Journal Pre-proof Toxicological effects of polystyrene microplastics on earthworm (Eisenia fetida) Xiaofeng Jiang, Yeqian Chang, Tong Zhang, Yu Qiao, Göran Klobučar, Mei Li PII:
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Received Date: 7 August 2019 Revised Date:
13 December 2019
Accepted Date: 28 December 2019
Please cite this article as: Jiang, X., Chang, Y., Zhang, T., Qiao, Y., Klobučar, Gö., Li, M., Toxicological effects of polystyrene microplastics on earthworm (Eisenia fetida), Environmental Pollution (2020), doi: https://doi.org/10.1016/j.envpol.2019.113896. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Toxicological effects of polystyrene microplastics on earthworm
Xiaofeng Jianga, Yeqian Changa, Tong Zhanga, Yu Qiaoa, Göran Klobučarb, Mei Lia,*
Affiliations of authors:
Environment, Nanjing University, Nanjing 210023, China
State Key Laboratory of Pollution Control and Resource Reuse, School of the
Department of Biology, Faculty of Science, University of Zagreb, 10000 Zagreb, Croatia
Mei Li (Address: 163 Xianlin Ave., Nanjing University, Nanjing 210023, China)
Email address: [email protected]
Microplastics are plastic fragments of particle sizes less than 5 mm, which are widely
distributed in marine and terrestrial environments. In this study, earthworms Eisenia
fetida were exposed to 100 and 1000 µg of 100 nm and 1300 nm fluorescent
polystyrene microplastics (PS-MPs) per kg of artificial soil for 14 days. Uptake or
accumulation of PS-MPs in earthworm intestines, histopathological changes,
oxidative stress, and DNA damage were assessed to determine the toxicological
effects of PS-MPs on E. fetida. The results showed that the average accumulated
concentrations in the earthworm intestines were higher for 1300 nm PS-MPs (0.084 ±
0.005 and 0.094 ± 0.003 µg/mg for 100 and 1000 µg/kg, respectively) than for 100 nm
PS-MPs (0.015 ± 0.001 and 0.033 ± 0.002 µg/mg for 100 and 1000 µg/kg,
respectively). In addition, histopathological analysis indicated that the intestinal cells
were damaged after exposure to PS-MPs. Furthermore, PS-MPs significantly changed
glutathione (GSH) level and superoxide dismutase (SOD) activity. The GSH levels
were 86.991 ± 7.723, 165.436 ± 4.256–167.767 ± 18.642, and 93.590 ± 4.279–
173.980 ± 15.523 µmol/L in the control, 100 nm, and 1300 nm PS-MPs treatment
groups. In addition, the SOD activities were 10.566 ± 0.621, 9.039 ± 0.787–9.408 ±
0.493, and 7.959 ± 0.422–9.195 ± 0.327 U/mg protein for the control, 100 nm, and
1300 nm PS-MPs treatment groups, respectively, indicating that oxidative stress was
induced after PS-MPs exposure. Furthermore, the comet assay suggested that
exposure to PS-MPs induced DNA damage in earthworms. Overall, 1300 nm PS-MPs
showed more toxic effect than 100 nm PS-MPs on earthworms. These findings 2
provide new insights regarding the toxicological effects of low concentrations of
microplastics on earthworms, and on the ecological risks of microplastics to soil
Keywords: Microplastics; Earthworm; Histopathology; Oxidative damage; DNA
Capsule title： ：
DNA damage and histopathological changes in the earthworm Eisenia fetida after
exposure to low concentrations of PS-MPs were observed for the first time.
Microplastics (MPs), plastic fragments with particle size < 5 mm (Thompson et
al., 2009), belong to a group of emerging contaminants that have caused concern
because of their effects on the environment in recent years (Alimi et al., 2018).
Degradation of MPs and their removal from the environment is challenging owing to
their chemical inertia. MPs are ubiquitous and have been detected in different
ecosystems (Diepens and Koelmans, 2018). Microplastics can be divided into primary
and secondary MPs (Cole et al., 2011). Primary MPs mainly refer to man-made
plastic particles that are used as raw materials for industrial manufacture or
production of cosmetics (Cole et al., 2011). Secondary MPs, including large plastics 3
used in agricultural production, industrial production, and urban construction, can be
degraded in the environment by UV radiation or high temperature (Rillig et al., 2012;
Steinmetz et al., 2016). The sources of MPs in soil are varied, including the land
application of sewage sludge that carries MPs (Mintenig et al., 2017), agricultural
plastic film decomposition (Wang et al., 2013; Ramos et al., 2015; Steinmetz et al.,
2016), deposition of atmospheric MPs onto the soil (Dris et al., 2016), and surface
runoff (Gies et al., 2018; Lares et al., 2018).
Research on microplastic toxicity has mainly focused on aquatic environments,
while studies on terrestrial ecosystems are limited. Surveys in terrestrial environments
have focused on larger pieces of plastic, which are common in urban soils (Rillig,
2012). In addition, because of their adsorption characteristics, MPs entering the soil
do not adsorb only organic pollutants (Beckingham et al., 2017), but also act as
carriers of metals such as zinc, thereby improving their bioavailability (Hodson et al.,
2017). Owing to the feeding of soil animals, MPs accumulate in the soil food chain
(Huerta Lwanga et al., 2016, 2017). However, reports on the adverse effects of MPs
on soil organisms are still limited (Huerta Lwanga et al., 2016; Hodson et al., 2017;
Rodriguez-Seijo et al., 2017, 2019; Wang et al., 2019). Therefore, studies focusing on
the effect of MPs on terrestrial ecosystems and on soil organisms are important
(Horton et al., 2017).
Studies have shown that MPs can affect the growth, reproduction, and diversity
of soil animals (Rillig et al., 2017). Once MPs are ingested or accumulated by soil
animals, they can cause physical tearing of organs and tissues, and elicit an 4
inflammatory response to invasive heterogenic substances (Song et al., 2019).
Furthermore, MPs ingestion can also cause insufficient supply of nutrients and energy
to organisms (Huerta Lwanga et al., 2016; Rodriguez-Seijo et al., 2018; Yin et al.,
2018; Prata et al., 2020). In addition, the toxic substances released by MPs and the
toxic effects of adsorbed pollutants can adversely affect individual and species
diversity (von Moos et al., 2012; Wright et al., 2013; Hodson et al., 2017). The effect
of MPs on soil animals is related to particle size and concentration. So far, only few
studies have reported the concentrations of MPs in soils, which range from
55.5−67,500 mg/kg (Chae and An, 2018). MPs with particle size less than 1 mm can
be easily ingested by soil animals and can be further transported from the intestines to
other tissues via the intestinal wall (Farrell and Nelson, 2013). Browne et al. (2008)
observed that microplastics with particle size < 1 mm translocated more easily into
the circulatory system of the organism.
Earthworms are important animals in the soil food chain and play an essential
part in fertility, metabolism, and maintenance of the structure and function of soil
ecosystems (OECD, 2004). In addition, earthworms have high reproduction rate and
strong adaptability, but are also vulnerable to toxic and harmful substances in the
environment. Therefore, earthworms, especially Eisenia fetida, are widely used to test
the toxicity of pollutants. Although studies have investigated the toxic effects of MPs
on earthworms, they have mainly focused on growth, reproduction, movement, and
oxidative stress (Besseling et al., 2012; Rillig et al., 2017; Rodriguez-Seijo et al.,
2019; Wang et al., 2019), while histopathological changes, and DNA and oxidative 5
Prendergast-Miller et al., 2019). In this study, our aim was to examine the potential
toxic effects of widely used polystyrene microplastics (PS-MPs) on earthworms at
concentrations that are lower than those reported in polluted soils (Chae and An,
2018). Therefore, to improve our understanding of the ecological risk of PS-MPs in
soil ecosystems, we have measured the mortality, histopathology, and oxidative and
DNA damage in E. fetida exposed to fluorescent PS-MPs at concentrations of 100 and
1000 µg/kg soil.
2. Materials and methods
2.1. Preparation of microplastics test solution and earthworm culture
Fluorescent PS-MPs (100 nm and 1300 nm in size) with excitation and emission
wavelengths of 538 nm and 580 nm were purchased from a commercial company
(Tianjin BaseLine ChromTech Research Centre). The PS-MPs test solution was
prepared according to Lu et al. (2016) and Jiang et al. (2019) with slight modifications.
Briefly, the PS-MPs fluorescent microsphere emulsion solutions were ultrasonicated
for 10 min and dispersed in 10 mL deionized water with 100 mg/10 mL PS-MPs,
diluted to the final concentrations of 100 nm and 1300 nm PS-MPs (0, 100 µg/kg,
1000 µg/kg, respectively), and stored at 4℃. The 100 µg/kg concentration was
prepared by adding 5 µL PS-MPs emulsion solutions to 500 g artificial soil and
similarly, the 1000 µg/kg was prepared by adding 50 µL PS-MPs emulsion solutions 6
to 500 g artificial soil. The distribution characteristics of the PS-MPs were measured
using dynamic light scattering (DLS, ZEN1600, Malvern Instruments, Malvern, UK)
and scanning electron microscopy (SEM, Hitachi, S-3400, Japan). The DLS plots of
100 nm and 1300 nm PS-MPs were measured in triplicate. Furthermore, PS-MPs were
identified using Fourier transformed infrared spectrometry (FTIR, Bruker, TENSOR
The earthworms (E. fetida) used in this study were obtained from a breeding
farm in Jurong, China, and cultured under laboratory controlled conditions (20 ± 2 ℃)
according to OECD guidelines (2004). Prior to exposure, the earthworms were
acclimated to the artificial soil consisting of 70% industrial sand, 20% kaolin clay,
and 10% dried cow manure for at least one week, adjusted with CaCO3 to the pH 6.0
± 0.5 (OECD, 1984). For the tests, the adult earthworms that showed well developed
clitellum and showed body weight between 300 and 600 mg were selected, as
recommended by OECD and ISO guidelines (OECD, 2004; ISO, 2012).
2.2. Exposure of the earthworms to PS-MPs
Before the tests, the earthworms were cultured for 24 h under the same artificial
soil conditions. Ten earthworms were placed into 2 L glass beaker with 500 g of
artificial soil (dry weight) containing different concentrations of 100 nm and 1300 nm
PS-MPs (0, 100 µg/kg, or 1000 µg/kg) (Four replicates each group). The beakers
wrapped with gauze were then incubated at 20 ± 2 ℃ in the presence of 75% humidity 7
for 14 days. During the exposure, the soils were maintained at 40% maximum water
holding capacity (MWHC) with addition of ultrapure water. The earthworms were fed
with 5 g cow manure spread on the soil surface once a week. After 14 days of
exposure, the earthworms were rinsed with ultrapure water and depurated for 24 h
before further analyses.
2.3. Uptake and accumulation in earthworm intestines
The uptake and accumulation of PS-MPs in earthworm intestines was measured
according to Lu et al. (2016) with some slight modifications. Briefly, after 14 days of
exposure, three earthworms per treatment were depurated for 24 h on wet filter paper,
rinsed with ultrapure water, surface moisture removed with filter paper, and weighed.
Subsequently, the earthworms were transversely cut in the middle of the body and
then dehydrated and weighed. Intestinal tissues were digested with 1 mL HNO3 at
70 °C for 2 h and ultrapure water was added to a final volume of 5 mL. The contents
of PS-MPs in earthworm intestines were determined using a fluorescence
spectrophotometer (HITACHI F-7000) at excitation and emission wavelengths of 538
nm and 580 nm, respectively. A standard curve was obtained according to serial
dilutions of fluorescent PS-MPs suspensions (Figure S2). The background
fluorescence of the intestine from unexposed PS-MPs earthworms, which were
processed in the same way as exposed PS-MPs earthworm, was measured and
deducted from that of the treated samples. Each sample was measured in triplicate. 8
2.4. Biochemical assays
SOD activity and GSH levels were measured to assess oxidative damage in
earthworms exposed to PS-MPs. Briefly, after 14 days of exposure, earthworms from
the control group and those exposed to 100 nm or 1300 nm PS-MPs at concentrations
of 100 µg/kg or 1300 µg/kg were used for further analyses. Earthworms (three
individuals per treatment) were rinsed with ultrapure water, then rapidly cut into
pieces, and blended with ice-cold phosphate buffered saline (PBS). Subsequently, the
mixture solution was homogenized with an ultrasonic processor (JY-250, Zhejiang,
China) (5 x10s , intermittent 20s, 120W) in an ice bath and centrifuged at 4000 rpm,
4°C, for 10 min. The supernatants were used for GSH and SOD analyses. GSH levels
and SOD activity were determined using commercial kits (No. A006-1 and No.
A001-3, respectively) (Nanjing Jiancheng, China). Enzyme activity was normalized
by protein content (bicinchoninic acid assay) using a commercial kit (No. A045-4)
(Nanjing Jiancheng, China). Each test was performed in triplicate.
2.5. Histopathological analyses
Histopathological analyses were performed according to our previous publication
(Yang et al., 2018) with slight modifications. After 14 days exposure, 15 earthworms
(three earthworms per treatment) were placed on cold filter paper for 10 min before
dissection. The intestines of the earthworms were removed, transversely cut, and then 9
placed in 10% formalin buffer (pH 7.0 ± 0.2) for 24 h. The intestines were then
embedded in paraffin wax, cut with a microtome into 4-µm thick slices, and stained
with hematoxylin and eosin (H&E) for microscopic observation. Photos of freshly
dissected earthworm intestines (Figure S1) and detailed description of the
histopathological procedure are included in the supplementary materials.
2.6. Comet assay
Coelomocytes were obtained according to Eyambe et al. (1991), and the levels of
DNA strand breaks were determined according to the method of Singh et al. (1998).
Three earthworms were used per treatment. After electrophoresis, each slide was
washed three times with 0.5 M Tris buffer (pH 7.5) and the DNA was stained with
ethidium bromide. Slides were examined using a fluorescence microscope (BX41,
Olympus, Japan) and at least 50 cells were analyzed per slide. The extent of DNA
migration was determined as the percentage of tail DNA (% tDNA) and olive tail
moment (OTM) with the CASP software using the method of Collins et al. (1995).
2.7. Statistical analysis
The means and standard deviations (SD) for all treatments were calculated using
the SPSS 19.0 software. Differences between control and treated samples were
analyzed using the Mann-Whitney U-test. p < 0.05 was considered significant. Origin
9.0 mapping was used for drawing the figures.
3. Results and discussion
3.1. Characteristics of PS-MPs
The characteristics of the PS-MPs are shown in Figure 1. The DLS results
demonstrated that the average particle size of 100 nm PS-MPs was 105.4 nm and that
of 1300 nm PS-MPs was 1343.2 nm (Figure 1A and 1C). SEM images showed that
both 100 nm and 1300 nm PS-MPs had spherical structures and did not show
aggregates (Figure 1B and 1D). The FTIR spectrum of two sizes of PS-MPs showed
in Figure 1E and 1F. Briefly, the FTIR spectrum indicated that the peaks at 3080 cm-1,
3060 cm-1, and 3030 cm-1 were related to aromatic C–H stretching vibration. The
peaks at 2920 cm-1 and 2850 cm-1 were assigned to the stretching vibration of –CH2–.
The peaks at 1600 cm-1, 1490 cm-1, and 1450 cm-1 were attributed to C–C stretches in
the aromatic ring. The peaks at 698 cm-1 and 756 cm-1 were considered an aromatic
substitution pattern. The characteristic peaks and composition of two sizes of PS-MPs
were similar to that reported previously (Lu et al., 2016; Wu et al., 2019).
Figure 1. PS-MPs particle distribution characteristics. DLS of 100 nm PS-MPs (A), SEM of 100
nm PS-MPs (B), DLS of 1300 nm PS-MPs (C), and SEM of 1300 nm PS-MPs (D). FTIR
spectroscopy of 100 nm PS-MPs (E) and 1300 nm PS-MPs (F).
3.2. Growth change induced by PS-MPs
During the 14 days of exposure, the percent of mortality and the average loss of
earthworm body weight in the control group were 5 ± 0.250 % and 3.85 ± 0.513 %
(Figure 2), which was less than 10% and 20%, respectively. Therefore, our results are 12
considered to be valid according to the standard toxicity procedure (ISO, 2012).
Nevertheless, compared to the control, significant changes in mortality were observed
in exposed earthworms (except for earthworms exposed to 1000 µg/kg of 1300 nm
PS-MPs) (p < 0.05, Figure 2). Furthermore, the growth rate of the earthworms in each
treatment was higher than that of the control group (p < 0.05), with the growth rates
of 3.85 ± 0.513 %, 5.66 ± 0.456 %–11.82 ± 0.208 %, and 5.12 ± 0.186 %–12.29 ±
0.199 % in the control, 100 nm, and 1300 nm PS-MPs treatment groups, respectively.
The highest exposure concentration of PS-MPs (1000 µg/kg) in this study was still
lower than the concentrations used in studies on MPs toxicity to earthworms so far
(Wang et al, 2019; Rodriguez-Seijo et al., 2018, 2019). The observed increase in the
growth of the exposed earthworms (Figure 2) might reflect the hormetic effect caused
by low concentrations of MPs. Similar results regarding earthworm growth were also
observed by other researchers, where the chlorinated flame retardant Dechlorane Plus
did not inhibit the growth of earthworms (Yang et al., 2016). Therefore, we focused
more on changes at the molecular and histopathological levels to investigate the
potential toxic effects of PS-MPs on earthworms.
Control 100 nm 1300 nm
Growth rate (%)
Control 100 nm 1300 nm
PS-MPs (µg/kg soil)
PS-MPs (µg/kg soil)
Figure 2. Mortality (%) (A) and growth rate (%) (B) of earthworms in control and PS-MPs
treatment groups. Data represent mean ± SD (n = 3). Different letters indicate significant
differences between treatments (p < 0.05).
3.3. Antioxidant enzyme activities
In this study, the activities of SOD and levels of GSH in earthworms were
measured to assess the oxidative stress caused by PS-MPs after 14 days of exposure
(Figure 3). The activities of SOD in earthworms were significantly reduced (p < 0.05)
when exposed to 100 and 1300 nm PS-MPs. The SOD activities of earthworms were
10.566 ± 0.621, 9.039 ± 0.787–9.408 ± 0.493, and 7.959 ± 0.422–9.195 ± 0.327 U/mg
protein when exposed to control soil, and soil treated with 100 nm and 1300 nm
PS-MPs, respectively. In contrast, the levels of GSH in earthworms increased
significantly (p < 0.05) following treatment with 100 nm (100 µg/kg, 1000 µg/kg) and
1300 nm (100 µg/kg) PS-MPs, whereas no significant alteration was observed in the
1300 nm (1000 µg/kg) PS-MPs group. GSH levels in earthworms were 86.991 ±
7.723, 165.436 ± 4.256–167.767 ± 18.642, and 93.590 ± 4.279–173.98 ± 15.523
µmol/L when exposed to control soil, and that treated with 100 nm and 1300 nm
Enzymatic activities have been considered effective indicators of environmental
pollutants (Liang et al., 2013). SOD is an oxygen free radical scavenger and therefore
plays a crucial part in the enzymatic defense system (Liu et al., 2012). Although 14
compared to the control, SOD activity was significantly inhibited in all treatments (p
<0.05), the highest inhibition was observed when exposed to 1300 nm PS-MPs at
1000 µg/kg (Figure 3). SOD activity has been observed to increase as a direct
response to increasing levels of super oxide anion radicals, indicating that the
exposure of the earthworms to PS-MPs led to the production of reactive oxygen
species (ROS), which then promoted SOD biosynthesis to protect the cells from
oxidative damage (Zhang et al., 2014). In our study, the reason for decreased SOD
activity in all treatment groups might be the excess ROS production, which can lead
to overwhelming of the antioxidant defenses. Similar decreasing trend in SOD activity
has already been observed when E. fetida is exposed to nanoscale zerovalent iron
(Liang et al., 2017) and low-density polyethylene microplastics (Wang et al., 2019).
GSH is also part of the cellular antioxidant defense system that represents water
soluble reductants (Liu et al., 2012). We observed that the GSH level increased after
14 days exposure to 100 µg/kg of both 100 nm and 1300 nm PS-MPs, while at
concentration of 1000 µg/kg this was observed only for 100 nm PS-MPs. Overall,
GSH levels tended to increase with exposure of earthworms to 100 nm PS-MPs. The
increase in GSH levels indicated that the exposure to the PS-MPs might have
increased the production of ROS, which activated the biosynthesis of GSH to protect
the cells against oxidative damage. However, upon exposure to 1300 nm PS-MPs, the
GSH level in earthworms exposed to 1000 µg/kg was similar to that of the control.
This was due to the over-production of ROS in earthworms, which exceeded the
capacity of the antioxidant system and probably led to reduction in GSH levels (Liu et 15
al., 2012). These findings indicated that PS-MPs induced antioxidant defenses in
earthworms as a result of oxidative stress caused by PS-MPs exposure.
Control 100 nm 1300 nm
SOD (U/mg prot)
Control 100 nm 1300 nm
PS-MPs (µg/kg soil)
PS-MPs (µg/kg soil))
Figure 3. Superoxide dismutase (SOD) activity and glutathione (GSH) level in earthworms after
14 days of PS-MPs exposure. Data represent mean ± SD (n = 3). Different letters indicate
significant differences between treatments (p < 0.05).
3.4. Histopathological changes induced by PS-MPs in earthworm intestinal tissue
Uptake of 100 nm and 1300 nm fluorescent PS-MPs was detected in earthworm
intestines after 14 days of exposure (Figure 4). All tested PS-MPs were found to
concentrations were higher for 1300 nm PS-MPs (p < 0.05). We also observed that
accumulation of PS-MPs was significantly higher at 1000 µg/kg than at 100 µg/kg.
Average concentrations in earthworm intestines for 100 nm/100 µg/kg, 100 nm/1000
µg/kg, 1300 nm/100 µg/kg, and 1300 nm/1000 µg/kg PS-MPs were 0.015 ± 0.001,
0.033 ± 0.002, 0.084 ± 0.005, and 0.094 ± 0.003 µg/mg, respectively. Uptake of MPs
in a size-dependent manner has been well investigated in marine species, and the
results indicate that ingested MPs significantly depend on their size (Wright et al.,
2013). In addition, we also examined histopathological changes induced by PS-MPs
in the intestines of E. fetida after 14 days of exposure (Figure 5). In the control group,
all observed sections showed normal histology of earthworm intestines (visible cell
divisions, regular shape and cell nucleus size). However, intestines of earthworms
exposed to PS-MPs showed enlarged intestinal cells with irregular shapes and altered
size of cell nuclei (Figure 5). The aberrant histopathology was more pronounced in
the 1300 nm (1000 µg/kg) PS-MPs treated samples than in the 100 nm PS-MPs
treated samples. In addition, more intestinal cell lysis was observed in the 1300 nm
(1000 µg/kg) PS-MPs treated group. Lu et al. (2016) observed that 2000 µg/L of
PS-MPs (both 5 µm and 20 µm) induced inflammation in damaged zebrafish tissues
(gill, liver, and gut) and that 5 µm PS-MPs accumulated more than 20 µm PS-MPs. It
is well-known that the smaller MPs tend to be retained more in the earthworm
intestine due to their feeding selectivity (Curry and Schmidt, 2007; Huerta Lwanga et
al., 2016; Rodriguez-Seijo et al., 2017), which differs from our observations. This
could be due to the small size of the particles used in our study (100 and 1300 nm)
compared to other studies that have used larger particles (minimal particle size was 5
µm), where depuration rate might not depend on the particle size. Owing to
differences in the ways of exposure (artificial soil, OECD, 2004) and sizes of MPs
used (Huerta Lwanga et al., 2016; Rodriguez-Seijo et al., 2017), the obtained results
cannot be compared easily. However, higher accumulation and more aberrant 17
histopathology of earthworms caused by higher concentrations of MPs, was also
observed by other researchers (Rodriguez-Seijo et al., 2017). These findings are in
agreement with the observed higher mechanical damage of the earthworm intestines
caused by 1300 nm PS-MPs than those caused by 100 nm PS-MPs.
PS-MPs (µg/mg intestine)
PS-MPs (µg/kg soil)
Figure 4. Concentrations of PS-MPs in earthworm intestines. Data represent mean ± SD (n =3).
Different letters indicate significant differences between treatments (p < 0.05).
Figure 5. Intestinal damage caused by PS-MPs. I: control; II: 100 nm 100 µg/kg; III: 1300 nm
100 µg/kg; IV: 100 nm 1000 µg/kg; V: 1300 nm 1000 µg/kg; A0: cell division; B0: regular cell
shape; C0: regular nucleus size; A: enlarged cell space; B: irregular cell shape; C: irregular nucleus
size; D: cell lysis.
3.5. DNA damage
Comet assay is an effective method for assessing DNA damage induced by
environmental pollutants, and is widely considered a sensitive biomarker for
determining and evaluating the genotoxicity of pollutants on invertebrates (Reinecke
et al., 2004; Saez et al., 2015, Gajski et al. 2019). In our study, DNA damage in
earthworms depended on the PS-MPs particle size and their concentration. Higher
concentration of PS-MPs (1000 µg/kg) increased DNA damage irrespective of particle
size (Figure S3). Exposure to PS-MPs increased the percentage of tDNA and OTM in 19
a dose-dependent manner. The results showed that both 100 and 1300 nm PS-MPs
elicited high levels of DNA damage (p < 0.05) (Figure 6). In particular, 1300 nm
PS-MPs appeared to induce more damage to earthworm DNA than 100 nm PS-MPs.
At the same concentration the % tDNA and OTM in 1300 nm PS-MPs were
significantly higher than for the 100 nm PS-MPs. When exposed to the same particle
size the % tDNA and OTM were, as expected, significantly higher at 1000 µg/kg than
at 100 µg/kg. Higher concentrations and larger particle size of PS-MPs caused an
increase of % tDNA and OTM indicating severe DNA damage in earthworms, which
were consistent with the observed size-dependent histopathological changes and the
results of the oxidative stress assays (Wright et al., 2013). Therefore, we assumed that
the ROS accumulation in earthworm tissues may be responsible for the subsequent
DNA damage in earthworm coelomocytes (Song et al., 2019).
4 Control 100 nm 1300 nm
Control 100 nm 1300 nm
PS-MPs (µg/kg soil)
PS-MPs (µg/kg soil)
Figure 6. DNA damage in earthworms after 14 days of exposure to PS-MPs. The bars represent
standard deviation (n =3). Different letters indicate significant differences between treatments (p <
This is the first study to reveal the toxicological effects of low concentrations of
PS-MPs (≤1 mg/kg of soil) on earthworms (Eisenia fetida). According to the growth
measurements, histological changes, and the results of DNA damage and oxidative
stress analyses, the acute toxicity of 100 and 1300 nm PS-MPs to earthworms appear
to be extremely low. However, exposure to PS-MPs with particle size larger than 100
nm caused DNA damage and oxidative stress in earthworms. Furthermore, we also
observed that PS-MPs exposure can cause histopathological damage in earthworm
intestines, especially when exposed to 1300 nm particles at 1000 µg/kg. Moreover, the
1300 nm PS-MPs were more toxic and accumulated to higher concentrations in the
earthworm intestines than 100 nm PS-MPs. These findings provide new insights
regarding the toxicological effects of low concentrations of microplastics on
earthworms and on the ecological risks of microplastics to soil animals.
Conflicts of interest The authors declare that they have no conflicts of interest.
This research was supported by National Natural Science Foundation of China
(No. 41773115, 41571468), Science and Technology Support Program of Jiangsu 21
Province (No. BE2016736) and Nanjing University Innovation and Creative Program
for PhD candidate (No. CXCY19-61). We also thank the anonymous reviewers and
the handling editor for their positive and suggestive comments regarding our study.
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This study is the first to reveal the adverse effects of PS-MPs at low concentrations on earthworms.
100 and 1300 nm PS-MPs can induce oxidative stress, histopathological changes and DNA damage in earthworms.
1300 nm PS-MPs were more toxic and accumulated to a greater extent in the earthworm intestines than 100 nm PS-MPs.
Author Contribution Statement
Conceptualization: Mei Li. Data curation: Xiaofeng Jiang, Yeqian Chang, Mei Li. Formal analysis: Xiaofeng Jiang. Funding acquisition: Mei Li. Investigation: Xiaofeng Jiang, Tong Zhang, Yu Qiao. Methodology: Mei Li, Xiaofeng Jiang, Göran Klobučar. Project administration: Mei Li. Resources: Xiaofeng Jiang. Software: Xiaofeng Jiang, Yeqian Chang. Supervision: Mei Li. Validation: Mei Li. Visualization: Xiaofeng Jiang, Yeqian Chang. Writing - original draft: Mei Li, Xiaofeng Jiang, Göran Klobučar. Writing - review & editing: Mei Li, Xiaofeng Jiang, Göran Klobučar.
Declaration of interests ☒ 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. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: