE detector
in variable vacuum conditions. Qualitative and quantitative tests of the
chemical composition were performed using an EDS Element X-ray en-
ergy spectrometer and analyzed by APEX™ software using the eZAF
correction method and scanning electron microscope Phenom XL
(Phenom-World BV the Netherlands) using a backscattered electron
detector (BSD) at an electrical voltage of 10 kilovolts and a high vac-
uum). RD was analyzed using a Hitachi SU8010 ultra-high resolution
eld emission scanning electron microscope (Hitachi High-Tech Cor-
poration, Tokyo, Japan) coupled with the UltraDry EDS detector and
NORAN System 7 X-ray microanalysis system (Thermo Fisher Scientic
Inc.). A portion of the road dust sample was sprinkled onto double sided
carbon tape mounted on a SEM stub and coated with a thin layer of gold
(CCU-010 HV compact coating unit, Safematic GmbH, Zizers,
Switzerland). Images of RD particles were collected with the secondary
electron (SE) detector at an accelerating voltage of 5 kV. EDS data were
acquired using a NSS 3 software (Version 3.2, Thermo Fisher Scientic
Inc.) at an accelerating voltage of 15 kV and an acquisition time of 90 s
and 300 s (for spectra and maps, respectively). Spectra were collected
from the central part of randomly selected particles in the Point and
Shoot mode. Standardless quantitative analysis with the Proza (Phi-R-
ho-Z) matrix correction algorithm was used to determine the weight
percentage (weight %) of each element detected. To determine the size
of RD particles, at least 25 particles of each group were randomly
selected from the captured SEM images and measured along their long
axis. The measurements were made manually using the Hitachi SU8010
PC-SEM software (Version 2.7).
2.4. Sample preparation
Biochemical and physiological biomarkers used in this study were
measured in midgut and fat body samples. To collect the tissues, in-
dividuals were anaesthetized on ice until they got motionless, then the
cephalic and anal endings of the body were cut off, and midgut was
gently taken out. After removing the midgut, fat body was extracted by
gently pressing the body shells. Appearing fat body was gradually
collected with forceps. For one sample, one midgut or fat body from one
individual was used. Total of 10 individuals of each experimental groups
were used for preparation of samples, one per sample. Half of the in-
dividuals (5) were used for preparing samples for stress and immune
parameters, using one midgut or one fat body per samples. All four
tested proteins were detected using homogenates of 1 sample (the vol-
ume and protein concentration was sufcient for detection of 4 anti-
gens). The remaining individuals were used for dissection of midguts
(one per sample) for enzymatic activity assays. Again, 1 sample was
enough to measure the activity of 3 enzymes. Therefore, in total, 40
individuals were used for the tests. Tissues were placed in Eppendorf
tubes, with 300 µl 0.05 M PBS buffer (Phosphate buffer saline, Sigma-
Aldrich), pH 7,4, one per sample. Tissues were then homogenized on
ice with a hand homogenizer Tissue Mini Grinder (EURx, Poland), and
centrifuged at 4 ◦C, 10 min at 15 000 g. Supernatants were collected and
stored at −70 ◦C until measurements. Prior to the measurements, the
samples were let to defrost and were diluted adequately for the mea-
surements. For enzymatic activity, the total protein concentration did
not exceed 2 mg •mL
−1
, and for immunodetection tests, it was set at
1 mg •mL
−1
. The amount of the diluted samples was enough for per-
forming all the biomarker measurements.
2.5. Digestive enzyme activity measurements
2.5.1. The digestive enzyme activity measurements were performed in the
midgut samples, Protease activity
Protease activity was determined using the Protease Activity Assay
kit (Abcam, ab111750), according to the manufacturer’s protocol, using
50 µL of a diluted sample. The test uses casein labeled with uorescein
isothiocyanate (FITC) as a substrate for protease. Fluorescence is
measured at Ex/Em=485/530 nm. Measurements were made using the
spectrouorimeter HITACHI F-7. Protease activity was expressed as
nmol casein/min/
μ
g protein, referring to the standard curves prepared
based on the uorescein isothiocyanate (FITC) -labeled casein solutions
of known concentrations (Supplementary materials S4). Samples
without enzyme source were used as blanks (homogenate was replaced
with homogenization buffer).
2.5.2. Cellulase activity
Cellulase activity was determined using the Cellulase Activity Assay
kit (Fluorometric) (Abcam, ab189817), according to the manufacturer’s
protocol, using 50 µL of a diluted sample. After cleavage of the substrate
A. Babczy´
nska et al.
Ecotoxicology and Environmental Safety 298 (2025) 118289
3

by cellulases, the resorun compound present in the sample is released,
and the uorescence is readily detectable at Ex/Em =550/595 nm.
Fluorescence changes were measured using spectrouorimeter HITACHI
F-7. Cellulase activity is expressed as nmol resorun/min/
μ
g protein,
referring to the standard curves prepared based on the resorun solu-
tions of known concentrations (Supplementary materials S5) Samples
without enzyme source were used as blanks (homogenate was replaced
with homogenization buffer).
2.5.3. Amylase activity
Midgut amylase activity was determined using a modied procedure
described by Nakonieczny et al. (2006), which is based on the use of
α
-amylase enzymatic reaction products (i.e., dextrins and the reducing
disaccharide maltose), which reduce 3,5- dinitrosalicylic acid at 100 ◦C,
in alkaline conditions, and produce a stable orange color reaction.
Absorbance, directly related to
α
-amylase activity, was measured based
on 20 µL of a diluted sample at the wavelength of 540 nm using the
multimode microplate reader (TECAN Spark®, Tecan Group Ltd.,
Switzerland).
α
-amylase activity was expressed as
μ
mol maltose/min/
μ
g
protein, referring to the standard curves prepared based on the maltose
solutions of known concentrations (Supplementary materials S6). Sam-
ples without enzyme source were used as blanks (homogenate was
replaced with homogenization buffer).
2.6. Immunodetection of lysozyme, defensins, heat shock proteins 70 and
metallothioneins
Lys, Def, HSP70, and Mts levels were detected using Enzyme-Linked
Immunosorbent Assay ELISA, according to standard protocol. 100
μ
L of
each homogenate of both tissues dissolved to contain the same protein
amount, was placed in a 96-well microplate and left overnight at 4 ◦C to
coat the walls and bottom of the wells. After the incubation, the sample
was replaced with 5 % bovine serum albumin (BSA, protein content
>95 %, Fluka) solution and incubated 1 hour at 37 ◦C to block uncoated
well areas. After the removal of blocking solutions, the samples were
incubated rst with antigen-specic primary antibody solutions for
2 hours at 37 ◦C and then with solutions of adequate secondary antibody
conjugated with alkaline phosphatase (AP) for 1 hour at 37 ◦C. The
following primary antibodies for Lys, Def, HSP70 and Mts were used,
respectively: rabbit anti-lysozyme primary antibody (Abcam), rabbit
anti-B-defensin 3 antibody (Sigma–Aldrich), mouse anti-heat shock
protein monoclonal antibody (Sigma–Aldrich) and mouse metal-
lothionein monoclonal antibody (Stressgen), while secondary anti-
rabbit and ant-mouse antibodies were, respectively: AP conjugated
goat anti-rabbit IgG and AP conjugated goat anti-mouse IgG (both:
Sigma–Aldrich), all diluted in the 1:1000 ratio. Finally, color reaction
was initiated by adding a 10 mM solution of p-nitrophenyl phosphate
(pNpp, Phosphatase substrate, Sigma-Aldrich, St. Louis, MO, USA) in
diethanolamine buffer pH 9.5. (Diethanolamine Substrate Buffer 5x
concentrate; Thermo Fisher Scientic, Rockford, USA). After 30 min.
incubation at room temperature, the absorbance was measured photo-
metrically at 405 nm using the multimode microplate reader (TECAN
Spark®, Tecan Group Ltd., Switzerland), based on the linear relationship
of the absorbance of a solution on the concentration of the color reaction
product in that solution Supplementary materials S7-S9). Between each
step, the wells were washed 4 times (after blocking solution – 2 times)
with PBS buffer containing 0.1 M Tween20. Samples without protein
source were used as blanks (homogenate was replaced with homogeni-
zation buffer).
2.7. Protein concentration
The total protein concentration in the samples was determined
photometrically, using the dye-binding method of Bradford (1976) with
bovine serum albumin (protein content 95 %, Fluka) as the standard, at
595 nm using the multimode microplate reader (TECAN Spark®, Tecan
Group Ltd., Switzerland).
2.8. Statistical analyses
Statistical analyses were performed for N =5 in each experimental
group (5 samples prepared of a midgut or fat body of.one individual
within one group). Cluster analysis was performed using the hierarchical
tree method with Ward’s method estimating the Euclidean linkage
Fig. 1. Number frequency histograms showing particle size distribution of TR and RD rubber and non-rubber particles.
A. Babczy´
nska et al.
Ecotoxicology and Environmental Safety 298 (2025) 118289
4

distance. The statistical analyses and their graphical presentations were
prepared using TIBCO Software Inc. (2017) STATISTICA (data analysis
software system). Due to the nature of the data, the rst, second (me-
dian), and third quartiles were used as descriptive statistics. Non-
parametric one-way PERMANOVA with 9999 permutations, based on
Euclidean distance and pairwise post hoc PERMANOVA F analysis
(p <0.05) were conducted using PAST 4.03 (Hammer et al., 2001).
3. Results
3.1. TR and RD particle characteristics
TR particles have relatively regular shape in each dimension. They
tend to aggregate if not mixed with any other material (Fig. S1 I). The
representative particle sizes were within the range of 31 µm and 274 µm.
For the RD, two groups of particles were distinguished: rubber-like ones,
attributed to the tire wear particles, and non-rubber-like ones, that
possibly origin from road or non-tire car parts abrasion including,
among others, organic matter fragments, ash or soil particles etc.
(Fig. S1 II). The size of the particles were, respectively, 183 µm - 565 µm
and 153 µm - 587 µm. The particle size distribution was demonstrated in
Fig. 1. Chemical composition of the two tire brands was similar with the
higher contents of C, O, and Si, while the element contents of RD were
highly diversied, reecting the different origins of road dust particles
(Table 1, supplementary materials S2 and S3). Moreover, the SEM an-
alyses demonstrate that TR particles were detectable both in food and
faeces of the T. molitor larvae (S1 III-IV).
3.2. Stress marker levels
In this study, two stress biomarkers were tested: heat shock proteins
70 (HSP70) and metallothionein (Mts) levels. Both of them revealed
trafc material concentration dependence. The fat body HSP70 level
decreased signicantly in the RD-exposed groups compared to the
control, remaining unchanged in the TR-exposed groups (Fig. 2). Met-
allothionein levels in the RD-and TR-exposed groups, when signicantly
different from the control, were lower both in fat body and gut samples.
The only exception was found for the group exposed to 1 % RD, where
Mts level was measured in the gut samples (Fig. 3).
Table 1
Content of elements (% weight) in the materials used in the experiment.
element „X” TR „Y” TR RD*
C K 50.2 57,2 1.5 – 39.8
O K 33.8 25.4 8.7 – 51.1
Si K 11 11.8 1.6 – 63.2
Al K 0.6 0.5 0.7 – 9.8
S K 1.6 1.5 0.8 – 2.3
Ca K 1.1 1.6 0.2 – 13.1
Fe K 0.7 0.5 0.6 – 5.7
Zn K 1.1 1.5 
N K 2.4 – 7.9
Na K 0.7 – 2.3
Mg K 0.3 – 4.3
K K 0.3 – 1.7
Cl K 0.4 – 1.6
Ba K 0.7
Ti K 0.1 – 0.4
*
RD: due to high heterogeneity of the material, depending on the type of the
particle analyzed, the range of values was given. Detail data can be found in
Supplementary le 2
Fig. 2. HSP 70 level (median (dashed line); boxes: 1st and 3dr quartiles;, circles: individual values) in fat body and gut samples of T. molitor larvae from the control
(ctr) and experimental groups exposed to 1 %, 2 %, 4 % of road dust (dust) and tread rubber dust (rubber). Various letters (a, b) denote statistically signicant
differences between experimental groups (post hoc PERMANOVA F analysis p ≤0.05).
A. Babczy´
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Ecotoxicology and Environmental Safety 298 (2025) 118289
5

3.3. Immunity marker levels
To the immunity markers, defensin (Def) and lysozyme (Lys) levels
were included. In general, Def level was consistent in all tissues and
materials apart from the TR 2 % group where, in gut samples, it was
signicantly higher than in the control group (Fig. 4). Lysozyme levels in
the RD-and TR-exposed groups, when signicantly different from the
control, were higher both in fat body and gut samples. It was found in
the 4 % RD group for fat body and gut and in 2 % TR rubber for gut
samples (Fig. 5).
3.4. Digestive enzyme activity
In the group of digestive enzymes, amylase, cellulase, and protease
were included. No signicant concentration-dependent differences were
found among this group of markers for any of the materials tested
(Fig. 6).
3.5. Cluster analysis
Cluster analysis of the protective parameter levels in the fat body
revealed a clear separation between groups exposed to the two different
types of particles. Additionally, within the cluster of tread rubber (TR)
groups, the 2 % exposure group was distinct from the other experi-
mental concentrations. Similarly, for road dust (RD), the groups exposed
to 1 % and 2 % concentrations also separated from the remaining groups
(Fig. 7A, main graph). In contrast, for protective parameters measured in
the gut, the separation based on particle type was less distinct (Fig. 7B,
main graph). Cluster analysis of digestive enzyme activities, however,
showed grouping primarily according to the type of particulate material
(Fig. 7C). It is worth noting that when all parameters are pooled
together, clustering based on median values clearly distinguishes groups
exposed to TR particles (Fig. 7A, B, C, inserted graphs).
4. Discussion
The analysis of SEM images shows that the tested particles mixed
with food are ingested by the tested insects and then excreted together
with other undigested food residues. It can therefore be assumed that the
insect had direct contact with this type of food contamination, which
could potentially translate into changes in the level of stress, immunity
and digestive biomarkers. The analysis of the obtained results does not,
however, allow for an unambiguous indication of the mode of action of
TR and RD on the organisms of T. monitor larvae. They only enable us to
observe certain emerging trends, according to which the exposure of the
larval stages of the beetle T. molitor to trafc-connected dust caused
several signicant changes in the levels of markers selected for this
investigation. Three various tendencies were noticed. The levels of
protective proteins in the exposed groups, were generally lower than the
control. Unlike HSP70 and Mts, immunity marker levels tended to in-
crease. Digestive enzyme activities were not signicantly inuenced by
the stressing factors regarding of their concentration in food. The
different directions of changes may be connected with the roles of the
groups of proteins in the organism. The protective molecules bind metals
(Mts), participate in defensive red-ox processes (both), or play their role
as chaperon proteins (HSP70) (Roesijadi, 1996; Ruttkay-Nedecky et al.,
2013; Rosenzweig et al., 2019; Zhang et al., 2022). Usually, protective
protein levels are up-regulated under stress conditions (Farahani et al.,
2020; Fisker al., 2013; Zhou et al., 2020). In the present project, the road
connected material caused, in general, a decrease in stress markers. In a
few cases, the change was statistically signicant. Some studies specu-
latively refer to Zn as a tire component that may possibly cause Mts level
Fig. 3. Metallothionein (Mts) level (median; boxes:1st and 3dr quartiles; whiskers: Min-Max) in fat body and gut samples of T. molitor larvae from the control (ctr)
and experimental groups exposed to 1 %, 2 %, 4 % of road dust (dust) and tread rubber dust (rubber). Various letters (a, b, c) denote statistically signicant dif-
ferences between experimental groups. p ≤0.05 (post hoc PERMANOVA F analysis p ≤0.05).
A. Babczy´
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Ecotoxicology and Environmental Safety 298 (2025) 118289
6

increase in reaction to TRWP (Masset et al., 2021). Trace amounts of Zn
(and other metals) in this study may be too low to induce Mts biosyn-
thesis. An interesting explanation for the lack of protective protein
synthesis can be found in Martyniuk et al. (2022). Although a simple
translation of these two situations is not appropriate for two reasons: (i)
differences in the material to which the model animals were exposed and
(ii) differences in the environments and routes of exposure, the argu-
ment may be useful in interpreting the present results. The quoted au-
thors exposed Unio tumidus of clean or polluted sites to microplastic for
14 days. Mts levels in the individuals from the unpolluted site were
lower than in the control group, which was opposite to the individuals
from the polluted area. It indicates that microplastic may have a sup-
pressive effect on Mts in non-resistant individuals (Martyniuk et al.,
2022). It may also apply to the present study where T. molitor exposed to
TRWP and rubber dust, had no history of pollution exposure. Data for
HSP70 in relation to trafc-related dust for invertebrates are limited to
the report on gene upregulation in the aquatic insect Chironomus riparius
larvae (Carrasco-Navarro et al., 2021). The authors connect it with
metals present in the tire material, which exert their pressure, in general,
in similar manner, irrespectively of the form of administration and the
dispersion in the environment. Jaikumar et al. (2021) emphasize that
the conclusion should consider also the material the animals are exposed
to and the specicity of sampled tissue.
Like stress proteins, the levels of immunity markers in response to the
TRWP exposure of terrestrial invertebrates are poorly represented in the
literature. In our study, immune marker levels, in general, showed an
upward trend. It may suggest an immunostimulatory effect of this kind
of particle on the beetle organisms. Results reported by Dolar et al.
(2021) after the exposure of Porcellio scaber to tire particles for 21 days
indicated a signicant increase in total hemocyte count (THC; 0.05 % of
the tire dust in soil vs control group). Notably, higher rubber dust
concentrations did not cause any signicant THC changes. Also, another
immunity marker, phenoloxidase-like activity, remained unchanged. In
another study, Dolar et al. (2022a) noted a signicant upregulation of
some immune-related genes while downregulation of others in hemo-
cytes and hepatopancreas, with more pronounced differences in the
former tissue in the experiment with the same model species exposed to
tire rubber particles in the concentration of 1.5 % in soil for 4- and
14-day exposure.
Unlike protective and immunity markers, digestive enzyme activity
in response to the exposure to both materials remained unchanged.
Since the midgut is the rst barrier against xenobiotics that enter the
body through the alimentary tract, modication in its functions can be
expected. It is generally known that polymer microparticles cause
damage in the gut tissue in a particle size-dependent manner (Lei et al.,
2018; Khosrovyan et al., 2020; Hsieh et al., 2021; Li et al., 2021). Unlike
gut damage, the activity of digestive enzymes is rarely studied. An
interesting report can be found in Trestrail et al. (2021) in their exper-
iment on the mussel M. galloprovincialis exposed to polyethylene (PE) or
polystyrene (PS) in lower (1000 particles L-1) or higher (5000 particles
L-1) concentration for 7 days. The mussel amylase activity decreased,
and cellulase activity increased signicantly in the exposure to the lower
PS concentration, while the remaining concentrations and polymer type
did not cause any changes. Protease activity increased only in the higher
PS concentration group (Trestrail et al., 2021). The authors attribute
these effects, in general, to the toxicity of polymer leachates, tissue
damage, or specic polymer-type reactions.
The lack of specic reaction in the present study may be explained
directly by high intragroup variability which, in turn, may be induced by
specic reaction of individuals, reecting their unique biological char-
acteristics and adjustability to external factors. One of the experimental
reason for this situation is the exposure duration. In this project it
Fig. 4. Defensin (Def) level (median; boxes: 1st and 3dr quartiles; whiskers: Min-Max) in fat body and gut samples of T. molitor larvae from the control (ctr) and
experimental groups exposed to 1 %, 2 %, 4 % of road dust (dust) and tread rubber dust (rubber). Various letters (a, b) denote statistically signicant differences
between experimental groups (post hoc PERMANOVA F analysis p ≤0.05).
A. Babczy´
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Ecotoxicology and Environmental Safety 298 (2025) 118289
7

covered entire larval period of the insect. In this study, the insects were
exposed to RD and TR particles for, on average, 43 days. This time may
be long enough for the individuals to adjust to the new conditions. High
intragroup variability usually characterizes the rst generation of ani-
mals exposed to a new selection factor. It allows us to indicate the
tolerance range of the population as a whole, whose survival often de-
pends on the potential plasticity of individuals, which are the source of
extreme values in measurements. This is the situation we are dealing
with in this project. Laboratory studies suggest that it is phenotypic
plasticity (Gutierrez, 2020; Badejo et al., 2021) that may be helpful in
the tolerance of chronic intoxication by a substance within one gener-
ation range (Augustyniak et al., 2016). Also, this phenomenon was
regarded as a possible basis for tolerance to stressing factors, as we
discussed in our previous studies where the inherited genetic basis for
adaptation had to be excluded (Tarnawska et al., 2019; Babczy´
nska
et al., 2020). Phenotypic plasticity-connected energy-saving mecha-
nisms may universally explain heterogeneity in the levels of biomarkers
studied in this project because exposure duration might be long enough
to induce possible compensatory mechanisms. An interesting question of
the development of tolerance of dust exposure in time was partly
answered by Dolar et al. (2022b), who exposed P. scaber individuals to
tire dust and assessed THC and hemocyte viability after 1, 2, 4, 7, 14 and
21 days. While a signicant decrease in THC was found only on the 4th
day of exposure, hemocyte viability revealed a pronounced but not
signicant decreasing tendency, and then their condition was closer to
the control level, beginning with the 4th day of exposure. In the cited
study, high intragroup variability resulting in high standard deviation
values is noticeable. It is also remarkable in the present report. It may
indicate individual differences in the compensatory mechanisms, which,
in consequence, may lead to the selection of more tolerant individuals
and give the origin of the population possibly better prepared for
unfavorable conditions. High intragroup variability is usually typical of
the rst generation of animals exposed to a new selection factor. It re-
sults from the tolerance range of the population as a whole whose sur-
vival often depends on the potential individual plasticity, which is the
source of extreme values in measurements. This statement, however,
should be further studied. As mentioned in the H1.3 hypothesis, the
HSP70 and Lys levels were concentration-dependent. Usually, the
higher the concentration, the higher extent of changes found. Thus, the
hormetic effect, considered in the H1.3 hypothesis, has to be excluded.
Given the scarcity of literature data, some analogy can be found in a
study by Selonen et al. (2021), who examined the survival rate and
selected biochemical markers of soil animals (Enchytraeus crypticus,
Folsomia candida, and P. scaber) exposed to road dust. The authors found
that dust administered in soil or feed did not change the survival or
reproductive capacity of animals; only the highest concentration (1 %
vs. 0.01 % and 0.1 %) caused a signicant reduction in acetylcholines-
terase activity in P. scaber (Selonen et al., 2021).
In the environment, the concentration and the composition of the
particles may inuence the responses. In this study, the pattern of the
beetles’ reaction differed slightly concerning the type of material.
Cluster analysis conrmed the material-specic nature of the responses
observed in the levels of the studied biomarkers. Both the type of ma-
terial and its concentration signicantly inuenced biomarker levels,
particularly in the fat body, which may be related to the immune defense
functions of this tissue (Skowronek et al., 2021). To a lesser extent, these
factors also affected biomarker levels in the gut samples; however, the
material-specic variability in the tested parameters remains
non-negligible. Statistically signicant differences were found more
often in the RD than in the TR exposure. According to EDS analysis, RD
has a more complex composition and contains more potentially harmful
elements such as Al, Si, and Fe than tire rubber. On the other hand, tire
Fig. 5. Lysozyme (Lys) level (median; boxes: 1st and 3dr quartiles; whiskers: Min-Max) in fat body and gut samples of T. molitor larvae from the control (ctr) and
experimental groups exposed to 1 %, 2 %, 4 % (respectively: 1 prc, 2 prc, 4 prc) of road dust (dust) and tread rubber dust (rubber). Various letters (a, b) denote
statistically signicant differences between experimental groups (post hoc PERMANOVA F analysis p ≤0.05).
A. Babczy´
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Ecotoxicology and Environmental Safety 298 (2025) 118289
8

elastomers contain additives of ecotoxicological concerns (Rauert et al.,
2022). Since both materials elicited responses based on the markers used
in this study, it may be suggested that TR particles have a signicant
contribution to RD toxicity.
In terrestrial ecosystems, there are still many ecological niches where
the effects of particulate matter related to road transport remain at a
very low level of research. These include areas where terrestrial in-
vertebrates are exposed to this material through direct consumption,
such as omnivorous or herbivorous insects or their larvae. Due to the
possibility of long-distance transport of this dust, this exposure does not
only concern neighboring habitats, but also spreads the threat to non-
urbanized areas, creating a global environmental problem. Therefore,
it is necessary to conduct research to identify the far-reaching side ef-
fects of the development of land transport, even if exhaust emissions are
reduced due to the increased use of electric cars. In the present study, the
biomarkers’ responses, despite some signals of stress, do not mean direct
adverse effects of the exposure. Relatively long contact duration gave
time for possible compensatory reactions. Therefore, further studies
involving more time points and life history endpoints are necessary to
understand the threat posed on invertebrates inhabiting trafc-impacted
habitats. Further research should also include not only a wide range of
biomarkers, including behavioral responses, studies involving more
time points and life history endpoints and multi-generational studies to
predict the stability of the population in the area affected by particulate
matter related to transport. In situ studies are also necessary, taking into
account all factors, including noise and vibrations generated as a result
of using road infrastructure.
Fig. 6. Amylase (A), celulase (B) and protease (C) activity (median; boxes: 1st and 3dr- quartiles; whiskers: Min-Max) in gut samples of T. molitor larvae from the
control (ctr) and experimental groups exposed to 1 %, 2 %, 4 % (respectively: 1 prc, 2 prc, 4 prc) of road dust (dust) and tread rubber dust (rubber). Various letters (a,
b) denote groups of statistically signicant differences between experimental groups (post hoc PERMANOVA F analysis p ≤0.05).
A. Babczy´
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Ecotoxicology and Environmental Safety 298 (2025) 118289
9

Fig. 7. Dendrograms of cluster analysis results of protective (MTs, HSP70, Lis, Def) and digestive (protease, amylase, cellulase activities) biomarker levels, generated
using Ward’s method for the exposure to tread rubber (rubber) or road dust (dust) and their concentrations (1 %, 2 %, 4 %) in the T. molitor larvae diet. Main graphs:
clustering based on individual parameters. Inserted graphs: clustering based on median values of pooled parameters. (A) fat body – protective biomarkers; (B) gut –
protective biomarkers; (C) gut – digestive enzymes.
A. Babczy´
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Ecotoxicology and Environmental Safety 298 (2025) 118289
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5. Conclusions
RD and TR potentially interact with systems that protect organisms
under stress conditions. Taking into account the high intra-group vari-
ability, caused by the presence of outliers, the trends in the changes of
the studied parameters are not clearly outlined. The analysis suggests
that trafc-related materials may cause a decrease in HSP70 and Mts
levels through potential inhibition of their synthesis. In turn, RD and TR
may act as factors that potentially stimulate lysozyme-based immune
responses. The observed wide range of biomarker values may also be
attributed to phenotypic plasticity, individual adaptability, or recovery
processes occurring during the relatively long exposure period (i.e., the
entire larval stage). Digestive enzymes appear either resistant to the
effects of RD and TR or subject to similar adaptive phenomena
To minimize the inuence of adaptation mechanisms, which over the
long term may affect the assessment of TR or RD toxicity towards
T. molitor larvae, further studies should focus on the following ap-
proaches: (i) acute toxicity studies, which would allow identication of
response mechanisms before they are neutralized, (ii) toxicity studies in
the context of life history parameters — at time points that would enable
tracking possible variability in responses as a result of individual
adaptation over time or during onthogenesis, and (iii) multi-
generational tests to determine the directions of trait selection in
response to stress related to exposure to dust generated by road
transport.
CRediT authorship contribution statement
Sawadro Marta: Writing – review & editing, Conceptualization.
Babczy´
nska Agnieszka: Writing – original draft, Supervision, Project
administration, Methodology, Investigation, Funding acquisition,
Conceptualization. Potocka Izabela: Writing – review & editing, Visu-
alization, Software, Investigation. Kowalewska-Groszkowska Mag-
dalena: Writing – review & editing, Software, Methodology,
Investigation. Sujetovien ˙
e Gintar ˙
e: Writing – review & editing, Vali-
dation, Conceptualization. Mizera Katarzyna: Writing – review &
editing, Resources, Investigation. Giulianini Piero: Writing – review &
editing, Validation, Conceptualization. Tarnawska Monika: Writing –
review & editing, Methodology, Investigation, Conceptualization.
Czerwonka Agnieszka: Writing – review & editing, Conceptualization.
ˇ
Zaltauskait˙
e J¯
urat˙
e: Writing – review & editing, Validation, Concep-
tualization. Ba´
nska Michalina: Writing – review & editing, Resources,
Investigation. Łozowski Bartosz: Writing – review & editing, Visuali-
zation, Software, Data curation, Conceptualization. Bro˙
zek Jolanta:
Writing – review & editing, Visualization, Software, Investigation,
Conceptualization. Renzi Monia: Writing – review & editing, Valida-
tion, Conceptualization. Augustyniak Maria: Writing – review & edit-
ing, Validation, Methodology, Funding acquisition, Conceptualization.
Giglio Anita: Writing – review & editing, Validation, Conceptualization.
Rozpędek Katarzyna: Writing – review & editing, Supervision, Meth-
odology, Investigation.
Declaration of Competing Interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
Acknowledgements
This study was supported within the Green Horizon and POB5 grant
nanced by Research Excellence Initiative of the University of Silesia in
Katowice.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.ecoenv.2025.118289.
Data availability
Data will be made available on request.
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