Journal Pre-proof

Microplastic fibers transfer from the water to the internal fluid of the sea cucumber
Apostichopus japonicus

Mohamed Mohsen, Libin Zhang, Lina Sun, Chenggang Lin, Qing Wang, Hongsheng
Yang

PII: S0269-7491(19)34391-X
DOI: https://doi.org/10.1016/j.envpol.2019.113606
Reference: ENPO 113606

To appear in: Environmental Pollution

Received Date: 5 August 2019

Revised Date: 6 October 2019
Accepted Date: 10 November 2019

Please cite this article as: Mohsen, M., Zhang, L., Sun, L., Lin, C., Wang, Q., Yang, H., Microplastic
fibers transfer from the water to the internal fluid of the sea cucumber Apostichopus japonicus,
Environmental Pollution (2019), doi: https://doi.org/10.1016/j.envpol.2019.113606.

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 1 Microplastic fibers transfer from the water to
2 the internal fluid of the sea cucumber
3 Apostichopus japonicus
4 Mohamed Mohsen a,b,c,d,f Libin Zhang a,b,c,d Lina Sun a,b,c,d Chenggang Lin a,b,c,d Qing

5 Wang e Hongsheng Yang a,b,c,d *.

a
6 CAS Key Laboratory of Marine Ecology and Environmental Sciences, Institute of
7 Oceanology, Chinese Academy of Sciences, Qingdao 266071, China.

b
8 Laboratory for Marine Ecology and Environmental Science, Qingdao National
9 Laboratory for Marine Science and Technology, Qingdao 266237, China.

c
10 University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049,
11 China.

d
12 Center for Ocean Mega-Sciences, Chinese Academy of Sciences, Qingdao 266071,
13 China.

e
14 Research and Development Center for Efficient Utilization of Coastal Bioresources,
15 Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai
16 264003, P. R. China.

f
17 Department of Animal Production, Faculty of Agriculture, Al-Azhar University,
18 Nasr City, Cairo, Egypt.

19 *Corresponding authors:

20 Dr. Hongsheng Yang

21 Key Laboratory of Marine Ecology and Environmental Sciences, Institute of

22 Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao, 266071,

23 Shandong, China.

24 Email address: hshyang@qdio.ac.cn

1
25 Abstract

26 Microplastics (MPs) are small plastic particles less than 5 mm in diameter. MPs in the

27 form of microfibers (MFs) are widely detected in aquatic habitats and are of high

28 environmental concern. Despite many reports on the effects of MFs on marine

29 animals, their effect on sea cucumbers is still unclear. In addition, our previous filed

30 study has shown that MFs might transfer to the coelomic fluid of the sea cucumber

31 Apostichopus japonicus (A. japonicus). Here, we show how MFs transfer to the

32 coelomic fluid of the sea cucumber. We captured the MFs during their transfer from

33 the water to the coelomic fluid through the respiratory tree. A. japonicus ingested in

34 the MFs along with the water during respiration; the MFs got stuck in the respiratory

35 tree or transferred to the coelomic fluid. The transferred MFs increased during 72 h of

36 exposure and persisted for 72 h after the transfer to clean water. Among the immunity

37 indices, lysozyme (LZM) levels increased in response to the transferred MFs, which

38 confirms the defensive role of LZMs against strange substances. Additionally,

39 non-significantly decreased levels of total antioxidant capacity (T-AOC),

40 malondialdehyde (MDA), peroxidase (POD) and phenol oxidase (PPO) were

41 observed at 24 h and 48 h post-exposure, suggesting minimal oxidative imbalance.

42 Furthermore, there were no significant changes in the speed and the total distance

43 moved by A. japonicus post-transfer. This study revealed that MFs transfer and

44 bioaccumulate in the coelomic fluid of A. japonicus.

2
45 Capsule

46 Synthetic microfibers transfer from the water to the coelomic fluid of the sea

47 cucumber and may increase over time.

48 Keywords

49 Sea cucumber; Microplastic fiber; Physical hazard; Lysozyme; Oxidative stress;

50 Biological accumulation

51 1. Introduction

52 MPs are tiny plastic particles that can be harmful to biota (reviewed by Anbumani and

53 Kakkar, 2018). These MPs can be of various shapes, from pellets and spheres to

54 fragments and fibers (de Sá et al., 2018). Microfibers (MFs), which are threads that

55 are uniformly shaped throughout their entire length ( de Sá et al., 2018; Suran, 2018),

56 are the prominent form of MPs in animal habitats (Browne et al., 2011;Van

57 Cauwenberghe et al., 2015; Barrows et al., 2018; Henry et al., 2019), and their major

58 source is synthetic clothing (Browne et al., 2011; de Sá et al., 2018; Barrows et al.,

59 2018). Synthetic MFs are more hazardous than other MP particles (Au et al., 2015;

60 Qiao et al., 2019), and they could be a vector for transferring pollutants to animals

61 (Suran, 2018; Mohsen et al., 2019a). MFs are ingested by a wide range of animals,

62 such as mussels (Catarino et al., 2018), fish (Compa et al., 2018), marine turtles

63 (Duncan et al., 2019), benthic invertebrates (Courtene-Jones et al., 2019), corals

64 (Rotjan et al., 2019) and crustaceans (Bour et al., 2018). MFs exposure decreases the
3
65 filtration rate in the mussel Mytilus edulis (Woods et al., 2018), increases mortality in

66 the crustacean Daphnia magna (Jemec et al., 2016), reduces food consumption in the

67 crab Carcinus maenas (Watts et al., 2014) and reduces growth in the amphipod

68 Hyalella azteca (Au et al., 2015).

69 Sea cucumbers are epibenthic deposit feeders that pull the sediment by the tentacles

70 into the mouth to extract nutrients. Sea cucumbers breath from their anus through the

71 respiratory tree. The respiratory tree serves as lungs that allow gas exchange between

72 the water and the coelomic fluid of sea cucumbers (Lambert, 1997; Gao and Yang,

73 2015). Sea cucumbers, especially A. japonicus (Yang and Bai, 2015), not only have

74 high economical and nutritional values (Toral-Granda et al., 2008; Bordbar et al.,

75 2011) but also play a crucial role in marine ecosystems (Purcell et al., 2016). Their

76 sediment reworking and feeding activities help to improve sediment characteristics

77 (Pischedda et al., 2008), influence the productivity of many benthos (Uthicke, 2001),

78 enhance water chemistry (Wolfe et al., 2018), decrease algal blooms (Michio et al.,

79 2003) and remove aquaculture waste (Zamora et al., 2018). Sea cucumber A.

80 japonicus has been harvested for centuries and its natural resources are depleted.

81 Consequently, A. japonicus has been listed as an endangered species (Hamel and

82 Mercier, 2013). Moreover, A. japonicus sea cucumbers ingest MFs and MFs might

83 transfer to their coelomic fluid (Mohsen et al., 2019b). Despite many reports on the

84 effects of MFs on marine animals, their effect on sea cucumbers is unknown. With the

4
 85 increasing of MFs in the ocean, especially from anthropic activities (Belzagui et al.,

86 2019), together with their hazardous properties (Suran, 2018), their effects on such

87 endangered species need to be clarified. To know whether these hazardous materials

88 can influence the biological functions and the development of sea cucumbers, and

89 whether the ingestion of these materials by sea cucumbers has implications for food

90 web since the internal viscera of sea cucumbers can be consumed by humans (Purcell

91 et al., 2012; Mao et al., 2015).

92 Therefore, this work was designed to answer the following questions: Do MFs

93 transfer to the coelomic fluid of A. japonicus? If so, what are the biological

94 consequences concerning that transfer, and do MFs persist in the coelomic fluid after

95 transferring A. japonicus to clean water?

96 2. Materials and Methods

97 2.1. Experimental Animals

98 Sea cucumbers (n = 150) were obtained from a local farm in Rushan, China

99 (36°46'40.7"N 121°34'34.7"E). As the animals were obtained from a local farm, no

100 license was needed to collect them. The animals were acclimated in the laboratory

101 using sand-filtered seawater with 30 ppt salinity at 16 ± 1°C for one week. After two

102 days of starvation, the animals were selected randomly and separated into groups;

103 there were no significant differences between the groups (P < 0.05).

5
104 2.2. Synthetic MFs preparation

105 MFs are mainly detected from the habitat of A. japonicus (Mohsen et al., 2019b).

106 Therefore, MPs in the form of MFs were prepared from polyester threads that are

107 commonly used in clothing manufacturing. These threads were separated into small

108 MFs (length: 1-5 mm) using a cutting machine and scissors ((Supporting information

109 (S) Figure S1) to simulate a washing machine process. The MFs were red in color,

110 and thus, it was easy to distinguish them under the microscope. Observation of the

111 MFs in the coelomic fluid of A. japonicus was performed by making an opening on

112 the ventral surface that let the coelomic fluid flow freely into a clean petri dish

113 (Mohsen et al., 2019b). A dissecting microscope (SMZ-161-BLED, Motic, China) or

114 a fluorescence microscope (OLYMPUS IX51) was used to detect and visualize the

115 MFs. A hand tally counter was used to count the MFs.

116 2.3. A. japonicus exposure to MFs from the water

117 To investigate the possibility of MFs transfer from the water, an initial experiment

118 was conducted. The individuals were placed in 4.8 L circular covered tanks with a

119 diameter of 27 cm with three replicates; oxygen was supplied by air stones. The

120 average sea cucumber weight was 25 ± 0.76 g (Mean ± SE). Firstly, the animals

121 were exposed to a concentration of 0.023 MFs mL-1, which is reported from the Bohai

122 Sea in China. (Dai et al., 2018). However, using this concentration, a homogenous

123 suspension of the MFs could not be achieved, because some of the MFs were
6
124 aggregated, suspended or settled down on the bottom of the tank. MFs in the habitat

125 may lose buoyancy or move with the current, and then, approach the anus of the sea

126 cucumber. Therefore, we used concentrations of 0.003 gL-1 and 0.006 gL-1,

127 approximately 25 MFs mL-1 and 40 MFs mL-1. The animals were sampled 1 h, 3 h,

128 and 12 h postexposure to examine the possibility of MF transfer. Consequently, we

129 observed that MFs transfer from the water to the coelomic fluid of A. japonicus and

130 their transfer depends on their location within the anus opening possibly during the

131 first breath. The closer the MFs were to the anus, the greater the chance of transfer

132 was. This is likely because of an increased possibility of reaching the walls of the

133 respiratory tree and thus transferring. Hence, experimentally, an increase in the MFs

134 exposure rate can reveal the MFs pathway into the organism. Subsequently, an

135 experiment was conducted to examine the enzymatic responses associated with the

136 MFs transfer. The animals were exposed to the same MF concentrations and sampled

137 24 h, 48 h and 72 h postexposure. Then, we examined the persistence of the MFs after

138 transferring the animals to clean water. The animals were transferred to sand-filtered

139 water at 72 h postexposure. The water was changed daily, and the animals were

140 sampled at 72 h posttransfer.

141 2.4. A. japonicus exposure to MFs from the ingested sediment

142 To examine the probability of MFs transfer from the ingested sediment through the

143 gastrointestinal epithelium of A. japonicus, a diet containing 40 % algae powder and

7
144 60 % sea mud that mixed with MFs was prepared. A. japonicus does not differentiate

145 between MFs and sediment particles (Mohsen et al., 2019b), thus the MFs were

146 expected to pass through the intestines and reveal the probability of MFs transferring.

147 Therefore, the individuals were exposed to the MFs in the feed at concentrations of

148 0.6 MFs g -1 and 1.2 MFs g-1, which are reported from the field (Mohsen et al.,

149 2019b), or at a concentration of 10 MF g -1. The individuals were placed in tanks

150 (45.33.25 cm) with three replicates. The oxygen was supplied by air stones, the feed

151 was introduced once a day and the water in each tank was changed daily. The average

152 sea cucumber weight was 83.23 ± 0.48 g (Mean ± SE). The animals were sampled 1

153 h, 3 h,12 h, 72 h and 168 h postexposure to examine the possibility of MF uptake by

154 the gastrointestinal epithelium. The MFs were not found to transfer to the coelomic

155 fluid and this investigation was continued for 60 d for further confirmation.

156 2.5. Enzyme analysis of the coelomic fluid due to MF transfer

157 The coelomic fluid of sea cucumbers is somewhat like blood in the vertebrates. The

158 coelomic fluid transports nutrients and contains phagocytic coelomocytes, which is

159 similar in function to the blood cells in vertebrates (Lambert, 1997; Gao and Yang,

160 2015). Therefore, to examine the physiological status of A. japonicus, the activities

161 of thirteen enzymes that represent immunity and oxidative responses were analyzed.

162 The coelomic fluid was collected in test tubes and stored in liquid nitrogen for

163 preservation at - 80 until analysis. The specific tests used in this study were
8
164 performed with commercial kits from Nanjing Jianchen Biological Institute

165 (Nanjing, China). Further details are described in table S1.

166 2.5.1. Immune defense indices

167 The immunity indices, including lysozyme (LZM), myeloperoxidase (MPO), acid

168 phosphatase (ACP) and alkaline phosphatase (AKP) activities were measured.

169 2.5.2. Oxidative stress indices

170 The oxidative stress indices, including superoxide dismutase (SOD), glutathione

171 peroxidase (GSH-Px), catalase (CAT), succinate dehydrogenase (SDH), lactate

172 dehydrogenase (LDH), total antioxidant capacity (T-AOC), malondialdehyde (MDA),

173 peroxidase (POD) and phenol oxidase (PPO) activities were measured.

174 2.6. Behavior recording post MF transfer

175 Sea cucumber activity was filmed at 72 h postexposure for 12 h in clean water using

176 an overhead camera. The film was analyzed using Ethovision XT 10.1 (Noldus Inc.,

177 the Netherlands) software and evaluated to determine the velocity and total distance

178 moved by the individuals.

179 2.7. Statistical analysis

180 The Shapiro-Wilk normality test with a 95% confidence level was used to examine

181 the normality of the data. To detect significant differences between the treatments, a

9
182 one-way ANOVA followed by Tukey’s post hoc test was used for normally

183 distributed data; otherwise, the Kruskal-Wallis H test followed by the Mann-Whitney

184 test was used (P < 0.05). SPSS Statistics 20.0 statistical software (SPSS Inc., Chicago,

185 IL) was used to conduct all statistical analyses.

186 3. Results

187 3.1. A. japonicus exposure to MFs from the water

188 The MFs were captured during their transfer from the water through the branch of the

189 respiratory tree under a fluorescence microscope (Figure 1). In addition, we observed

190 that when MFs were present, A. japonicus ingested in the MFs along with water

191 during respiration. The MFs entered the anus with the water, and expulsion with the

192 water was attempted, but the MFs got stuck in the branches of the respiratory tree

193 (Figure S2) or were transferred to the coelomic fluid (Figure S3). Ingesting MFs with

194 the first respiratory intake of water by A. japonicus may increase the probability of

195 MFs being transferred to the coelomic fluid. Furthermore, the number of MFs

196 transferred was increased over time, except for one case in T2 at 48 h postexposure,

197 without a significant difference in both treatments (P < 0.05) (Figure 1). In addition,

198 at 72 h post-transfer to the sand-filtered water, the MFs were persisted in the coelomic

199 fluid and their number was comparable with those sampled at 72 h postexposure in

200 both treatments (Figure S4) (P < 0.05). No animals were died or eviscerated during

201 the experiment.

10
202 3.2. A. japonicus exposure to MFs from the ingested sediment

203 A. japonicus ingested the feed that mixed with MFs, however, the MFs were not

204 found to transfer from the water to the coelomic fluid through the gastrointestinal

205 epithelium even up to 60 d of exposure.

206 3.3. Enzymatic responses of the coelomic fluid due to MF transfer

207 3.3.1. Immune defense indices

208 The activity of the LZMs in the treated animals was higher than that in the control

209 animals at 24 h, 48 h and 72 h postexposure in both treatments. The activity in the

210 treated animals showed a significant difference from that in the control animals at 48

211 h postexposure in both treatments (P < 0.05) (Figure 1). The activities of AKP, ACP,

212 and MPO in the treated animals were comparable with those in the controls (Figures

213 S5- S7).

214 3.3.2. Oxidative stress indices

215 The concentrations of T-AOC MDA, PPO and POD showed non-significant

216 decreased levels at 24 h and 48 h but not at 72 h postexposure in both treatments

217 (Figure 1 and S8-10). Also, the concentrations of SOD, GSH, CAT, LDH, and SDH

218 in the treated animals were not significantly different from those in the control

219 animals in either treatment and their levels did not show a regular tendency (Figures

220 S11-S15).
11
221 3.4. Behavior recording after MFs transfer

222 After 72 h of exposure, the total distance moved by treated A. japonicus was not

223 different from that of the controls during the 12 h of observation in clean water

224 (Figure S16) (P < 0.05). In addition, the average velocity of treated A. japonicus was

225 not different from the controls (Figure S17) (P < 0.05). The average velocity of A.

226 japonicus was 0.7 mm s-1.

227 4. Discussion

228 4.1. A. japonicus exposure to MFs from the water

229 MFs are widely detected in aquatic habitats and are of high environmental concern,

230 likely because of their size (Henry et al., 2019). In our previous field study, we

231 speculated that MFs might transfer to the coelomic fluid of A. japonicus. MFs were

232 found in the coelomic fluid of A. japonicus specimens that collected from eight sites

233 along the Bohai Sea and the Yellow Sea in China (Mohsen et al., 2019b). Therefore,

234 here we investigated the probability of MFs transfer in the laboratory. We detected

235 MFs that transferred to the coelomic fluid of A. japonicus from the water through the

236 respiratory tree; this process has not been previously shown. The respiratory tree

237 serves as lungs that is responsible for gas exchange between the water and the

238 coelomic fluid. A. japonicus breathes by drawing water through the anus and then

239 expelling it (Gao and Yang, 2015). Therefore, the MFs entered the respiratory tree

12
240 through the process of breathing. The MFs increased by time exposure except in one

241 case in T2 at 48 h postexposure, because the MFs were stuck in the respiratory tree

242 (Figure S2). Thus, MFs can physically harm the respiratory tree if they fit to enter the

243 anus. The shape of the MFs has an important role in penetrating the walls of the

244 respiratory tree since a single MF can pass through (Figure S18). This process is

245 likely to happen in nature over the lifetime of A. japonicus because MFs were

246 detected from the coelomic fluid of A. japonicus in farms, with an average weight of

247 53 g to 144 g (Mohsen et al., 2019b). In addition, the MFs might accumulate in the

248 coelomic fluid over time, since the haemal system of sea cucumbers is a closed

249 system (Lambert, 1997; Gao and Yang, 2015). After transfer the sea cucumber to

250 clean water for 72 h, the number of the transferred MFs was comparable with those

251 sampled at 72 h postexposure, suggesting that the transferred MFs had no way out of

252 the coelomic fluid. This accumulation is different from that in mussels. The mussel

253 Mytilus edulis accumulate the MFs during the filter-feeding but these MFs can finally

254 be ejected. The mussel Mytilus edulis can decrease an average of 63 % of the

255 accumulated MFs within 6 h posttransfer to clean water (Woods et al., 2018).

256 Increasing the MFs accumulation in the coelomic fluid of A. japonicus will not likely

257 result in mortality but might disrupt the biological functions and the development of A.

258 japonicus, which worth further investigation. Additionally, MFs accumulation in the

259 coelomic fluid of A. japonicus might provide a route for subsequent transport of

260 contaminants to the internal viscera of sea cucumbers, since MFs adsorb contaminants
13
261 from the surrounding environment (Suran, 2018; Mohsen et al., 2019a). The internal

262 viscera of sea cucumbers can be consumed by humans. In Japan, the intestine is

263 processed into salted product and the gonad is used to make soup or tea (Mao et al.,

264 2015). In Malaysia, the coelomic fluid of the sea cucumber Stichopus horrens is used

265 to prepare medicinal products (Purcell et al., 2012).

266 4.2. A. japonicus exposure to MFs from the ingested sediment

267 Sea cucumbers are deposit feeders that ingest the sediment to extract nutrients (Gao

268 and Yang 2015). Sea cucumbers ingest sediment that mixed with MFs in laboratory

269 experiments (Graham and Thompson, 2009), in deep water (Taylor et al., 2016), in

270 the wild (Renzi et al., 2018) and in farms (Mohsen et al., 2019b). In the current

271 investigation, the ingested MFs passed through the intestines without transferring to

272 the coelomic fluid through the gastrointestinal epithelium. This is likely because of

273 that the synthetic MFs cannot be taken up by the gastrointestinal epithelium or

274 penetrate the thick walls of the intestines.

275 5. Enzymatic responses of the coelomic fluid due to MF transfer

276 5.1.1. Immune defense indices

277 The immune system of the sea cucumber is an innate system that includes a

278 nonspecific defense mechanism to attack foreign cells (Xue et al., 2015). The

279 activities of typical enzymes, including LZM, ACP, AKP and MPO, which are

14
280 commonly used as indicators for evaluating the immunity status of A. japonicus, were

281 measured (Wang et al., 2008; Chi et al., 2014; Xue et al., 2015; Liu et al., 2016; Jiang

282 et al., 2016; Huo et al., 2018). Among the immunity indices, LZM showed a response

283 to the transferred MFs and appeared to have a defensive role against the transferred

284 MFs in the coelomic fluid of A. japonicus. Significantly increased levels of LZM

285 were observed at 48 h postexposure, while LZM levels were also higher in the treated

286 animals than in the control animals at 24 h and 72 h postexposure, but without

287 significant difference. This might be ascribed to destabilization in the LZMs of cells

288 due to different growth phases (Von Moos et al., 2012). LZMs are extremely common

289 enzymes that play a defensive role against foreign substances in the sea cucumber

290 (Canicatti and Roch, 1989). In A. japonicus, LZMs respond to stress challenges such

291 as low oxygen levels (P < 0.05) (Huo et al., 2018), salinity changes (P < 0.05) (Bai et

292 al., 2015), temperature fluctuations (P < 0.05) (Wang et al., 2008; Bai et al., 2018),

293 and the injection of saccharides (P < 0.05) (Li et al., 2009). Thus, MFs transfer to the

294 coelomic fluid caused a physiological stress to A. japonicus that induced LZM

295 activities. High LZM activities was also noticed in the bivalve Mytilus

296 galloprovincialis that exposed to cationic polystyrene nanoparticles (Canesi et al.,

297 2015). On the other hand, MP exposure decreases LZM levels in the crab Eriocheir

298 sinensis (P <0.05) (Liu et al., 2019). MPO enzyme is also involved in the immunity

299 response (Klebanoff, 2005), but did not show a significant difference from the control.

300 This might suggest that A. japonicus follows a specific mechanism to defend against
15
301 the transferred MFs, elevating LZM levels. For ACP and AKP enzymes, they are two

302 types of phosphatase enzymes that cause dephosphorylation, which can be correlated

303 with inflammatory conditions (Sacco et al., 2012); they have been used in the

304 assessment of the immunity status of A. japonicus (Chi et al., 2014; Liu et al., 2016;

305 Jiang et al., 2016; Huo et al., 2018). Neither ACP nor AKP levels in the treated

306 animals were significantly different from those in the control animals (P < 0.05).

307 However, ACP and AKP levels significantly change in A. japonicus under low

308 oxygen conditions (P < 0.05) (Huo et al., 2018) and under dietary probiotic

309 supplementation conditions (P < 0.05) (Chi et al., 2014; Liu et al., 2016). In addition,

310 previous studies reported a significant difference in AKP and ACP levels between

311 treated and control animals after MP exposure in the discus fish Symphysodon

312 aequifasciatus (P < 0.05) (Wen et al., 2018) and in the crab Eriocheir sinensis (P <

313 0.05) (Liu et al., 2019). Thus, this suggests that A. japonicus responds differently to

314 the effect of the transferred MFs. Similarly, the exposure to MPs does not

315 significantly alter ACP levels in the worm Hediste diversicolor (P < 0.01) (Revel et

316 al., 2018).

317 5.1.2. Oxidative stress indices

318 Oxidative stress is an imbalance between antioxidants and oxidants in favor of

319 oxidants that can cause damage to cells. Antioxidant enzymes such as SOD, MDH,

16
320 GSH, CAT, T-AOC, LDH, SDH, PPO, and POD evaluate the oxidative response of A.

321 japonicus ( Yu et al., 2016; Bai et al., 2017; Huo et al., 2018).

322 Non significantly decreased levels of MDH, PPO, POD, and T-AOC were observed at

323 24 h and 48 h post-exposure but not at 72 h post-exposure, which might indicate that

324 the oxidative stress caused by the transferred MFs is minimal and might be recovered.

325 The oxidative status of A. japonicus changes significantly under the conditions of

326 salinity challenge (P < 0.05) (Bai et al., 2015), ascorbic acid intake (P < 0.05) (Bai et

327 al., 2017), temperature fluctuation (P < 0.05) (Bai et al., 2018), hypoxia (P < 0.05)

328 (Huo et al., 2018) and after saccharide injection (P < 0.05) (Li et al., 2009), which

329 indicates that the oxidative stress caused by the transferred MFs is minimal in

330 comparison to the effect of other stressors on A. japonicus. Similarly, the translocated

331 MPs to the gut cavity of the mussel Mytilus edulis do not disturb the oxidative status

332 (Browne et al., 2008).

333 5.1.3. Behavior recording

334 Behavioral assessment is used to characterize the effect of pesticides, metals, drugs,

335 and gases. In addition, assessing motor activity can be suitable to examine

336 neurotoxicity (Kulig et al., 1996). In A. japonicus, locomotor behavior is affected by

337 melatonin exposure (Ding et al., 2019), reproduction in females (Ru et al., 2017),

338 substrate type (Qiu et al., 2014) and water velocity (Pan et al., 2015). However, in the

339 current study, there was no effect of the transferred MFs during 72 h of exposure on
17
340 the locomotor behavior of A. japonicus, including the velocity and total distance

341 moved (P < 0.05). Similarly, the swimming speed and total distance moved were not

342 affected by MP exposure in the tadpole Xenopus laevis (De Felice et al., 2018). On

343 the other hand, nano plastics affect the locomotion behavior of zebrafish, Danio rerio

344 (Chen et al., 2017).

345 In conclusion, here we show that synthetic MFs can cause physical harm and

346 bioaccumulate in the internal fluid of an endangered species. The transferred MFs

347 altered LZM levels, but neither the oxidative stress indices nor the locomotion

348 behavior of A. japonicus were significantly affected. Given the observed fragility of

349 the respiratory tree, other shapes of MPs may transfer to the coelomic fluid as well.

350 This process might disrupt the physiological processes and the development of A.

351 japonicus, or it might be a route for the transport of pollutants to the internal viscera

352 of sea cucumbers, which worth further investigation. In addition, A long-term

353 experiment will be required to determine whether A. japonicus can adapt to MFs

354 accumulation over time.

355 Acknowledgments

356 We would like to thank the anonymous reviewers for their guidance. We also would

357 like to thank Da Huo and Xin Xiaoke for their comments during the experiment.

358 This study was supported by the National Natural Science Foundation of China

359 (41876157, 41776161,41576122), the International Partners Program of the Chinese

18
360 Academy of Sciences (133137KYSB20180069), Youth Innovation Promotion

361 Association CAS (2016196, 2015163), and Taishan Scholars Program.

362

363

364

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Figure captions

Figure 1. a) Microfibers (MFs) crossing a branch of the respiratory tree,

b) average MFs number in the coelomic fluid (line chart) and the

lysozyme (LZM) responses of the coelomic fluid (U/ml) (column chart) at

24 h, 48 h and 72 h postexposure (Mean ± SE), c) total antioxidant

capacity (T-AOC) activities (nmol/L) at 24 h, 48 h and 72 h postexposure.

Different letters indicate a significant difference and columns without

letters indicate no significant difference (Mean ± SE) (P < 0.05).

 100 12.0 0.2
90
10.0
T-AOC concentrations
LZM concentrations

80 b
b
MFs number

70 8.0
60
50 a 6.0 0.1
40
30 4.0
20 2.0
10
0 0.0 0.0
24 h 48 h 72 h 24 h 48 h 72 h
Time exposure Time exposure
C T1 T2
Highlights

• Microfibers (MFs) transfer to the coelomic fluid of the sea cucumber.

• MFs increased over time and persisted in the coelomic fluid of A. japonicus.
• MFs altered the lysozyme responses in the coelomic fluid of A. japonicus.
• MFs did not affect the speed and the total distance moved by A. japonicus.
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: