Microplastic Size-Dependent Toxicity, Oxidative Stress Induction, and p JNK and p p38 Activation in the Monogonont Rotifer (Brachionus koreanus)

Transcription

1 pubs.acs.org/est Microplastic Size-Dependent Toxicity, Oxidative Stress Induction, and p JNK and p p38 Activation in the Monogonont Rotifer (Brachionus koreanus) Chang-Bum Jeong,, Eun-Ji Won,, Hye-Min Kang, Min-Chul Lee, Dae-Sik Hwang, Un-Ki Hwang, Bingsheng Zhou, Sami Souissi, # Su-Jae Lee, and Jae-Seong Lee*, Department of Biological Science, College of Science, Sungkyunkwan University, Suwon 16419, South Korea Department of Chemistry, College of Natural Sciences, Hanyang University, Seoul 04763, South Korea Marine Chemistry and Geochemistry Research Center, Korea Institute of Ocean Science and Technology, Ansan 15627, South Korea Marine Ecological Risk Assessment Center, West Sea Fisheries Research Institute, National Fisheries Research & Development Institute, Incheon 46083, South Korea State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan , China # Universite de Lille, CNRS, Universite Littoral Cote d Opale, UMR 8187, LOG, Laboratoire d Oceánologie et de Geósciences, F Wimereux, France Department of Life Sciences, College of Natural Sciences, Hanyang University, Seoul 04763, South Korea *S Supporting Information ABSTRACT: In this study, we evaluated accumulation and adverse effects of ingestion of microplastics in the monogonont rotifer (Brachionus koreanus). The dependence of microplastic toxicity on particle size was investigated by measuring several in vivo end points and studying the ingestion and egestion using 0.05-, 0.5-, and 6-μm nonfunctionalized polystyrene microbeads. To identify the defense mechanisms activated in response to microplastic exposure, the activities of several antioxidant-related enzymes and the phosphorylation status of mitogen-activated protein kinases (MAPKs) were determined. Exposure to polystyrene microbeads of all sizes led to significant sizedependent effects, including reduced growth rate, reduced fecundity, decreased lifespan and longer reproduction time. Rotifers exposed to 6-μm fluorescently labeled microbeads exhibited almost no fluorescence after 24 h, while rotifers exposed to and 0.5-μm fluorescently labeled microbeads displayed fluorescence until 48 h, suggesting that 6-μm microbeads are more effectively egested from B. koreanus than or 0.5-μm microbeads. This observation provides a potential explanation for our findings that microbead toxicity was sizedependent and smaller microbeads were more toxic. In vitro tests revealed that antioxidant-related enzymes and MAPK signaling pathways were significantly activated in response to microplastic exposure in a size-dependent manner. INTRODUCTION Since its first description in 1972, plastic pollution has become one of the most concerning environmental problems due to its detrimental effects on ecosystems. 1 3 In the marine environment, one of the important problems is associated with degradation of plastic debris into smaller particles, which are difficult to collect and remove from the aquatic environment. Size degradation can mainly occur by weathering through biodegradation, thermooxidative degradation, thermal degradation, hydrolysis, and photodegradation. 4 Microplastics (less than 5 mm) resulting from the degradation of larger plastic debris have been shown to constitute up to 60 80% of all marine garbage. 2,4 6 The deleterious effects of plastics on marine organisms are evidently increasing; the relatively small plastic debris are easier to be absorbed into biological processes due to their large surface/volume ratio. Also, the capacity to adsorb persistent organic pollutants allows their ingestion and the accumulation of plastics in the aquatic food web to threaten the ecosystem further. 7 However, little attention has been paid Received: March 25, 2016 Revised: June 12, 2016 Accepted: July 20, 2016 XXXX American Chemical Society A

2 to date on the adverse effects of nanosized and microsized plastics due to the methodological difficulties in detecting and quantifying these microplastics. Microplastic uptake has been investigated in a wide range of animals, including mammals and invertebrates Specifically, microplastic uptake has been investigated in plankton, 8 10 the blue mussel Mytilus edulis, and the lugworm Arenicola marina. 14 However, the fate of microplastics after ingestion is still unknown. For instance, it remains an outstanding question whether ingested microplastics can translocate into other organs through the circulatory system. However, the accumulation of ingested microplastics in digestive organs has been reported. 8,9 Accumulated nanoparticles (NPs) taken up by phagocytosis have been shown to induce cytotoxicity by generating reactive oxygen species (ROS) in mammalian cell lines and aquatic invertebrates. 18,19 Increased ROS levels have also been measured in the rotifer Brachionus koreanus after exposure to carbon nanotubes (CNTs), indicating that CNT induces ROS-related mitochondrial dysfunction. 20 Similarly, in the algae Chlorella and Scenedesmus, polystyrene microbeads have been shown to generate ROS and inhibit photosynthetic ability. 18 Despite growing concern regarding the deleterious effects of microplastics on marine organisms, particularly on filter feeding plankton at the bottom of the food chain, the defense mechanisms of marine organisms in response to microplastic ingestion have not yet been reported. Rotifers play a prominent role in energy transfer in the aquatic food chain and their ability to carry pollutants to higher trophic levels by digestion and accumulation. 21,22 Also, rotifers have several advantages as experimental species such as their small size ( 200 μm), short generation cycle ( 24 h), simple structure, genetic homogeneity, high fecundity, and easy laboratory maintenance. 21,22 These advantages have facilitated their use in ecophysiology, ecotoxicology, and environmental genomics studies. As filter feeding organisms, the habitat and eating behavior of rotifers allows them to float along waves and currents with other small particles such as microplastics. These behaviors also allow the ingestion of small particles with poor feeding selectivity. Cumulatively, these properties make rotifers very suitable species for studying the effects of microplastic ingestion. The aim of this study was to investigate the effects of nanosized and microsized microplastics on rotifers. We used three different sizes of polystyrene microbeads (0.05, 0.5, and 6 μm) to check the size-dependent toxicity of microplastics and examined the effect of microplastic sizes on the defense mechanisms in rotifers at the molecular and biochemical levels. To our knowledge, this is the first study with an explanation of defense mechanisms at molecular and biochemical levels in response to microplastics, thereby providing a better under- of the impact of microplastics on marine organisms. standing MATERIALS AND METHODS Rotifer Culture. The monogonont rotifer B. koreanus was collected at Uljin ( N, E), South Korea. A single individual rotifer was isolated, reared, and maintained in filtered artificial seawater (TetraMarine Salt Pro, Tetra, Cincinnati, OH, U.S.). Rotifers were cultured at 25 C under a light:dark 12:12 h photoperiod with 15 practical salinity units (psu). Green alga Chlorella sp. were used as a live diet (approximately cells/ml). Species identification was confirmed via morphological analysis and sequencing of the mitochondrial DNA gene CO1. 23,24 Polystyrene Microbeads. Polystyrene was chosen because it is one of the most abundant polymers in marine debris. 25 As concerns about smaller-sized plastic particles are increasing, 26 we have used nanosized and microsized polystyrene microbeads. Nonfunctionalized polystyrene microbeads with diameters of 0.05, 0.5, and 6 μm were purchased from Polyscience (Warrington, PA, U.S.). According to the manufacturer, the average sizes of the microbeads in each stock solution were 0.05, 0.5, and 6 μm, respectively. We defined 0.05-μm polystyrene microbeads as nanosized microplastics, while 0.5- and 6-μm microbeads were defined as microsized microplastics. For ingestion experiments, fluorescently labeled polystyrene microbeads (Warrington, PA, U.S.; excitation and emission wavelengths at 441 and 486 nm, respectively) of the same diameters were used. All the stock solutions were sonicated before use for dispersion. In Vivo Toxicity Tests. For size-dependent and concentration-dependent toxicity tests of microplastics in B. koreanus, rotifers were exposed to 0.05-, 0.5-, and 6-μm polystyrene microbeads at concentrations of 0.1, 1, 10, and 20 μg/ml. To examine the effects of microplastic exposure on B. koreanus growth, 12 individual rotifers were added to 4 ml artificial seawater (ASW) in each well of a 12-well culture plate (SPL, Seoul, South Korea). The rotifers were exposed to 0.1, 1, 10, and 20 μg/ml polystyrene microbeads and placed in an incubator at 25 C. The numbers of rotifers were counted daily under a stereomicroscope (SZX-ILLK200, Olympus Corporation, Tokyo, Japan) for 12 days. To examine the effects of microplastics on B. koreanus fecundity and lifespan, 12 individual rotifers were placed in each well of a 12-well culture plate, exposed to 0.1, 1, 10, and 20 μg/ ml polystyrene microbeads, and incubated at 25 C. The numbers of newborn rotifers were counted every 12 h until the matured rotifer died. After that, the newborn rotifers were removed. Rotifer lifespan was determined as the number of days that the rotifers stayed alive, and the experiments were continued until the founding rotifers died. The time needed for a just-hatched rotifer to grow to a matured rotifer was measured as the reproduction time. Rotifer maturation was defined as the time at which the rotifer first produced eggs. The rotifers were observed in each well every 2 h under the stereomicroscope. All experiments were initiated with just-hatched rotifers within 2 h of their collection from the pool of rotifer eggs. During all experiments, the green alga Chlorella sp. (approximately cells/ml) was supplied as a diet and approximately 50% of the testing solution was renewed daily. All exposures were for 12 days and performed in triplicate. Polystyrene Microbead Ingestion and Egestion. To examine the kinetics of microplastic ingestion in B. koreanus, fluorescently labeled polystyrene microbeads were used. Briefly, rotifers (50 individual organisms) were exposed to 10 μg/ml of 0.05-, 0.5-, and 6-μm fluorescently labeled polystyrene microbeads and then incubated at 25 C in the dark for 24 h. After exposure, rotifers were washed with clean ASW and fixed with 4% formaldehyde. Fluorescently labeled polystyrene microbeads in the digestive organs of the rotifers were observed with a fluorescence microscope (Olympus IX71, Olympus Corporation). Microplastic egestion from rotifer digestive organs was also observed. Rotifers were exposed to 10 μg/ml of 0.05-, 0.5-, and 6-μm fluorescently labeled polystyrene microbeads for 24 h, transferred to clean ASW, and then incubated for 6, 12, 24, or 48 h (50 individual organisms were used for each treatment). B

3 Figure 1. Effects of exposure to polystyrene microbeads of different diameters (0.05, 0.5, and 6 μm) on B. koreanus (A) growth rate, (B) fecundity, and (C) life span. Rotifers were exposed to microbeads for 24 h. Differences between groups were analyzed for significance using Tukey s multiple comparison test. Different letters above columns indicate significant differences (P < 0.05). After incubation, each group of rotifers was sampled at different times to examine the egestion of the microplastics. Briefly, the sampled rotifers were washed with clean ASW and then fixed. The remaining microbead fluorescence in the rotifer digestive organs was examined using a fluorescence microscope. During the experiment, no alga food was provided. To exclude the possibility that decreased fluorescence in the rotifer digestive organs was due to fluorescent microbead degradation, the fluorescence stability of the labeled polystyrene microbeads was measured. To this end, 10 μg/ml of 0.05-, 0.5-, and 6-μm fluorescently labeled polystyrene microbeads were added to ASW and their fluorescence intensities were measured for 48 h with a fluorescence spectroscope (Thermo Varioskan Flash, Thermo Fisher Scientific, Vantaa, Finland). Antibodies, Western Blot Analysis, NAC Treatment, Measurements of ROS Level, GSH Content, GSH-Related Enzyme Activities, and Mitochondrial Membrane Integrity. A detailed description of all materials and methods is incorporated in the Supporting Information. Briefly, to examine the effect of different sizes of microplastics on intracellular reactive oxygen species (ROS) induction, antioxidant enzymatic activities, and the activation of MAPK signaling pathways (e.g., phosphorylation patterns of ERK, JNK, and p38) were checked after 24 h in response to 0.05-, 0.5-, and 6-μm microbeads. B. koreanus (approximately 6,000 individuals) were homogenized in a lysis buffer (40 mm Tris HCl [ph 8.0], 120 mm NaCl, and 0.1% Nonidet-P40) containing a complete protease inhibitor cocktail (Roche; South San Francisco, CA, U.S.) and used for ROS and GSH-related enzyme activities measurement and Western blot analysis. For ROS scavenging, 0.5 mm of N-acetyl-L-cysteine (NAC) was treated in the presence of different sizes of microbeads. Statistical Analysis. The SPSS ver (SPSS Inc.; Chicago, IL, U.S.) software package was used for all statistical analysis. Data are expressed as means ± SDs. The significance of differences between the control and experimental groups was analyzed using Student s paired t-test and one-way and/or multiple-comparison ANOVA followed by Tukey s test. Any difference with P value < 0.05 was considered significant. RESULTS In Vivo Effects of Polystyrene Microbead Exposure. Microbeads exhibited significant size-dependent and concen- C

4 tration-dependent toxicity with negative correlation with particle size (Figure 1). Interestingly, 0.05-μm microbeads were significantly more toxic to rotifers than either 0.5- or 6-μm microbeads. Rotifers exposed to 6-μm microbeads displayed only slightly retarded growth, and no significant differences were observed in any of the other in vivo toxicity tests. Similar to the effects on growth, rotifer reproduction time was affected by different sizes of microbeads (Figure 2). Consistent with the toxicity data, 0.05-μm microbeads exerted the most deleterious effects on reproduction time. Figure 2. Effects of exposure to polystyrene microbeads of different diameters (0.05, 0.5, and 6 μm) for 24 h on the reproduction time of B. koreanus. All polystyrene microbeads were used at 10 μg/ml. Polystyrene Microbead Ingestion and Egestion. All sizes of microbeads were ingested by the rotifers, as exhibited by the microbead fluorescence in the rotifer digestive organs (Figure 3). Efflux was measured by analyzing the decrease in rotifer fluorescence over time (Figure 4). Particularly striking was the finding that rotifers exposed to fluorescent 6-μm microbeads did not exhibit any fluorescence by 24 h postingestion, while rotifers exposed to and 0.5-μm fluorescent microbeads continued to fluoresce up to 48 h postingestion. To ensure that the decreases in fluorescence did not correspond to microbead degradation, we assessed the stability of the fluorescent microbeads. After 48 h of incubation, the fluorescence intensity had decreased by only 20% compared with the initial fluorescence (0 h), suggesting that the decreased rotifer fluorescence over time was not due to microbead degradation (data not shown). ROS Levels and Phosphorylation of MAPKs. Intracellular ROS levels exhibited a significant size-dependent increase, and the phosphorylation status of different MAPKs showed different expression patterns in rotifers exposed to 0.05-, 0.5-, and 6-μm microbeads (10 μg/ml) for 24 h (Figure 5A). Rotifers showed increased phosphorylation of c-jun N- terminal kinase (p-jnk) and p38 (p-p38); moreover, these patterns correlated with the increases in intracellular ROS levels (Figure 5B). However, extracellular signal-regulated kinase phosphorylation (p-erk) was not affected by the different doses of microbeads. These findings indicate that smaller microbeads activate more proteins that may mediate their toxic effects. To determine whether microplastic-induced ROS generation is activated by p-jnk and p-p38, rotifers were treated with the ROS scavenger NAC (0.5 mm). NAC treatment blocked the Figure 3. Images of fluorescently labeled polystyrene microbeads of different diameters ingested by B. koreanus: (A C) control, and (D F) 0.05, (G I) 0.5, and (J L) 6 μm. All fluorescently labeled polystyrene microbeads were used at 10 μg/ml. D

5 Figure 4. Time course images of fluorescently labeled polystyrene microbead egestion by B. koreanus:(a D) 0, (E H) 6, (I L) 12, (M P) 24, and (Q T) 48 h. All fluorescently labeled polystyrene microbeads were used at 10 μg/ml. generation of intracellular ROS (Figure 5C) and also prevented the phosphorylation of p38 and JNK (Figure 5D). GSH Content and Enzymatic Activities of GPx, GR, GST, and SOD. There were patterns of increased activities of GPx, GR, GST, and SOD in a size-dependent manner in rotifers exposed to different sizes of microbeads (10 μg/ml) for 24 h, while it was similar level with the control group in 6- μm microbeads-exposed rotifers (Figure 6). However, the concentration of total GSH was slightly increased after exposure to 0.05-μm microbeads, whereas it was slightly decreased after exposure to 0.5- and 6-μm microbeads compared with the control group (Figure 6). DISCUSSION In Vivo Toxicity Tests. We found that exposure of the rotifer B. koreanus to polystyrene microbeads induced physiological alterations that led to significant repercussions on various life cycle parameters (population growth, fecundity, lifespan, reproduction rate, and individual growth). Consistent with our findings, exposure of the freshwater cladoceran Daphnia magna to nanosized polystyrene beads also resulted in negative effects on growth rate, mortality, and reproduction. 27 In polystyrene microbead-exposed B. koreanus, the extent of growth inhibition depended on the polystyrene microbead size. In addition to growth inhibition, exposed rotifers showed reduced fecundity and shortened lifespan, all of which could contribute in reducing rotifer populations. Of these variables, rotifer fecundity is highly likely to be associated with population growth. With respect to fecundity, the number of offspring was significantly reduced in response to exposure to 0.05-μm polystyrene microbeads. Most likely explanations for these findings are linked to insufficient nutrition due to ingestion of microplastic instead of diet T. suecica during experiments. In a previous study on a copepod, decrease in fecundity was measured in test groups that exposed to 0.5- and 6-μm diameter plastic beads. 9 Also in several species, fecundity and/ or the reproduction rate have been shown to be sacrificed in energy trade-offs as adaptation mechanisms in response to environmental stresses. For example, reproduction was inhibited in the soft coral Lobophytum compactum in response to environmental stressors. These stressors were shown to cause the reallocation of the cellular energy budget from reproduction to the expression of defensome components. 28 Environmental stresses were also shown to reduce the energy allocation toward reproduction in the copepods Tigriopus japonicus and Paracyclopina nana, resulting in lowered fecundity. 29,30 Also, the shortened lifespan of polystyrene microbead-exposed rotifers can certainly limit their spawning periods, which are essential for generating new individuals. The increased reproduction time is related to a low rotifer growth rate in response to polystyrene microbeads, since individual growth is directly associated with rotifer maturation. E

6 Figure 5. Effects of exposure to polystyrene microbeads of different diameters (0.05, 0.5, and 6 μm) on ROS production and phosphorylation of MAPK signaling proteins: (A, C) ROS levels w/o NAC treatment, (B, D) phosphorylation of MAPK proteins without NAC treatment. All polystyrene microbeads were used at 10 μg/ml. Levels of ROS are represented as percentage of controls. Different letters above columns indicate significant differences, defined as P < These results imply that polystyrene microbead exposure is associated with a range of repercussions on B. koreanus. These repercussions include growth retardation, lowered fecundity, shortened life span, and decreased population. With respect to polystyrene microbead size, and 0.5-μm microbeads exerted clear negative effects on the rotifers, while only slight growth retardation was measured upon exposure to 6-μm microbeads. Thus, exposure to relatively small microbeads in particular significantly affects the growth rate, fecundity, lifespan, and reproduction time of rotifers. Size-dependent toxicity has also been demonstrated in in vivo toxicity tests in the marine copepod T. japonicus. 9 Specifically, and 0.5-μm polystyrene microbeads significantly slowed the developmental time and decreased the copepod survival rate, while copepods exposed to 6-μm polystyrene microbeads did not exhibit any significant growth inhibition. These findings imply that the toxicity of polystyrene microbeads is likely to be due to differences in the ingestion of the various particles. In fact, ingestion is a most important factor as the transfer of microplastic is totally based on feeding behaviors. 8 Bioavailability of microplastic is known to increase as particle size decreases since the smaller particles have higher surface/ volume ratios and increased abilities to be taken up by cells or in interaction with other living organisms. These attributes may explain the increased toxicity of the smaller particles The preferential uptake of different sizes of polystyrene microbeads by rotifers and copepods may affect their physiological alterations as fewer particles are taken up since copepods tend to ingest larger particles than rotifers. 9 Thus, rotifers are likely to be more sensitive to nanosized particles than copepods in selective feeding polystyrene microbead experiments. Ingestion/Egestion of Microbeads by Rotifers. The digestive organs of B. koreanus were strongly fluorescent after exposure to microbeads of all diameters. In several species of F

7 Figure 6. Effects of exposure to polystyrene microbeads of different diameters (0.05, 0.5, and 6 μm) on antioxidant-related enzymes: (A) total GSH, (B) SOD, (C) GST, (D) GR, and (E) GPx. All polystyrene microbeads were used at 10 μg/ml. Enzyme activities are represented as percentage of controls. Different letters above columns indicate significant differences, defined as P < copepods, fluorescently labeled microbeads have also been detected after uptake; however, microbead uptake was shown to vary according to taxon, life stage, and bead size. 8 Interestingly, uptake did not depend on particle size, although size-dependent toxicity was observed. 9 In B. koreanus, the fluorescence intensity in the digestive tract decreased after uptake via egestion of the fluorescently labeled microbeads to the clean seawater. In rotifers exposed to 6-μm microbeads, the fluorescence intensity of the digestive tract was greatly reduced after 24 h, while fluorescence persisted up to 48 h in rotifers exposed to and 0.5-μm microbeads. These findings suggest that retention time in rotifers is likely to be associated with microbead size. Generally, nanoparticle toxicity has also been shown to be significantly associated with particle size, with small particles associated with prolonged retention times and high bioavailability Thus, we hypothesize that the and 0.5-μm microbeads have longer retention times in rotifers, which would allow them to have more time to exert negative effects. This explanation is consistent with our in vivo polystyrene microbead toxicity data. Molecular Responses in Response to Microplastic Exposure. In B. koreanus, high ROS levels were observed after exposure to 0.05-μm microbeads (Figure 5A). Also, the negative correlation between ROS levels and microbead size supports the size-dependent toxicity that has been shown by others in nanoparticles 31,32 and our in vivo toxicity tests. Similarly, 0.02-μm polystyrene microbeads have been shown to generate ROS and inhibit the photosynthetic ability of the algae Chlorella and Scenedesmus. 18 We also exposed B. koreanus to microbeads and determined the relationship between oxidative stress and toxicity by examining the phosphorylation status of different MAPK signaling proteins. The MAPK pathway is an important signal transduction pathway that links extracellular signals to intracellular processes and is highly associated with apoptosis and inflammation. 35 We found that JNK and p38 MAPK were both phosphorylated in response to exposure to 0.05-μm microbeads. In the copepod T. japonicus, p38 MAPK has been shown to be activated in response to ultraviolet B (UVB) radiation and copper exposure. 36,37 In sea urchins, p38 MAPK has also been shown to be activated by titanium dioxide nanoparticles. 38 Thus, the strong activation of p-jnk and p-p38 MAPK in rotifers exposed to 0.05-μm microbeads suggests that these two MAPK-activating proteins are involved in the subsequent signal transduction that generates the oxidative stress response. Indeed, the highest intracellular ROS level was observed in rotifers exposed to 0.05-μm microbeads. Immunoblot analysis of MAPK phosphorylation patterns after treatment with the ROS scavenger NAC revealed significantly decreased phosphorylation in response to 0.05-μm microbead exposure. Also, mitochondrial membrane integrity in B. koreanus was decreased, showing a negative correlation with particle size in its mitochondrial dysfunction. A detailed discussion about mitochondrial membrane integrity is incorporated in the Supporting Information. This finding indicates that ROS are the key component for activating the MAPK pathway and that microplastic-induced ROS are the key toxicity factor in response to microplastic exposure. Thus, polystyrene microbead exposure is strongly associated with ROS generation in B. koreanus. We observed that the enzymatic activities of the antioxidants SOD, GST, GR, and GPx were showing increased patterns in and 0.5-μm microbead-exposed rotifers compared to the control group and that these effects were negatively correlated with the bead size. In contrast, the level of total GSH did not show any significant changes after exposure to any of the beads. In marine organisms, the level of the nonenzymatic antioxidant GSH and the levels of GSH-related enzymes have been shown to increase in response to UVB radiation, 36 metal, 37 and nanoparticle exposure. 39 These results indicate that these antioxidants play a role in protection against various environmental stressors. The levels of both the reduced form (GSH) and oxidized form (GSSG) of glutathione are tightly regulated by antioxidant enzymes such as GR and GPx in response to various stressors. 39 Although the level of GSH varies due to its involvement in nonenzymatic detoxification as a chelator for ROS scavenging, no significant correlations have been observed between the levels of ROS and GSH. 40 Taken together, our data indicate that exposure to microplastics significantly increases the level of ROS in a size-dependent manner and that the activation of antioxidant-related enzymes including SOD, GST, GR, and GPx is directly related to a defense mechanism against microplastic-induced oxidative stress. In conclusion, in vivo toxicity tests revealed that nanosized and microsized plastic particles cause adverse effects on normal physiological responses on growth, hatching, and reproduction in the rotifer and also indirectly may affect energy flow in the aquatic ecosystem. Also, in vivo tests demonstrated sizedependent toxicity of microplastics to the rotifer B. koreanus. Moreover, microplastic exposure up-regulated ROS production and triggered MAPK signaling pathways, and several antioxidant-related enzymes were also significantly induced. These results inform our understanding of the effects of microplastic pollution on marine microorganisms and build a foundation for marine environmental studies on mechanistic and biochemical responses to microplastics. ASSOCIATED CONTENT *S Supporting Information The Supporting Information is available free of charge on the ACS Publications website at. Detailed description of all materials and methods, and detailed discussion about mitochondrial membrane integrity (PDF) G

8 Environmental Science & Technology AUTHOR INFORMATION Corresponding Author *Tel.: Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS We thank Prof. Kevin Chipman (University of Birmingham, United Kingdom) for his comments on the manuscript, and we also thank three anonymous reviewers for their valuable comments. This work was supported by grants from the Development of Techniques for Assessment and Management of Hazardous Chemicals in the Marine Environment ( ) of the Ministry of Oceans and Fisheries, Kore, and from the Korea-Polar Ocean Development: K-POD (project no. PM15050) of the Ministry of Oceans and Fisheries, Korea funded to Jae-Seong Lee. REFERENCES (1) Carpenter, E. J.; Anderson, S. J.; Harvey, G. R.; Miklas, H. P.; Peck, B. B. Polystyrene spherules in coastal waters. Science 1972, 178, (2) Cole, M.; Lindeque, P.; Halsband, C.; Galloway, T. S. Microplastics as contaminants in the marine environment: a review. Mar. Pollut. Bull. 2011, 62, (3) Ivar do Sul, J. A.; Costa, M. F. The present and future of microplastic pollution in the marine environment: A review. Environ. Pollut. 2014, 185, (4) Andrady, A. L. Microplastics in the marine environment. Mar. Pollut. Bull. 2011, 62, (5) Gregory, M. R.; Ryan, P. G. Pelagic plastics and other seaborne persistent synthetic debris: a review of Southern Hemisphere perspectives. In Marine Debris. Sources, impacts, and solutions; Coe, J. M., Rogers, D. B., Eds.; Springer-Verlag: 1997; pp (6) Barnes, D. K. A.; Galgani, F.; Thompson, R. C.; Barlaz, M. Accumulation and fragmentation of plastic debris in global environments. Philos. Trans. R. Soc., B 2009, 364, (7) Zarfl, C.; Matthies, M. Are marine plastic particles transport vectors for organic pollutants to the Arctic? Mar. Pollut. Bull. 2010, 60, (8) Cole, M.; Lindeque, P.; Fileman, E.; Halsband, C.; Goodhead, R.; Moger, J.; Galloway, T. S. Microplastic ingestion by zooplankton. Environ. Sci. Technol. 2013, 47, (9) Lee, K. W.; Shim, W. J.; Kwon, O. Y.; Kang, J. H. Size-dependent effects of micro polystyrene particles in the marine copepod Tigriopus japonicus. Environ. Sci. Technol. 2013, 47, (10) Cole, M.; Lindeque, P. K.; Fileman, E.; Halsband, C.; Galloway, T. The impact of microplastics on feeding, function and fecundity in the copepod Calanus helgolandicus. Environ. Sci. Technol. 2015, 49, (11) 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, (12) von Moos, N.; Burkhardt-Holm, P.; Kohler, A. Uptake and effects of microplastics on cells and tissue of the blue mussel Mytilus edulis L. after an experimental exposure. Environ. Sci. Technol. 2012, 46, (13) Wegner, A.; Besseling, E.; Foekema, E. M.; Kamermans, P.; Koelmans, A. A. Effects of nanopolystyrene on the feeding behavior of the blue mussel (Mytilus edulis L.). Environ. Toxicol. Chem. 2012, 31, (14) Besseling, E.; Wegner, A.; Foekema, E. M.; van den Heuvel- Greve, M. J.; Koelmans, A. A. Effects of microplastic on fitness and PCB bioaccumulation by the lugworm Arenicola marina (L.). Environ. Sci. Technol. 2013, 47, H (15) Lee, H. M.; Shin, D. M.; Song, H. M.; Yuk, J. M.; Lee, Z. W.; Lee, S. H.; Hwang, S. M.; Kim, J. M.; Lee, C. S.; Jo, E. K. Nanoparticles up-regulate tumor necrosis factor-[alpha] and CXCL8 via reactive oxygen species and mitogen-activated protein kinase activation. Toxicol. Appl. Pharmacol. 2009, 238, (16) Chairuangkitti, P.; Lawanprasert, S.; Roytrakul, S.; Aueviriyavit, S.; Phummiratch, D.; Kulthong, K.; Chanvorachote, P.; Maniratanachote, R. Silver nanoparticles induce toxicity in A549 cells via ROS-dependent and ROS-independent pathways. Toxicol. In Vitro 2013, 27, (17) Manke, A.; Wang, L.; Rojanasakul, Y. Mechanisms of Nanoparticle-Induced Oxidative Stress and Toxicity, a review. BioMed Res. Int. 2013, 2013, 1. (18) Bhattacharya, P.; Lin, S. J.; Turner, J. P.; Ke, P. C. Physical adsorption of charged plastic nanoparticles affects algal photosynthesis. J. Phys. Chem. C 2010, 114, (19) Li, S.; Pan, X.; Wallis, L. K.; Fan, Z.; Chen, Z.; Diamond, S. A. Comparison of TiO2 nanoparticle and graphene TiO2 nanoparticle composite phototoxicity to Daphnia magna and Oryzias latipes. Chemosphere 2014, 112, (20) Lee, J.-W.; Kang, H.-M.; Won, E.-J.; Hwang, D.-S.; Kim, D.-H.; Lee, S.-J.; Lee, J.-S. Multi-walled carbon nanotubes (MWCNTs) lead to growth retardation, antioxidant depletion, and activation of the ERK signaling pathway but decrease copper bioavailability in the monogonont rotifer Brachionus koreanus. Aquat. Toxicol. 2016, 172, (21) Snell, T. W.; Janssen, C. R. Rotifers in ecotoxicology, a review. Hydrobiologia 1995, , (22) Dahms, H.-U.; Hagiwara, A.; Lee, J.-S. Ecotoxicology, ecophysiology, and mechanistic studies with rotifers. Aquat. Toxicol. 2011, 101, (23) Hwang, D.-S.; Suga, K.; Sakakura, Y.; Park, H.-G.; Hagiwara, A.; Rhee, J.-S.; Lee, J.-S. Complete mitochondrial genome of the monogonont rotifer, Brachionus koreanus (Rotifera, Brachionidae). Mitochondrial DNA 2014, 25, (24) Hwang, D.-S.; Dahms, H.-U.; Park, H.-G.; Lee, J.-S. A new intertidal Brachionus and intrageneric phylogenetic relationships among Brachionus as revealed by allometry and CO1-ITS1 gene analysis. Zool. Stud. 2013, 52, 13. (25) Plastics Europe. Analysis of Plastics Production, Demand and Recovery in Europe; Association of Plastic Manufacturers: Brussels, 2006; pp (26) da Costa, J. P.; Santos, P. S. M.; Duarte, A. C.; Rocha-Santos, T. (Nano)plastics in the environment-sources, fates and effects. Sci. Total Environ. 2016, , (27) Besseling, E.; Wang, B.; Lurling, M.; Koelmans, A. A. Nanoplastic affects growth of S. obliquus and reproduction of D. magna. Environ. Sci. Technol. 2014, 48, (28) Michalek-Wagner, K.; Willis, B. L. Impacts of bleaching on the soft coral Lobophytum compactum. I. Fecundity, fertilization and offspring viability. Coral Reefs 2001, 19, (29) Han, J.; Won, E.-J.; Lee, M.-C.; Seo, J.-S.; Lee, S.-J.; Lee, J.-S. Developmental retardation, reduced fecundity, and modulated expression of the defensome in the intertidal copepod Tigriopus japonicus exposed to BDE-47 and PFOS. Aquat. Toxicol. 2015, 165, (30) Won, E.-J.; Lee, J.-S. Gamma radiation induces growth retardation, impaired egg production, and oxidative stress in the marine copepod Paracyclopina nana. Aquat. Toxicol. 2014, 150, (31) Pan, Y.; Neuss, S.; Leifert, A.; Fischler, M.; Wen, F.; Simon, U.; Schmid, G.; Brandau, W.; Jahnen-Dechent, W. Size-dependent cytotoxicity of gold nanoparticles. Small 2007, 3, (32) Choi, O.; Hu, Z. Q. Size dependent and reactive oxygen species related nanosilver toxicity to nitrifying bacteria. Environ. Sci. Technol. 2008, 42, (33) Shang, L.; Nienhaus, K.; Nienhaus, G. U. Engineered nanoparticles interacting with cells: size matters. J. Nanobiotechnol. 2014, 12, 5.

9 (34) Choi, S.; Kim, S.; Bae, Y.-J.; Park, J.-W.; Jung, J. Size-dependent toxicity of silver nanoparticles to Glyptotendipes tolunagai. Environ. Health Toxicol. 2015, 30, e (35) Malik, S.; Suchal, K.; Gamad, N.; Dinda, A. K.; Arya, D. S.; Bhatia, J. Telmisartan ameliorates cisplatin-induced nephrotoxicity by inhibiting MAPK mediated inflammation and apoptosis. Eur. J. Pharmacol. 2015, 748, (36) Kim, B.-M.; Rhee, J.-S.; Lee, K.-W.; Kim, M.-J.; Shin, K.-H.; Lee, S.-J.; Lee, Y.-M.; Lee, J.-S. UV-B radiation-induced oxidative stress and p38 signaling pathway involvement in the benthic copepod Tigriopus japonicus. Comp. Biochem. Physiol., Part C: Toxicol. Pharmacol. 2015, 167, (37) Rhee, J.-S.; Yu, I.-T.; Kim, B.-M.; Jeong, C.-B.; Lee, K.-W.; Kim, M.-J.; Lee, S.-J.; Park, G.-S.; Lee, J.-S. Copper induces apoptotic cell death through reactive oxygen species triggered oxidative stress in the intertidal copepod Tigriopus japonicus. Aquat. Toxicol. 2013, , (38) Pinsino, A.; Russo, R.; Bonaventura, R.; Brunelli, A.; Marcomini, A.; Matranga, V. Titanium dioxide nanoparticles stimulate sea urchin immune cell phagocytic activity involving TLR/p38 MAPK-mediated signaling pathway. Sci. Rep. 2015, 5, (39) Halliwell, B.; Gutteridge, J. Free Radicals in Biology and Medicine; Clarendon: Oxford, U.K., (40) Monserrat, J. M.; Rosa, C. E.; Sandrini, J. Z.; Marins, L. F.; Bianchini, A.; Geracitano, L. A. Annelids and nematodes as sentinels of environmental pollution. Comments Toxicol. 2003, 9, I