Toxicological effects of polystyrene microplastics on earthworm (Eisenia fetida)

Toxicological effects of polystyrene microplastics on earthworm (Eisenia fetida)

Journal Pre-proof Toxicological effects of polystyrene microplastics on earthworm (Eisenia fetida) Xiaofeng Jiang, Yeqian Chang, Tong Zhang, Yu Qiao, ...

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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:

S0269-7491(19)34434-3

DOI:

https://doi.org/10.1016/j.envpol.2019.113896

Reference:

ENPO 113896

To appear in:

Environmental Pollution

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.

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Toxicological effects of polystyrene microplastics on earthworm

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(Eisenia fetida)

3

4

Authors:

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Xiaofeng Jianga, Yeqian Changa, Tong Zhanga, Yu Qiaoa, Göran Klobučarb, Mei Lia,*

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Affiliations of authors:

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a

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Environment, Nanjing University, Nanjing 210023, China

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b

State Key Laboratory of Pollution Control and Resource Reuse, School of the

Department of Biology, Faculty of Science, University of Zagreb, 10000 Zagreb, Croatia

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Corresponding authors:

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Mei Li (Address: 163 Xianlin Ave., Nanjing University, Nanjing 210023, China)

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Email address: [email protected]

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1

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Abstract

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Microplastics are plastic fragments of particle sizes less than 5 mm, which are widely

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distributed in marine and terrestrial environments. In this study, earthworms Eisenia

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fetida were exposed to 100 and 1000 µg of 100 nm and 1300 nm fluorescent

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polystyrene microplastics (PS-MPs) per kg of artificial soil for 14 days. Uptake or

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accumulation of PS-MPs in earthworm intestines, histopathological changes,

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oxidative stress, and DNA damage were assessed to determine the toxicological

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effects of PS-MPs on E. fetida. The results showed that the average accumulated

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concentrations in the earthworm intestines were higher for 1300 nm PS-MPs (0.084 ±

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0.005 and 0.094 ± 0.003 µg/mg for 100 and 1000 µg/kg, respectively) than for 100 nm

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PS-MPs (0.015 ± 0.001 and 0.033 ± 0.002 µg/mg for 100 and 1000 µg/kg,

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respectively). In addition, histopathological analysis indicated that the intestinal cells

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were damaged after exposure to PS-MPs. Furthermore, PS-MPs significantly changed

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glutathione (GSH) level and superoxide dismutase (SOD) activity. The GSH levels

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were 86.991 ± 7.723, 165.436 ± 4.256–167.767 ± 18.642, and 93.590 ± 4.279–

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173.980 ± 15.523 µmol/L in the control, 100 nm, and 1300 nm PS-MPs treatment

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groups. In addition, the SOD activities were 10.566 ± 0.621, 9.039 ± 0.787–9.408 ±

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0.493, and 7.959 ± 0.422–9.195 ± 0.327 U/mg protein for the control, 100 nm, and

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1300 nm PS-MPs treatment groups, respectively, indicating that oxidative stress was

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induced after PS-MPs exposure. Furthermore, the comet assay suggested that

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exposure to PS-MPs induced DNA damage in earthworms. Overall, 1300 nm PS-MPs

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showed more toxic effect than 100 nm PS-MPs on earthworms. These findings 2

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provide new insights regarding the toxicological effects of low concentrations of

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microplastics on earthworms, and on the ecological risks of microplastics to soil

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animals.

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Keywords: Microplastics; Earthworm; Histopathology; Oxidative damage; DNA

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damage

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Capsule title: :

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DNA damage and histopathological changes in the earthworm Eisenia fetida after

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exposure to low concentrations of PS-MPs were observed for the first time.

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1. Introduction

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Microplastics (MPs), plastic fragments with particle size < 5 mm (Thompson et

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al., 2009), belong to a group of emerging contaminants that have caused concern

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because of their effects on the environment in recent years (Alimi et al., 2018).

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Degradation of MPs and their removal from the environment is challenging owing to

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their chemical inertia. MPs are ubiquitous and have been detected in different

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ecosystems (Diepens and Koelmans, 2018). Microplastics can be divided into primary

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and secondary MPs (Cole et al., 2011). Primary MPs mainly refer to man-made

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plastic particles that are used as raw materials for industrial manufacture or

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production of cosmetics (Cole et al., 2011). Secondary MPs, including large plastics 3

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used in agricultural production, industrial production, and urban construction, can be

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degraded in the environment by UV radiation or high temperature (Rillig et al., 2012;

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Steinmetz et al., 2016). The sources of MPs in soil are varied, including the land

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application of sewage sludge that carries MPs (Mintenig et al., 2017), agricultural

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plastic film decomposition (Wang et al., 2013; Ramos et al., 2015; Steinmetz et al.,

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2016), deposition of atmospheric MPs onto the soil (Dris et al., 2016), and surface

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runoff (Gies et al., 2018; Lares et al., 2018).

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Research on microplastic toxicity has mainly focused on aquatic environments,

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while studies on terrestrial ecosystems are limited. Surveys in terrestrial environments

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have focused on larger pieces of plastic, which are common in urban soils (Rillig,

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2012). In addition, because of their adsorption characteristics, MPs entering the soil

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do not adsorb only organic pollutants (Beckingham et al., 2017), but also act as

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carriers of metals such as zinc, thereby improving their bioavailability (Hodson et al.,

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2017). Owing to the feeding of soil animals, MPs accumulate in the soil food chain

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(Huerta Lwanga et al., 2016, 2017). However, reports on the adverse effects of MPs

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on soil organisms are still limited (Huerta Lwanga et al., 2016; Hodson et al., 2017;

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Rodriguez-Seijo et al., 2017, 2019; Wang et al., 2019). Therefore, studies focusing on

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the effect of MPs on terrestrial ecosystems and on soil organisms are important

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(Horton et al., 2017).

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Studies have shown that MPs can affect the growth, reproduction, and diversity

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of soil animals (Rillig et al., 2017). Once MPs are ingested or accumulated by soil

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animals, they can cause physical tearing of organs and tissues, and elicit an 4

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inflammatory response to invasive heterogenic substances (Song et al., 2019).

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Furthermore, MPs ingestion can also cause insufficient supply of nutrients and energy

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to organisms (Huerta Lwanga et al., 2016; Rodriguez-Seijo et al., 2018; Yin et al.,

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2018; Prata et al., 2020). In addition, the toxic substances released by MPs and the

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toxic effects of adsorbed pollutants can adversely affect individual and species

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diversity (von Moos et al., 2012; Wright et al., 2013; Hodson et al., 2017). The effect

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of MPs on soil animals is related to particle size and concentration. So far, only few

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studies have reported the concentrations of MPs in soils, which range from

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55.5−67,500 mg/kg (Chae and An, 2018). MPs with particle size less than 1 mm can

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be easily ingested by soil animals and can be further transported from the intestines to

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other tissues via the intestinal wall (Farrell and Nelson, 2013). Browne et al. (2008)

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observed that microplastics with particle size < 1 mm translocated more easily into

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the circulatory system of the organism.

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Earthworms are important animals in the soil food chain and play an essential

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part in fertility, metabolism, and maintenance of the structure and function of soil

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ecosystems (OECD, 2004). In addition, earthworms have high reproduction rate and

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strong adaptability, but are also vulnerable to toxic and harmful substances in the

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environment. Therefore, earthworms, especially Eisenia fetida, are widely used to test

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the toxicity of pollutants. Although studies have investigated the toxic effects of MPs

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on earthworms, they have mainly focused on growth, reproduction, movement, and

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oxidative stress (Besseling et al., 2012; Rillig et al., 2017; Rodriguez-Seijo et al.,

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2019; Wang et al., 2019), while histopathological changes, and DNA and oxidative 5

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damage

have

rarely

been

investigated

(Rodriguez-Seijo

et

al.,

2017;

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Prendergast-Miller et al., 2019). In this study, our aim was to examine the potential

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toxic effects of widely used polystyrene microplastics (PS-MPs) on earthworms at

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concentrations that are lower than those reported in polluted soils (Chae and An,

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2018). Therefore, to improve our understanding of the ecological risk of PS-MPs in

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soil ecosystems, we have measured the mortality, histopathology, and oxidative and

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DNA damage in E. fetida exposed to fluorescent PS-MPs at concentrations of 100 and

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1000 µg/kg soil.

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2. Materials and methods

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2.1. Preparation of microplastics test solution and earthworm culture

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Fluorescent PS-MPs (100 nm and 1300 nm in size) with excitation and emission

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wavelengths of 538 nm and 580 nm were purchased from a commercial company

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(Tianjin BaseLine ChromTech Research Centre). The PS-MPs test solution was

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prepared according to Lu et al. (2016) and Jiang et al. (2019) with slight modifications.

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Briefly, the PS-MPs fluorescent microsphere emulsion solutions were ultrasonicated

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for 10 min and dispersed in 10 mL deionized water with 100 mg/10 mL PS-MPs,

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diluted to the final concentrations of 100 nm and 1300 nm PS-MPs (0, 100 µg/kg,

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1000 µg/kg, respectively), and stored at 4℃. The 100 µg/kg concentration was

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prepared by adding 5 µL PS-MPs emulsion solutions to 500 g artificial soil and

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similarly, the 1000 µg/kg was prepared by adding 50 µL PS-MPs emulsion solutions 6

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to 500 g artificial soil. The distribution characteristics of the PS-MPs were measured

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using dynamic light scattering (DLS, ZEN1600, Malvern Instruments, Malvern, UK)

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and scanning electron microscopy (SEM, Hitachi, S-3400, Japan). The DLS plots of

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100 nm and 1300 nm PS-MPs were measured in triplicate. Furthermore, PS-MPs were

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identified using Fourier transformed infrared spectrometry (FTIR, Bruker, TENSOR

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27, Germany).

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The earthworms (E. fetida) used in this study were obtained from a breeding

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farm in Jurong, China, and cultured under laboratory controlled conditions (20 ± 2 ℃)

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according to OECD guidelines (2004). Prior to exposure, the earthworms were

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acclimated to the artificial soil consisting of 70% industrial sand, 20% kaolin clay,

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and 10% dried cow manure for at least one week, adjusted with CaCO3 to the pH 6.0

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± 0.5 (OECD, 1984). For the tests, the adult earthworms that showed well developed

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clitellum and showed body weight between 300 and 600 mg were selected, as

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recommended by OECD and ISO guidelines (OECD, 2004; ISO, 2012).

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2.2. Exposure of the earthworms to PS-MPs

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Before the tests, the earthworms were cultured for 24 h under the same artificial

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soil conditions. Ten earthworms were placed into 2 L glass beaker with 500 g of

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artificial soil (dry weight) containing different concentrations of 100 nm and 1300 nm

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PS-MPs (0, 100 µg/kg, or 1000 µg/kg) (Four replicates each group). The beakers

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wrapped with gauze were then incubated at 20 ± 2 ℃ in the presence of 75% humidity 7

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for 14 days. During the exposure, the soils were maintained at 40% maximum water

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holding capacity (MWHC) with addition of ultrapure water. The earthworms were fed

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with 5 g cow manure spread on the soil surface once a week. After 14 days of

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exposure, the earthworms were rinsed with ultrapure water and depurated for 24 h

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before further analyses.

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2.3. Uptake and accumulation in earthworm intestines

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The uptake and accumulation of PS-MPs in earthworm intestines was measured

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according to Lu et al. (2016) with some slight modifications. Briefly, after 14 days of

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exposure, three earthworms per treatment were depurated for 24 h on wet filter paper,

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rinsed with ultrapure water, surface moisture removed with filter paper, and weighed.

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Subsequently, the earthworms were transversely cut in the middle of the body and

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then dehydrated and weighed. Intestinal tissues were digested with 1 mL HNO3 at

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70 °C for 2 h and ultrapure water was added to a final volume of 5 mL. The contents

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of PS-MPs in earthworm intestines were determined using a fluorescence

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spectrophotometer (HITACHI F-7000) at excitation and emission wavelengths of 538

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nm and 580 nm, respectively. A standard curve was obtained according to serial

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dilutions of fluorescent PS-MPs suspensions (Figure S2). The background

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fluorescence of the intestine from unexposed PS-MPs earthworms, which were

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processed in the same way as exposed PS-MPs earthworm, was measured and

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deducted from that of the treated samples. Each sample was measured in triplicate. 8

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2.4. Biochemical assays

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SOD activity and GSH levels were measured to assess oxidative damage in

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earthworms exposed to PS-MPs. Briefly, after 14 days of exposure, earthworms from

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the control group and those exposed to 100 nm or 1300 nm PS-MPs at concentrations

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of 100 µg/kg or 1300 µg/kg were used for further analyses. Earthworms (three

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individuals per treatment) were rinsed with ultrapure water, then rapidly cut into

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pieces, and blended with ice-cold phosphate buffered saline (PBS). Subsequently, the

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mixture solution was homogenized with an ultrasonic processor (JY-250, Zhejiang,

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China) (5 x10s , intermittent 20s, 120W) in an ice bath and centrifuged at 4000 rpm,

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4°C, for 10 min. The supernatants were used for GSH and SOD analyses. GSH levels

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and SOD activity were determined using commercial kits (No. A006-1 and No.

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A001-3, respectively) (Nanjing Jiancheng, China). Enzyme activity was normalized

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by protein content (bicinchoninic acid assay) using a commercial kit (No. A045-4)

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(Nanjing Jiancheng, China). Each test was performed in triplicate.

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2.5. Histopathological analyses

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Histopathological analyses were performed according to our previous publication

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(Yang et al., 2018) with slight modifications. After 14 days exposure, 15 earthworms

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(three earthworms per treatment) were placed on cold filter paper for 10 min before

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dissection. The intestines of the earthworms were removed, transversely cut, and then 9

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placed in 10% formalin buffer (pH 7.0 ± 0.2) for 24 h. The intestines were then

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embedded in paraffin wax, cut with a microtome into 4-µm thick slices, and stained

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with hematoxylin and eosin (H&E) for microscopic observation. Photos of freshly

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dissected earthworm intestines (Figure S1) and detailed description of the

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histopathological procedure are included in the supplementary materials.

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2.6. Comet assay

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Coelomocytes were obtained according to Eyambe et al. (1991), and the levels of

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DNA strand breaks were determined according to the method of Singh et al. (1998).

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Three earthworms were used per treatment. After electrophoresis, each slide was

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washed three times with 0.5 M Tris buffer (pH 7.5) and the DNA was stained with

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ethidium bromide. Slides were examined using a fluorescence microscope (BX41,

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Olympus, Japan) and at least 50 cells were analyzed per slide. The extent of DNA

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migration was determined as the percentage of tail DNA (% tDNA) and olive tail

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moment (OTM) with the CASP software using the method of Collins et al. (1995).

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2.7. Statistical analysis

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The means and standard deviations (SD) for all treatments were calculated using

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the SPSS 19.0 software. Differences between control and treated samples were

10

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analyzed using the Mann-Whitney U-test. p < 0.05 was considered significant. Origin

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9.0 mapping was used for drawing the figures.

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3. Results and discussion

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3.1. Characteristics of PS-MPs

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The characteristics of the PS-MPs are shown in Figure 1. The DLS results

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demonstrated that the average particle size of 100 nm PS-MPs was 105.4 nm and that

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of 1300 nm PS-MPs was 1343.2 nm (Figure 1A and 1C). SEM images showed that

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both 100 nm and 1300 nm PS-MPs had spherical structures and did not show

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aggregates (Figure 1B and 1D). The FTIR spectrum of two sizes of PS-MPs showed

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in Figure 1E and 1F. Briefly, the FTIR spectrum indicated that the peaks at 3080 cm-1,

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3060 cm-1, and 3030 cm-1 were related to aromatic C–H stretching vibration. The

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peaks at 2920 cm-1 and 2850 cm-1 were assigned to the stretching vibration of –CH2–.

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The peaks at 1600 cm-1, 1490 cm-1, and 1450 cm-1 were attributed to C–C stretches in

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the aromatic ring. The peaks at 698 cm-1 and 756 cm-1 were considered an aromatic

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substitution pattern. The characteristic peaks and composition of two sizes of PS-MPs

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were similar to that reported previously (Lu et al., 2016; Wu et al., 2019).

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Figure 1. PS-MPs particle distribution characteristics. DLS of 100 nm PS-MPs (A), SEM of 100

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nm PS-MPs (B), DLS of 1300 nm PS-MPs (C), and SEM of 1300 nm PS-MPs (D). FTIR

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spectroscopy of 100 nm PS-MPs (E) and 1300 nm PS-MPs (F).

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3.2. Growth change induced by PS-MPs

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During the 14 days of exposure, the percent of mortality and the average loss of

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earthworm body weight in the control group were 5 ± 0.250 % and 3.85 ± 0.513 %

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(Figure 2), which was less than 10% and 20%, respectively. Therefore, our results are 12

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considered to be valid according to the standard toxicity procedure (ISO, 2012).

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Nevertheless, compared to the control, significant changes in mortality were observed

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in exposed earthworms (except for earthworms exposed to 1000 µg/kg of 1300 nm

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PS-MPs) (p < 0.05, Figure 2). Furthermore, the growth rate of the earthworms in each

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treatment was higher than that of the control group (p < 0.05), with the growth rates

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of 3.85 ± 0.513 %, 5.66 ± 0.456 %–11.82 ± 0.208 %, and 5.12 ± 0.186 %–12.29 ±

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0.199 % in the control, 100 nm, and 1300 nm PS-MPs treatment groups, respectively.

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The highest exposure concentration of PS-MPs (1000 µg/kg) in this study was still

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lower than the concentrations used in studies on MPs toxicity to earthworms so far

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(Wang et al, 2019; Rodriguez-Seijo et al., 2018, 2019). The observed increase in the

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growth of the exposed earthworms (Figure 2) might reflect the hormetic effect caused

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by low concentrations of MPs. Similar results regarding earthworm growth were also

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observed by other researchers, where the chlorinated flame retardant Dechlorane Plus

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did not inhibit the growth of earthworms (Yang et al., 2016). Therefore, we focused

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more on changes at the molecular and histopathological levels to investigate the

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potential toxic effects of PS-MPs on earthworms.

10.0

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(A)

Control 100 nm 1300 nm

a

a

12

c

Growth rate (%)

7.5

Mortality (%)

(B)

Control 100 nm 1300 nm

c

5.0

b

b

2.5

a

8

b

b

c 4

0.0

0 Control

100

100

1000

1000

Control

PS-MPs (µg/kg soil)

100

100

1000

PS-MPs (µg/kg soil)

251 13

1000

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Figure 2. Mortality (%) (A) and growth rate (%) (B) of earthworms in control and PS-MPs

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treatment groups. Data represent mean ± SD (n = 3). Different letters indicate significant

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differences between treatments (p < 0.05).

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3.3. Antioxidant enzyme activities

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In this study, the activities of SOD and levels of GSH in earthworms were

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measured to assess the oxidative stress caused by PS-MPs after 14 days of exposure

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(Figure 3). The activities of SOD in earthworms were significantly reduced (p < 0.05)

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when exposed to 100 and 1300 nm PS-MPs. The SOD activities of earthworms were

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10.566 ± 0.621, 9.039 ± 0.787–9.408 ± 0.493, and 7.959 ± 0.422–9.195 ± 0.327 U/mg

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protein when exposed to control soil, and soil treated with 100 nm and 1300 nm

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PS-MPs, respectively. In contrast, the levels of GSH in earthworms increased

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significantly (p < 0.05) following treatment with 100 nm (100 µg/kg, 1000 µg/kg) and

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1300 nm (100 µg/kg) PS-MPs, whereas no significant alteration was observed in the

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1300 nm (1000 µg/kg) PS-MPs group. GSH levels in earthworms were 86.991 ±

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7.723, 165.436 ± 4.256–167.767 ± 18.642, and 93.590 ± 4.279–173.98 ± 15.523

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µmol/L when exposed to control soil, and that treated with 100 nm and 1300 nm

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PS-MPs, respectively.

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Enzymatic activities have been considered effective indicators of environmental

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pollutants (Liang et al., 2013). SOD is an oxygen free radical scavenger and therefore

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plays a crucial part in the enzymatic defense system (Liu et al., 2012). Although 14

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compared to the control, SOD activity was significantly inhibited in all treatments (p

274

<0.05), the highest inhibition was observed when exposed to 1300 nm PS-MPs at

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1000 µg/kg (Figure 3). SOD activity has been observed to increase as a direct

276

response to increasing levels of super oxide anion radicals, indicating that the

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exposure of the earthworms to PS-MPs led to the production of reactive oxygen

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species (ROS), which then promoted SOD biosynthesis to protect the cells from

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oxidative damage (Zhang et al., 2014). In our study, the reason for decreased SOD

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activity in all treatment groups might be the excess ROS production, which can lead

281

to overwhelming of the antioxidant defenses. Similar decreasing trend in SOD activity

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has already been observed when E. fetida is exposed to nanoscale zerovalent iron

283

(Liang et al., 2017) and low-density polyethylene microplastics (Wang et al., 2019).

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GSH is also part of the cellular antioxidant defense system that represents water

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soluble reductants (Liu et al., 2012). We observed that the GSH level increased after

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14 days exposure to 100 µg/kg of both 100 nm and 1300 nm PS-MPs, while at

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concentration of 1000 µg/kg this was observed only for 100 nm PS-MPs. Overall,

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GSH levels tended to increase with exposure of earthworms to 100 nm PS-MPs. The

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increase in GSH levels indicated that the exposure to the PS-MPs might have

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increased the production of ROS, which activated the biosynthesis of GSH to protect

291

the cells against oxidative damage. However, upon exposure to 1300 nm PS-MPs, the

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GSH level in earthworms exposed to 1000 µg/kg was similar to that of the control.

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This was due to the over-production of ROS in earthworms, which exceeded the

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capacity of the antioxidant system and probably led to reduction in GSH levels (Liu et 15

295

al., 2012). These findings indicated that PS-MPs induced antioxidant defenses in

296

earthworms as a result of oxidative stress caused by PS-MPs exposure.

12

200

(A)

Control 100 nm 1300 nm

c

SOD (U/mg prot)

(B)

a

a

a

Control 100 nm 1300 nm

160

b

b

b a

9

GSH (µmol/L)

15

6

120

b

b 80

40

3

0

0 Control

100

100

1000

1000

Control

100

100

1000

1000

PS-MPs (µg/kg soil)

PS-MPs (µg/kg soil))

297

298

Figure 3. Superoxide dismutase (SOD) activity and glutathione (GSH) level in earthworms after

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14 days of PS-MPs exposure. Data represent mean ± SD (n = 3). Different letters indicate

300

significant differences between treatments (p < 0.05).

301

302

3.4. Histopathological changes induced by PS-MPs in earthworm intestinal tissue

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Uptake of 100 nm and 1300 nm fluorescent PS-MPs was detected in earthworm

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intestines after 14 days of exposure (Figure 4). All tested PS-MPs were found to

305

accumulate

306

concentrations were higher for 1300 nm PS-MPs (p < 0.05). We also observed that

307

accumulation of PS-MPs was significantly higher at 1000 µg/kg than at 100 µg/kg.

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Average concentrations in earthworm intestines for 100 nm/100 µg/kg, 100 nm/1000

309

µg/kg, 1300 nm/100 µg/kg, and 1300 nm/1000 µg/kg PS-MPs were 0.015 ± 0.001,

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0.033 ± 0.002, 0.084 ± 0.005, and 0.094 ± 0.003 µg/mg, respectively. Uptake of MPs

in

earthworm

intestines.

16

However,

the

average

accumulated

311

in a size-dependent manner has been well investigated in marine species, and the

312

results indicate that ingested MPs significantly depend on their size (Wright et al.,

313

2013). In addition, we also examined histopathological changes induced by PS-MPs

314

in the intestines of E. fetida after 14 days of exposure (Figure 5). In the control group,

315

all observed sections showed normal histology of earthworm intestines (visible cell

316

divisions, regular shape and cell nucleus size). However, intestines of earthworms

317

exposed to PS-MPs showed enlarged intestinal cells with irregular shapes and altered

318

size of cell nuclei (Figure 5). The aberrant histopathology was more pronounced in

319

the 1300 nm (1000 µg/kg) PS-MPs treated samples than in the 100 nm PS-MPs

320

treated samples. In addition, more intestinal cell lysis was observed in the 1300 nm

321

(1000 µg/kg) PS-MPs treated group. Lu et al. (2016) observed that 2000 µg/L of

322

PS-MPs (both 5 µm and 20 µm) induced inflammation in damaged zebrafish tissues

323

(gill, liver, and gut) and that 5 µm PS-MPs accumulated more than 20 µm PS-MPs. It

324

is well-known that the smaller MPs tend to be retained more in the earthworm

325

intestine due to their feeding selectivity (Curry and Schmidt, 2007; Huerta Lwanga et

326

al., 2016; Rodriguez-Seijo et al., 2017), which differs from our observations. This

327

could be due to the small size of the particles used in our study (100 and 1300 nm)

328

compared to other studies that have used larger particles (minimal particle size was 5

329

µm), where depuration rate might not depend on the particle size. Owing to

330

differences in the ways of exposure (artificial soil, OECD, 2004) and sizes of MPs

331

used (Huerta Lwanga et al., 2016; Rodriguez-Seijo et al., 2017), the obtained results

332

cannot be compared easily. However, higher accumulation and more aberrant 17

333

histopathology of earthworms caused by higher concentrations of MPs, was also

334

observed by other researchers (Rodriguez-Seijo et al., 2017). These findings are in

335

agreement with the observed higher mechanical damage of the earthworm intestines

336

caused by 1300 nm PS-MPs than those caused by 100 nm PS-MPs.

0.12

PS-MPs 0.10

PS-MPs (µg/mg intestine)

b

a

0.08

0.06

c

0.04

0.02

d

0.00 100nm/100

100nm/1000

1300nm/100

1300nm/1000

PS-MPs (µg/kg soil)

337 338

Figure 4. Concentrations of PS-MPs in earthworm intestines. Data represent mean ± SD (n =3).

339

Different letters indicate significant differences between treatments (p < 0.05).

18

340 341

Figure 5. Intestinal damage caused by PS-MPs. I: control; II: 100 nm 100 µg/kg; III: 1300 nm

342

100 µg/kg; IV: 100 nm 1000 µg/kg; V: 1300 nm 1000 µg/kg; A0: cell division; B0: regular cell

343

shape; C0: regular nucleus size; A: enlarged cell space; B: irregular cell shape; C: irregular nucleus

344

size; D: cell lysis.

345

346

3.5. DNA damage

347

Comet assay is an effective method for assessing DNA damage induced by

348

environmental pollutants, and is widely considered a sensitive biomarker for

349

determining and evaluating the genotoxicity of pollutants on invertebrates (Reinecke

350

et al., 2004; Saez et al., 2015, Gajski et al. 2019). In our study, DNA damage in

351

earthworms depended on the PS-MPs particle size and their concentration. Higher

352

concentration of PS-MPs (1000 µg/kg) increased DNA damage irrespective of particle

353

size (Figure S3). Exposure to PS-MPs increased the percentage of tDNA and OTM in 19

354

a dose-dependent manner. The results showed that both 100 and 1300 nm PS-MPs

355

elicited high levels of DNA damage (p < 0.05) (Figure 6). In particular, 1300 nm

356

PS-MPs appeared to induce more damage to earthworm DNA than 100 nm PS-MPs.

357

At the same concentration the % tDNA and OTM in 1300 nm PS-MPs were

358

significantly higher than for the 100 nm PS-MPs. When exposed to the same particle

359

size the % tDNA and OTM were, as expected, significantly higher at 1000 µg/kg than

360

at 100 µg/kg. Higher concentrations and larger particle size of PS-MPs caused an

361

increase of % tDNA and OTM indicating severe DNA damage in earthworms, which

362

were consistent with the observed size-dependent histopathological changes and the

363

results of the oxidative stress assays (Wright et al., 2013). Therefore, we assumed that

364

the ROS accumulation in earthworm tissues may be responsible for the subsequent

365

DNA damage in earthworm coelomocytes (Song et al., 2019).

4 Control 100 nm 1300 nm

25

b

ab

Control 100 nm 1300 nm

a (B)

(A)

15

d

OTM

% tDNA

b

3

20

c

b

a

c

2

10 1

d

5

0

Control

0 100

100

1000

1000

PS-MPs (µg/kg soil)

Control

100

100

1000

1000

PS-MPs (µg/kg soil)

366 367

Figure 6. DNA damage in earthworms after 14 days of exposure to PS-MPs. The bars represent

368

standard deviation (n =3). Different letters indicate significant differences between treatments (p <

369

0.05).

370 20

371

Conclusions

372

This is the first study to reveal the toxicological effects of low concentrations of

373

PS-MPs (≤1 mg/kg of soil) on earthworms (Eisenia fetida). According to the growth

374

measurements, histological changes, and the results of DNA damage and oxidative

375

stress analyses, the acute toxicity of 100 and 1300 nm PS-MPs to earthworms appear

376

to be extremely low. However, exposure to PS-MPs with particle size larger than 100

377

nm caused DNA damage and oxidative stress in earthworms. Furthermore, we also

378

observed that PS-MPs exposure can cause histopathological damage in earthworm

379

intestines, especially when exposed to 1300 nm particles at 1000 µg/kg. Moreover, the

380

1300 nm PS-MPs were more toxic and accumulated to higher concentrations in the

381

earthworm intestines than 100 nm PS-MPs. These findings provide new insights

382

regarding the toxicological effects of low concentrations of microplastics on

383

earthworms and on the ecological risks of microplastics to soil animals.

384 385

386

Conflicts of interest The authors declare that they have no conflicts of interest.

387

388

Acknowledgements

389

This research was supported by National Natural Science Foundation of China

390

(No. 41773115, 41571468), Science and Technology Support Program of Jiangsu 21

391

Province (No. BE2016736) and Nanjing University Innovation and Creative Program

392

for PhD candidate (No. CXCY19-61). We also thank the anonymous reviewers and

393

the handling editor for their positive and suggestive comments regarding our study.

394

395

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29

Highlights



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: