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Uptake and accumulation of polystyrene microplastics in zebrafish (Danio rerio) and toxic effects in liver Yifeng Lu, Yan Zhang, Yongfeng Deng, Wei Jiang, Yanping Zhao, Jinju Geng, Lili Ding, and Hong-qiang Ren Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b00183 • Publication Date (Web): 07 Mar 2016 Downloaded from http://pubs.acs.org on March 7, 2016

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Environmental Science & Technology

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Title page

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Title:

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Uptake and accumulation of polystyrene microplastics in zebrafish (Danio rerio) and toxic

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effects in liver

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Authors:

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Yifeng Lu1, Yan Zhang*1, Yongfeng Deng1, Wei Jiang1, Yanping Zhao2, Jinju Geng1, Lili

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Ding1, Hongqiang Ren*1

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Affiliations:

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

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Nanjing University, Nanjing, Jiangsu 210023, China

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2 Nanjing Normal University, Jiangsu Key Lab Environmental Change & Ecological

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Construct, School of Geography Science, Nanjing, Jiangsu 210023, China

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Corresponding author:

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tel: +86 25 89680160; email: [email protected] (Yan Zhang) and [email protected]

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(Hongqiang Ren)

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1

ACS Paragon Plus Environment


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TOC Art

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Abstract

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Microplastics have become an emerging contaminant and caused widespread concern

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about its potential toxic effects. In this study, the uptake and tissue accumulation of

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polystyrene microplastics (PS-MPs) in zebrafish were detected, as well as the toxic effects in

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liver were investigated. The results showed that after 7 days of exposure, 5-µm diameter MPs

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accumulated in fish gill, liver, and gut, while 20-µm diameter MPs only accumulated in fish

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gill and gut. Histopathological analysis showed that both sizes of 5-µm and 70-nm PS-MPs

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caused inflammation and lipid accumulation in fish liver. PS-MPs also induced significantly

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increased activities of SOD and CAT, indicating oxidative stress was induced after MPs

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treatment. In addition, metabolomic analysis suggested that MPs exposure induced alterations

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of metabolic profiles in fish liver and disturbed the lipid and energy metabolism. These

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findings provide new insights into the toxic effects of MPs on fish.

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Introduction

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In recent years, microplastics (MPs, diameter < 5 mm) have been frequently detected in

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oceans, sediments, rivers, and sewages1-3. Due to its small size and poor biodegradation, MPs

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can be ingested by organisms and accumulate for a long time. Furthermore, it can also be

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transported into food chains and threaten the environmental health and ecological safety4-6.

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MPs have become emerging contaminations which warrant further study.

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Once MPs accumulate in the organisms, they have the potential to cause a lot of adverse

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effects, including mortality, reduced feeding activity, inhibited growth and development,

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endocrine disruption, energy disturbance, oxidative stress, immunity and neurotransmission

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dysfunction and even genotoxicity6-11. Many studies have been conducted to investigate the

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mechanisms of the toxic effects of MPs. It was found that MPs could induce reactive oxygen

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species (ROS) and oxidative stress in lugworms12 and mussels9. MPs could induce decrease of

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the phagocytic activity of immune cells in worms12. Some studies also found that MPs could

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cause neurotoxic effects on organisms. For example, polyethylene microplastics (PE-MPs)

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adversely disturbed the neurofunction of gobies13 and PS-MPs could also lead to similar

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consequences in mussels by depressing the acetylcholinesterase (AChE) activity9. However,

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the mechanisms of toxic effects of MPs are still largely unclear and need further study.

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Tissue section and enzymatic analysis are widely used to determine the toxic effects of

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MPs14, 15. Moreover, transcriptomics was used to study the differential regulation of genes

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induced by MPs6,

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signatures of biochemical activities and biological responses due to environmental

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contamination. Metabolomics has been used to study the toxic effects of MPs5 and

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. In contrast to transcriptomics, metabolomics can provide direct

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nanomaterials16-18. However, the toxic mechanisms remain unclear.

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Polystyrene (PS) is not only one kind of plastics with high production 19 but also one of

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the primary compositions of plastic debris observed in the environment. In this study, three

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different sizes of commercially available PS beads were selected as model MPs. Zebrafish

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(Danio rerio) was used as a model organism. The purpose of this study is not only to

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investigate the uptake and accumulation of MPs in fish but also to reveal the toxic effects

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MPs in liver.

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Material and Methods

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PS-MPs used in this study

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PS particles stocks as dispersion (2.5% w/v, 10 mL) were purchased from Tianjin

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BaseLine ChromTech Research Centre (Tianjin, China) and costumed to have three different

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primary nominal sizes of 70 nm, 5 µm and 20 µm, respectively (Supporting Information,

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Figure S1). Two types of beads were used in this study. One type is fluorescent PS particles

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with encapsulated fluorescent dyes (with an excitation of 418 nm/emission of 518 nm and the

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diameter of 5 µm and 20 µm). These PS beads were stained by gradual solvent evaporation

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method using NBD-Cl as the fluorescent dye20. This type of beads were used for the uptake

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and accumulation test. Another type is virgin PS particles (with the diameter of 70 nm and 5

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µm), which is used for the toxicity test. The PS suspensions were prepared using

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UV-sterilized aerated water and sonicated prior to use. The composition of the virgin PS beads

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was confirmed by FTIR spectroscopy and there was no differences between different sizes of

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particles (Figure S2). The aggregation of 70 nm particles in water was determined by dynamic

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light scattering (DLS) (Malvern, ZEN1600, UK) and no significant aggregation was observed

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(Figure S3).

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Zebrafish maintenance

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Adult healthy zebrafish (Danio rerio, 5-month-old and 0.29 ± 0.022 g in wet weight) were

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maintained at 24 ± 1 oC with a 14-hour-light-cycle in culture water (UV-sterilized and

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well-aerated water, pH: 7.2 ± 0.5, dissolved oxygen: 6.6 ± 0.3 mg/L, electrical conductivity:

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0.256 ± 0.005 mS/cm, water hardness: 185 ± 9 mg/L CaCO3) and acclimated in 15 L glass

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tanks for 2 weeks before the experiment. Fish were fed 1.0% body-weight twice daily with

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commercial food (Inch-gold, China). All of the experimental researches were in accordance

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with the National Institutes of Health Guide for the Care and Use of Laboratory animals.

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Uptake and accumulation of PS-MPs in fish tissues

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After starved for 24 h, fish were randomly placed into 30 glass tanks, each tank

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containing 6 fish and 500 mL test solution. Test solutions were prepared by adding two sizes

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of fluorescent PS-MPs (5-µm and 20-µm diameter, respectively) to culture water with final

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concentration of 20 mg/L (2.9 × 105 particles/mL for 5-µm PS-MPs, 4.5 × 103 particles/mL

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for 20-µm PS-MPs). For the experiment, the test solution in each tank was refreshed every 48

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h. During the experiment, the tanks were continuously aerated to keep the dispersion of the

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particles in water. No aggregation was observed in the tanks with aeration (Figure S3). Three

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replicate tanks were used for each of the 5 following sampling time: 4h, 12h, 1d, 2d, and 7d.

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At each sampling time, fish were rinsed to remove the particles from skin, and then six fish of

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each replicate were pooled in case of liver, gill, and gut. Pooling was necessary due to the

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small tissue sizes and to control for interindividual variability.

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After lyophilization for 72 h, the tissues were digested in nitric acid (1 mL) at 70 oC for 2

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h, and then diluted with deionized (DI) water to a final volume of 5 mL. The concentration of

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PS-MPs in different tissues of fish was measured by fluorescent spectrophotometer

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(HITACHI F-7000) with excitation at 418 nm and emission at 518 nm. The standard curve

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was generated by using serial dilutions of fluorescent PS-MPs suspensions (Figure S4). The

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background fluorescence of the tissues of unexposed fish, which were processed in the same

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way as that for exposed fish, was detected and subtracted from that of treatment samples.

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Each detection was run in triplicate. The tissues of fish were also fixed in 10% formalin for 1

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h, embedded in paraffin wax, sectioned at 4 µm thickness, and stained with hematoxylin and

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eosin (H&E) for final observation. Ingestion and distribution of particles were ascertained by

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a fluorescence microscope (Zeiss Axio Imager A1) to determine the presence of PS-MPs

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(fluorescing yellow-green) within the gill, liver, and gut of the zebrafish.

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Toxic exposure of PS-MPs

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For toxicity test, acclimated fish were randomly assigned to control group and

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PS-MPs-treated groups. For treatment groups, 70-nm and 5-µm virgin PS-MPs were used and

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the exposure concentrations were 20 µg/L (1.1 × 108 particles/mL for 70-nm PS-MPs, 2.9 ×

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102 particles/mL for 5-µm PS-MPs), 200 µg/L (1.1 × 109 particles/mL for 70-nm PS-MPs, 2.9

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× 103 particles/mL for 5-µm PS-MPs), and 2000 µg/L (1.1 × 1010 particles/mL for 70-nm

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PS-MPs, 2.9 × 104 particles/mL for 5-µm PS-MPs), which are according to environmental

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concentration21 and previous literatures about the toxicity of MPs on aquatic organisms 6, 7, 11.

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For each size of PS-MPs, 60 fish were used for oxidative stress analysis and histopathological

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analysis. These fish were randomly assigned into 12 tanks (5 fish each tank) and 3 tanks were

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used for each treatment groups (exposed to 20, 200, and 2000 µg/L PS-MPs, respectively) and

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control (culture water without particles). In addition, 400 fish were used for metabolomics

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analysis. These fish were randomly placed into 20 tanks (20 fish each tank) and 5 tanks were

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used for each treatment groups and control. These fish were exposed for 3 weeks. The test

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solutions preparation and fish maintenance are the same as mentioned above. After the

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exposure, fish were caught with a steel grid and rinsed to remove MPs-containing water from

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the skin. Afterward, they were anaesthetized in a saturated benzocaine solution in DI water.

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Depending on the aim of the experiment, the liver of the fish was dissected and immediately

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frozen in liquid nitrogen and stored at -80 oC for metabolomic analysis and oxidative stress

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analysis, or fixed in 10% formalin for histopathological analysis.

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Oxidative stress analysis

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The activities of superoxide dismutase (SOD) and catalase (CAT) in fish livers were

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determined to identify the oxidative stress of PS-MPs in zebrafish. Livers of five fish of each

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replicate were pooled for one sample and 20 mg homogenized tissue was used for detection.

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The activities of SOD and CAT were determined by using commercial kits (Jiancheng Bioeng.

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Inst., China) and experimental operations were conducted according to the manufacturer

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protocol. Each assay was run in triplicate.

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Histopathological analysis

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The livers of fish from 2000 µg/L PS-MPs treated group and control group were fixed in

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10% formalin, embedded in paraffin wax, sectioned at 4 µm thickness, and stained with H&E

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for microscopic observation.

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Metabolomic analysis

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Livers of 20 fish in each group were pooled together and used for hepatic metabolites

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extraction. The extraction process referred to previous studies with some modifications

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In brief, 50 mg tissues were homogenized with 500 µL methanol-water (4:1, v/v) and 600 µL

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chloroform-water (2:1, v/v). The mixture was centrifuged at 10000 × g for 5 min at 4oC. The

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supernatant phase (hydrophilic metabolites) and lower phase (lipophilic metabolites) were

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separated and lyophilized. All samples were kept at -80 oC. The hydrophilic and lipophilic

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extracts were reconstituted in 600 µL D2O containing 0.05% trisilylpropionic acid (TSP) and

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600 µL of CDCl3 containing 0.05% tetramethylsilane (TMS), respectively, and then

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transferred into NMR tubes for analysis. The metabolites were analyzed by a Bruker 600

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MHz spectrometer (Bruker, Germany). The detailed detection was conducted according to the

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methods described previously24 .

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The metabolite resonances were identified according to the information from the previous 25

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studies

and the Human Metabolome Database (HMDB). PLS-DA was used to explore the

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main effects in the NMR data sets and calculated with MetaboAnalyst 3.0. The significantly

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changed metabolites between the control and treatment groups were identified following the

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criteria that p < 0.05, fold change ≥ ± 1.2 and significantly changed in at least two dose

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groups. In order to visualize the variations of metabolites between different groups, heatmap

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was generated based on z-scores. The z-scores of the altered metabolites were calculated with

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the following formula: z-score = (treatment metabolite abundance – control mean)/standard

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deviation of control.

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Statistical Analysis

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The results were expressed as mean ± standard deviation (SD). One-way ANOVA test

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was used to evaluate the statistical differences of biological parameters between control group

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and treatment groups. All analysis was undertaken by SPSS 19.0 software (SPSS Inc.). P-

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value < 0.05 was accepted as significance.

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Results

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Up take and accumulation of PS-MPs in tissues of Zebrafish

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In the uptake experiment, fluorescently labeled PS-MPs of 5-µm diameter accumulated

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in fish gill, liver, and gut after 7 days of exposure (Figure 1). While large-sized PS-MPs of

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20-µm diameter only accumulated in fish gill and gut, and no similar particles were found in

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fish liver (Figure 1).

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Within exposure process, the accumulation of both sizes of polystyrene particles were

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quantified by fluorescence spectroscopy with external standard calibrations (Figure S4) and

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the results are showed in Figure 2. It is found that PS-MPs quickly accumulated in fish tissues

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overtime. For small-sized particles (5-µm diameter), steady state was reached within 48 h in

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different tissues (gill, liver and gut), although the variability between replicates was high. The

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maximum concentrations of PS-MPs accumulated in gill, liver, and gut were 0.566, 1.251,

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and 0.895 µg/mg fish dry weight on average, respectively. In addition, it is found that 5-µm

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PS-MPs mainly accumulated in the alimentary system of zebrafish and the concentrations of

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PS-MPs retained in internal tissues (liver and gut) were higher than that in external tissues

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(gill).

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Histopathological changes induced by PS-MPs treatment

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According to the accumulation experiments, 20-µm PS-MPs were not observed in fish

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liver. In order to evaluate the potential toxicity of PS-MPs in liver, another two sizes of

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particles (5-µm and 70-nm diameter PS-MPs) were used for the toxicity test. Within three

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weeks, no death was found in PS-MPs treated groups. Representative histological maps of

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liver from fish exposed to 2000 µg/L PS-MPs are shown in Figure 3. Compared with control,

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necrosis, infiltration, and lipid droplets were observed in hepatocytes in PS-MPs treated fish,

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indicating that high dose of PS-MPs (both size of 5-µm and 70-nm diameter) caused

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inflammation and lipid accumulation in liver.

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Oxidative stress induced by PS-MPs treatment

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In this study, the activities of SOD and CAT in zebrafish liver were detected to evaluate

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the oxidative stress induced by PS-MPs treatment, and the results are shown in Figure 4.

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Generally, the activities of SOD and CAT significantly increased in 5-µm PS-MPs treated

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fish with a dose-response manner (p < 0.05). Under the doses of 200 µg/L and 2000 µg/L, the

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levels of SOD and CAT in 5-µm PS-MPs treated fish were both higher than that in 70-nm

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PS-MPs treated fish. Only the high dose (2000 µg/L) of 70-nm PS-MPs induced increase of

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SOD and CAT activities significantly (p < 0.05).

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Metabolomic alterations induced by PS-MPs Treatment

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Alterations of metabolomic profiles in fish liver due to PS-MPs exposure were

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determined by 1H-NMR, and PLS-DA was used for the NMR data sets to characterize the

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variations (Figure 5). The pattern recognition results showed that 5-µm PS-MPs treated

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groups could be clearly separated with control group under all three exposure dosages, which

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suggested that 5-µm PS-MPs induced significant alterations of hepatic metabolome in

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zebrafish. Besides, samples in 5-µm and 70-nm PS-MPs treated groups were also successfully

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separated under all three exposure dosages, which suggested that these two sizes PS-MPs

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induced different metabolomic profiles in fish liver. While separation between 70-nm

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PS-MPs treated group and control group was only observed in high-dose group (2000 µg/L)

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(Figure 5C).

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In addition, significantly changed metabolites in treatment groups were identified and

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listed in Table S1. As a result, 5-µm and 70-nm PS-MPs treatment induced 21 and 11 altered

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metabolites respectively, and 7 metabolites were common between the treated groups (Figure

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6A). These altered metabolites were also demonstrated in the heat-map (Figure 6B). As a

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result, we noticed a trend that fatty acids were up-regulated and amino acids were

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down-regulated with increasing doses of PS-MPs.

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Discussion

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Size dependent uptake of MPs in marine species has been studied which indicates that

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MPs ingestion significantly depends on the particle size26. This is largely attributed to the

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similar size of prey and special filter organs. For instance, plastics with the size 11-700 µm,

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may readily be ingested by amphipods27. While 1 µm is a common size to be intercepted by

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the gastric filters of crustaceas28. In addition, particle size also influences the distribution of

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MPs in biological systems. For example, 10-µm plastics can be transported into the

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circulatory system in mussels29, whereas 8-10 µm PS-MPs mainly accumulate in gill and gut

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of crabs30. Accumulation and distribution of MPs in different tissues of zebrafish has not been

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reported before.

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In this study, 5-µm and 20-µm MPs were both found in the gill and gut of zebrafish. In

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addition, 5-µm plastics were observed and quantified in liver, while 20-µm plastics were not

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found. On one hand, this is probably because the 5-µm particles could enter into the

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circulatory system and be transferred to liver. It has been demonstrated that submicrometer

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metal particles could accumulate in the liver and spleen of human31. PS nanoparticles could be

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taken up through gills and transferred to several organs of fish via the blood32. On the other

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hand, the differences seen between the larger particles and the smaller particles for uptake may

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be due to the concentration and chance that one particle comes in contact with tissue more often,

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after all the count of particles per unit volume for 5-µm PS-MPs is two orders of magnitude

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higher than that of 20-µm PS-MPs. Moreover, fluorescence spectroscopy was used to

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determine the concentrations of MPs accumulated in tissues of zebrafish. Although this

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method relies on the fluorescent dyes encapsulated in plastic beads, it is very useful to

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quantify the accumulation of MPs in different species using fluorescent MPs as a model for

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future ecotoxicological research.

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Although MPs can be ingested and accumulate in aquatic organisms, the potential toxic

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effects are largely unclear. In this study, early inflammatory responses, such as vacuolation,

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infiltration and necrosis, were observed in hepatocytes in MPs treated groups. Similar results

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in zebrafish has not yet been reported, although many hepatotoxicity studies have suggeasted

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that liver may be the major target organ for the nano materials33, 34. Inflammatory responses

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and lysosomal membrane destabilization have been found in mussels exposed to PS- and

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PE-MPs9,14. No conspicuous differences of histopathological changes in tissues were

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observed between fish treated with the two sizes of particles (5-µm and 70-nm diameter).

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Based on the oxidative stress analysis, large-sized MPs (5-µm diameter) induced

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increased activities of SOD and CAT in this study. However, we can not simply conclude that

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5-µm MPs is more toxic than 70-nm MPs. Actually, it is generally speculated that small-sized

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particles would be more toxic than large-sized particles owing to the increase of specific

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surface area35, 36. Our report on this phenomenon is the first, and this may be attributed to

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insufficient nutrition or the inhibition of food digestion due to more large-sized MPs were

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ingested by fish. Many studies have demonstrated that low feeding activity or limited food

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intake can change oxidative stress profiles37. Another explanation may be that 70-nm particles

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were aggregated in fish to a greater extent than the 5-µm particles, although it can not be

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aggregated in water. We were unable to determine the accumulation and aggregation of 70-nm

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particles (the size is too small) in fish tissues but deem this an important consideration for

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future research efforts.

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MPs exposure also disturbed the metabolomic profiles in the liver of fish, which

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provided additional insights on molecular mechanisms of toxic effects induced by MPs.

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These altered metabolites involved in lipid metabolism and energy metabolism, appeared as a

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primary response to PS-MPs exposure.

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In this study, lipid metabolites of triglycerides and fatty acids (monounsaturated fatty

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acid (MUFA), linoleic acid, FA-αH2, FA-ω-CH3, and fatty acyl chains) were significantly

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changed. MPs also induced alterations of choline, phosphorycholine and cholesterol, which

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are all related to lipid metabolism. Choline and phosphorycholine are both essential

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substances in the synthesis and transportation of phospholipids and promote lipid

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metabolism38. Choline has been considered to have a positive effect on cholesterol level in the

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digestive system39. Isoleucine, valine and leucine are branched-chain amino acids (BCAAs),

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which can promote fatty acids metabolism and prevent fat accumulation40. MPs exposure

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induced decreases of these BCAAs. Alterations of these metabolites indicated that PS-MPs

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exposure induced a disruption of lipid metabolism in zebrafish, which can also be confirmed

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by the lipid droplets observed in fish liver. It has been demonstrated that PS nanoparticles

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could affect lipid metabolism in fish5, 41.

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On the other hand, isoleucine, leucine, valine and lysine can regulate energy

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metabolism42. Disruption of energy metabolism was confirmed by the altered metabolite of

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ATP/ADP/AMP. Similar results have been reported that ingested MPs depleted energy

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reserves in marine worms and copepods8, 43, and affected the feeding activity of fish

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reasonable explanation may be that the large amount of ingested MPs, which have no

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nutritional value, affected the normal absorption of food. Alteration of food absorption can

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also lead to changes in lipid and energy metabolism44, 45.

41

.The

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In summary, fluorescent spectrophotometry was firstly used for the quantification of

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MPs in zebrafish and size-dependent uptake and tissue accumulation of MPs was observed.

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5-µm and 70-nm PS-MPs could induce histological changes and oxidative stress in fish liver.

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In addition, metabolomics was used to reveal the toxic effects of MPs and our findings show

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that MPs disturbed the metabolism of lipid and energy in fish liver.

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Supporting Information

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Supporting information includes Figure S1-S4, Table S1. This material is available free of

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charge via the Internet at http://pubs.acs.org.

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Acknowledgements

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This work was financially supported by the National Key Technology Support Program

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(2014BAC08B07), Jiangsu Natural Science Foundation (BK20130559), National Natural

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Science Foundation of China (21507058 and 21407076), and Specialized Research Fund for

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the Doctoral Program of Higher Education (20130091120012).

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Nanotubes on the Cellular Division, Microstructure, Uptake, Oxidative Stress, and Metabolic Profiles. Environ. Sci. Technol. 2015, 49, (18), 10825-10833. 17. Feng, J. H.; Li, J. Q.; Wu, H. F.; Chen, Z., Metabolic responses of HeLa cells to silica nanoparticles by NMR-based metabolomic analyses. Metabolomics 2013, 9, (4), 874-886. 18. Bo, Y.; Jin, C. Y.; Liu, Y. M.; Yu, W. J.; Kang, H. Z., Metabolomic analysis on the toxicological effects of TiO2 nanoparticles in mouse fibroblast cells: from the perspective of perturbations in amino acid metabolism. Toxicol. Mech. Methods 2014, 24, (7), 461-469. 19. Sadri, S. S.; Thompson, R. C., On the quantity and composition of floating plastic debris entering and leaving the Tamar Estuary, Southwest England. Mar. Pollut. Bull. 2014, 81, (1), 55-60. 20. Zhang, Q.; Han, Y.; Wang, W. C.; Zhang, L.; Chang, J., Preparation of fluorescent polystyrene microspheres by gradual solvent evaporation method. Eur. Polym. J. 2009, 45, (2), 550-556. 21. Zhao, S. Y.; Zhu, L. X.; Wang, T.; Li, D. J., Suspended microplastics in the surface water of the Yangtze Estuary System, China: First observations on occurrence, distribution. Mar. Pollut. Bull. 2014, 86, (1-2), 562-568. 22. Li, J.; Zhao, Z.; Feng, J.; Gao, J.; Chen, Z., Understanding the metabolic fate and assessing the biosafety of MnO nanoparticles by metabonomic analysis. Nanotechnology 2013, 24, (45), 455102. 23. Wu, B.; Liu, S.; Guo, X. C.; Zhang, Y.; Zhang, X. X.; Li, M.; Cheng, S. P., Responses of Mouse Liver to Dechlorane Plus Exposure by Integrative Transcriptomic and Metabonomic Studies. Environ. Sci. Technol. 2012, 46, (19), 10758-10764. 24. Samuelsson, L.; Förlin, L.; Karlsson, G.; Adolfsson-Erici, M.; Larsson, D. G. J., Using NMR metabolomics to identify responses of an environmental estrogen in blood plasma of fish. Aquat. Toxicol. 2006, 78, (4), 341-349. 25. Vinaixa, M.; Rodriguez, M. A.; Rull, A.; Beltran, R.; Blade, C.; Brezmes, J.; Canellas, N.; Joven, J.; Correig, X., Metabolomic Assessment of the Effect of Dietary Cholesterol in the Progressive Development of Fatty Liver Disease. J. Proteome Res. 2010, 9, (5), 2527-2538. 26. Wright, S. L.; Thompson, R. C.; Galloway, T. S., The physical impacts of microplastics on marine organisms: A review. Environ. Pollut. 2013, 178, 483-492. 27. Chua, E. M.; Shimeta, J.; Nugegoda, D.; Morrison, P. D.; Clarke, B. O., Assimilation of polybrominated diphenyl ethers from microplastics by the marine amphipod, Allorchestes Compressa. Environ. Sci. Technol. 2013, 48, 8127-8134. 28. Hamer, J.; Gutow, L.; Kohler, A.; Saborowski, R., Fate of Microplastics in the Marine lsopod Idotea emarginata. Environ. Sci. Technol. 2014, 48, (22), 13451-13458. 29. Browne, M. A.; Dissanayake, A.; Galloway, T. S.; Lowe, D. M.; Thompson, R. C., Ingested microscopic plastic translocates to the circulatory system of the mussel, Mytilus edulis (L.). Environ. Sci. Technol. 2008, 42, 5026-5031. 30. Watts, A. J. R.; Lewis, C.; Goodhead, R. M.; Beckett, S. J.; Moger, J.; Tyler, C. R.; Galloway, T. S., Uptake and retention of microplastics by the shore crab Carcinus maenas. Environ. Sci. Technol. 2014, 48, (15), 8823-8830. 31. Urban, R. M.; Tomlinson, M. J.; Hall, D. J.; Jacobs, J. J., Accumulation in liver and spleen of metal particles generated at nonbearing surfaces in hip arthroplasty. J. Arthroplasty 2004, 19, (8), 94-101. 32. Kashiwada, S., Distribution of nanoparticles in the see-through medaka (Oryzias latipes). Environ. Health Perspect. 2006, 114, (11), 1697-1702. 33. Sharma, V.; Anderson, D.; Dhawan, A., Zinc oxide nanoparticles induce oxidative DNA damage and ROS-triggered mitochondria mediated apoptosis in human liver cells (HepG2). Apoptosis 2012, 17,

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422

Figure caption

423

Figure 1. Fluorescently labeled PS-MPs (arrows) of 5-µm and 20-µm diameter accumulated

424

in gill, liver, and gut of zebrafish.

425

Figure 2. Concentration of PS-MPs (5-µm and 20-µm diameter) in different tissues of

426

zebrafish (A, gill; B, liver; C, gut) at dfferent exposure times (R2, determination coefficient of

427

fitting curve).

428

Figure 3. Repressentative liver sections from zebrafish exposed for 3 weeks to 2000 µg/L

429

PS-MPs (5-µm and 70-nm diameter). Necrosis, infiltration, and fat droplets were observed in

430

hepatocytes (red arrow).

431

Figure 4. Activities of CAT and SOD in zebrafish livers due to PS-MPs exposure. Star marks

432

significantly difference (p < 0.05).

433

Figure 5. Partial least-squares discriminant analysis (PLS-DA) for hepatic metabolic profiles

434

between PS-MPs treated groups under different concentrations (A, 20 µg/L; B, 200 µg/L; C,

435

2000 µg/L).

436

Figure 6. (A) Venn diagram representing significantly changed metabolites between

437

treatment groups. (B) Heat map for altered metabolites between PS-MPs treated groups and

438

control group (CK, control; SL, 20 µg/L 70 nm PS-MPs; SM, 200 µg/L 70 nm PS-MPs; SH,

439

2000 µg/L 70 nm PS-MPs; LL, 20 µg/L 5 µm PS-MPs; LM, 200 µg/L 5 µm PS-MPs; LH,

440

2000 µg/L 5 µm PS-MPs; *, p < 0.05 and fold change ≥ ± 1.2).

441

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442

Figure 1. Fluorescently labeled PS-MPs (arrows) of 5-µm and 20-µm diameter accumulated

443

in gill, liver, and gut of zebrafish.

444 445

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446

Figure 2. Concentration of PS-MPs (5-µm and 20-µm diameter) in different tissues of

447

zebrafish (A, gill; B, liver; C, gut) at dfferent exposure times (R2, determination coefficient of

448

fitting curve).

449 450

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Page 24 of 27

451

Figure 3. Repressentative liver sections from zebrafish exposed for 3 weeks to 2000 µg/L

452

PS-MPs (5-µm and 70-nm diameter). Necrosis, infiltration, and fat droplets were observed in

453

hepatocytes (red arrow).

454 455 456

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457

Figure 4. Activities of CAT and SOD in zebrafish livers due to PS-MPs exposure. Star marks

458

significantly difference (p < 0.05).

459 460 461

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462

Figure 5. Partial least-squares discriminant analysis (PLS-DA) for hepatic metabolic profiles

463

between PS-MPs treated groups under different concentrations (A, 20 µg/L; B, 200 µg/L; C,

464

2000 µg/L). These ellipses represent the 95% confidence region.

465 466 467

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468

Figure 6. (A) Venn diagram representing significantly changed metabolites between

469

treatment groups. (B) Heat map for altered metabolites between PS-MPs treated groups and

470

control group calculated by z-scores (CK, control; SL, 20 µg/L 70 nm PS-MPs; SM, 200 µg/L

471

70 nm PS-MPs; SH, 2000 µg/L 70 nm PS-MPs; LL, 20 µg/L 5 µm PS-MPs; LM, 200 µg/L 5

472

µm PS-MPs; LH, 2000 µg/L 5 µm PS-MPs; *, p < 0.05 and fold change ≥ ± 1.2).

473 474

(A)

475 476

(B)

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Uptake and Accumulation of Polystyrene ... - ACS Publications

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