Nanoplastic Ingestion Enhances Toxicity of Persistent Organic

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Ecotoxicology and Human Environmental Health

Nanoplastic Ingestion Enhances Toxicity of Persistent Organic Pollutants (POPs) in the Monogonont Rotifer Brachionus koreanus via Multixenobiotic Resistance (MXR) Disruption Chang-Bum Jeong, Hye-Min Kang, Young Hwan Lee, Min-Sub Kim, Jin-Sol Lee, Jung Soo Seo, Minghua Wang, and Jae-Seong Lee Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b03211 • Publication Date (Web): 07 Sep 2018 Downloaded from http://pubs.acs.org on September 9, 2018

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Environmental Science & Technology 1

-revised

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Nanoplastic Ingestion Enhances Toxicity of Persistent Organic

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Pollutants (POPs) in the Monogonont Rotifer Brachionus

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koreanus via Multixenobiotic Resistance (MXR) Disruption

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Chang-Bum Jeonga,#, Hye-Min Kanga,#, Young Hwan Leea, Min-Sub Kima,

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Jin-Sol Leea, Jung Soo Seob, Minghua Wangc, and Jae-Seong Leea,*

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a

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16419, South Korea

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b

13

c

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of the Environment & Ecology, Xiamen University, Xiamen 361102, China

Department of Biological Science, College of Science, Sungkyunkwan University, Suwon Pathology Division, National Institute of Fisheries Science, Busan 46083, South Korea

Key Laboratory of the Ministry of Education for Coastal and Wetland

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#

33

*

These authors contributed equally to this manuscript.

Corresponding author. E-mail: [email protected] (J.-S. Lee)

1

ACS Paragon Plus Environment

Ecosystems/College

Environmental Science & Technology

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ABSTRACT: Among the various materials found inside microplastic pollution,

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microplastics are of particular concern due to difficulties in quantification and detection;

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moreover, they are predicted to be abundant in aquatic environments with stronger toxicity

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than micro-sized microplastics. Here, we demonstrated a stronger accumulation of nano-sized

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microbeads in the marine rotifer Brachionus koreanus compared to micro-sized ones, which

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was associated with oxidative stress-induced damages on lipid membranes. In addition,

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multixenobiotic resistance (MXR) conferred by P-glycoproteins (P-gps) and

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resistance proteins (MRPs), as a first line of membrane defense, was inhibited by nanoplastic

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pre-exposure, leading to enhanced toxicity of 2,2′,4,4′-tetrabromodiphenyl ether

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and triclosan (TCS) in B. koreanus. Our study provides a molecular mechanistic insight into

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the toxicity of nano-sized microplastics towards aquatic invertebrates, and further implies the

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significance of synergetic effects of microplastics with other environmental persistent organic

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pollutants.

47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68

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nano-sized

multidrug (BDE-47)

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ABSTRACT ART

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■ INTRODUCTION

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Plastic pollution is considered one of the most concerning environmental problems due to its

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abundance and persistence in the aquatic environment.1,2 The biggest issue regarding plastic

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pollution is ‘microplastic,’ which generally refers to plastic particles less than 5 mm. These

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particles are associated with larger plastic debris in aquatic environments, accounting for

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to 80% of marine debris as they shatter into smaller plastic particles via natural weathering

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and aging processes.3 These plastic particles are more bioavailable when their sizes decrease

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with an increased surface area to the volume ratio, and therefore are considered more toxic to

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aquatic organisms.4 Furthermore, as the size of these particles can vary and become similar to

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that of food sources for aquatic organisms, organisms with low feeding selectivity 5-8

up

ingest

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these plastic particles, which is likely accompanied with negative effects.

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among aquatic invertebrates, various zooplanktons have shown the ability to ingest

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polystyrene microbeads ranging from 1.7 to 30.6 µm with decreased algal feeding in the

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copepod Centropages typicus.6 The rotifer Brachionus manjavacas was also shown to be

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capable of ingesting the polystyrene microbeads within the range of 0.05 to 3 µm.9 In another

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study, size-dependent negative effects of 0.05, 0.5, and 6 µm microplastic ingestion were

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shown on reproduction and growth rates in the rotifer Brachionus koreanus and copepod

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Paracyclopina nana due to microplastic-induced oxidative stress.4,10

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For example,

One particular concern of microplastic pollution is related to the issues

regarding

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nanoplastics. While the abundance of nanoplastics in nature via nanofragmentation of larger

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plastic particles and their release from plastic products has been suggested by previous

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studies

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difficulties in detection and quantification. Assuming the worst-case scenario, the number of

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plastic particles increases inversely with the radius of the particle. While in-vivo

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effects of microplastics on aquatic organisms have been relatively well

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molecular responses and subsequent consequences in aquatic organisms have not been

11,12

, there is no available information on nanoplastic concentration in nature due to adverse

documented,

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studied, particularly for nanoplastics. Therefore, in the present study, we focused

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multixenobiotic resistance (MXR) to investigate the molecular effects of microplastic and/or

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nanoplastic on marine zooplanktons with further assessment of synergetic effects

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nanoplastic in the rotifer B. koreanus in response to 2,2′,4,4′-tetrabromodiphenyl ether (BDE-

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47) and triclosan (TCS) as model persistent organic pollutants (POPs). MXR is a

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phenomenon conferred by ATP-binding cassette (ABC) transporters, namely, p-glycoprotein

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(P-gp), multidrug resistance protein (MRP), and breast cancer resistance protein (BCRP),

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on of

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which play a first line of defense in aquatic invertebrates in response to xenobiotics including

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environmental pollutants. 10

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Rotifers occupy an important ecological niche in the aquatic ecosystem, linking the energy

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flow from primary producers to organisms at higher levels. Rotifers are planktonic, floating

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along water currents and ingesting ambient particles (e.g., algae) with low feeding selectivity

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as a filter feeder13,14, which possibly increases the opportunities for rotifers to

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microplastics. As a laboratory experimental species, they have a small body size (~ 170 µm

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for adults), short generation cycle (~ 24 h), high fecundity, and are easy to culture.13,14 Based

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on these advantages, a genome database has been recently constructed that facilitates rotifers

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as a model species for ecotoxicological studies.15-17

ingest

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In the present study, we demonstrate the inhibition of MXR by ingestion of nanoplastics

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(0.05 µm [50 nm]), resulting in enhanced toxicity of BDE-47 and TCS on the rotifer B.

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koreanus. This study provides a better understanding of the synergic effects of microplastics

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and POPs.

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■ MATERIALS AND METHODS

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Rotifer Culture. The monogonont rotifer B. koreanus was collected at Uljin, South Korea

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(36°58′43.01″N, 129°24′28.40″E) and maintained in the laboratory since 2011. A single

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rotifer was isolated, reared, and maintained in filtered artificial seawater (ASW; TetraMarine

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Salt Pro, Tetra, Cincinnati, OH, USA). The seawater temperature was maintained at 25°C and

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the rotifers were maintained under a light:dark 12:12 h photoperiod with 15 practical salinity

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units (psu). The green alga Tetraselmis suecica was used as a live diet (approximately 6 × 104

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cells/mL). Species identification of the rotifer was confirmed via morphological analysis and

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sequencing of the mitochondrial DNA gene CO1.18,19

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Microbeads and chemicals. Three different sizes of non-functionalized

polystyrene

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microbeads (0.05, 0.5, and 6 µ m) were purchased from Polysciences (Warrington, PA, USA).

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Polystyrene was chosen since it is one of the most abundant polymers in marine plastic

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debris.20 For ingestion experiments, fluorescently labeled polystyrene microbeads (excitation

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of 441 nm/emission of 486 nm) of the same diameters were used.

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BDE-47 and TCS were dissolved in 99.5% dimethylsulfoxide (DMSO; Sigma-Aldrich, St.

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Louis, MO, USA) to prepare the stock solution. To conduct experiments, stock solutions of

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all chemicals were re-dissolved in DMSO. The solvent concentration in control and treatment

140

groups did not reach over 0.001% DMSO (v/v).

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Microbeads Ingestion. To demonstrate microplastic ingestion in B. koreanus,

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approximately 200 adult rotifer individuals were exposed to 10 µg/mL of fluorescently

143

labeled microbeads for 24 h. After exposure, the rotifers were washed with clean ASW to

144

remove the residual microbeads, and then fixed in 4% formaldehyde. The ingested

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microbeads remaining in the rotifers were observed using confocal laser scanning microscopy

146

(LSM 510 META; Zeiss, Oberkochen, Germany) with a 20x lens at excitation of 441 nm and

147

emission of 486 nm.

148

Microplastic Accumulation Rate. To measure the accumulation rate of microbeads in

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rotifers, rotifers (n ≈ 2,000) were exposed to different sizes of fluorescently

150

microbeads (0.05, 0.5, and 6 µ m) for 24 h and transferred into clean seawater for 24 h to

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allow egestion. For the control groups, rotifers were exposed to different sizes of

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fluorescently labeled microbeads for 24 h and used for the analysis without the egestion

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process. Rotifers were homogenized with a Teflon homogenizer to measure the fluorescence

154

of accumulated microbeads and then subsequently sonicated for 1 min to dissociate any

155

possible microbead aggregations. Homogenized samples were used for the

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detection using Varioskan Flash (Thermo Electron, Vantaa, Finland) at an excitation of 441

157

nm and emission of 486 nm. For the normalization, homogenized samples were centrifuged

158

at 10,000 g for 10 min and the supernatants were used for the protein quantification by the

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Bradford method.21 The accumulation rate was expressed by the relative percentage with

160

respect to the control groups. The experiment was performed in triplicate.

161

labelled

microbeads

Reactive Oxygen Species (ROS) and Malondialdehyde (MDA) Assays. Approximately

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2,000 adult rotifer individuals were exposed to different concentrations of 0.05

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microbeads (0, 0.1, 1, 10, and 20 µ g/mL) for 24 h, washed, and then homogenized in a buffer

164 165

containing 0.32 M sucrose, 20 mM HEPES, 1 mM MgCl2, and 0.5 mM PMSF (pH 7.4) with a Teflon homogenizer. Homogenates were centrifuged at 10,000 g for 10 min and supernatants

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were used for the analysis.

µm

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Intracellular reactive oxygen species (ROS) levels were measured using 2′7′-

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dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes, Eugene, OR, USA), which was oxidized to fluorescent dichlorofluorescein (DCF) by intracellular ROS. A 96-well

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black plate was filled with 170 µL PBS buffer, 10 µL probe (H2DCFDA at a final concentration of 40 µM), and 20 µL supernatant fraction to a final volume (200 µL).

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Measurements were obtained with an excitation wavelength at 485 nm and

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wavelength at 520 nm using Varioskan Flash (Thermo Electron).

emission

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The lipid peroxidation level was measured using a commercially available kit (Abcam,

175

Cambridge, UK). Briefly, lipid peroxidation was detected by measuring the level of

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malondialdehyde (MDA), a final product of lipid peroxidation. To detect

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thiobarbituric acid (TBA) was used to generate MDA-TBA adducts, which were quantified

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with an excitation wavelength at 532 nm and emission wavelength at 553 nm using Varioskan

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Flash (Thermo Electron). All experiments were performed in triplicate. Measured ROS and

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MDA levels were normalized by total protein and represented as a percentage relative to the

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control group. Total protein was determined by the Bradford method.21

MDA,

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MXR Activity Measurements. The activity of P-gp and MRP was measured according to

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Rhee et al. (2012) with a minor modification.22 To examine whether microbead ingestion

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influences MXR activity in B. koreanus, rotifers (n ≈ 200) were exposed to 10 µg/mL of

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different sizes of microbeads (0.05, 0.5, and 6 µm) for 24 h, and then

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transferred into clean ASW containing 0.5 µM rhodamine B (Sigma-Aldrich) or 1 µM calcein

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AM (Sigma-Aldrich), which are fluorescent substrates for P-gp and MRP, respectively. After

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exposure for 2 h in the dark, the rotifers were washed with clean ASW and fixed by 4%

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formalin. The fluorescence of substrate dyes were observed using confocal laser scanning

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microscopy (LSM 510 META; Zeiss) with a 20x lens at excitation of 535 nm and emission of

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590 nm for rhodamine B, and excitation of 485 nm and emission of 535 nm for calcein AM.

subsequently

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To further investigate the effects of nanoplastic pre-exposure on the MXR activity in

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response to BDE-47 and TCS, rotifers (n ≈ 2,000) were exposed to 25 µg/L BDE-47 or 50

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µg/L TCS for 24 h with or without pre-exposure to nanoplastic (10 µg/mL) for 24 h, and

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incubated for 2 h in clean ASW containing 0.5 µM rhodamine B (Sigma-Aldrich) or 1 µM

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calcein AM (Sigma-Aldrich), which are fluorescent substrates for P-gp and MRP,

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respectively.22 After incubation, the rotifers were washed and homogenized

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buffered saline (PBS), and then centrifuged at 10,000 g for 10 min. Approximately 200 µL of

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supernatant was used for the measurement of accumulated fluorescence in the rotifers using

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Varioskan Flash (Thermo Electron) over a calibration curve of rhodamine B (excitation 535

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nm; emission 590 nm) and calcein AM (excitation 485 nm; emission 535 nm).

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fluorescent intensities were normalized by the protein quantity of each sample determined by

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the Bradford method.21

in phosphate-

The

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In vivo experiments. To compare the acute toxicity of BDE-47 and TCS in rotifers pre-

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exposed to nanoplastic versus healthy rotifers, ten healthy individuals as well as the rotifers

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pre-exposed to 10 µg/mL of 0.05 µm microbeads for 24 h were transferred into a 12

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culture plate (SPL, Seoul, South Korea) with 4 mL of ASW containing 0, 100, 250, 500, 750,

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and 1,000 µg/L BDE-47 or 0, 100, 200, 300, and 400 µg/L TCS. After 24 h exposure, the

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numbers of dead and live rotifers were counted under a stereomicroscope (SZX-ILLK200,

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Olympus Corporation, Tokyo, Japan).

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well


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To measure population growth and reproduction rates as a chronic exposure test, an

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individual of rotifer pre-exposed to nanoplastic (10 µg/mL) and healthy rotifer

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transferred into 2 mL ASW containing 25 µg/L BDE-47 or 50 µg/L TCS. The numbers of

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rotifers were counted daily under a stereomicroscope (Olympus Corporation) for population

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growth rate measurement, and the numbers of newborn rotifers were counted every 12 h until

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the matured rotifer died as a readout of fecundity, after which the newborn rotifers were

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removed. During the chronic experiments, half of the media was renewed daily with the

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supply of the green alga T. suecica as a diet (approximately 6 × 104 cells/mL). Three

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biological replicates were performed for all experiments.

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was

Statistics. SPSS ver. 17.0 (SPSS Inc., Chicago, IL, USA) was used for all statistical

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analyses. Data are expressed as the mean ±

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the control and experimental groups was analyzed using Student’s paired t-test and one-way

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and/or multiple-comparison analysis of variance (ANOVA) followed by Tukey’s test. Any

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difference with a P value < 0.05 was considered significant.

SD. The significance of the differences between

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■ RESULTS

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Microplastic Accumulation. Fluorescence of all microbead sizes was observable in the

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rotifers exposed to different sizes of microbeads for 24 h (Fig. 1). Particularly,

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fluorescence of 0.5 and 6 µm microbeads were observed in the digestive tracts, whereas 0.05

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µm microbeads were dispersed in the surrounding organs, indicating that 0.05 µm microbeads

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are possibly membrane permeable, leading to bioaccumulation in rotifers. This hypothesis

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was further supported by the size-dependent egestion rates of the microbeads. Compared to

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0.05 µm microbeads, 6 and 0.5 µm microbeads were more easily egested, as the accumulated

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amount of microbeads in the rotifers increased in a size-dependent manner with respect to the

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microbeads (Fig. 2).

the

237

Oxidative Stress and Lipid Peroxidation Measurements. The ROS and MDA levels

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increased with a concentration-dependent tendency up to 10 µg/ml of nanoplastic, and then

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decreased at the highest concentration (20 µg/ml) (Fig. 3). The strongest oxidative stress and

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lipid peroxidation were induced at 10 µg/ml, indicating that nanoplastic generates oxidative

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stress and leads to the induction of oxidative damages on lipid components.

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MXR Activities. The activities of P-gp and MRP were both decreased in rotifers exposed

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to 0.05 µm microbeads (Fig. 4), while the fluorescent intensity of accumulated substrates

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(rhodamine B for P-gp; calcein AM for MRP) was the most increased by 0.05 µm microbeads

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compared to the control. The red rhodamine B signal was observed in the entire body, while

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the green calcein AM signal was mostly observed in the digestive organs.

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To verify whether MXR is capable of effluxing BDE-47 and TCS out of rotifers, the

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activities of P-gp and MRP were measured as described in Rhee et al. (2012) with a minor

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modification. The activities of P-gps and MRPs were significantly increased (P < 0.05) in

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response to BDE-47 and TCS exposures compared to the control (Fig. 5A), suggesting that

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BDE-47 and TCS are likely a substrate for efflux activity of P-gps and MRPs. In contrast,

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their activities were significantly decreased (P < 0.05) when rotifers were pre-exposed to the

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nanoplastics (Fig. 5B). In addition, these transporters had different substrate specificities for

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BDE-47 and TCS, as the detected intensity differed depending on the transporter and

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chemical tested.

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In vivo effects. Since the nanoplastics showed the most destructive effects on MXR

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functions, the acute and chronic toxicity of BDE-47 and TCS was measured with rotifers pre-

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exposed to nanoplastics and compared with healthy rotifers. The GC/MS analysis

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the actual concentration of BDE-47 and TCS in the exposure media as 22.8 and 23.0 µg/L,

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respectively, which is lower than nominal values owing their high lipophilicity.23 Rotifers in

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each group exhibited different survival rates, showing higher tolerance to each pollutant in

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the non-pre-exposed rotifers (i.e., healthy rotifers) (Fig. 6A). The estimated median lethal

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concentration (LC50) values for BDE-47 and TCS were 205.03 and 307.42 µg/L, respectively,

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with lower values in rotifers pre-exposed to nanoplastics (146.01 µg/L for BDE-47 and

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179.19 µg/L for TCS) (Table 1). The acute toxicity values for the healthy rotifers were lower

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than those previously reported,24,25 probably due to the food restrictions of healthy rotifers

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during the 24 h exposure time in the pre-exposure group. In addition to acute toxicity,

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population growth and reproduction rates were measured as a chronic toxicity test. Rotifers

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exposed to BDE-47 and TCS alone did not show significant differences, compared to the

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control, whereas rotifers exposed to BDE-47 and TCS, treated with nanoplastic pre-exposure,

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have shown the decreased tendency (Fig. 6B). These results indicate that the pre-exposure of

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nanoplastics negatively affected the tolerance against BDE-47 and TCS in the rotifers.

detected

the

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■ DISCUSSION

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Ingestion of microplastics in marine zooplanktons has been demonstrated in many previous

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studies and is considered as a primary exposure pathway of microplastics to zooplanktons,

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leading to adverse effects on their life parameters such as growth and reproduction.4,6,7,10

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Consistent with previous studies, the fluorescence of microbeads was strongly detected in the

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digestive tracts of rotifers, indicating that all different sizes of microbeads were ingested.

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Interestingly, the smallest microbeads (0.05 µm) were dispersed in the ambient organs of

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digestive tracts, possibly leading to longer retention time of microbeads in the rotifers.

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Similar result was also shown by Snell and Hicks, as the nano-sized polystyrene microbeads

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(0.37 µm) were distributed in whole body with their ability to penetrate the gut wall, while

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larger microbeads were remained in the digestive tracts.9 According to previous studies, a

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stronger tendency for nano-sized particles to penetrate into cells has been shown as particle

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size decreases compared to micro-sized particles.26 For example, in mouse red blood cells,

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nano-sized (0.078 µm) polystyrene microbeads accumulated much more via membrane

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penetration compared to micro-sized (1 and 2 µm) polystyrene microbeads.27 Therefore, the

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dispersion of nanoplastics in the rotifers observed in the present study is

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with the membrane permeability of nanoplastics. Furthermore, the nanoplastics seemed to

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have longer retained time in the rotifers compared to 0.5 and 6 µm microbeads, probably due

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to the escape of nanoplastics out of digestive tracts, which act as a pathway for egestion via

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their capacity to penetrate. In order to verify cellular accumulation of nanoplastic

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membrane penetration, we applied transmission electron microscopy (TEM) image analysis,

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but it was not possible to distinguish polystyrene microbeads from the embedding resins

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because of their similar physico-chemical properties. Nanoparticles are known to

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more ‘bioreactive’ as their size decreases, as the increased total surface area facilitates

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chemical reactivity with higher surface area to volume ratios.28 In this regard, it has been

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reported that the degree of adverse outcomes (e.g., retardation in growth rate and

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reproduction) of polystyrene microbead exposure is closely related to particle size,4,9,10

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implying the potential nanotoxicity of retained nanoplastics in the rotifers in the present study.

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Based on these results, we focused on the nanotoxicity of nanoplastics with subsequent

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experiments, as they cause more serious influences at the cellular level than micro-sized

305

microbeads, leading to more potential harmful impacts at the individual level.

possibly associated

via

become

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One of the principal mechanisms behind the toxicity of microplastics is related to the

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generation of oxidative stress, which negatively influences biological homeostasis in an

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organism.29,30 In the present study, nanoplastic exposure to rotifers significantly increased (P

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< 0.05) the level of intracellular ROS as a possible consequence of the strong accumulation

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of nanoplastics observed in the present study. Along with ROS induction, the level of MDA

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for lipid peroxidation also increased with the level of ROS. MDA is a final product of lipid

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peroxidation, which is related to oxidative damage on the lipid membranes. Therefore, our

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results suggest that oxidative stress was induced by nanoplastics, which is associated with

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dysfunction of lipid membranes. In consistent, multiple evidences have been reported for

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oxidative damages on membranes of human cell lines from silica nanoparticles exposure.31

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These results suggest that nano-sized particles are capable of inflicting direct oxidative

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damages on lipid membranes, causing deleterious effects on biological processes including

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defense. In addition to lipid peroxidation as a main pathway for nanoplastic-induced damage

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on the membranes, the direct interaction between the membranes and particles may be

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another important route leading to membrane damage. Using a model membrane system,

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Rossi et al. (2014) stimulated the effects of nano-sized polystyrene particles on

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membranes and altered membrane structures due to particle accumulation and penetration

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along the membrane accompanied with dysfunction.32 Therefore, the dispersed fluorescence

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and stronger accumulation of nanoplastic shown in the present study imply that nanoplastics

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cause membrane dysfunction in chemical and/or physical ways.

lipid

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MXR is a phenomenon of xenobiotic efflux out of cells conferred by a set of ABC

327

transporters, including P-gp and MRPs. Since these transporters are located in the cellular

328

membrane with important roles as efflux transporters, we further examined the activity of

329

MXR-mediated ABC transporters, namely P-gp and MRPs. The conserved function of P-gps

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and MRPs conferring MXR in aquatic invertebrates, including the rotifer B. koreanus, have

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been proposed as a first line of defense against environmental toxicants33,34; this suggests that

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nanoplastic-induced membrane disruption would cause inhibition of MXR activities, leading

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to decreases in tolerance to environmental toxicants. In particular, lipid

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increased membrane permeability and fluidity, leading to disorders in MXR functions.35,36 To

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verify this hypothesis, we measured the activities of P-gps and MRPs with or without

336

different sizes of microbead pre-exposure to determine whether the efflux capacity of P-gps

337

and MRPs is affected by microbead ingestion. As a result, the fluorescence of P-gp and MRP

338

substrates was most strongly accumulated in rotifers exposed to nanoplastics, indicating that

339

the activity of P-gps and MRPs was inhibited in a size-dependent manner with respect to the

340

microbeads, in parallel to the stronger accumulation of nanoplastics in the rotifers compared

341

to micro-sized microbeads, as shown in our study.

peroxidation

342

The inhibitions of P-gp and/or MRPs have previously enhanced the toxicity of

343

environmental toxicants to organisms. For example, in the mosquito Aedes aegypti, co-

344

treatments of P-gp inhibitor and RNA interference of P-gp encoding gene have increased the

345

toxicity of temephos by 24 and 57%, respectively.37 In addition, the survival rate of the rotifer

346

B. koreanus was significantly decreased by inhibition of P-gp and MRPs in response to four

347

different biocides exposure, implying the critical role of P-gp and MRPs in

348

systems.34 In the present study, the protective role of P-gp and MRPs in response to BDE-47 11


defense


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and TCS was confirmed as their efflux activity was induced by BDE-47 and TCS. The

350

decreased activities observed with pre-exposure to nanoplastic suggest that nanoplastic is

351

able to disrupt MXR functions in response to BDE-47 and TCS. Furthermore, the survival

352

rate and lethal concentration values (LC10 and LC50) with further population growth and

353

reproduction rates of rotifers were decreased in the nanoplastic pre-exposure

354

compared to the chemical only exposure groups. As the increased sensitivity of rotifers to

355

BDE-47 and TCS was further demonstrated using inhibitors specific to P-gp and MRP as the

356

negative controls (Fig. S1), our results clearly imply the enhanced toxicity of BDE-47 and

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TCS to the rotifers via MXR disruption. In nature, complex mixtures of toxicants are present

358

and can be transferred by absorption on microplastics, causing synergic interactions between

359

them to enhance toxicity in the organism. In fact, there are several reports that microplastics

360

can be a vector for environmental toxicants and provide synergic effects. For example, in the

361

amphipod Allorchestes compressa, polybrominated diphenyl ethers (PBDEs) derived from

362

microplastic assimilated into amphipod tissues via microplastic ingestion.38 The combined

363

exposure of microplastics and phamarceuticals, including procainamide and doxycycline,

364

reduced the growth rate and chlorophyll a concentrations in the algae Tetraselmis chuii, even

365

though microplastics alone did not exhibit any toxicity.39 Similarly, in the common goby

366

Pomatoschistus microps, the activity of isocitrate dehydrogenase was decreased by combined

367

exposure to pyrene and polyethylene microbeads, whereas no significant changes were

368

observed when the two stressors were separately exposed.40 These results suggest that

369

toxicological interactions between microplastics and other environmental toxicants lead to

370

enhanced toxicity of environmental toxicants or at least disruption of biological processes. In

371

this regard, to our knowledge, we firstly provide a mechanistic view that provides a better

372

understanding of the synergetic toxicity of microplastics and POPs. Taken

373

nanoplastics had longer retention time in the rotifer B. koreanus and induced membrane

374

dysfunction via oxidative damages and penetration, leading to disruption of MXR

375

(Fig. 7). As the increased mortalities in response to BDE-47 and TCS have shown a possible

376

consequence of those subsequent molecular events, we have shown how

377

microplastics affect aquatic invertebrates at the molecular level with a mechanistic insight

378

into a joint toxicity of microplastics and environmental toxicants.

379 380

■ AUTHOR INFORMATION

381

Corresponding Author

382

*E-mail: [email protected] 12


groups

together, functions nano-sized

Page 13 of 25


383

ORCID

384

Jae-Seong Lee: 0000-0003-0944-5172

385

Notes

386

The authors declare no competing financial interest.

387

388

■ ACKNOWLEDGEMENTS

389

This work was supported by the Postdoctoral Research Program of Sungkyunkwan

390

University (2017).

391

392

■ SUPPORTING INFORMATION

393

Supporting data is included in Supporting Information. Supporting Information is available

394

with free of the charge on the website at http://pubs.acs.org.

395

396

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397

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517

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519

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520

16


Pomatoschistus

Page 17 of 25


521

522

Table 1. LC10, LC50, and 95% confidence intervals (CI) for BDE-47 and TCS in B.

523

koreanus in the presence or absence of pre-exposure to 0.05 µm microbeads. Chemical

BDE-47

TCS

Pre-exposure

LC10 (µg/L)

LC50 (µg/L)

None

53. 31 (28.77-98.78)

205.03 (153.63-273.62)

0.05 µm microbeads

48.97 (25.90-92.56)

146.01 (105.10-202.85)

None

248.53 (212.25-284.82)

307.42 (267.88-355.90)

0.05 µm microbeads

93.83 (11.74-147.34)

179.19 (82.83-257.21)

524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549

17



Page 18 of 25

550

551

Figure legends

552 553 554

Figure 1. Images of ingested fluorescently labeled polystyrene microbeads (0.05, 0.5, and 6

µm) according to rotifer.

555 556

Figure 2. Quantification of accumulated polystyrene microbeads (0.05, 0.5, and 6 µm) in the

557

rotifers after egestion. Zero h egestion groups were considered as the control group.

558

Asterisk above columns indicates significant differences (P < 0.05).

559 560

Figure 3. Level of ROS (A) and MDA (B) after exposure to different sizes of polystyrene

561

microbeads (0.05, 0.5, and 6 µ m). Different letters above columns

562

significant differences (P < 0.05).

indicate

563 564 565

Figure 4. Fluorescence images of accumulated fluorescent substrates for P-gp (A) and MRP

(B) in response to different sizes of polystyrene microbeads (0.05, 0.5, and 6 µ m).

566 567

Figure 5. Relative P-gp and MRP activities in response to 25 µg/L BDE-47 and 50 µg/L TCS

568

in the absence (A) or presence (B) of nanoplastic pre-exposure. Different letters

569

above columns indicate significant differences (P < 0.05).

570 571

Figure 6. Acute (A) and chronic (B) toxicity of BDE-47 and TCS in the rotifers with or

572

without nanoplastic pre-exposure. Asterisk and different letters above columns

573

indicate significant differences (P < 0.05).

574 575 576

Figure 7. Schematic representation of the proposed mechanism for MXR disruption via

nanoplastic ingestion in the rotifer B. koreanus. Modified from Lee et al. (2018).33

577

18


Page 19 of 25


Fig. 1

Bright light

Fluorescence

Control

0.05 μm

0.5 μm

6 μm


Merged


Page 20 of 25

Fig. 2


Page 21 of 25


Fig. 3

A) Intracellular ROS level 140

c

120

b

b

0.1

1

b

% ROS level

a 100 80 60 40 20 0 Control

10

20

Concentration (g/ml)

B) Lipid peroxidation 140

c b

120

% MDA level

a

b

a

100 80 60 40 20 0 Control

0.1

1

10

Concentration (g/ml)


20


Page 22 of 25

Fig. 4

A) P-gp

BL

FL

M

BL

FL

M

Control

0.05 μm

0.5 μm

6 μm

B) MRP Control

0.05 μm

0.5 μm

6 μm


Page 23 of 25


Fig. 5

A)

Efflux activity

250

c MRPs P-gps

200

150

100

b a

a

c

b

50

0 Control

BDE-47

TCS

B) 120

a

a

MRPs P-gps

Efflux activity

100 80

b

60

c

40

b

b

20 0

Control

BDE-47


TCS


Page 24 of 25

Fig. 6

A) Acute exposure * 1.2 a

0.8

b b

b

0.6

c c

0.4 0.2

d

d

0.0 0

100

250

a

1.0

Survival rate

Survival rate

BDE-47 BDE-47 + 0.05 m MP

a

1.0

1.2

500

a

TCS TCS + 0.05 m MP

a

* 0.8 b

0.6

b c

0.4 0.2

d d

d

750

1000

c d d d

0.0 0

100

200

300

400

b

b

TCS (g/L)

BDE-47 (g/L)

B) Chronic exposure 200

a

30

Reproduction

150

Population

35

Control BDE-47 TCS MP + BDE-47 MP + TCS

100 50 0

ab

25

ab

20 15 10 5 0

1

2

3

4

5

6

7

Control

BDE-47

TCS

BDE-47

Exposure

Day


+ MP

TCS

Page 25 of 25


Fig. 7

Nanoplastics

MXR disruption

Normal function i) Oxidative damages

P‐gp

MRP

P‐gp

MRP

ii) Membrane penetration

ATP

ADP

ATP

ADP

Toxicant accumulation


Nanoplastic Ingestion Enhances Toxicity of Persistent Organic

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