Ecotoxicological effects of microplastics and cadmium on the earthworm Eisenia foetida

Ecotoxicological effects of microplastics and cadmium on the earthworm Eisenia foetida

Journal Pre-proof Ecotoxicological effects of microplastics and cadmium on the earthworm Eisenia foetida Yanfei Zhou (Writing - original draft) (Metho...

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Journal Pre-proof Ecotoxicological effects of microplastics and cadmium on the earthworm Eisenia foetida Yanfei Zhou (Writing - original draft) (Methodology) (Software), Xiaoning Liu (Investigation) (Data curation), Jun WangWriting review and editing) (Supervision)

PII:

S0304-3894(20)30261-2

DOI:

https://doi.org/10.1016/j.jhazmat.2020.122273

Reference:

HAZMAT 122273

To appear in:

Journal of Hazardous Materials

Received Date:

2 June 2019

Revised Date:

29 January 2020

Accepted Date:

10 February 2020

Please cite this article as: Zhou Y, Liu X, Wang J, Ecotoxicological effects of microplastics and cadmium on the earthworm Eisenia foetida, Journal of Hazardous Materials (2020), doi: https://doi.org/10.1016/j.jhazmat.2020.122273

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Ecotoxicological effects of microplastics and cadmium on the earthworm Eisenia foetida Yanfei Zhoua,c, Xiaoning Liua, Jun Wanga,b* a

Key Laboratory of Aquatic Botany and Watershed Ecology, Wuhan Botanical Garden, Chinese

Academy of Sciences, Wuhan 430074, China b

University of Chinese Academy of Sciences, Beijing, 100049, China

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c

College of Marine Sciences, South China Agricultural University, Guangzhou 510642, China

Jun Wang, Key Laboratory of Aquatic Botany and Watershed Ecology, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China. Tel/fax: +86 027 87700859;

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E-mail address: [email protected]

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Graphical abstract

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Highlights 

Microplastics and the synergic contamination of microplastics and heavy metal can induce adverse effects on Eisenia foetida



Combined exposure to microplastics and cadmium enhances the negative effects of cadmium.



MPs particles are retained within Eisenia foetida, and retained particle numbers increase



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with exposure duration

The presence of microplastics increase cadmium accumulation in Eisenia foetida

Abstract

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As microplastics (MPs) have become ubiquitous in both aquatic and terrestrial

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environments, there has been a growing concern about these new anthropogenic stressors. However, comparatively little is known about the negative effects of MPs, co-contamination

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of MPs and heavy metals on terrestrial organisms. The objective of this study was performed to understand the adverse effects of exposure to MPs and co-exposure to MPs and cadmium

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(Cd) on the earthworm Eisenia foetida (E. foetida). Results showed that exposure to MPs only or to a combination of MPs+Cd decreased growth rate and increased the mortality (>300

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mg·kg-1) after exposure for 42 d, with MPs+Cd (>3000 mg·kg-1) posing higher negative influence on the growth of E. foetida. Exposure to MPs might induce oxidative damage in E.

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foetida, and the presence of Cd accelerates the adverse effects of MPs. Furthermore, the MPs particles can be retained within E. foetida, with values of 4.3 to 67.2 particles·g-1 earthworm, and can increase the accumulation of Cd in earthworm from 9.7% to 161.3%. Collectively, the results of this study demonstrate that combined exposure to MPs and Cd poses higher negative effects on E. foetida, and that MPs have the potential to increase the bioaccessibility of heavy metal ions in the soil environment. 2

Keywords:Microplastics, Eisenia foetida, Heavy metals, Combined exposure, Oxidative stress

1. Introduction Microplastics (MPs) pollution has been gradually increasing as a global environment issue [1-3]. MPs are usually defined as plastic debris with diameters smaller than 5 mm. This new anthropogenic ubiquituous stressor occurs in aquatic and terrestrial environments[1-5]. They

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may be specifically produced within a micrometre size range (e.g., microbeads and abrasive scrubbers in cosmetic and personal products) [6, 7] or derived from the breakdown of large plastic products [8, 9]. MPs can be deposited on environment via discharging of sewage sludge,

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degradation of large plastic items, stormwater runoff, atmospheric fallout, and other sources [10, 11]. Until recently, MPs were mainly found widely distributed in oceans, freshwater,

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beaches, and sediments [2, 12-14]. Although less widely reported, MPs also occur in terrestrial environments and have been detected both in industrial and remote areas at concentrations as

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high as 67 g·kg-1 [3, 5, 15-17]. The negative biological effects of MPs are thought to be induced by two mechanisms [18]. First, small-sized MPs are accidently ingested, causing direct

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damages to organisms, and may eventually be accumulated along food webs [19, 20]. Secondly, MPs provide high specific surface area upon which, other environment pollutants may adsorb;

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thus, the MPs act as vectors transferring these contaminants [21-23].

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Some implications of MPs ingestion have been demonstrated in previous studies, such as increase of mortality and oxidative stress [24-26] and decrease of energy reserves and growth rate [25, 27]. For example, in a terrestrial environment, Huerta Lwanga et al. [25] reported that the mortality and growth rate of Lumbricus terrestris (L. terrestris) were negatively impacted after exposure to MPs. In another study, Yu et al. [26] showed that oxidative stress was induced in the liver of Eriocheir sinensis by ingestion of MPs, when the concentration of MPs exceeded 3

4 mg·L-1. It’s highly likely that these harmful impacts are related to the retention of MPs within organisms [28]. As shown in a previous study, MPs particles could be retained inside of lugworm collected alone French−Belgian−Dutch coastline, an average of 1.2±2.8 particles·g-1 tissue was detected [29]. In addition, Watt et al. [30] observed that polystyrene (PS) microspheres (8-10 μm) were mainly retained in the foregut of crab after exposure for 20 d. In consideration of the vital role played by the earthworm in soil processes and ecosystem services [32], further research on the impacts of ingestion of MPs and the synergic pollution of MPs

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and other contaminants in terrestrial environment are clearly necessary. In addition to the demonstrated direct effects of ingested MPs on organisms, MPs might also act as vectors for transferring other contaminants [21, 23, 31]. These adsorbed pollutants

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would be desorbed within the organism post-ingestion, due to the existence of gut surfactants, such as linear alkylbenzene sulfonate and humic acid [32], and this process would presumably

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lead to higher toxic effects [19, 24]. Currently, most studies have considered the deleterious

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effects resulting from co-exposure to MPs and organic pollutants [32-34]. Yet, the toxic effects induced by simultaneous exposure to MPs and heavy metals are comparatively underreported, especially for terrestrial organisms. Recently, Lu et al. [24] provided the evidence of toxic

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effects from combined exposure of MPs and Cd in aquatic environments. They detected oxidative damage in the tissues of zebrafish; in addition, the accumulation of Cd was clearly

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enhanced after zebrafish were exposed to both MPs and Cd for three weeks. Likewise, MPs in

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terrestrial environments can act as well as vectors for increasing metal exposure to soil organisms [7]. It is reasonable to assume that MPs combining with none-essential elementary metals pose higher associated risks. Cadmium is one of the most toxic heavy metals and is commonly encountered in soil; its typical background concentrations are in the range of 2 to 20 mg·kg-1 [35]. MPs in soil are highly likely to form combined pollutants with heavy metals, and thus, it is important to consider the synergic pollution of MPs and heavy metals when 4

assessing the risk of MPs to soil fauna. Accordingly, the specific objectives of the current study were to ascertain: (1) whether adverse effects on Eisenia foetida (E. foetida) would be induced by polypropylene microplastic (hereinafter denoted as MPs) and MPs+Cd; (2) whether MPs particles could be retained within E. foetida; and (3) whether the accumulation of Cd in E. foetida would be enhanced by the presence of MPs.

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2. Materials and methods 2.1. Preparation of artificial soil, microplastics, and test organisms

Artificial soils were prepared to conduct a laboratory scale exposure experiment. Soils

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were mixed mainly according to OECD guidelines with moderate modification [36, 37]. The specific components were as follow: 20% kaolinite clay (Aladdin, China), 70% quartz sand

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(Greagent-Reagent, China), 10% sphagnum peat (Jiffy substrates, purchased from a local gardening store). Analytical reagent CaCO3 was used to adjust the initial pH of the soil. Organic

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carbon content was 5%, total organic nitrogen content was 0.0978%, initial pH was 6.7, and the cation exchange capacity (CEC) was 14.6 cmol·kg-1. Organic carbon was determined by

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the Walkley-Black method [38]. Total nitrogen content was measured according to the Kjeldahl method [39], and pH was determined using a pH-meter (Mettler Toledo FE20) with a ratio of

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water and soil of 2.5:1. The method for analysing CEC follows Hendershot et al. [40].

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Analytical grade CdCl2 (99.9% purity), obtained from Sigma (USA), was used as the Cd source in this experiment. Prepared CdCl2 solution was sprayed into the soil at a concentration of 8 mg·kg-1 Cd2+ dry soil (denoted as mg·kg-1 hereinafter, the actual measured Cd concentration was 8.4 mg·kg-1). We selected this Cd exposure dose in order to mimic the ambient concentration of Cd in many farmland soils [35, 41, 42]. Polypropylene (PP) was considered as a model MPs in this study, as it is one of the most 5

commonly produced synthetic polymers and is prevalent in the environment [2, 16]. PP pellets (C3H6)n purchased from Sigma-Aldrich (density 0.90 g·cm-3 at 25℃) were mechanically ground to a diameter of < 150 μm. The average number of particles of MPs was estimated as (1.3±0.05) × 105 particles·g-1 (dry weight). Ground MPs particles were characterized by DXR 2 Raman (Thermo Fisher Scientific, USA), and a typical Raman spectrum was shown in Fig. 1a. Optic images of ground MPs particles were captured by a stereomicroscope (Nikon SMZ25) with a high-resolution camera (Fig. 1a). The typical surface morphology of MPs

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particles was obtained by using a Quanta 250 scanning electron microscope (SEM) (Fig. 1b). Four MPs concentration levels were utilized in this experiment: 300, 3000, 6000, and 9000 mg·kg-1, dry soil (i.e., 0.03%, 0.3%, 0.6%, 0.9% MPs, dry weight, dw).

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The E. foetida individuals used in this experiment were obtained from an earthworm breeding company in Jiangsu, China. After a week of acclimation in artificial soil, 16 healthy

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adult E. foetida with obvious clitellum and body weight between 0.3 and 0.4 g were picked out

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for each experimental beaker. Each E. foetida was gently washed with micro-filtrated ultrapure water, and then kept in a petri dish covered with moisturized filter papers for 48 h. The body

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weight of each individual was recorded before being transferred into experimental beakers. 2.2. Exposure experiment

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The pretreated E. foetida were randomly transferred into glass beakers, each beaker containing 16 E. foetida and 500 g soil. Four experimental groups were set up: CK, control

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group (without MPs and Cd); T1, MPs+Cd group (T1C1 with 300 mg·kg-1 MPs+8 mg·kg-1 Cd; T1C2 with 3000 mg·kg-1 MPs+8 mg·kg-1 Cd; T1C3 with 6000 mg·kg-1 MPs+8 mg·kg-1 Cd; T1C4 with 9000 mg·kg-1 MPs+8 mg·kg-1 Cd); T2, MPs group (T2C1 with 300 mg·kg-1 MPs; T2C2 with 3000 mg·kg-1 MPs; T2C3 with 6000 mg·kg-1 MPs; T2C4 with 9000 mg·kg-1 MPs); T3, Cd group (8 mg·kg-1 Cd alone). Each treatment was prepared in triplicate. To prevent the 6

introduction of other pollutants into the experimental groups, we did not feed the E. foetida during this experiment; however, 2 g dried (105 ℃) horse manure per 100 g soil was incorporated into each beaker during soil preparation [37]. The beakers were placed in a culture chamber in the dark with a temperature of 21 °C±0.5 °C, and soil moisture was measured (measured with a mobile soil-moisture sensor) every two days. If water evaporated, microfiltrated ultrapure water was added into the beakers to keep soil moisture constant at 25% during the trial.

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The exposure experiment was carried out for 6 weeks (42 d); soils and E. foetida were collected at the second (14 d), fourth (28 d), and sixth week (42 d). Cast collection was performed as follows: at each sampling time each E. foetida in its beaker was picked out and

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carefully washed, then kept in a glass petri dish covered with moisturized filer paper for 48 h. The moisturized filter paper in each petri dish was changed every 5 h, in case of repeated

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ingestion of casts, and then casts were collected and dried to constant weight. At each sampling

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time (14, 28, and 42 d), five E. foetida (starved for 48 h) were randomly selected for recording body weight. For measuring lipid peroxides (LPO) levels and glutathione (GSH) content and for extracting MPs, three E. foetida were picked out and thoroughly washed with cold normal

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saline (0.86%), then immediately killed by freezing. The rest of the E. foetida specimens were

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returned to their individual beakers. 2.3. Extraction of MPs

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MPs in artificial soil and casts were extracted according to the method suggested by

Enders et al. [43]. KOH:NaClO solution (30 mL 30% ) was added into each soil sample (20 g soil/cast), then digested at 50 °C for 24 h to remove organic matters. After digestion was completed, micro-filtrated ultrapure water was applied to extract the MPs, and the supernatants were then collected. This extraction process was repeated several times until there were no 7

obvious particles found. Extracted MPs on filter membranes were placed in a petri dish and kept at 40 °C to constant weight. Before isolating MPs from earthworm, every E. foetida were thoroughly washed by saturated NaCl to remove soil and MPs particles sticking on skins. Subsequently, KOH:NaClO solution (30 mL 50%) was added and then digested at 50 °C for 48 h [43]. Finally, digestion solutions were filtered (0.45 μm pore size, 47 mm diameter) to collect MPs particles from E. foetida. To ensure that all extracted particles were MPs particles, a DXR2 Raman microscope (Thermo Fisher Scientific, USA) was used to identify polymeric

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compositions. Each suspected MPs particle was manually located with a 10× lens of DXR2 Raman. All obtained Raman spectra were compared with the libraries in the OMNIC database

2.4. Determination of Cd content in earthworms

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(Thermo Fisher Scientific, USA) with at least 80% matching score for spectra.

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At the end of 14 d, 28 d, and 42 d, E. foetida was taken from every experiment beakers of the MPs+Cd group and Cd group for determining the accumulation of Cd. All E. foetida

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were starved for 48 h to induce the excretion of casts, and then thoroughly washed with cold normal saline (0.86%). Each animal sample was transferred into digestion tubes, then a 3:1

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mixture solution of HNO3 and H2O2 was added into the digestion tube with a 1:10 (m/v) ratio of earthworm weight (g) and mixture solution (mL) [24, 44]. Subsequently, E. foetida was

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digested at 105 °C for 3 h; afterward, cold digestion solutions were filtered and diluted, and the diluted solutions were kept at 4 °C until analysis. Inductively Coupled Plasma-Mass

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Spectrometry (ICP-MS, Thermo Scientific X Series 2) was used to measure the content of Cd. The Cd content in the whole body of E. foetida was represented as mg·kg-1 wet weight (wt.). All labware was soaked with HCl (10% v/v) solution for 24 h, and thoroughly rinsed with microfiltered ultrapure water prior of use [21, 24]. 2.5. Calculation of mortality and growth rate 8

The mortality was calculated according to the percentage of dead earthworms in each treatment at the end of 14 d, 28 d, and 42 d. Growth rate is an important indicator of stress and extensively applied as an index for relevant environmental monitoring and risk assessment [45, 46]. In this study, the growth rate was calculated as follow: (𝑀𝑜𝑟𝑔,1 −𝑀𝑜𝑟𝑔,2 )

𝐾𝑔𝑟 (𝑚𝑔 · 𝑤𝑜𝑟𝑚−1 · 𝑑𝑎𝑦 −1 ) =

𝑀𝑜𝑟𝑔,1

𝑡

Where Morg,1 and Morg,2 refer to initial body weights (g) and final body weights (g) of E. foetida,

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respectively, and t refers to the exposure days (d). This calculation method was performed in accordance with Huerta Lwanga et al.[25]. 2.6. Biochemical parameters

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As suggested in previous studies, both MPs and heavy metals can cause oxidative distress and histopathological in animal organisms [24, 47, 48]. The production of LPO in organisms

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can reflect the degree of intracellular damage. LPO levels usually be measured as an indicator

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of lipid peroxidation and of oxidative stress in organisms and has been extensively applied to ascertain the oxidative damage in studies of earthworm toxicology [24, 47, 49, 50]. GSH, a low

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molecular mass thiol compound and no-enzymatic antioxidant, is widely distributed in organisms. This most common biological thiol was found positively correlated with metal

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stress and directly involved in the cellular metabolism of genotoxic agents [51-53]. GSH is known to play a critical role in the detoxification mechanism against oxidation stress [54, 55].

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Moreover, GSH can reduce the availability of Cd in the cytoplasm through chelating Cd, and significantly enhance organisms defence mechanisms against oxidative stress [51, 52]. It also has been considered to play a critical role in coping with reactive oxygen species (ROS) stress with the function of detoxification and antioxidant action [56, 57]. Therefore, in this study, lipid peroxides (LPO) levels were detected to assess the extent of oxidative injury in E. foetida, and GSH contents were measured to evaluate the detoxification 9

and antioxidant action in E. foetida. During the experimental period, LPO and GSH level were measured after exposure for 14 d, 28 d, and 42 d. The whole E. foetida were homogenized on ice in 50 mM phosphate buffer (including 0.1 mM EDTA, pH=7.0). Homogenates were centrifuged at 5000rmp for 15 min at 4 °C. The supernatants were collected for analyzing LPO levels and GSH contents. Assay kits (Jiancheng Bioeng Inst., China) were used to assess LPO levels and GSH contents. All experimental operations were performed following the manufacturer's protocols.

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2.6. Statistical methods

Univariate analysis was performed by using SPSS 22. Data sets used for one-way ANOVA were tested for normality (Shapiro-Wilk) and homogeneity of variances (Levene’s test);

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logarithmic transformation and rank cases (Blom) were performed in advance, if assumptions

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of parametric tests could not be fulfilled. A nonparametric test (Kruskal-Wallis) was applied to analyse variance. One-way ANOVA was used to determine MP-related data significant

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differences among groups (post-hoc test with Tukey multicomparison). For assessing

3. Results

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relevance, a significance level of 0.05 was set.

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3.1. Effects of MPs and MPs+Cd on E. foetida growth rate, mortality Co-exposure to MPs+Cd or singly exposure to MPs (>3000 mg·kg-1) both decreased

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growth rate, and the presence of Cd significantly enhanced the negative impacts imposed by MPs (Fig. 2a). For example, on day 28, the growth rate of individuals in the T1C4 treatment was 81.6% lower than that in T2C4 treatment (Fig. 2a). In each treatment, the growth rate on day 42 was significantly lower (P< 0.05) than that on day 14 and day 28 (P<0.05, supplementary Table 2), and body mass dwindled (except in CK) when compared with the body weight on day 28 (Table 1). 10

Higher mortality was induced by exposure to high MPs concentrations (>300 mg·kg-1) after exposure for 42 d, but the existence of Cd did not obviously raise the mortality (Fig. 2b). As illustrated in Fig. 2b, the mortality in the MPs+Cd group and the MPs group increased from 12% to 38.4% and from 12.7% to 36.7%, respectively. No significant difference of mortality was found after 28 d among each treatment (Fig. 2b). The mortality significantly raised with increase in exposure time (P<0.05, supplementary table 2, except in T2C1 treatment), and mortality on day 42 was significantly higher than that on day 28 and day 14 (P<0.05,

3.2. The adverse effects of MPs and MPs+Cd on E. foetida

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supplementary Table 2).

Exposure to high MPs concentrations resulted in the increase of LPO levels in E. foetida

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in both MPs and MPs+Cd groups (Fig. 3a). The combined exposure to Cd and MPs induced

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higher LPO levels than that MPs alone induced after exposure 14 d (Fig. 3a). For example, on day 42, LPO levels in T1C1, T1C2, T1C3 and T1C4 treatments were higher 114.8%, 254%, 61.7%,

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and 55.3% than that in T2C1, T2C2, T2C3 and T2C4 treatments, respectively. Similar results were observed between MPs+Cd and MPss group after exposure 28 d (except between T1C1 and

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T2C1, Fig. 3a). The LPO levels were greatly increased after combined exposure to MPs+Cd compared with exposure MPs or Cd alone. For example, on day 28, the LPO levels observed

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in T1C4 (combined exposure) treatment were 44.1% and 191.9% higher than that in T2C4 (MPs alone) and T3 (Cd alone) treatment, respectively (Fig. 3a).

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Exposure to high MPs concentrations increased the GSH content in E. foetida both in MPs

and MPs+Cd group (Fig.3b). Significantly higher GSH content was obtained after co-exposure to MPs+Cd (P<0.05). For instance, on day 42, the GHS content in T1C3 and in T1C4 were significantly higher than that in T2C3 and T2C4, and higher up to 41.3% and 35.6%, respectively (Fig. 3b). Similar results were also detected on day 14 and day 28. The GSH contents in high 11

MPs concentration treatments (>300 mg·kg-1) groups tended to increase with the duration of exposure (Fig. 3b). Overall, GSH content was increased in an MPs dose-dependent manner, and the existence of Cd augmented GSH content (Fig. 3b). 3.3. Microplastics ingestion MPs particles retained within E. foetida significantly increased with the increase of MPs concentration in soil (Fig. 4a). After exposure for 42 d, a total of 4.3, 42.3, 53.1, and 67.2

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particles·g-1 earthworm was extracted from treatments with MPs concentration of 300, 3000, 6000, and 9000 mg·kg-1, respectively (Fig. 4a). Additionally, in a high MPs pollution environment more MPs particles could be egested by E. foetida (Fig. 4b). For example, a total of 48500 mg·kg-1 of MPs particles in cast was recorded at high-dose MPs treatment (9000

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mg·kg-1). What’s more, MPs concentration in the egestion is higher (up to 5.4-fold,

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approximately) than that in the soil, and significantly differences were showed between treatments (except between T2C1 and T2C2 treatment). The percentage of MPs in cast on day

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14 and 28 ranged from 2.8% to 21.7% and 3.1% to 25.6%, respectively (Fig. 4b).

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3.4. Effect of MPs on accumulation of Cd in E. foetida Our results showed that the accumulation of Cd in E. foetida significantly increased with

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exposure to MPs+Cd compared with exposure to Cd alone. After 28 d of combined exposure, Cd accumulation in E. foetida increased significantly in relation to the MPs content in the soil

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(Fig. 5a). In T1C1, T1C2, T1C3, and T1C4 treatments, the Cd content in E. foetida was found to be 3.4, 5, 6.7, and 8.1 mg·kg-1, respectively, after exposure for 42 d (Fig. 5a). These concentrations are greater by 9.7%, 61.3%, 116.1% and 161.3% than the result of the T3 treatment (Cd alone), respectively (Fig. 5a). 4. Discussion

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4.1. Effects of MPs and MPs+Cd on E. foetida growth rate, mortality The growth rate in our study was markedly lower at high doses of MPs (>300 mg·kg-1) after 28 d of exposure, which is in accordance with earlier reports for earthworm [25, 33, 46]. For example, Huerta Lwanga et al. [25] exposed L. terrestris to different doses of polyethylene (PE) microplastics, and found that growth rate significantly declined after exposure to >28% MPs in the top soil lay. On day 42, the growth rate in high MPs concentration treatments (T2C3, T2C4, T1C3, and T1C4) was sharply decreased to -10.5 (in T2C4 treatment) and -14 mg·worm·d-1 (in T1C4 treatment). Huerta Lwanga et al. [25, 46] also observed negative growth rate (-

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0.5 mg·worm-1·d-1) and body weight decrease after exposure to 60% MPs in the top soil layer (similar to 6000 mg·kg-1 of our study), which is in agreement with our results. Up to a point,

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the response mechanism of ingestion of MPs within terrestrial and aquatic organisms may be

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alike [19, 20, 25]; direct physical abrasion and blockage caused by MPs inside organisms may dilute and limit the bioavailability of nutrients [33].

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As indicated in previous studies, MPs have great potential to accumulate metal contaminants on their surfaces from the ambient environment due to strong hydrophobicity and

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higher specific surface area [14, 31, 58, 59], and pollutants can easily be released inside of organisms after ingestion [32]. For example, the bioconcentration and bioaccumulation of

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mercury were greatly increased in Dicentrarchus labrax by combined exposure to MPs [60]. Thus, the Cd concentration in E. foetida and bioaccessibility of Cd might have been enhanced

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by MPs, an explanation for the lower growth rate recorded in MPs+Cd group in the current study. The sharp decrease of growth rate in the control group suggests that the growth of earthworms was presumably influenced by insufficient nutrients in experimental soils [45]. However, overall, we observed that higher concentrations of MPs and combined exposure to MPs and Cd in soil caused even lower growth rates. Exposure duration and MPs concentration may interact in E. foetida mortality, as 13

suggested in previous studies [25, 46]. Low MPs doses and short exposure duration were believed to have no negative effects on the survival and energy reserves of experimental organisms [46, 48, 61]. For example, Rodriguez-Seijo et al. [48] have demonstrated that no significant effect on mortality of earthworm (Eisenia andrei) was induced after exposure for 28 d to a low dose of PE microplastics (<1000 mg·kg-1 MPs soildw). A similar result was recorded in this study (Fig. 1b, supplementary Table 3). However, with increasing concentration of MPs in soil and exposure duration, the damage impacts (abrasion and

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blockage) inside the gut of earthworms may be boosted, and directly lead to weight loss and possibly mortality [25]. Huerta Lwanga et al. [46] reported that the mortality of earthworms was significantly higher on day 60 than on day 14 (≥28% w/w in top litter layer), which is in

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accordance with our results. As detected in a previous study, no significant mortality of E. foetida occurred after exposure to Cd (1~40 mg·kg-1) for 14 weeks [50]. The low content of

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Cd and relatively short exposure duration might be the reason why higher mortality was not

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observed after combined exposure to MPs+Cd.

4.2. The adverse effects of MPs and MPs+Cd on E. foetida

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Not only was the presence of MPs correlated with decreased growth rate and increased mortality of E. foetida, it also amplified the negative effects. In this study, the LPO levels were

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greatly increased after combined exposure to MPs+Cd compared with single exposure to either MPs or Cd. Previous studies have indicated that MPs can induce oxidative stress in organisms

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[24, 54, 62], and Cd exposure can increase the level of LPO [47, 50]. In addition, it was reported that MPs can alter the immune system and cause gastrointestinal tract inflammation of soil faunas [48, 63]. This might be related to the higher LPO levels after exposure to high doses of MPs in this study. The upward trend of LPO levels in high-dose MPs treatments after 14 d and the significant increase of LPO levels in MPs+Cd (>300 mg·kg-1) suggest that abrasion and 14

toxic impact might be induced by MPs and Cd, and MPs may accelerate the oxidative damages provoked by Cd. Consequently, E. foetida might suffer from sustained oxidative stress, intracellular damage and histopathological damage [24, 48, 50, 60]. As most plastic additives (such as brominated flame retardants and antioxidants) were mechanically incorporated into polymer without formation of chemical bounds, they can leach from plastics over time after ingestion [64, 65]. This might be an explanation for that LPO levels in E. foetida had a tendency to increase with exposure duration in high MPs concentration treatments (except T2C2 with

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3000 mg·kg-1 MPs on day 42) (Fig. 3a). The toxicity of MPs in organisms primarily derives from oxidative stress via the generation of ROS. The cumulate ROS subsequently provokes series biological reaction, e.g.

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signaling pathways elicited by oxidative stress, apoptosis, and inflammation [52, 54]. Previous studies suggested that GSH-related antioxidant enzymes could be augmented in response to

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oxidative stress imposed by xenobiotics, such as MPs [54, 55]. GSH plays as ROS scavenger,

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and its related antioxidant enzymes take a crucial role in detoxification mechanism against oxidative stress [54]. As indicated in previous studies that antioxidant enzymes activities could be largely increased due to the presence of metal stressors and micro(nano)particles exposure.

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In this study, GSH contents were significantly higher after exposure to MPs (>300 mg·kg-1), this is in agreement with the study of Jeong et al. [54]. They showed GSH contents in

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Paracyclopina nana were increased after exposure to polystyrene microbead.

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In the case of the measured GSH contents in E. foetida of this work, it differed from the results reported by Lu et al. [24]. They showed that GSH contents in zebrafish was significantly lower in the presence of PS microplastic (20 and 200 μg·L-1). However, taking into consideration the important role of GSH in diverse biological processes (e.g., maintaining normal cellular redox state, detoxification and antioxidant action, Cd chelation, and the synthesis of DNA and function in immune system), the content of GSH may differ between 15

species and under different experimental conditions [51, 52, 54, 66]. In our work, GSH contents increased after exposure to Cd (Fig. 3b), this is in accordance with previous studies on E. foetida [55, 67]. Furthermore, higher GSH contents were detected after combined exposure to MPs+Cd (Fig. 3b). Which may suggesting that the rise in GSH contents may be closely related to the adaptive mechanism of organisms to mitigate oxidative stress or detoxifying oxidative stresses [54, 55, 68]. As shown above, E. foetida might suffer from sustained oxidative stress, which may induce the synthesis of GSH to alleviate oxidative damages. This may be an

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explanation for why the trend in GSH contents were similar to that of LPO levels. 4.3. Microplastics ingestion

MPs retention has been discovered in aquatic worms [28, 29]. For instance, Hurley et al.

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[28] identified on average 129±65.4 particles·g-1 tissue retained within Tubifex worm.

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However, retention of MPs in terrestrial organisms has seldom been reported. In a similar exposure experiment, Hodson et al. [7] did not detect MPs particles in earthworm digestate

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after exposure to PE (on average of 920 μm, 3500 mg·kg-1 dw.) for 28 d. As suggested in previous studies, earthworms tend to ingest small MP particles (<100 μm) [25] and the ecotype

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of earthworm may influence their reaction to external pollutants [42]. The exposure manner, doses of MPs, MPs particles size and the ecotype of earthworm may be explanations for the

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differences between our study and previous studies [7, 25, 48]. In addition, in high MPs concentration treatments, particles retained inside of E. foetida were observed to be

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significantly higher after exposure for 42 d than on days 14 and 28 (supplementary Table 3). Our results showed that MPs particles within earthworms increased with exposure duration, except in the lowest dose of MPs treatment (supplementary Table 3). This may indicate that when MPs concentration exceed a certain value in soil environment, E. foetida may accumulate more MPs particles inside the body. 16

MPs percentage in cast significantly increased with the doses of MPs concentration in the soil, but no distinct differences were found on days 14 and 28 (supplementary Tables 4 and 5). The higher MPs concentration in egestion indicates that MPs pollution in the soil environment may account for greater ecological effects than we had previously assumed. Furthermore, the typical surface morphology of MPs particles extracted from E. foetida was clearly different from that of MPs particles recovered from soil (Fig. 4c and d). There were noticeable changes in the surface of extracted particles, i.e., holes indicating biodegradation (Fig. 4d). In contrast,

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no holes were detected on the surface of particles recovered from soil; rather, we found that countless smaller particles or nanoparticles adsorbed on the surface of MPs particles from the soil (Fig. 4c). However, with respect to the ingestion and retention of MPs in soil fauna, further

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studies are necessary to investigate whether the retained particles will be egested or transferred

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to other tissues.

4.4. Effect of MPs on accumulation of Cd in E. foetida

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Cd accumulation in E. foetida was significantly raised after co-exposure to MPs+Cd, compared with exposure to Cd alone. Thus, it is possible that E. foetida uptakes metal from the

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soil, and that MPs can accelerate the accumulation of metal in the body of E. foetida, such accumulation increasing with the increase of exposure duration. To the best of our knowledge,

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there is only one study focused on the effects of MPs and metal on L. terrestris [7]. Our findings with E. foetida are not in agreement with the study of Hodson et al. [7], in which no metal (Zn)

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accumulation in L. terrestris was found during the 28-day exposure experiment. Two possible explanations for the differences may be that Zn, as an essential metal for organisms, is likely well regulated by metabolic processes of L. terrestris [7], and, secondly, that the different ecotypes of the earthworms may also influence the accumulation of heavy metals [42]. Moreover, it was indicated that MPs can adsorb and transport heavy metal contaminants 17

due to specific surface characteristics [7, 22], the accumulated metal ions on the surface of MPs are highly likely to be released inside organisms after ingestion [7, 32]. Our result showed that the content of Cd in E. foetida is positively correlated with the MPs particles in E. foetida. Hence, MPs particles adsorb Cd ions on its surfaces, and then Cd ions desorb inside E. foetida due to gut surfactants maybe an explanation for our result. In these cases, as shown in the embedded figure (Fig. 5a), the Cd concentration in E. foetida increased with exposure duration. The accumulated Cd content of each treatment was significantly different between day 14 and

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day 42. Notably, we found statistically significant increases in Cd concentration in E. foetida after exposure to 14 d, 28 d, and 42 d and with high MPs concentrations (6000 and 9000 mg·kg1

) (Fig. 5a). The ability of MPs to increase the accumulation of heavy metals in aquatic

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organisms has been widely reported [24, 60, 69]. For instance, Lu et al. [24] found that after zebrafish was exposed to combined MPs (200 μg·L-1) and Cd (10 μg·L-1), the Cd content in

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zebrafish was 85% higher than that in the Cd treatment. However, further studies are needed

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to evaluate the biological consequences of different polymers and heavy metals on a wide range of soil faunas.

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5. Conclusion

Overall, exposure to MPs alone or co-exposure to MPs+Cd for 42 d both can decrease the

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growth rate and increase the mortality of E. foetida. We found that the presence of MPs (>3000 mg·kg-1) might induce oxidative damage and that MPs enhance the toxic effect of Cd. The

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results demonstrate that MPs particles could be retained within E. foetida and the number of particles retained increased with the dose of MPs and exposure duration. The accumulation of Cd in E. foetida could be augmented by the existence of MPs, and Cd content was increased with the duration of exposure. These findings provide a further understanding of the adverse effects of MPs and the synergic pollution of MPs and heavy metals on soil fauna. 18

Author Contributions Section

Zhou Yanfei: Writing-Original draft preparation, Methodology, Software;

Liu Xiaoning: Investigation, Data curation; Wang Jun: Reviewing and Editing , Supervision.

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Declaration of interests

☐ √ The authors declare that they have no known competing financial interests or personal

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relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

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This work was supported by the Funding Project of National Key Research and

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Development Program of China (2018YFD0900604), Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (2018), and Sino-Africa Joint Research Center,

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Chinese Academy of Sciences (Y623321K01).

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(2019) 215-225. http://doi. org/10.1016/j.watres.2018.12.017. [66] D.E. Hutter, B.G. Till, J.J. Greene, Redox state changes in density-dependent regulation of proliferation, Exp. Cell Res., 232 (1997) 435-438. http://doi. org/10.1006/excr.1997.3527. [67] Y.S. Li, H. Tang, Y.X. Hu, X.H. Wang, X.J. Ai, L. Tang, C. Matthew, J. Cavanagh, J.P. Qiu, Enrofloxacin at environmentally relevant concentrations enhances uptake and toxicity of cadmium in the earthworm Eisenia fetida in farm soils, J. Hazard. Mater., 308 (2016) 312-320. http://doi. org/10.1016/j.jhazmat.2016.01.057. [68] Y.G. Xue, X.Y. Gu, X.R. Wang, C. Sun, X.H. Xu, J. Sun, B.G. Zhang, The hydroxyl radical generation and oxidative stress for the earthworm Eisenia fetida exposed to tetrabromobisphenol A, Ecotoxicology, 18 (2009) 693-699. http://doi. org/10.1007/s10646-009-0333-2. [69] L.G.A. Barboza, L.R. Vieira, V. Branco, N. Figueiredo, F. Carvalho, C. Carvalho, L. Guilhermino, Microplastics cause neurotoxicity, oxidative damage and energy-related changes and interact with the bioaccumulation of mercury in the European seabass, Dicentrarchus labrax (Linnaeus, 1758), Aquat. Toxicol., 195 (2018) 49-57. http://doi. org/10.1038/s41598-018-34125-z.

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Fig 1. (a) Roman spectrum of the PP microplastics pellets (a). (b) Typical surface morphology of PP

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microplastics before incorporating into soil.

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Fig. 2. Growth rate (a) and mortality (b) of E. foetida exposed to MPs and MPs+Cd (four MPs concentrations: C1, C2, C3, and C4 refer to 300, 3000, 6000 and 9000 mg·kg-1, respectively. Cd

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concentration: 8 mg·kg-1;T1C1~4, MPs +Cd; T2C1~4, MP alone; T3, Cd alone; CK: without MPs and Cd).

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Values represent means ± SD (n=5), diff erent letters indicate significant diff erences (p < 0.05).

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Fig. 3. The LPO levels (nmol·mg protein-1) (a) and GSH content (mg GSH· g protein-1) (b) in E. foetida exposed to MPs and MPs+Cd, (four MPs concentrations: C1, C2, C3, and C4 refer to 300, 3000, 6000 and

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9000 mg·kg-1, respectively. Cd concentration: 8 mg·kg-1;T1C1~4, MPs +Cd; T2C1~4, MP alone; T3, Cd

alone; CK: without MPs and Cd). Values represent means ± SD (n=3), diff erent letters indicate significant

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lP

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diff erences (p < 0.05).

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Fig. 4. (a) MP particles retained within E. foetida, values represent means ± SD (n=3), diff erent letters indicate significant diff erences (p < 0.05) (four MPs concentrations: C1, C2, C3, and C4 refer to 300,

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3000, 6000 and 9000 mg·kg-1, respectively. Cd concentration: 8 mg·kg-1;T1C1~4, MPs +Cd; T2C1~4, MP alone). (b) MP concentration and the percentage of MP in the cast of E. foetida (airstrike means significant

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differences, p<0.05). (c) The typical surface morphology of the PP microplastic particle in soil without E.

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foetida. (d) The typical surface morphology of the MP particle retained within E. foetida.

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Fig. 5. (a) Cd accumulation in E. foetida after singly exposed to Cd and jointly exposed to MPs+Cd. (four MPs concentrations: C1, C2, C3, and C4 refer to 300, 3000, 6000 and 9000 mg·kg-1, respectively. Cd

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concentration: 8 mg·kg-1;T1C1~4, MPs +Cd; T2C1~4, MP alone; T3, Cd alone; CK: without MPs and Cd). Values represent means ± SD (n=3), diff erent letters indicate significant diff erences (p < 0.05). (b) The

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correlation between concentrations of Cd and MP particles in E. foetida. (after 42 d of combined exposure

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to MP and Cd).

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Table 1. Fresh weight (g, Mean ± SD) of E. foetida. After exposure of 14 d, 28 d, and 42 d in various Treatmentsa. Experimental

Treatment

group

Duration of exposure (days) 14 d 0

14

28

42

Control

28 d 0.33±0.02c

0.44±0.02bc

0.5±0.02ab

0.51±0.01a

Cd

T3

48 d 0.32±0.01c

0.41±0.01bc

0.45±0.02a

0.43±0.01ab

T2C1

0.31±0b

0.42±0.04a

0.47±0.06a

0.46±0.06a

T2C2

0.31±0.01b

0.42±0.02a

0.47±0.07a

0.45±0.07a

T2C3

0.33±0.02a

0.35±0.03bc

0.47±0.04a

0.42±0.03ab

T2C4

0.34±0.02c

0.36±0.04bc

0.46±0.03a

0.39±0.02bc

T1C1

0.32±0.01b

0.39±0.02b

T1C2

0.31±0.01b

0.37±0.01a

T1C3

0.32±0.01a

0.33±0.01a

T1C4

0.31±0.02b

0.33±0.02a

MPs

MPs+Cd

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CK

0.41±0.02a

0.39±0.02b

0.39±0.02a

0.36±0.02a

0.41±0.05a

0.35±0.04a

0.42±0.01a

0.33±0.01a

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Different letters indicate significant differences amongst treatments; a > b >c MPs concentrations: C1, C2, C3, and C4 refer to 300, 3000, 6000 and 9000 mg·kg-1, respectively. Cd concentration: 8 mg·kg-1;T1C1~4, MPs +Cd; T2C1~4, MP alone; T3, Cd alone; CK: without MPs and Cd.

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