Science of the Total Environment 783 (2021) 147016

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Effects of polystyrene microplastics on uptake and toxicity of

phenanthrene in soybean
Guanghui Xu a,b, Yang Liu a,b, Yong Yu a,⁎
a
Key Laboratory of Wetland Ecology and Environment, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130102, China
b
University of Chinese Academy of Sciences, Beijing 100049, China

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

• Microplastics (MPs) decreased the up-

take of phenanthrene in soybean.
• MPs and phenanthrene co-exposure
caused higher toxicity to soybean tissue.
• Micron-size MPs and phenanthrene ex-
hibited higher genotoxicity to soybean.
• Micron-size MPs inhibited relative
abundance of Proteobacteria in
rhizosphere soil.

a r t i c l e i n f o a b s t r a c t

Article history: Microplastics (MPs) can influence the availability of contaminants in the soil and have adverse effects on plants.
Received 23 February 2021 Up to now, the effects of MPs on the uptake of organic pollutants by leguminous plants are still unclear. In this
Received in revised form 4 April 2021 study, we explored the impacts and mechanisms of polystyrene MPs of different sizes on the uptake of phenan-
Accepted 4 April 2021
threne (Phe) by soybean seedlings. The results showed that MPs decreased the uptake of Phe in soybean roots
Available online 10 April 2021
and leaves. Micron-size MPs showed a higher inhibition of Phe uptake in roots than nano-size MPs (4.83
Editor: Jay Gan mg/kg) at the beginning with concentrations of 1.89 mg/kg, 3.40 mg/kg, and 0.72 mg/kg in groups 1 μm, 10
μm, and 100 μm MPs/Phe, respectively. The combined toxicity of micron-size MPs and Phe to soybean plants
was higher than that of nano-size MPs and Phe, and 100 μm MPs and Phe co-contaminant show the highest tox-
Keywords: icity to soybean. The activities of antioxidative enzymes and their gene expression showed that micron-size MPs
Microplastics induced higher genotoxic and oxidative damage to soybean roots than nano-size MPs, which decreased the ac-
Phenanthrene tivity of roots, thus leading to the lower uptake of Phe by soybean roots and leaves. This study highlights that
Soybean the combined exposure to MPs and Phe causes harmful effects on soybean plants and MPs inhibit the uptake
Rhizospheric microorganism
of organic pollutants by higher plants.
Toxicity
© 2021 Elsevier B.V. All rights reserved.

1. Introduction et al., 2019). It has been estimated that about 63,000–430,000 and
44,000–300,000 tons of MPs enter farmland soils per year with the ap-
Microplastics (MPs) are a diverse group of high-molecular polymer plication of sewage sludge in Europe and North America, respectively
particles (<5 mm) (Wright et al., 2013), which are widely distributed (Nizzetto et al., 2016). They may also enter the farmland by the frag-
in marine and terrestrial ecosystems (Collard et al., 2019; Lenaker mentation of agricultural films, sewage irrigation and application of or-
ganic fertilizer (Ng et al., 2018).
⁎ Corresponding author. So far, a few works have studied effects of MPs on plants. They could
E-mail address: [email protected] (Y. Yu). be adsorbed on the root surface of plants (Urbina et al., 2020), and

https://doi.org/10.1016/j.scitotenv.2021.147016
0048-9697/© 2021 Elsevier B.V. All rights reserved.
G. Xu, Y. Liu and Y. Yu Science of the Total Environment 783 (2021) 147016

reduce the water conductivity in roots, which hinder the transpiration, Seed Company, Changchun, China. ELISA kits and root activity Kits were
nutrient uptake, and growth of plants. Polyethylene (PE) MPs were purchased from Shanghai MLBIO Biotechnology Co. Ltd. and Shanghai
also reported to impact the germination of seed by blocking the pores Yuanye Biotechnology Co., Ltd. (Shanghai, China), respectively.
of seed capsule (Bosker et al., 2019). The size of MPs is a critical factor
in eco-toxicological risk assessments for plants. Growth limitations 2.2. Exposure of soybean to MPs
and biomass decrement are observed in wheat after exposure to
micron-size MPs (Qi et al., 2018). Nano-size MPs can be taken up by The soil was collected from farmland in Northeast Institute of Geog-
plants and have adverse impacts on their seed germination and growth. raphy and Agroecology, Chinese Academy of Sciences, Changchun,
They can penetrate cell wall, enter the cell and cause adverse effects to China. The physical and chemical properties of the soil are shown in
root-tip cells (Jiang et al., 2019). Bandmann et al. revealed that tobacco Table S1. Phe was dissolved with acetone and added into the soil to
cells could incorporate 20–40 nm MPs in the process of cell culture achieve 1 mg/kg. After that, the soil was placed in the fume cupboard
(Bandmann et al., 2012). In a previous study, nano-size MPs exhibited for 48 h to evaporate the solvent. Then, the soil was amended with fluo-
size-dependent growth inhibition rates, but 5 μm MPs had little effect rescent MPs to achieve 10 mg/kg. Ten groups were set up: T1, Phe +
on the growth of Chlorella pyrenoidosa (Li et al., 2020). Smaller MPs sig- 100 nm MPs; T2, Phe + 1 μm MPs; T3, Phe + 10 μm MPs; T4, Phe +
nificantly affect fertility and growth of algae and photosynthesis is also 100 μm MPs; T5, Phe alone; T6, no Phe or MPs (CK); T7, 100 nm MPs;
disturbed (Chen et al., 2020). In addition, plants can take up and trans- T8, 1 μm MPs; T9, 10 μm MPs; T10, 100 μm MPs.
locate nano-size MPs (Dong et al., 2021; Huang et al., 2021; Khalid et al., Eight soybean seeds were planted in a 5000 ml box with 5000 g of
2020). MPs in the hundred-micrometer range have no significant toxic spiked soil and placed in a greenhouse at 25 °C and 50% humidity for
effects to diatom (Guo et al., 2020), whereas nano-size MPs can accu- 30 days after sprouting. The soybean plants were sampled on day 10,
mulate in roots and induce higher genotoxic and oxidative damage to 20, 30, and cut into root, stem and leaf. The samples were lyophilized
broad bean than micron-size MPs (Jiang et al., 2019). and stored in a refrigerator (−20 °C) until further analysis.
Higher plants are widely used to detect and evaluate the toxicity of
pollutants, such as heavy metals, nanomaterials and MPs (Dong et al., 2.3. Sample analysis
2021; Ge et al., 2018; Urbina et al., 2020). Soybean is an important cash
crop and the fifth largest crop in agricultural production in the world One gram soybean plant samples (root, stem and leaf; dry weight)
(Yusefi-Tanha et al., 2020). Studies on potential toxic effects and mecha- were placed in 10 ml of HNO3 at 70 °C for 5 h until the samples were
nisms of MPs have not been well explored in cash crops. Due to the large digested completely. After that, the samples were added with 1%
surface area and functional groups, MPs can adsorb organic pollutants HNO3 solution to 10 ml and the concentrations of MPs were determined
and impact their translocation in aquatic plants (Kalcikova et al., 2017). by a fluorescence spectrophotometer (RF-5301PC, Shimadzu, Japan) at
Until now, effects of MPs with different sizes on the toxicity of organic excitation and emission wavelengths of 570 nm and 526 nm, respec-
contaminants to cash crops are relatively scarce. tively. The concentrations of Phe in root, stem and leaf of soybean
Polycyclic aromatic hydrocarbons (PAHs) are a class of organic pol- were measured by a gas chromatography–mass spectrometry, (GC–
lutants and have become a worldwide environmental problem. PAHs MS, Trace 1300 ISQ, Thermo Fisher, USA). The details are shown in the
can be taken up by plants and cause toxicity to plants (Zhan et al., Supplementary Material.
2015). The accumulation of PAHs in plants decreases the chlorophyll The soybean plant samples were weighed and disrupted with ho-
content, plant height, fresh weight and induces oxidative stress (Meng mogenizer in phosphate buffer saline (PBS) solution (pH = 7.4). The
and Chi, 2015; Shen et al., 2017). A previous study found that MPs de- mixture was put into a tube and centrifuged at 3000 r/min, 4 °C for 20
creased the toxicity of phenanthrene (Phe) to diatom (Guo et al., min. The supernatant was collected to measure physiological indexes,
2020). In addition, Tang et al. reported that (Tang et al., 2020) the tox- including peroxidase (POD), superoxide dismutase (SOD), catalase
icity of PAHs was generally aggravated by nano-size MPs and mitigated (CAT), malondialdehyde (MDA) and Reactive Oxygen Species (ROS)
by micron-size MPs. However, effects of MPs on the uptake and accu- by ELISA kits. The root activity was determined by the Kit.
mulation of PAHs by higher plants and mechanisms have not been clar-
ified. Their combined toxicity on higher plants has also rarely been 2.4. Analysis of gene expression and high-throughput sequencing
studied at the gene expression level.
Higher plants are the primary basis of many food chains, and the ac- Soybean roots were ground in an ice bath with a glass homogenizer.
cumulation of PAHs in higher plants may have adverse impacts on the Total RNA of soybean roots were extracted with 1 ml TRIzol Reagent
production, quality and safety of foods. The aims of this study were to (Invitrogen, Carlsbad, CA, USA). The absorbance was determined by an
study the uptake and toxicity of Phe in different parts of soybean in ELIASA (Synergy H1, Biotek, USA). cDNA was synthesized by the testing
the presence of polystyrene (PS) MPs with different sizes, and to reveal kit (FSQ-101; TOYOBO, Shanghai, China) and then subjected to a quan-
their individual and combined genotoxicity. Our study will contribute to titative polymerase chain reaction (q-PCR, Mx3000p, Agilent, USA).
a comprehensive understanding toward the toxic effects of MPs on Fluorescence Quantitative Detection Kits (TIANGEN, Beijing, China)
higher plants. were employed for q-PCR analysis. High-throughput 16S rRNA sequenc-
ing was performed to detect the microbial community in rhizosphere
2. Materials and methods soil. Details of q-PCR reaction system, gene primers (Table S2), 16S
rRNA gene amplification, high-throughput sequencing, and bioinfor-
2.1. Materials matic analysis are shown in the Supplementary Material. The relative
expression levels were measured by the 2−ΔΔCt method.
Fluorescent PS MPs (100 nm, 1 μm, 10 μm and 100 μm) were pur-
chased from Tianjin BaseLine ChromTech Research Centre, Tianjin, 2.5. Statistical analysis
China. The functional groups, elemental composition and morphology
of MPs were measured by Fourier transform infrared spectroscopy All the samples were analyzed in triplicate. Data were presented as
(FT-IR, Nicolet NEXUS-670, Thermo Fisher, USA), transmission electron the mean ± standard deviation (SD) and analyzed by SPSS 22.0 soft-
microscope (TEM, JEM-2100F, JEOL, Japan) and scanning electronic mi- ware. The analysis of variance (ANOVA) and LSD test were performed
croscopy, (SEM, SU-8010, Hitachi, Japan). Phe (purity 97.0%) was pur- to examine the differences among groups. The differences were consid-
chased from Macklin Industrial Corporation, Shanghai, China). ered significant at p < 0.05. Origin 8.0 software was used to draw the
Soybean seeds (Glycine max L. Merrill) were obtained from Jilin Province figures.

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G. Xu, Y. Liu and Y. Yu Science of the Total Environment 783 (2021) 147016

3. Results 3.2. Oxidative stress of Phe and MPs to soybean plants

3.1. Occurrence of Phe in soybean tissues Biomarkers were measured to evaluate the toxic effects of contami-
nants to soybean plants (Figs. 2, S2, S3). The contents of ROS and MDA
The characteristics of the PS MPs are shown in the Supplementary (Fig. 2A and B) in roots increased significantly (p < 0.05) in all the treat-
Material (Fig. S1). MPs in soybean plants were not detected. As shown ment groups, and the highest content was found in group T4. CAT activity
in Fig. 1A, Phe in soybean roots in groups T5, T1 and T3 (Phe alone, in roots showed ascending trends with exposure time in all treatment
100 nm, and 10 μm MPs/Phe) showed the same trend, which decreased groups except T4, where CAT activity decreased slightly after 20 days
at day 30 compared with day 20. In groups M2 and M4 (1 μm and 100 and then increased significantly (Fig. 2C). Similarly, SOD (Fig. 2D) and
μm MPs/Phe), concentrations of Phe in soybean roots increased POD (Fig. 2E) activity also increased with exposure time in all treatment
significantly on day 30. The highest level of Phe was found in T5 com- groups except T4 and T5, respectively. The activity of SOD in group T4 in-
pared with other groups on day 20, indicating that MPs may restrain creased significantly at the beginning and then decreased. POD activity in
the uptake of Phe in soybean roots. As shown in Fig. 1b, Phe in soybean group T5 decreased significantly with exposure time.
stems in groups T5, T2 and T3 (Phe alone, 1 μm, and 10 μm MPs/Phe) The contents of ROS, MDA and CAT (Fig. S2A, S2B and S2C) in stems
exhibited the same trend, and the highest concentrations of Phe were increased significantly (p < 0.05) in groups T1-T6 with exposure time.
found on day 20. In groups T1 and T4 (100 nm and 100 μm MPs/Phe), The activities of SOD and POD increased significantly in groups T1-T6
the concentrations of Phe in soybean stems increased significantly with exposure time except group T3, where they decreased slightly at
with exposure time. The concentrations of Phe in soybean leaves are the beginning and then increased significantly. The biomarkers in leaves
shown in Fig. 1c. Phe in leaves in groups T5 and T1 (Phe alone, increased significantly, and showed higher values in group T5 compared
100 nm MPs/Phe) decreased significantly on day 30. In groups T2, T3 with other groups (Fig. S3). However, the biomarkers in MPs alone ex-
and T4, the concentrations of Phe in soybean leaves increased posure groups had no obvious change in soybean stems and leaves ex-
significantly at the end of exposure time. Generally, MPs decreased cept CAT and SOD in leaves, where they showed an ascending trend.
the accumulation of Phe in soybean roots and leaves, and increased
the accumulation of Phe in soybean stems. The occurrence of Phe in 3.3. Effects of MPs and Phe on root activity and gene expression
stems and leaves revealed its acropetal translocation in soybean. The
distribution of Phe in soybean plants followed the descending order The activity of soybean roots is shown in Fig. 3. In general, root activ-
roots > stems > leaves, and the uptake of Phe by soybean plants can ity increased with exposure time, and was significantly affected by indi-
be attributed to root uptake and acropetal translocation. vidual or combined MPs and Phe treatment. The activity of soybean

Fig. 1. The concentration of phenanthrene in different parts of soybeans, (A) roots, (B) stems, (C) leaves. Different letters indicate significant differences in same day (p < 0.05).

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G. Xu, Y. Liu and Y. Yu Science of the Total Environment 783 (2021) 147016

Fig. 2. Antioxidant enzyme activities in soybean roots. (A) ROS, (B) MDA contents, and (C) CAT, (D) SOD, (E) POD activity. Different letters indicate significant differences between
treatments in the same day.

roots was lower in all treatment groups compared with CK, with the 3.4. Effects of MPs and Phe on rhizosphere soil microbial community
lowest activity in group T4.
The relative expression levels of three antioxidant enzyme genes are The results of 16S rRNA genes in rhizosphere soil characterized
shown in Fig. 4. The relative expression levels of SOD (Fig. 4A) gene in all by high-throughput sequencing are shown in Figs. 5, 6, S4 and S5.
treatments were significantly higher than that in CK group (p < 0.05) Venn and flower diagrams show that 2558 OTUs were shared in
except group T4 (0.76), with the highest expression in group T2 rhizosphere soil from all the groups, and 2969 OTUs were shared
(1.88). The relative expression of CAT gene (Fig. 4B) was inhibited in in rhizosphere soil from co-exposure groups (Fig. 5). The numbers
groups T1-T5 (0.88, 0.81, 0.45, 0.64, and 0.65), and significantly lower of unshared OTUs in rhizosphere soils of co-exposure groups
than that in CK group. In contrast, the relative expression of CAT gene were lower than the CK group. The relative abundance of
was stimulated after exposure to MPs individual treatments. The rela- Proteobacteria decreased significantly in rhizosphere soils from
tive expression of POD gene (Fig. 4C) was also stimulated after exposure co-exposure groups (groups T1-T4), whereas the relative abun-
to pollutants. The relative expression levels in co-exposure groups were dance of cyanobacteria, and chloroflexi increased significantly
higher than other groups. (Fig. 6A).

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G. Xu, Y. Liu and Y. Yu Science of the Total Environment 783 (2021) 147016

to Phe and MPs compared with CK and showed the lowest abundance
in group T4 (100 μm MPs/Phe). The abundance of Cyanobacteria in-
creased significantly in groups T2, T3 and T4 compared with other
groups.
The results of PCoA plots based on the Bray-Curtis distance (Fig. S4)
indicated that the rhizosphere soil community structures among differ-
ent groups were significantly separated (P < 0.01). The PCoA primary
(PC1) and secondary axis (PC2) displayed the separation of the rhizo-
sphere soil, explaining 35.6% and 16.7% inter sample variation. After
co-exposed to contaminants, the Alpha-diversity in groups T1-T4 in-
creased significantly in soybean rhizosphere soils compared with
other groups (Fig. 6B), which can be confirmed by the rarefaction curves
of OTUs (Fig. S5).

4. Discussion

4.1. Effects of MPs on Phe uptake and translocation in soybean plants

Nano-size (40 nm) and micron-size (1 μm) PS-MPs can accumulate

Fig. 3. The activity of soybean roots. Different letters indicate significant differences at root surface of wheat, especially at the root tip (Taylor et al., 2020).
between treatments in the same day.
Micron-size MPs affect the root growth by mechanical blocking
(Kalcikova et al., 2017). In this study, MPs inhibited the uptake of Phe
by soybean roots from soil, which can be attributed to the adsorption
The structure and abundance of the rhizosphere soil microbiotas of of MPs on the soybean roots and blocking the connections between
soybean plants were affected by the co-exposure of Phe and MPs with cells or cell wall pores for transport of contaminants (Jiang et al., 2019).
different sizes. At the phylum level, Proteobacteria, Bacteroidota, The uptake of Phe by roots was significantly different in treatment
Actinobacteriota and Firmicutes were the most abundant phyla in rhizo- groups due to the changes in root activity. In contaminated soils, Phe
sphere soil microbiotas, accounting for 90% of all sequence reads. The is adsorbed on the root surface upon activation in rhizosphere, and
abundance of Proteobacteria decreased dramatically after co-exposure transported to the other parts through apoplastic and symplastic uptake

Fig. 4. Relative mRNA levels of (A) SOD, (B) CAT and (C) POD in soybean roots on day 30. Different letters indicate significant differences between treatments (p < 0.05).

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G. Xu, Y. Liu and Y. Yu Science of the Total Environment 783 (2021) 147016

Fig. 5. Venn and flower diagrams showing the number of bacterial OTUs shared in the rhizosphere soils among different treatments. (A) The number of shared OTUs was counted between
samples for each of the MPs and phenanthrene co-exposure groups. (B) The number of shared OTUs was observed between the CK and treatment groups.

(Redjala et al., 2010). Due to their insolubility, PS-MPs can be adsorbed was related to the MP sizes, consistent with the results of the oxidative
onto the surface of soybean roots (Dong et al., 2020). In soils, MPs were stress assays (ROS, MDA, CAT, SOD and POD enzyme). Gene injury could
adsorbed on the surface of soybean roots, and they may compete with be related to oxidative stress induced by ROS (Jiang et al., 2019). There-
Phe for adsorption sites and affect the absorption of Phe by influencing fore, damages in soybean tissues partially contributed to the accumula-
the root activity and transpiration (Li et al., 2021a; Li et al., 2021b). tion of Phe by soybean stems.
MPs can reduce the photosynthetic rate of plants by changing the re- The leguminous plant root cell wall pores have an average radius of
sponse of guard cells, resulting in partial closure of stomata and de- 7–20 nm (Hylmö, 1958), MPs accumulate at the root surface of soybean
crease in stomatal conductance (Dong et al., 2020). The decrease of and barely enter the plant due to their larger size. Therefore, the uptake
root activity would decrease the transpiration rate, thus reducing the of nutrients and water through plant root tips were blocked and the ac-
ability to absorb water. Phe could induce the thylakoid membrane tivity of roots was inhibited (Jiang et al., 2019). Due to the inhibition of
damage, destroy chlorophyll structure and reduce activity of MPs on root activity, the ability of taking up Phe was restricted, there-
photosynthesis-related enzymes (Guo et al., 2020), thus affecting fore, the concentrations of Phe in soybean roots and leaves were re-
plant growth and elimination of contaminants. duced. A previous study has also found that MPs can induce the
accumulation of As in rice by decreasing the root activity (Dong et al.,
4.2. Toxicity to soybean plants by Phe and MPs 2020). These results revealed that the size of MPs is an important factor
in determining the uptake and distribution of Phe in crops.
MPs inhibited the accumulation of Phe in soybean roots and leaves. MPs in soils enhanced the toxicity of Phe to soybean plants, and the
Meanwhile, the presence of MPs amplified the negative effects caused micron-size MPs led to more oxidative damage and affected the gene
by Phe. ROS would be generated in plant tissue when the plant is ex- expression of soybean roots. The relative gene expression levels of
posed to pollutants. In this study, the activities of SOD, POD and CAT SOD, POD, CAT and contents of MDA and ROS indicated the chronic tox-
were inhibited under Phe stress, leading to the accumulation of ROS icity of micron-size MPs to soybean roots and leaves, which may explain
and damage of cell membrane. The contaminants could change the ter- the decrease of Phe in roots and leaves in groups T2-T4. To further ex-
tiary structure of enzyme proteins and result in the decrease of antiox- plore its mechanism, microbial community structures in soybean rhizo-
idant enzyme activity (Jobby et al., 2016). In addition, the expression of sphere soil were measured by high-throughput 16S rRNA sequencing
relative gene was inhibited, indicating that Phe caused the damage to analysis.
enzyme genes and further led to the decrease of antioxidant enzyme ac-
tivity. The mechanism of oxidative stress induced by MPs is different 4.3. Co-contaminant effects on soybean rhizosphere soil microbiota
from Phe. MPs are not able to enter plant cells, but they may cause me-
chanical damage to plant roots (Minibayeva et al., 2015) and increase The microbiota community composition and structure of soybean
the activity of antioxidant enzymes. ROS contents and SOD and CAT ac- rhizosphere soils were significantly changed after co-exposure of MPs
tivities in treatment groups were higher than CK group, suggesting that and Phe. MPs can affect soybean root activity, and exposure to MPs
soybean plants responded to excess ROS by increasing enzyme activity. may alter root exudates and thus lead to changes in the rhizosphere
However, overload of ROS may exceed the tolerance of plant cells, caus- soil microflora. Previous studies have also revealed that MPs impacted
ing the cell damage and inhibiting the expression of genes. The increase rhizosphere soil microbiota via changing soil physical properties and
of MDA revealed that MPs and Phe induced the membrane lipid perox- nutrient conditions in agricultural soils (Huang et al., 2019; Ren et al.,
idation in soybean tissues. 2020; Wang et al., 2020). Bacteroidetes are considered to be specialized
MPs increased the accumulation of Phe in soybean stems at the end in degrading complex organic matter in biosphere, which are widely
of experiment. MPs and Phe co-exposure treatment damaged the distributed in various ecosystems (Wolińska et al., 2017). Proteobacteria
soybean plant cells and micron-size MPs (100 μm) caused the highest prefer to colonize in nutrient-rich and low bulk density soils. Recently,
toxicity to cells, which is consistent with the higher accumulation of researchers found that PE MPs increased the rates of soil water evapora-
di-butyl phthalate in lettuce by micron-size MPs (Gao et al., 2021). In tion and decreased the soil moisture contents (Wan et al., 2019), which
this study, we further demonstrated that gene injury in soybean roots could change microbial community in soil. In this study, rhizosphere soil

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G. Xu, Y. Liu and Y. Yu Science of the Total Environment 783 (2021) 147016

Fig. 6. Relative abundance of bacterial at phylum (A) and clustering map of species abundance (B) in the rhizosphere soil among different treatments. Categories with relative abundance
<1% were clustered into “Other”.

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G. Xu, Y. Liu and Y. Yu Science of the Total Environment 783 (2021) 147016

diversity decreased significantly after exposure to MPs and Phe communities during the reproductive growth phase than during vegetative growth.
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Declaration of competing interest Nizzetto, L., Futter, M., Langaas, S., 2016. Are agricultural soils dumps for microplastics of
urban origin? Environ. Sci. Technol. 50, 10777–10779.
Qi, Y., Yang, X., Pelaez, A.M., Huerta Lwanga, E., Beriot, N., Gertsen, H., Garbeva, P., Geissen,
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