Chemosphere 289 (2022) 133212
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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere
Interactions of microplastics and organic compounds in aquatic
environments: A case study of augmented joint toxicity
Andrey Ethan Rubin a, Ines Zucker a, b, *
a
b
Porter School of Earth and Environmental Studies, Faculty of Exact Sciences, Tel Aviv University, Tel Aviv, 69978, Israel
School of Mechanical Engineering, Faculty of Engineering, Tel Aviv University, Tel Aviv, 69978, Israel
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
• This study examines the role of micro­
plastics (MPs) as a contaminant vector
to the human body.
• Triclosan (TCS) sorbed onto polystyrene
MPs of various surface functionalities.
• Desorption of TCS from polystyrene MP
occurred in cellular conditions.
• Augmented joint toxicity toward Caco-2
cells was observed for TCS-sorbed MPs.
• MP risk assessment must consider its
inevitable interaction with co-existing
contaminants.
A R T I C L E I N F O
A B S T R A C T
Handling Editor: Willie Peijnenburg
High levels of persistent contaminants such as microplastics (MPs) and trace organic compounds (TrOCs) in the
aquatic environment have become a major threat on the ecosystem and human health. While MP’s role as a
vector of environmental TrOCs is widely discussed in the literature, the corresponding implications of the
interaction between these two compounds on human health (i.e., their joint toxic effect) have not been illus­
trated. Using a TrOCs model (Triclosan, TCS) and primary MPs (polystyrene microbeads), this work evaluates the
sorption and desorption potential of TCS and MPs in simulated environmental and cellular conditions, respec­
tively, and estimates the single and joint toxicity of these interactions toward human cells (Caco-2). Surface
functionality of the microbeads highly increased their adsorption capacity of TCS, from 2.3 mg TCS for non­
− functionalized microbeads to 4.6 mg and 6.1 mg TCS per gram of microbeads for amino- and carboxylfunctionalized MPs, respectively. Using non-functionalized MPs, non-specific “hydrophobic-like” interactions
and π-π interactions dominated the sorption mechanism of TCS; however, the addition of hydrogen interactions
between functionalized microbeads and TCS increased the microbeads’ overall sorption capacity. TCS was
desorbed from both functionalized and non-functionalized MPs when changing from environmental conditions to
cellular conditions. Desorption was found to be dependent on the matrix complexity and protein content as well
as microbead functionality. Finally, toxicity tests suggested that while low concentrations of TCS and MPs
(separately) have minor toxic effect toward Caco-2 cells, TCS-sorbed MPs at similar concentrations have an order
of magnitude higher toxicity than pristine MPs, potentially associated with the close interaction of both MP and
TCS with the cells. Overall, this study not only elucidates the role of MPs as a TrOC vector, but also demonstrates
a realistic scenario in which co-presence of these environmental contaminants poses risks to the environment and
human health.
Keywords:
Microplastic
Polystyrene microbeads
Environmental pollutants
Triclosan
Adsorption
Toxicity
Viability test
Caco-2
* Corresponding author. Tel Aviv University, Tel Aviv, 69978, Israel.
E-mail address: ineszucker@tauex.tau.ac.il (I. Zucker).
https://doi.org/10.1016/j.chemosphere.2021.133212
Received 17 October 2021; Received in revised form 5 December 2021; Accepted 6 December 2021
Available online 7 December 2021
0045-6535/© 2021 Elsevier Ltd. All rights reserved.
A.E. Rubin and I. Zucker
Chemosphere 289 (2022) 133212
humans of 39,000 to 52,000 MP particles annually (Cox et al., 2019).
Oral consumption of MP- by contaminated food and water is one of the
main human MP exposure routes and therefore, the intestinal cells
(especially the epithelial cells) are considered to be the first barrier
between MPs and the human body (Huang et al., 2021). However, due to
their small size, MPs have recently been reported to cross the intestinal
barrier and get into our bodies (Ragusa et al., 2021).
Although MPs can cross the epithelial barrier and get into the
bloodstream, their potential hazard is still unclear; while some studies
suggest MP as a harmful substance toward human health (Prüst et al.,
2020; Wang et al., 2021), others claim that MP has little toxicity (Hwang
et al., 2020). To evaluate the potential hazard arising from oral con­
sumption of MPs, scientists often use a single cell model, such as the
Caco-2 cell line (Liu et al., 2020; B. Wu et al., 2019a). Through such an in
vitro method, MPs were shown to stimulate inflammatory reactions and
cause death in the tested cell lines (Dong et al., 2020; Hwang et al.,
2019). However, the potential sorption of co-existing TrOCs in the
environment is rarely taken into consideration when evaluating MP
toxicity toward human cells. The potential joint toxic effect of MP and
TrOCs has been demonstrated in aquatic organisms, suggesting
increased toxic effects in the presence of both (Z. Li et al., 2020b; Yang
et al., 2020; Zhang et al., 2020; Zhu et al., 2019). Beyond better eval­
uation of such joint effects toward human cells, research should consider
the surrounding conditions in which those interactions take place. While
sorption of TrOCs occurs in environmental conditions, oral uptake and
transfer to cellular conditions may result in desorption of the TrOCs from
the MP surface which may largely affect the overall toxicity of the
original TrOC-MP complex.
In this study, we evaluate the sorption and desorption potential of a
TrOC model (Triclosan, TCS) onto primary MPs as well as their single
and joint toxic effects. By providing an outlook of MP fate during
transport from environmental to cellular conditions, we aim to shed
light on MP role as a vector of environmental contaminants. Sorption
levels of TCS onto polystyrene (PS) microbeads of various surface
functionalities were evaluated under simulated environmental condi­
tions, with results suggesting that oxidized MPs have a higher affinity
towards TCS. Upon the change in surrounding conditions (from envi­
ronmental to cellular conditions), 27− 65% desorption of TCS was
observed depending on MP surface functionality. Toxicity tests sug­
gested that TCS and carboxylated (COOH) MPs have the highest joint
toxic effect toward Caco-2 cells, which was related to close bonding
patterns of sorbed, functionalized microbeads with cells. Overall, this
study suggests that surface functionalization of MPs and surrounding
conditions play a significant role in the interaction of MPs with TrOCs,
which highly dictates the risk associated with oral exposure to MPs.
1. Introduction
Microplastic (MP) pollution is a growing environmental concern
according to its persistent nature and associated risks to the environ­
ment and human health. In the aquatic environment, for example, these
plastic particles in size scales smaller than five mm accumulate in coastal
and deep waters (Lindeque et al., 2020), sediment (Cauwenberghe et al.,
2015), and even in various aquatic living forms such as shrimp and fish
(Ribeiro et al., 2020). The source of these MPs can be either from plastics
originally synthesized in micron-scale for specific applications (primary
MPs) or from degraded larger plastic waste (secondary MPs). In aquatic
environments, various co-existing substances may interact with MPs
which often results in sorption of those substances onto the MP surface.
Of particular interest is MPs’ adsorptive potential towards various
organic contaminants in sub− ppm concentrations (Gong et al., 2019; T.
Wang et al., 2020b), also known as trace organic compounds (TrOCs).
TrOCs—such as pharmaceuticals, personal care products, pesticides,
herbicides, and hormones—are typically released to the aquatic envi­
ronment directly or through discharge of untreated or treated waste­
water (as they persist during conventional wastewater treatment)
(Bernot et al., 2016; Zucker et al., 2015). Such contaminants are
therefore detected in a variety of water bodies such as groundwater,
rivers, and even oceans (Moore et al., 2002). Both the nature and extent
of sorption are dictated by many environmental factors (including so­
lution pH, presence of organic matter, salinity, and temperature) (Mei
et al., 2020) as well as sorbent and sorbate properties (Mei et al., 2020).
For instance, MP properties such as polymer type (Hai et al., 2020),
particle size (Wang et al., 2019), and crystallinity (F. Wang et al., 2020c;
Xu et al., 2019) have been shown to have a strong effect on the sorption
potential of organic substances.
Due to continuous exposure to weathering conditions, bulk and
surface properties of both primary and secondary MPs are constantly
changing (Rubin et al., 2021; P. Wu et al., 2019b). One of the most
common weathering-driven surface modifications of MPs is the addition
of carbonyl functionalities (Liu et al., 2019; Sarkar et al., 2021), which
play a key role in sorption processes (Rubin et al., 2021). Other MP
physico-chemical modifications that can be driven by weathering
include increased surface-area-to-volume ratios, eco-corona and biofilm
additions (Mei et al., 2020; Rubin et al., 2021), and elevated hydro­
philicity (Mei et al., 2020)—all of which have a direct influence on
sorption interactions and affinity towards co-existing substances.
The interactions of MPs with TrOCs have various environmental
implications. First, sorption of TrOCs may further alter the surface
properties of the MPs, and thus impact the MPs’ environmental fate
including their interactions with microorganisms and overall may
contribute to their sinking under aquatic conditions (Chen et al., 2019).
Second, MP transport in the aquatic environment (as a suspended ma­
terial) will increase the probability of interactions with hydrophobic
TrOCs and concentrate the TrOCs on top of the particle surface finally
increase their potential toxicity toward living forms (T. Wang et al.,
2020a). Because of these interactions and their potential joint toxic ef­
fects on the environment and human health, the role of MPs as a vector
of environmental TrOCs has been widely discussed in recent literature
(Koelmans et al., 2016; M. Li et al., 2020a). However, there is no
agreement regarding the significance of such phenomenon, due to the
high variability in the MP and TrOC properties which may drive trans­
port of TrOC to cells by MPs, as well as lack of experimental evidence to
joint toxicity by TrOC-sorbed MPs.
Humans are exposed to MP contamination through a variety of food
sources—such as fruit and vegetables (Oliveri et al., 2020a, b), seafood
(Ribeiro et al., 2020), food containers (Kedzierski et al., 2020), salt
(Peixoto et al., 2019), and even tea bags (Hernandez et al., 2019)—
which results in MP accumulation in the digestive system. Drinking
water sources, including both bottled water (Mason et al., 2018) and tap
water (Tong et al., 2020), are also reported to contain a significant
number of MPs. Overall, scientists estimate an average exposure to
2. Material and methods
2.1. Polystyrene microbeads and triclosan
1− μm PS microbeads of various functionalities (i.e., pristine nonfunctionalized, carboxyl (COOH)- and amino (NH2)-modified) were
purchased from Spherotech (Spherotech Inc, U.S.A) in suspended form.
Presence of functional groups was verified using Fourier transform
infrared - Attenuated Total Reflection, FTIR-ATR analysis (Figure S1),
Supplementary Information (SI). The microbeads size and surface
charge were measured using Zetasizer Nano with DTS 1070 capillary
cuvette (Malvern Panalytical Ltd, UK). Triclosan (TCS, 5-Chloro-2-(2,4dichloro phenoxy) phenol) was purchased from Sigma-Aldrich (Merck
KGaA, Germany). As TCS has low solubility in water, 200 mg/L stock
solution was prepared in ethanol (100% absolute, USP-grade), which
was then diluted with aqueous buffered solution (10 mM phosphate
buffer, pH 7.7) to a final concentration of 20 mg/L. Sodium dihydrogen
phosphate dihydrate (NaH2PO4 ⋅ 2H2O, ≥99.0%) and sodium phosphate
dibasic dihydrate (Na2HPO4 ⋅ 2H2O, ≥98.0%) were purchased from
Sigma-Aldrich. Deionized (DI) water was obtained from a Milli-Q
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A.E. Rubin and I. Zucker
Chemosphere 289 (2022) 133212
ultrapure water purification system (Merck KGaA, Germany).
2.4. Triclosan quantification
2.2. Adsorption experiments
TCS concentration was determined using high-performance liquid
chromatography (HPLC, Agilent 1260 Infinity Series) coupled with a
photodiode array UV–Vis detector. A sample volume of 100 μL was
injected into a Fast-guard Poroshell 120 (4.6 mm, 4 μm) pre-column and
Poroshell 120 EC C18 (100 mm × 4.6 mm, 4 μm) column (Agilent
Technologies, U.S.A) at 30 ◦ C. The mobile phase was an isocratic
mixture comprising 70% HPLC-grade acetonitrile (Bio-Lab Ltd, Israel)
and 30% DI water with 1% acetic acid (Merck KGaA, Germany) at a flow
rate of 0.6 mL/min. TCS was detected at a retention time of 6 min with
an absorption wavelength of 200 nm.
The TCS adsorption kinetics and isotherms onto the PS microbeads
were studied under simulated environmental conditions. Prior to
adsorption experiments, the PS microbead suspension was washed using
a 10 mM phosphate buffer to eliminate sodium azide (NaN3) residues
from the purchased suspension. The PS microbead suspension was then
re-suspended using a sonication bath (Elmasonic S-30, Elma Schmid­
bauer GmbH, Germany). To avoid plastic cross-contamination, all the
experiments were conducted in glass vials.
In a typical adsorption kinetic experiment, 500 μL of the rinsed
microbeads suspension, 2.5 mL of TCS stock solution, and 10 mM
phosphate buffer at a total volume of 50 mL were mixed in borosilicate
bottles for final concentrations of 50 μg/L microbeads and 1 mg/L TCS.
The kinetic experiments were performed in duplicate for 164 h under
controlled conditions (25 ◦ C, dark conditions, and mixing at 180 rpm).
Aliquots (500 μL) were taken periodically and centrifuged (5000×g for
10 min) to separate the microbeads from the supernatant. Then, 450 μL
of supernatant was transferred to 2 mL glass HPLC vials and stored in the
refrigerator prior to TCS analysis. The isotherm experiments were con­
ducted in a similar manner, where TCS initial concentrations ranged
between 0.1 to 4 mg/L.
The sorption capacity q (mg TCS to g MP) was calculated using Eq.
(1) where C0 (mg/L) is the TCS initial concentration, Ce (mg/L) is the
equilibrium TCS concentration in the supernatant, V is the solution
volume in L, and m is the adsorbent mass (i.e., microbeads) in grams.
q=
(C0 − Ce )⋅V
m
2.5. Caco-2 cell cultivation
We used human colon epithelial cells of Caco-2 (passage 10) for all
toxicity experiments. Prior to the experiment, the cells were unfrozen
and cultivated for seven days in a 25 cm2 cell culture flask. The culture
was cultivated in Dulbecco’s Modified Eagle Medium (DMEM pH− 7.5,
Sigma Aldrich Inc, Israel) with supplements including 10% fetal bovine
serum (FBS), 1% L-Glutamine, and 0.1% Penicillin – Streptomycin
(Biological Industries LTD, Israel) with 5% CO2 at 37 ◦ C.
2.6. Viability test using PrestoBlue assay
The toxicities of microbeads and TCS was investigated using a singlecell model of Caco-2 cells. The experiment was performed in a 96-well
plate (Costar 3596, Corning Inc., U.S.A) with 100 μL of cells in each
well (106 cells per mL). The cells were incubated with the microbeads,
TCS, or TCS-sorbed microbeads for 24 h. Cell viability was examined
using the Prestoblue cell viability kit (Thermo Fisher Scientific, U.S.A).
The PrestoBlue assay is a commonly used viability test based on redox
reactions and transformation of a weakly fluorescent Resazurin reagent
(blue) into highly fluorescent Resorufin (pink) (Sittampalam et al.,
2016). In brief, incubated Caco-2 cells were washed with DMEM media,
and 100 μL of diluted Prestoblue reagent (1/10 in DMEM media) were
added to each of the wells for 3 h. Control wells contained only Pres­
toblue to account for the spectroscopic background. The changes in cell
viability were quantified using a fluorescence plate reader Tecan− Spark
(Tecan Group Ltd, Switzerland) at excitation and emission wavelengths
of 560 and 590 nm, respectively. The cell viability percentage was
calculated as the relative difference in emission signal of toxin-exposed
and non-exposed (control) cells (Sittampalam et al., 2016). Each toxicity
test was conducted in five replicates. The significance of toxicity tests
was analyzed with a two-tailed t-test (alpha: 0.05). A summary of the
experimental setup—including sorption, desorption, and toxicity
tests—is presented in Fig. 1.
(1)
Non-linear fitting of the isotherm curves was performed using the
Origin V9.0 (Origin Lab Corporation, U.S.A) fitting tool to Langmuir
isotherm based on Eq. (2), where qmax is the max adsorption capacity of
1 g of microbeads, and b is the adsorption capacity in mg/L.
q=
qmax ⋅b⋅Ce
1 + b⋅Ce
q = Kf ⋅Ce1/n
(2)
(3)
Fitting to Freundlich isotherm based on Eq. (3) was also conducted,
where Kf and n represent the equilibrium constants.
2.3. Desorption experiments
TCS-sorbed microbeads were exposed to biological conditions to
evaluate TCS desorption. In a realistic scenario by which MP is orally
consumed, interaction with intestinal cells will occur only after MP
travels through the digestive tract. To simulate pre-exposure to cellular
media prior to interaction with intestinal cells, TCS-loaded microbeads
at equilibrium (~0.1 mg/L) were re-suspended in 5 mL of DMEM cell
culture media for 48 h, which has been previously suggested as the
median whole gut transit time of food (Lee et al., 2014). To eliminate
interferences during TCS analysis by the complex cellular suspen­
sion—which contains a large number of organic and inorganic sub­
stances—TCS was extracted from the supernatant through solid-phase
extraction. Briefly, 3 mL of the supernatant was passed through StrataX
polymeric reverse phase 30 mg/3 mL cartridges (Phenomenex Inc, U.S.
A) using a GenieTouch syringe pump (Kent Scientific Corporation, U.S.
A) at a flow rate of 0.1 mL/min. Then, the cartridge was washed with 1
mL 1% MeOH solution, and TCS was then eluted using 1 mL 70/30 (v/v)
acetonitrile and DI water and stored in the refrigerator prior to analysis.
Recovery during solid-phase extraction was calculated based on refer­
ence experiments at known concentrations and was found to be 80%.
Desorption experiments were completed in triplicate. Error is reported
as standard propagated error.
3. Results and discussion
3.1. Sorption and desorption of triclosan onto PS microbeads
TCS was sorbed onto both functionalized and non-functionalized
microbeads (Fig. 2A). Kinetics of adsorption were fitted to a pseudosecond order kinetic model (Table S1), similar to kinetics reported for
TCS adsorption onto MPs in previous studies (Chen et al., 2021; Li et al.,
2019). Non-functionalized PS microbeads reached equilibrium faster
(k2 = 2.22 M s− 1, r2 = 0.92) than NH2-functionalized (k2 = 1.72 M s− 1,
r2 = 0.94) and COOH-functionalized (k2 = 0.34 M s− 1, r2 = 0.86)
microbeads. Other than a strong dependence on experimental conditions
(Li et al., 2019) (including temperature, ionic strength, salinity, and pH),
sorption kinetics are known to also depend on structural properties of
both the sorbate and the sorbent (Torres et al., 2021). Here, we see how
at similar experimental conditions, the surface functionality of the sor­
bent highly affects the sorption kinetics.
The TCS sorption capacity also varied depending on the microbeads’
3
A.E. Rubin and I. Zucker
Chemosphere 289 (2022) 133212
Fig. 1. Schematic illustration of the experimental
setup. (A) Microbeads (black dots) of various func­
tionalities and TCS (yellow) were mixed in buffered
solution under controlled temperature in the orbital
shaker until equilibrium was reached. (B) Then, solids
(TCS-sorbed microbeads) were separated from the
buffered solution through centrifugation and the su­
pernatant was taken for analysis of remaining TCS.
(C) The solids were resuspended in biological media
(DMEM) to evaluate toxicity (using Prestoblue re­
agent) toward Caco-2 cells with and without a 48-h
desorption period in cell culture media (D). (For
interpretation of the references to colour in this figure
legend, the reader is referred to the Web version of
this article.)
Fig. 2. Triclosan adsorption and desorption onto polystyrene microplastics (MPs) of various functionalities. (A) Removal kinetics of 1 mg/L of triclosan by
microbeads. (B) Sorption isotherms by microbeads in initial triclosan concentration range of 0.1–4 mg/L. Langmuir and Freundlich fittings are shown in solid and
dashed lines, respectively. The adsorption rate is presented as a mass ratio of adsorbed pollutant (triclosan) to adsorbing material (MPs). All experiments were
conducted in a buffered solution at pH 7.7 with a 10 mM phosphate buffer. Error bars represent standard deviation from an average of three experiments. (C)
Triclosan desorption from 0.1 mg/L TCS-sorbed MPs at DMEM media (in percentage).
surface functionality (Fig. 2B). While non-functionalized microbeads
had the lowest adsorption capacity of 2.26 mg/g (mg TCS per g of
microbeads), NH2-functionalized and COOH-functionalized microbeads
demonstrated elevated adsorption capacity of 4.63 mg/g and 6.15 mg/g,
respectively. This trend stands in line with previous studies demon­
strating an increasing adsorption capacity of weathered PS particles
(which are typically oxidized with COOH-functionalized surfaces)
compared to non-functionalized particles (Li et al., 2019). As both
Langmuir (r2 = 0.99) and Freundlich (r2 = 0.98) models could be used to
fit the isotherm results (Table 1), it is difficult to draw conclusions on the
dominant sorption mechanism. The maximum potential sorption ca­
pacity (qmax) of TCS was found to be 12 mg/g for COOH-functionalized
microbeads. This elevated TCS sorption capacity onto carboxylated
PS—the PS model most representative of environmentally weathered
PS—suggest that sorption potential is expected to increase once MPs are
aged.
The dominant sorption mechanism may differ depending on the
surface functionality of the sorbate. Potential sorption mechanisms are
electrostatic interactions, π-π interactions, and hydrogen bonding. The
microbeads’ zeta potential at the adsorption test conditions (i.e., solu­
tion pH of 7.7) were − 20.7 mV, − 35.0 mV, − 42.1 mV for NH2-func­
tionalized,
non-functionalized,
and
COOH-functionalized
PS
microbeads, respectively (Table S2). According to the microbeads
manufacturer, the amino and carboxyl groups were grafted using func­
tionalized monomers after the polymerization process, which suggest
calculated pKa values of 10.5 ± 0.4 for amino functional groups and 4.9
± 0.4 for carboxyl functional groups (Figure S2).
At solution pH of 7.7 approximately 30% of TCS is negatively
charged (Li et al., 2019)—and together with the zeta and pKa values of
the microbeads—if electrostatic attraction plays a large role in the
sorption mechanism, one would expect that the NH2-functionalized
microbeads would attract TCS while COOH-functionalized microbeads
would repeal TCS charged molecules. However, the opposite phenom­
enon is true and COOH-functionalized microbeads demonstrate the
highest TCS adsorption capacity. Therefore, electrostatic interaction
might not be the dominant sorption mechanism as was recently sug­
gested in a study on adsorption of TrOCs onto weathered plastics
(Munoz et al., 2021). Furthermore, sorption in presence of higher ionic
strength (0.1 M NaCl) (Figure S3) was similar to that obtained in 0.01 M
phosphate buffer, demonstrating the limited effect of both buffer and
ionic strength on the sorption potential. It is worth mentioning that all
three microbeads had similar hydrodynamic radius of 1600 nm
(Table S2), which together with their non-porous nature suggest that
differences in specific surface area are also not responsible for to the
differences in sorption capacities obtained for the PS microbeads.
Based on the highly hydrophobic nature of both PS and TCS, all
microbeads types may create hydrophobic and π-π interactions with TCS
Table 1
Fitting parameters of TCS sorption isotherms for Langmuir and Freundlich
models.
Model
Parameter
Nonfunctionalized
PS
COOHfunctionalized
PS
NH2functionalized
PS
Langmuir
qmax
b
R2
Kf
n
R2
6.32
0.19
0.99
0.91
1.16
0.99
12.0
1.17
0.99
6.30
1.40
0.99
8.61
0.67
0.99
3.58
1.29
0.98
Freundlich
4
A.E. Rubin and I. Zucker
Chemosphere 289 (2022) 133212
(Li et al., 2019). However, while such interactions dominated sorption of
TCS onto pristine PS microbeads, functionalized PS microbeads may also
adsorb TCS through hydrogen bonding (Mei et al., 2020). The difference
in TCS sorption capacities of COOH and NH2 groups can be explained by
the availability and strength of hydrogen bonds; while protonated TCS
(70% of TCS in solution) can create hydrogen bonds with
COOH-functionalized microbeads (O–H–O), the rest of the TCS (i.e.,
deprotonated TCS) can create hydrogen bonds (N–H–O) with
NH2-functionalized microbeads. The strength of hydrogen bonds is also
expressed in a change of bond distances, where the COOH groups have
been reported to create stronger hydrogen bonds (average bond dis­
tance: 0.263–0.270 nm) compared to the medium-strength bonds of NH2
groups (average bond distance: 0.288 nm) (Haynie, 2008).
Following sorption under environmental conditions, TCS-sorbed MP
can transport farther in water bodies, increasing their likelihood of
eventual exposure to humans. The uptake of the TCS-MP complex can
occur, for example, through consumption of contaminated seafood
(Smith et al., 2018) or water (Danopoulos et al., 2020). Once consumed,
the TCS-sorbed MP will be in new cellular conditions and thus initiate a
new equilibrium sequence of sorption and desorption of TCS. To un­
derstand the nature of desorption of TCS from PS microbeads and the
fate of the TCS-MP complex once transferred to cellular conditions, the
original buffered water media used was replaced with DMEM media.
Desorption experiments were conducted with TCS-sorbed MPs at similar
concentrations (0.1 mg/L loaded TCS), to assure that desorption is not
affected by the initial concentration of sorbed TCS. After a 48 h
desorption period (Lee et al., 2014), pristine, COOH-functionalized, and
NH2-functionalized PS released an average of 26.5%, 27.3%, and 64.6%
of sorbed TCS, respectively (Fig. 2C). Surprisingly, desorption did not
follow a similar trend to that observed during sorption, suggesting
different mechanisms for sorption and desorption phenomena.
Compared to the buffered solution used in sorption experiments,
DMEM media is highly enriched with organic substances such as
L− Glutamine amino acids (1% of the total solution). In solution pH of
7.7, the L− Glutamine amino acids will be negatively charged (pKa: 2.17,
pKr: 4.3) (Ma et al., 2013) and will have the potential to create elec­
trostatic interactions with other positively charged molecules in the
media. Therefore, it can be expected that negatively-charged
L− Glutamine
will
be
electrostatically
attracted
to
the
positively-charged NH2-functionalized MPs at higher affinities than
those of TCS molecules. Such electrostatic exchanges will result in
desorption of TCS from NH2 functionalized MPs. However, this
replacement may occur at lower rates on the surface of pristine (no
charged groups) and carboxylated (negatively charged) MPs which both
negatively-charged under experimental conditions (Table S2). Similar
phenomena have been observed by others in recent literature, in which
organic substances—like humic acid and NOM—encouraged desorption
of TrOCs from MP surfaces (Chen et al., 2021; Munoz et al., 2021).
3.2. Toxicity towards human intestinal cells by triclosan and
microplastics in single and joint tests
A series of individual toxicity tests were performed with microbeads
and TCS (separately). First, we evaluated the toxicity of pristine and
functionalized PS microbeads in a concentration range of 25 μg/L to 100
μg/L (107 to 108 particles/mL). We observed close interactions of the
microbeads with the cells (Figure S4) by overlaying images in trans­
mission with fluorescence to indicate colocation of the Caco2 cells and
microbeads. In addition, we measured the viability of cells following
interaction with pristine, COOH-functionalized, and NH2-functionalized
microbeads (Fig. 3A). Interactions with COOH- and NH2-functionalized
microbeads caused statically significant changes in viability of cells after
interaction as compared to the control (two-tailed t-test, alpha: 0.05)
and increasing concentrations of COOH- and NH2-functionalized
microbeads resulted in decreased cell viability (i.e., higher toxicity). No
statistically significant change in viability was observed in cell in­
teractions with pristine MPs, which is similar to other research on MP
toxicity (He et al., 2020).
Functionalized microbeads were previously reported to be more
toxic than non-functionalized microbeads, due to the functional groups’
interferences in the cell anti-oxidative processes (He et al., 2020). Other
studies suggest additional toxicity mechanisms for MPs towards cells
such as MP-induced mechanical stress to cell membranes (Fleury and
Baulin, 2021) and generation of reactive oxygen species (Banerjee and
Shelver, 2021). It should be noted that although both functionalized
microbeads in this study show similar toxic effects at 100 μg/L, the
number of functional groups on the carboxylated microbeads is three
orders of magnitude smaller than the NH2-enabled microbeads (~4 ×
106 and 4 × 109 groups/microbead for COOH and NH2 microbeads,
respectively), suggesting carboxyl groups’ enhanced toxicity compared
to amino functional groups.
When exposing cells to TCS, increasing concentrations of TCS from
0.01 to 2 mg/L resulted in a decrease of cell viability, as illustrated in
Fig. 3B. We found an approximate 20% decrease in viability at 1 mg/L
TCS, while other studies found similar toxic effect at much higher TCS
concentrations (~43 mg/L) (Oliver et al., 2020a, b). However, large
differences in toxicity values may arise from changes in experimental
conditions—such as use of older passage cells in the current study­
—emphasizing the importance of holding experimental conditions
constant. Using these results, the joint toxicity effect was tested with 50
μg/L of microbeads and 0.1 mg/L of TCS because microbeads and TCS at
these concentrations separately demonstrate only a slight decrease in
Fig. 3. Viability of Caco-2 cells in presence of (A) polystyrene microbeads in a concentration range of 25–100 μg/L and (B) triclosan in concentration range of 0.01–2
mg/L p values from two-tailed (**) t-tests are reported for viability tests conducted in the selected conditions for joint toxicity tests.
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Chemosphere 289 (2022) 133212
cell viability (up to 13%), and thus any appearance of joint toxicity will
be more pronounced and can be associated with a hybrid effect of the
two.
To evaluate the joint toxic effect of TCS and MPs, all three types of PS
microbeads were pre-loaded with 0.1 mg/L of TCS. We exposed the
Caco-2 cells to the TCS-sorbed microbeads with and without a desorp­
tion stage (i.e., with and without 48 h of pre-exposure to cellular media
conditions) to account for the role of TCS desorption on the cell viability.
The COOH-functionalized PS microbeads without a desorption stage
result in a 9% decrease in cell viability, while the same microbeads with
a desorption stage resulted in a 25% decrease in cell viability. A similar
trend was found for NH2-functionalized and pristine PS microbeads,
where desorption elevated cell mortality to 22% and 13%, respectively.
However, desorbing TCS from pristine MPs did not significantly influ­
ence their toxicity toward the cells as compared to the control (black
bar, Fig. 4).
Elevated toxicity following the desorption stage, can be explained by
the change in surface properties of microbeads in such conditions. Under
cellular conditions, microbeads tend to sorb organic substances (i.e.,
build-up of protein-corona coating) which intensifies their affinity to­
ward cells (Yin Win and Feng, 2005) and therefore may also increase
Fig. 4. Joint toxic effect on Caco-2 cells of 0.1 mg/L TCS pre-loaded onto
pristine, COOH-functionalized, and NH2-functionalized PS microbeads. Exper­
iments were conducted with and without pre-desorption stage of 48 h in DMEM
media. Two-tail (**) t-test p-values are reported for viability tests conducted
with pre-desorption stage.
Fig. 5. Evolution of MP particles in the experimental conditions: (A) sorbed 0.1 mg/L of TCS (in yellow) under simulated environmental conditions and potential
sorption mechanisms, including π-π interactions, hydrophobic interactions and hydrogen bonds. (B) Following desorption and coating with protein-corona (in red)
from DMEM substances with a (C) final effect of cellular joint toxicity. (For interpretation of the references to colour in this figure legend, the reader is referred to the
Web version of this article.)
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Chemosphere 289 (2022) 133212
their overall toxicity. On the other hand, presence of protein-corona
coating by its own (i.e., in the absence of TCS) does not result in
increased toxicity toward Caco-2 cells (Figure S5), suggesting that both
presence of protein-corona coating and co-presence of TCS and MPs
(which experienced adsorption-desorption) are required to intensify
toxicity. A possible explanation for this phenomenon is that a
protein-corona coating contribute to re-sorption of TCS onto microbeads
surface as part of the new equilibrium, and through that re-sorption
increase its overall toxicity toward cells (Jin et al., 2018). Therefore,
pre-sorbed microbeads without desorption time and lacking
protein-corona coating might experience less intimate attachment to the
cell membrane, which lowers their overall toxicity rates.
The enhanced joint toxic effect was observed for TCS and function­
alized MPs which experienced adsorption-desorption at higher extent
compared to TCS and pristine MPs. This observation agrees well with
toxicity results from single toxicity tests for microbeads only—where
functionalized MPs demonstrated higher toxicity compared to pristine
MP ones—possibly through cell membrane bonding with MP functional
groups (Barbul et al., 2018). Overall, TCS-sorbed COOH-functionalized
MPs at non-toxic concentrations of the TCS resulted in similar toxicity to
that obtained for 1 mg/L of TCS alone, suggesting an order of magnitude
increased toxicity by TCS sorption-desorption phenomena. These com­
plex realistic sorption-desorption-toxicity scenarios was outlined in
Fig. 5.
inflammatory response which in the future might translate to cancer
(Misra et al., 2018). As MP toxicity is directly associated with its size, the
toxic effect is expected to be yet more significant for nanoscale plastic
particles (Jeong et al., 2016; B. Wu et al., 2019a) with an elevated
surface area and therefore elevated sorption capacities as well as
increased permeability and uptake of the particles into the cells.
Credit author statement
Andrey Ethan Rubin: Conceptualization, Methodology, Investiga­
tion, Writing – original draft preparation. Ines Zucker: Conceptualiza­
tion, Resources, Supervision, Writing – review & editing.
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
AER would like to acknowledge the Aaron Frenkel Pollution Initia­
tive at Tel Aviv University for their funding provided for this research.
AER and IZ would like to thank Prof. Rafi Korenstein for assistance in
cell toxicity experiments. Human colon epithelial cells were kindly
provided by Dr. Tsaffrir Zor from the Department of Biochemistry &
Molecular Biology, Life Sciences Faculty, Tel Aviv University. We would
like to also thank Dr. Yinon Yecheskel, Dr. Amit Kumar-Sarkar, and Dr.
Igal Gozlan for the assistance in mechanistic assessment of adsorption/
desorption phenomena. Finally, we would like to thank Guy Shimel, for
the assistance in laboratory work and Ben Karni for paper editing.
Graphical abstract, Fig. 1, and Fig. 5 were created using Bio-render
software.
4. Conclusions and outlook
Although toxicity of isolated MP and TrOCs toward humans is largely
discussed in literature, there still exists a scientific gap in understanding
their joint toxicity in aqueous solution (T. Wang et al., 2020a). Assess­
ment of such joint effects should take into consideration the character­
istic changes the MP may experience in environmental, and later
cellular, conditions. This study offers a close look at realistic
sorption-desorption-toxicity scenarios through environmentally
-relevant interaction of TCS with MPs and post-exposure to cellular
conditions. We used a simple model system to demonstrate the phe­
nomena of enhanced joint toxicity between MP and TCS. Particularly,
we found that COOH-functionalized MPs which sorbed TCS in simulated
environmental conditions, partly desorbed TCS in cellular conditions,
and resulted in mortality of 25% of epithelial cells to which the TCS-PS
matrix was exposed. This joint toxic effect was higher for
COOH-functionalized MPs than for pristine and NH2-functionalized
MPs, possibly through carboxyl-membrane bonding. These results of
augmented toxicity through TrOC-MP pairing suggest that MPs may not
only act as a vector for TrOCs to cells, but exacerbate toxicity concerns.
Under environmental conditions, MPs tend to age, resulting in
oxidation of the MP surface. Such aging and change in surface properties
of MPs largely affect their interactions with the surrounding substances
such as TrOCs. Therefore, we suggest COOH-functionalized PS with
sorbed TCS as a relevant model for weathered primary MP risk assess­
ment in this context. Later, MPs with pre-loading of TrOCs can be
exposed to cellular conditions upon their consumption by living forms,
resulting in new equilibrium with their environment and formation of
protein corona layer. Then, the complex may interact with living cells
and change their viability.
Even though primary MPs (i.e., microbeads) are not the main type of
MPs in the environment, the risks associated with their presence should
be evaluated due to their high resistance to environmental weathering
and persistence in the environment (Chamas et al., 2020). As MPs have
already shown to penetrate the intestinal barrier of the human body
(Huang et al., 2021), this effect may have detrimental effect on cell
viability once the MPs are loaded with TrOCs. Indeed, damage to the
integrity of intestinal barriers may increase not only the number of MPs
in our bloodstream but also pave the way for pathogens and toxic
molecules to enter bloodstream in a much simpler manner. Furthermore,
constant exposure to low amounts of MPs might trigger a local
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.chemosphere.2021.133212.
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