Environmentally Relevant Concentrations of the Flame Retardant Tris

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Ecotoxicology and Human Environmental Health

Environmentally Relevant Concentrations of the Flame Retardant Tris(1,3-dichloro-2-propyl) Phosphate Inhibit the Growth and Reproduction of Earthworms in Soil Ya Zhu, Jianying Zhang, Yaoxuan Liu, Gangping Su, Lizhong Zhu, and Daohui Lin Environ. Sci. Technol. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.estlett.9b00227 • Publication Date (Web): 17 Apr 2019 Downloaded from http://pubs.acs.org on April 18, 2019

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Environmental Science & Technology Letters

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Environmentally Relevant Concentrations of the Flame Retardant Tris(1,3-

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dichloro-2-propyl) Phosphate Inhibit the Growth and Reproduction of

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Earthworms in Soil

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Ya Zhu†, Jianying Zhang†‡, Yaoxuan Liu†, Gangping Su†, Lizhong Zhu†‡, Daohui Lin* †‡

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† Department

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‡ Zhejiang

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University, Hangzhou 310058, China

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*Author for correspondence:

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of Environmental Science, Zhejiang University, Hangzhou 310058, China

Provincial Key Laboratory of Organic Pollution Process and Control, Zhejiang

Tel: 86 571 88982582; Fax: 86 571 88982590. Email: [email protected] (D. Lin)

ACS Paragon Plus Environment 1


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Abstract

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Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is a synthetic organophosphate flame retardant

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that has been used for decades in various products. Its environmental occurrence and toxicity to

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aquatic organisms has been reported. However, no study to date has been performed on the toxicity

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of TDCIPP to terrestrial invertebrates. In this study, earthworms (Eisenia fetida) were exposed to 0,

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50, 500, and 5000 ng/g TDCIPP in soil for 28 d. The 500 and 5000 ng/g TDCIPP treatments

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significantly lowered (4.2% and 9.2%, respectively) the average body mass of earthworms and the

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5000 ng/g TDCIPP treatments significantly decreased (25.0%) the number of juveniles born per

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earthworm. Moreover, seminal vesicle injury, skin lesions, and muscle atrophy were observed. It is

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worth noting that E. fetida accumulated TDCIPP mainly via epidermal adsorption. Using a

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combination of transcriptomics and metabolomics, detailed molecular information was provided on

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the responses of earthworms to TDCIPP exposure. After 5000 ng/g TDCIPP exposure, the contents

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of L-malic acid, cholesterol, scyllo-inositol, D-galactose, and maltose significantly changed and

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transcriptomic analysis identified 80 upregulated and 55 downregulated differentially expressed

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genes compared with the control group. TDCIPP exposure affected the hormone biosynthesis

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pathway, leading to the inhibition of earthworm reproduction. The inhibition of growth can be

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attributed to an affected phosphatidylinositol signaling system and the arachidonic acid metabolism

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pathway. These findings provide new insight into the molecular mechanisms by which earthworms

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respond to TDCIPP exposure and offer important information for the environmental risk assessment

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of organophosphate flame retardants.

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Key Words: Environmental toxicology; organophosphate flame retardant; earthworm; soil


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1. Introduction

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As the knowledge about environmental risks associated with brominated flame retardants has

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improved, the manufacture and application of some of these compounds have been steadily prohibited,

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such as hexabromocyclododecane, polybrominated biphenyl, and polybrominated diphenyl ethers.

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Organophosphate flame retardants are increasingly used as an alternative (1). However, their

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environmental risks are largely unknown and need to be evaluated.

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Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is one of the main organophosphate flame

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retardants. According to the United States Environmental Protection Agency (USEPA), between

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2011 and 2015, the annual production volume of TDCIPP was 4500–23 000 tons in the United States

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(2). Like brominated flame retardants, organophosphate flame retardants have now been widely found

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in water, outdoor air, indoor air, soil, and biota (3). Soil is a vital sink for organic and inorganic

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pollutants, including organophosphate flame retardants. For example, a study of soil in Shenyang

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City, China showed that the maximum concentrations of 13 total organophosphate esters reached 950

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ng/g dry weight (dw), of which TDCIPP was one of the most abundant organophosphate esters with

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a range of 2 to 41 ng/g dw (4). The concentrations of TDCIPP in the surface soil of Nepal were

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reported to be 12.1-390 ng/g dw (5). TDCIPP in outdoor settled dust from a multiwaste recycling area

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in Tianjin, China was up to 4460 ng/g (6). Therefore, soil TDCIPP has reached a considerable

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concentration and its environmental risk needs to be evaluated.

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A few studies have reported that TDCIPP exposure at concentrations of 65 ng/L to 1.4 mg/L

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could cause developmental, reproductive, and/or neurological toxicity to aquatic organisms including

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zebrafish and daphnia (7-15). However, thus far, no available study has been conducted to evaluate

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the risk of TDCIPP towards terrestrial species. To fill this gap, in this study, a chronic exposure

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experiment was performed to study the mechanisms of toxicity of TDCIPP to the soil sentinel species



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Eisenia fetida. The results of this study are believed to provide important information for the

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environmental risk assessment of organophosphate flame retardants.

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2. Materials and Methods

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2.1 Animals and exposure protocols

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Information related to chemicals and reagents is provided in the Supporting Information (SI).

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Soil samples were collected from a rice field in Hangzhou, China (30°22′19.52″ N, 119°58′55.71″

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E). The soil samples were air-dried and sieved to 1 mm, and the maximum water holding capacity

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was analyzed with the method based on the Organisation for Economic Co-operation and

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Development (OECD) guideline 222 (16) and described in the SI. The properties of the soil were as

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follows: silty clay loam; maximum water holding capacity (59%); pH (5.9); organic matter content

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(2.0%). E. fetida were purchased from an earthworm farm (Green Grass Garden Co., Jiaxing, China).

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Earthworms were kept in a soil culture at 20 ± 1 °C and fed with cow manure every 7 d in the lab.

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After two weeks of adaptation in the lab environment, mature earthworms with obvious clitellum

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were used for exposure experiments.

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The earthworm growth and reproduction assays were carried out according to the OECD

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guideline 222 (16). Fifty milliliters of 0, 0.5, 5, and 50 mg/L TDCIPP acetone solutions were added

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to 500 g soil, resulting in 0, 50, 500, and 5000 ng TDCIPP per gram of dry soil, respectively. The 0

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mg/L TDCIPP acetone solution-treated soil was used as a control. The spiked soils were placed into

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the blender and vigorously mixed for a few minutes as described in a previous study (17). Complete

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mixing was ensured by turning any compressed soil in the blade with a glass spatula, blending was

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then resumed. Turning/mixing was repeated twice. Then, the soil samples were volatilized in a fume

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hood for 7 d to evaporate the acetone. Ultrapure water was added to adjust the water content to 60%

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of the maximum water holding capacity. Ten mature earthworms were exposed to each soil sample


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in each of four replicate glass beakers for each concentration. The body mass of the individual

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earthworms was between 400 and 600 mg. The capacity of the glass beaker was 2 liters. The exposure

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lasted for 28 d, and the earthworms were fed with 10 g cow manure in each glass beaker every 7 d.

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The cow manure was moistened with ultrapure water and spread on the soil surface in each beaker.

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During the exposure period, the earthworms were maintained at 20 °C under controlled light-dark

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cycles (16 h light/ 8 h dark) with an illumination of 600 lux. Soil moisture was maintained at 60% of

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the maximum water holding capacity by adding water regularly. To study the potential degradation

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of TDCIPP in the soil with the earthworms, 2 grams of soil was sampled from each beaker 0, 1, 2, 3,

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4, 5, 14, 21, and 28 d after TDCIPP spiking. After the 28 d exposure, the earthworms were collected

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and placed onto a humid filter paper for 48 h to empty their gut contents (18, 19), and the body

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weights were then measured. The earthworm-produced cocoons remained in the soil without feeding,

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and the number of hatched juveniles after another 28 d was recorded.

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2.2 Determination of TDCIPP in soil and earthworm tissues

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Earthworms were dissected into skin, gut, and other tissues (earthworm body after removal of

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the skin and gut). Acetonitrile was used to extract TDCIPP from the soil and earthworms (20, 21).

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TDCIPP was analyzed by using a Waters ACQUITY UPLC I-Class system (Milford, MA, USA)

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coupled with an AB Sciex QTrap 5500 system (Foster City, CA, USA) (22). Detailed protocols are

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provided in the SI. TDCIPP was undetectable (< 0.14 ng/g) in any of the control groups (soil or

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earthworm).

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2.3 Electron microscopy observations

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Earthworms for ultrastructural observation were fixed in 2.5% glutaraldehyde overnight,

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followed by staining with osmium tetroxide, dehydration, embedding, and ultrathin sectioning (23,

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24). The obtained sections were observed with transmission electron microscopy (TEM; JEM-1230,



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Japan).

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2.4 Histological examination

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Histological examination of the seminal vesicles was performed according to previously

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published methods (13). The details are provided in the SI.

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2.5 Transcriptomic sequencing and quantitative real-time polymerase chain reaction (qRT-

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PCR) validation

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Transcriptomic sequencing was performed by Shanghai Majorbio Bio-pharm Technology Co.

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(Shanghai, China). qRT-PCR was performed using SYBR Green kits (Takara, Kusatsu, Japan).

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Detailed protocols are provided in the SI. The data were analyzed on the online Majorbio I-Sanger

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Cloud Platform.

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2.6 Metabolomic analyses

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Gas chromatography-mass spectrometry (GC-MS)-based metabolomics was used to analyze the

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metabolites in the earthworms according to previous reports (25, 26). The details are provided in the

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SI.

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2.7 Statistical analyses

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All data were analyzed using SPSS statistics 19.0 software and are represented as the means ±

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standard errors. Kolmogorov-Smirnov and Levene’s tests were used to analyze the normality and the

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homoscedasticity, respectively. The differences between the control and treatment groups were

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evaluated by one-way analysis of variance (ANOVA) and post hoc analysis using the Tukey’s test.

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Statistical significance was determined by p < 0.05.

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3. Results and Discussion

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3.1 TDCIPP contents in earthworms and soil

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The measured concentrations of TDCIPP in the exposure soil samples with nominal


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concentrations of 50, 500, and 5000 ng/g were 33 ± 3, 359 ± 11, and 5390 ± 60 ng/g, respectively, at

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0 d, which gradually decreased to 11 ± 2, 141 ± 9, and 4808 ± 78 ng/g, respectively, at 28 d (Figure

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S1). After the 28 d exposure and 48 h depuration, the concentrations of TDCIPP in the earthworms

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were 1.06 ± 0.09, 9.95 ± 1.20, and 138.2 ± 9.89 ng/g wet weight (ww) in these three exposure groups,

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respectively (Figure 1C). The concentration of TDCIPP in the skin was significantly higher (1.69 ~

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1.96-fold) than that in the gut (Figure 1C). The highest concentration of TDCIPP was detected in the

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skin of the exposed earthworms, indicating that E. fetida mainly accumulated TDCIPP via epidermal

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absorption rather than gut processes. Previous studies have shown that earthworms take up chemical

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contaminants through two major routes: absorption via intestinal digestion from soil particles or

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passive diffusion through the skin from soil interstitial water (27, 28). For instance, atrazine

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bioaccumulation in epigeic E. fetida was mainly through dermal absorption, whereas bioaccumulation

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in anecic Metaphire guillelmi was largely affected by the gut processes. Gut processes of M. guillelmi

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facilitated desorption of atrazine from soil, thereby greater biouptake was observed for M. guillelmi

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than for E. fetida (28). The relative contributions of these two paths depend on the feeding behavior

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of earthworm species (29) and the hydrophobicity of chemicals (30). Previous study have indicated

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that when the chemical logKow is larger than 6, the gut ingestion becomes the dominant uptake route

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(30). Since E. fetida ingests a small amount of soil (28) and the logKow of TDCIPP is 3.65 (10), the

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accumulation of TDCIPP could be mainly through epidermal absorption.

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3.2 TDCIPP exposure decreased the growth and reproduction of earthworms

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Although all earthworms survived, the exposure to TDCIPP caused a dose-dependent inhibition

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of earthworm growth (Figure 1A). The 500 and 5000 ng/g TDCIPP treatments significantly lowered

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(4.2% and 9.2%, respectively) the body mass per earthworm in each container, whereas the 50 ng/g

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treatment had no significant effect. The number of juveniles born per earthworm during the 28 d



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exposure significantly decreased (25.0%) in the 5000 ng/g group (Figure 1B).

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3.3 TDCIPP exposure induced seminal vesicle injury, skin lesion and muscle atrophy

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As shown in Figure 2A, seminal vesicle focal necrosis and cytoplasmic vacuolation appeared in

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the earthworms exposed to 5000 ng/g TDCIPP. However, no significant effect was observed in the

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seminal vesicles exposed to 50 or 500 ng/g TDCIPP. Figure 2B shows the ultrastructure of earthworm

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skin. In the control and 50 ng/g groups, the epicuticle was closely arranged, while in the 500 ng/g

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group, a small amount of epicuticle shed. In the 5000 ng/g group, the epicuticle was damaged and the

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cuticle layer was severely thickened. The exposure to high concentrations of TDCIPP (500 and 5000

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ng/g) obviously enlarged the gap between earthworm muscles, with muscle atrophy observed in the

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5000 ng/g group (Figure 2C).

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3.4 TDCIPP exposure affected transcriptome and metabolism in earthworms

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To reveal the underlying mechanism, transcriptomic and metabolomic analyses were performed.

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Eighty upregulated (fold change ≥ 2, p-adjust < 0.05) and 55 downregulated (fold change ≤ 0.5, p-

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adjust < 0.05) differentially expressed genes were identified in the 5000 ng/g exposure group

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compared with the control group (Figure S2). To validate the reliability of the transcriptomic

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sequencing analyses, 10 differentially expressed genes were randomly selected for qRT-PCR

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verification (Table S1). As shown in Table S2, the data from both methods were largely consistent,

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suggesting that the RNA-seq results were credible. Kyoto Encyclopedia of Genes and Genomes

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(KEGG) pathway enrichment analysis on the downregulated genes demonstrated that the hormone

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biosynthesis, phosphatidylinositol signaling system, and arachidonic acid metabolism pathway were

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significantly enriched after exposure to 5000 ng/g TDCIPP (Figure 3A), while no KEGG pathways

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among the list of upregulated genes were significantly enriched (p-adjust > 0.05).

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GC-MS-based nontarget metabolomics enabled the identification and quantification of 47


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metabolites in earthworms (Table S3). The contents of L-malic acid, cholesterol, scyllo-inositol, D-

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galactose, and maltose were significantly changed (p < 0.05) after 5000 ng/g TDCIPP exposure

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compared with the control (Figure 3B). KEGG pathway analysis of these significantly changed

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metabolites showed that the steroid biosynthesis pathway was significantly enriched after exposure

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to 5000 ng/g TDCIPP (Figure 3C).

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Arachidonic acid is a polyunsaturated fatty acid found in the phospholipids of the cell membrane

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and is abundant in muscle. It has been indicated that arachidonic acid can enhance myonuclear

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accretion and myotube growth during myogenesis (31). Moreover, the eicosanoid product has been

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shown to play critically important functional roles in various basic biological processes, such as cell

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proliferation, intracellular signaling, and inflammation (32). Therefore, arachidonic acid metabolism

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is crucial to muscle development. The observed muscle atrophy after TDCIPP exposure in this study

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could be caused by the interruption of arachidonic acid metabolism and could have contributed to the

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inhibition of earthworm growth. Transcriptomic sequencing showed that the phosphatidylinositol

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signaling system pathway was significantly affected by 5000 ng/g TDCIPP exposure. The results of

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the metabolomics supported this finding, with scyllo-inositol being downregulated in response to

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TDCIPP exposure. A previous study indicated that phosphatidylinositols could be involved in a

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number of physiological processes including cell proliferation, death, motility, cytoskeletal regulation,

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intracellular vesicle trafficking, and cell metabolism (33). This may be another cause of earthworm

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growth inhibition. Decreased carbohydrate contents were observed in earthworms or other

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invertebrates exposed to toxins, followed by decreased growth (34). In the present study, the

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metabolomics analysis showed a decrease in carbohydrate (maltose and galactose) concentrations and

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an increase in L-malic acid in the TDCIPP-exposed earthworms. L-Malic acid is an important organic

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acid produced during the metabolism of organisms. It is a key intermediate in the tricarboxylic acid



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cycle (TCA cycle) and can enter the mitochondria rapidly through the membrane and directly

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participate in energy metabolism (35). Therefore, the observed higher concentration of L-malic acid

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in the exposed earthworms indicate the TCA cycle was enhanced to offset the energy deficiency due

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to TDCIPP exposure. However, an adequate energy supply was essential to maintain normal

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physiological function, and therefore, a reduction in carbohydrate contents could also contribute to

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growth inhibition.

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The reproduction of annelids is regulated by steroid hormones (36). The transcriptomics results

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showed that the hormone biosynthesis pathway was affected by TDCIPP exposure, which was

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consistent with the metabolomics results. Since cholesterol is essential for the synthesis of steroid

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hormones (36), higher concentrations of cholesterol observed by the metabolomics analysis indicated

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that hormone synthesis was blocked. As a result, the reproductive capacity of earthworms was

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inhibited.

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Supporting Information

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Chemicals and reagents, determination of the maximum water holding capacity of the soil,

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determination of TDCIPP in soil and earthworm tissues, histological examination, protocol of

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transcriptomic sequencing and qRT-PCR validation, metabolomic analysis, changes in the TDCIPP

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concentrations in soil (Figure S1), number of differentially expressed genes in earthworms (Figure

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S2), score plot of PCA and OPLS-DA (Figure S3), sequences of primers for the test genes (Table

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S1), validation of the RNA-seq analysis by qRT-PCR (Table S2), and the list of identified metabolites

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(Table S3). The Supporting Information is available free of charge on the ACS Publications website

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at DOI: XX.XX.XXXX.

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Acknowledgements

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This work was supported by the National Natural Science Foundation of China (21621005,


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21525728, and 21777139) and the National Key Research and Development Program of China

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(2017YFA0207003).

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Figure 1. Body mass (A), the number of juveniles number produced per earthworm (B), and

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TDCIPP contents in earthworms and their different tissues (C) after 28 d of exposure to various

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concentrations of TDCIPP. Values represent the mean ± SE (n=4). *p < 0.05.


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Figure 2. Histological examination of seminal vesicles (A; optical microscope images),

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ultrastructural observations of skin (B; TEM images), and longitudinal muscles (C; TEM

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images) of earthworms after exposure to various concentrations of TDCIPP for 28 d. SV:

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seminal vesicle. Yellow arrows in A indicate cytoplasmic vacuolation and focal necrosis. The

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blue arrow in B indicates the epicuticle shed. Red arrows in C indicate the gaps between

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muscles. ACS Paragon Plus Environment 17


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Figure 3. KEGG enrichment analysis of downregulated genes from transcriptomics (A), fold

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changes of differentiated metabolites (B), and a summary of the pathway analysis of

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metabolomics (C) in earthworms exposed to 5000 ng/g TDCIPP. * p < 0.05.


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Environmentally Relevant Concentrations of the Flame Retardant Tris

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