Subchronic exposure to pristine and aged microplastics causes no impairment of survival or growth in a marine mysid (Americamysis bahia)

Subchronic exposure to pristine and aged microplastics causes no impairment of survival or growth in a marine mysid (Americamysis bahia) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Subchronic exposure to pristine and aged microplastics causes no impairment of survival or growth in a marine mysid (Americamysis bahia) Nobuyuki Ohkubo, Mana Ito, Chisato Kataoka, Toshimitsu Onduka, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9144095/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract There are increasing concerns that µicroplastics (MPs) will be ingested by marine organisms that feed on small prey, such as plankton. In this study, we examined the biological effects (survival and growth) of exposure to pristine polyethylene MP and aged MP (aMP; length ≤ 53 µm) prepared by maintaining the MP under laboratory conditions at 20°C for 28 days to create biofouling that closely resembles that found in the natural environment. To determine the type of biofilm formed during the aging process, microbial community analysis of aMP was conducted. Rhodobacteraceae and Flavobacteriaceae were detected from the aMP, and these same bacterial members were associated with MP collected from actual marine environments, suggesting that we successfully produced aMP under conditions that simulated those in the field. We then evaluated the subchronic toxicity (7 d) of MP by using marine mysid ( Americamysis bahia ) larvae. After 10 mg/L pristine and aMP exposure, no significant differences in survival rate or body weight were observed between the solvent control group and MP exposure groups ( p > 0.05), though aMP exposure slightly increased total length ( p = 0.02). Overall, no remarkable adverse effects on marine mysid larvae were observed after pristine MP and aMP exposure at the tested sizes, materials, and concentrations. This suggests that the biological impact of aMP on marine planktonic crustaceans will be minimal at current levels of MP contamination, and that the small impact will make detection difficult in subchronic toxicity tests. Polyethylene Aged MP Biofouling Marine mysid Subchronic toxicity Figures Figure 1 Figure 2 1. Introduction Plastic waste of various sizes affects organisms in different ways (Koelmans et al., 2022 ). The necessity of controlling marine plastic waste is gaining increasing international attention (e.g., G20 Implementation Framework for Actions against Marine Plastic Litter, 2021 ). There is an urgent need to understand the biological impacts of marine plastic waste (Isobe et al., 2019 ) as a basis for determining what action should be taken. Microplastics (MPs), which are variously defined as plastic fragments smaller than 5 or 1 mm, are a relatively new threat to marine organisms and ecosystems (e.g., Andrady 2011 ; Chae and An, 2017 ; Galloway and Lewis, 2016 ). They are mainly formed when plastic waste breaks down into small pieces and becomes scattered throughout the ocean (e.g., Morales-Caselles et al., 2021). Presently, ocean monitoring efforts primarily focus on MPs larger than 300 µm (Isobe et al., 2019 ). However, recent reports have detected finer MPs (20–300 µm, “micro-MP”), with numbers reported to be hundreds to thousands of times greater than that of larger MPs (Song et al., 2018 ; Ueda et al., 2025 ; Yang. et al., 2025). In particular, Jacob et al., ( 2020 ) reports that micro-MPs smaller than 50 µm are more toxic to fish. There are concerns that micro-MP is ingested by marine organisms that feed on small prey, such as plankton (Gao et al., 2025 ; Jeyavani et al., 2022 ). The available information on the biological effects of MP is compromised by biases in the materials studied (e.g., Koelmans et al., 2022 ; Paul-Pont et al., 2018 ; de Ruijter et al., 2023 ). For example, polystyrene (PS) is rarely found in the ocean (e.g., Yang et al., 2025 ), yet it is the material most often used in biological toxicity assessments (Paul-Pont et al., 2018 ). The effects of MP itself on marine organisms are still unclear. MPs have a variety of shapes (spherical, fragmented, fibrous, etc.), contain a variety of materials (e.g., PS; polyethylene, PE; polypropylene, PP; polyethylene terephthalate), and occur in various sizes (e.g., Auta et al., 2017 ; Yang et al., 2025 ). Moreover, once released into the marine environment, MPs inevitably experience aging processes (e.g., UV radiation, biofouling, and hydrolysis) (Alimi et al., 2018 ; Auta et al., 2017 ). MPs are sometimes covered in a biofilm (Carson et al., 2013 ; Zettler et al., 2013 ), which affects their properties (Harrison et al., 2018 ; Tu et al., 2020 ; Wang et al., 2021 ), including buoyancy, that could affect their interaction with various organisms (Gaylarde et al., 2023 ; Harrison et al., 2018 ; Oberbeckmann et al., 2015 ). For these reasons, it is likely insufficient to assess only the effects of MPs of various sizes and shapes. To assess effects of MP in a more environmentally relevant manner, researchers have proposed conducting exposure tests with MP collected from the ocean (e.g., de Ruijter et al., 2023 ). However, MP collected from environment can carry attached hazardous chemicals or pathogens; because of this, exposure testing using field-collected MP runs the risk of overestimating the effect of the MP itself (Cormier et al., 2021 ; 2022 ; Feng et al., 2020 ; Oberbeckmann et al., 2015 ; Pannetier et al., 2020 ; Rochman et al., 2014 ; Wang et al., 2021 ), making it challenging to identify the source of any biological effects and ensure the reproducibility of test results. In this study, we examined the biological effects of aged MP (aMP) by stimulating biofouling of micro-MP in a laboratory that closely resembles that found in the natural environment. By preparing aMP modeled on natural MP and conducting exposure tests, we can evaluate the biological effects of the MP itself in a biologically relevant manner. Importantly, the adhesion of harmful chemicals can be minimized by performing MP aging treatment in the laboratory. A microbial community analysis was conducted to determine the type of biofilm formed during the aging process, and the results were then compared to MP collected from the ocean. Furthermore, to investigate the biological effects based on the presence or absence of bacterial flora adhering to the MP, MP samples were aged in seawater containing bacteria and in seawater from which bacteria had been removed by 0.2-µm filtration. In addition, a separate exposure test was conducted using pristine MP for comparison. Polyethylene was selected as the material because it is one of the most frequently detected plastics in the ocean (e.g., Yang et al., 2025 ), yet it has been the focus of only a minority of impact assessment studies (Paul-Pont et al., 2018 ). Additionally, the exposure concentration was set at 10 times the highest anticipated marine MP concentration in 2060 (1 mg/L, as reported by Isobe et al., 2019 ), and exposure was conducted at this elevated level. For the evaluation, the juvenile stage of crustaceans, which has been reported to be affected in some cases (e.g., Jeyavani et al., 2022 ; Lee et al., 2021 ; Wang et al., 2020 ), was used. Our study evaluated the toxic effects of aMP on a marine mysid ( Americamysis bahia ), because it is a typical marine crustacean with a crucial role in marine food webs as a link between producers and high-trophic-level consumers (e.g., fish) with high ecological and economic value (Verslycke et al., 2007 ). Despite its important role in marine ecosystems (Oliveira et al., 2023 ), this species has only been included in a few MP impact studies to date (Biefel et al., 2024 ; Lee et al., 2021 ; Lee et al., 2024 ; Wang et al., 2020 ). 2. Materials and Methods 2.1. MP preparation for the aging process We used fluorescent yellow PE microspheres of diameter 45–53 µm. The MPs had a density of 1.00 g/cm 3 , and were purchased from Cospheric (Santa Barbara, CA, USA). Seawater collected on 26 August 2024 in our laboratory was sand- and then active-carbon filtered at 20°C, then pre-filled into bottles and allowed to stand at room temperature for two days to allow any suspended sand particles to settle out. The decanted filtered seawater (2 L) was then filtered through a 10-µm mesh filter. One liter of the seawater was filtered again through a 0.2-µm bottle-top filter (Thermo Fisher; Nalgene Rapid-Flow sterile disposable bottle top filters with polyethersulfone membranes, Mexico). Aged MP was prepared by adding MP at a concentration of 20 mg/L (converted value: 6.0 × 10 5 particle/L) to the 10-µm and 0.2-µm mesh-filtered seawater (to create 10-µm-filtered and 0.2-µm-filtered aMP, respectively). The mixture was stirred continuously for 28 days in an incubator (LH-241PFDT-SP, NKsystem, Osaka, Japan,) under constant temperature (20.5 ± 0.2°C) and light cycle (12-h light:12-h dark). The 0.2-µm-filtered aMP served as a negative control for biofilm formation. The aMP was collected using a 47-mm-diameter PTFE filter (Omnipore 5.0 µm PTFE membrane; Merk Millipore, Ireland), placed in 4 L of artificial seawater (Marine Art SF-1, Osakayakken. Co. Ltd, Japan) adjusted to a salinity of 25 and pH of 8.0 and containing 0.5 ppm of Tween 80, and resuspended by processing with an ultrasonic agitator for 2 min. The shape and size of MP particles were determined by fluorescence microscopy (IX73; Olympus, Japan) with an Olympus model DP73 Color Fidelity Microscope Digital Camera. To stain the peptidoglycan layer of any bacteria, crystal violet staining was performed by slightly modifying established protocols (Merritt et al., 2005 ), as follows. The aged MP was stained with a 0.1% crystal violet (FUJIFILM Wako Pure Chemical Corporation, Japan) solution for 30 s. Then, it was washed with distilled water and examined under a stereomicroscope (SZX7; Olympus) equipped with an Olympus model DP21 Color Fidelity Microscope Digital Camera. 2.2. DNA extraction and MiSeq sequencing To evaluate the microbial community formed on the aMP surface, 10-mL samples of both the 10-µm-filtered and 0.2-µm filtered aMP solutions ( n = 2 each) were filtered onto the PTFE membrane (Merck Millipore) at the end of the aging process. DNA was extracted from the MP before and after aging with a DNeasy PowerSoil Pro kit (QIAGEN, Hilden, Germany). The hypervariable V3–V4 region of bacterial 16S rRNA was amplified on an Illumina Miseq platform. Obtained sequences were clustered into OTUs (97% similarity) against the Greengenes database (ver. 13_8) using QIIME2 software (ver. 2024.10). Quantitative PCR (qPCR) assays targeting genes encoding 16S rRNA (337F-797R) were carried out with a SsoAdvanced universal SYBR green supermix (Bio-Rad, United Kingdom) on a CPX96 Real-Time System (Bio-Rad). 2.3 Subchronic toxicity test Mysids ( A. bahia ) were purchased from the National Institute for Environmental Studies (Tsukuba, Japan). The culture has been maintained in our laboratory for more than 5 years. The culture medium was prepared using sand-filtered seawater, diluted with aerated deionized tap water to a final salinity of 25. A 16-h-light:8-h-dark photoperiod was used during culturing and the water temperature was maintained at 25°C. The mysids were fed every two days with Artemia sp. nauplii (24 h after hatching) ad libitum. Survival and growth tests were performed using A. bahia by following a procedure from the US EPA on 7-day-old juveniles (US EPA, 2002 ) with minor modifications. For each test, 150–250-mL samples of artificial seawater adjusted to a salinity of 25 and pH of 8.0 were placed in 300-mL glass beakers, and each treatment consisted of 5 individuals per beaker with triplicate (aMP exposure, 15 mysids total per treatment) or quadruplicate (pristine MP exposure, 20 mysids total) trials. The larvae were exposed to MP for 7 days in an incubator at 25°C with a 16-h-light:8-h-dark photoperiod. During the test, the artificial seawater was completely replaced every 2–3 days with a semi-static exposure method. The main exposure concentration was 10 mg/L, which is 10× the predicted maximum concentration of MP in the ocean in 2060 (1 mg/L; Isobe et al., 2019 ). Additionally, 3-mg/L and 1-mg/L treatments were established in the pristine MP exposure test. The number of MPs in the exposure medium was counted on days 0 and 2 of the experiment with a fluorescence microscope (BZ-X800; Keyence Corp., Osaka, Japan). The solvent control group was exposed only to the solvent carrier (Tween80, 0.5 ppm) in the artificial seawater. We confirmed in a separate experiment that this concentration of Tween 80 does not exhibit toxicity to marine mysids. Approximately 200 Artemia nauplii were provided to each beaker on each day of the test except the fourth. The total number of molts (i.e., shedding of the old exoskeleton [exuvia]) and mortality in each treatment group were counted daily. At the end of the test, test specimens were fixed in 70% ethanol (v/v) for 24 h, and measured for total length under a stereomicroscope equipped with a digital camera and for body weight with an electronic balance (CPA224S; Sartorius AG, Germany). The number of individuals showing evidence of MP uptake (MP uptake frequency) was then counted under a fluorescence microscope (IX73). 2.4. Statistical analysis We used the Cochran–Armitage test to determine the significance of differences in survival rate among treatments. Total length, body weight, and total number of molts at the end of the test were subjected to one-way analysis of variance (ANOVA) to identify significant differences among treatments, and a Bonferroni correction was applied for multiple tests. If the data were heteroscedastic, a Kruskal–Wallis test and a post-hoc Steel’s test were performed. Differences were regarded as significant if the type I error rate ( p ) was less than 0.05. The Microsoft Excel add-in software Excel Tokuei for Windows (SSRI Inc., Tokyo, Japan) was used for all statistical analyses. 3. Results and Discussion 3.1. Characteristics of aMP Stirring caused the aMP to fragment (Fig. 1 A and B), resulting in an average long diameter of 25 µm and a short diameter of 16 µm in the 0.2-µm-filtered aMP and an average long diameter of 24 µm and a short diameter of 15 µm in the 10-µm-filtered aMP. The 10-µm-filtered aMP stained more intensely in crystal violet stain (Fig. 1 D) compared to the 0.2-µm-filtered aMP (Fig. 1 C), indicating that more biofilm was attached. These results suggest that the ageing treatment produced micro-MPs that were fragmented and coated with biofilm. The copy number of bacterial 16S rRNA genes was higher in the 10-µm-filtered aMP (3.3 × 10 8 copies/mg MP) than in the 0.2-µm-filtered aMP (1.9 × 10 7 copies/mg MP) (Fig. 2 ), showing that a large number of bacteria proliferated on the MP surface in the 10-µm-filtered treatments. The composition and distribution of biofilm on the surface of MPs tends to differ from those in the surrounding seawater, and are enriched with certain bacteria (Feng et al., 2020 ). Rhodobacteraceae (Elifantz et al., 2013 ) and Flavobacteriaceae (Feng et al., 2020 ), which are major biofilm components in various regions, were detected from the 10-µm-filtered aMP (Fig. 2 ), and have also been associated with MP (both PP and PE) collected from actual marine environments (Zettler et al., 2013 ). This suggests that we were able to produce aged MP that successfully simulated that produced under natural conditions. Of the bacterial sequences obtained from the 10-µm-filtered aMP, 67% were homologous to bacteria detected in MP from natural marine environments. Previous studies have artificially formed biofilms on MP and evaluated their biological effects (Amariei et al., 2022 ; Vroom et al., 2017 ). The present study is the first to analyze the microbial community and verify its similarity to natural conditions. Our technique for artificially aging MP is likely to be important for evaluating the impact of MP on marine organisms. Furthermore, the technique could prove useful for experimentally simulating the adsorption of chemicals onto the plastic particles and their subsequent uptake by marine organisms, which is another major issue concerning marine plastic debris (e.g., Carson et al., 2013 , Wang et al., 2021 ). 3.2. Subchronic toxicity of aMP The results of the pristine MP exposure test are shown in Table S1 . The Cochran–Armitage test revealed no significant difference in survival rates between the solvent control and pristine MP exposure groups ( p > 0.05). The MP uptake frequency was higher at higher MP concentrations; however, no significant differences were observed. A one-way ANOVA showed no significant differences in the total number of molts, body weight, or total length ( p > 0.05). Measurements of the number of pristine MP particles in solutions of 1, 3, and 10 mg/L yielded values 77%, 230%, and 40%, respectively, of the predicted number of particles calculated from the nominal weight concentration, average MP size (50 µm), and specific gravity. The difficulty of controlling the concentration of MP in water has been well established (Sun and Wu, 2023 ), resulting in variations between the actual and nominal concentrations. The results of the aMP exposure test are shown in Table 1 . The Cochran–Armitage test revealed no significant difference in survival rates between the solvent control and MP exposure groups ( p > 0.05). The count of aMP particles in solution was slightly higher for the 10-µm-filtered aMP, however, the two were almost identical. Most mysid individuals had ingested MP. However, a one-way ANOVA showed no significant differences in the total number of molts or body weights (Table 1 ). Although the 10-µm-filtered aMP exposure group had significantly greater total length ( p = 0.02) than the solvent control group at the end of the experiment (Table 1 ), the difference was slight (about 10%). The 10-µm-filtered aMP had more attached biofilm (Fig. 1 D), and crustaceans use aquatic biofilms as an important nutrient source (Abreu et al., 2007 ; Tang et al., 2021 ), suggesting that this may have slightly promoted growth in the 10-µm-filtered group, as has been reported for D. magna (Amariei et al., 2022 ). In addition, an exposure study using naturally derived MP has also reported positive effects on survival (de Ruijter et al., 2023 ), and these data suggested that even if MP exposure has negative effects on growth due to the inhibition of food assimilation, such effects can be counterbalanced by the ingestion of attached biofilms in the natural environment. Table 1 Survival rate, total number of molts, microplastic (MP) uptake frequency, total length, and body weight of mysids at the end of the aged MP (aMP) exposure test Experimental group (mean measured MP concentration) Survival rate (%) Total number of molts MP uptake frequency (%) Total length(mm) Body weight(mg) Solvent control 93 (14/15) 3 - 5.4 ± 0.1 1.3 ± 0.1 0.2-µm-filtered aMP (9.5 × 10 4 particle/L) 100 (15/15) 5 12/15 (80) 5.5 ± 0.2 1.3 ± 0.1 10-µm-filtered aMP (1.6 × 10 5 particle /L) 93 (14/15) 0 14/14 (100) 6.0 ± 0.1* 1.4 ± 0.1 -, n.d.; *, p < 0.05 compared to solvent control Our results show no adverse effects on mysid larvae from pristine or aMP exposure for the tested sizes ( \(\:\le\:\) 53 µm), materials (PE), and concentrations (10 mg/L). As we previously mentioned, the biological effects of pristine MP exposure can differ from those of exposure to MP in the marine environment (Carson et al., 2013 ; Wang et al., 2021 ). Therefore, we prepared an aMP sample that was closer to the MPs that are found in the field and conducted exposure tests at a concentration 10× higher (10 mg/L) than that predicted for 2060 (Isobe et al., 2019 ). As also mentioned above, planktonic crustaceans are the taxonomic group most frequently reported to be adversely affected by MP (e.g., Jeyavani et al., 2022 ; Lee et al., 2021 ; Wang et al., 2020 ). The lack of observed harmful effects on the mysid larvae suggests that the biological impact on marine planktonic crustaceans will be minimal at current levels of MP concentration, and that the small magnitude of any potential impact will make detection difficult in subchronic toxicity tests. On the other hand, there are reports of adverse effects on the survival of other crustaceans, particularly juveniles, following prolonged exposure at higher concentrations of 1- or 10-µm PS (Lee et al., 2021 ), or 10–45-µm PP (Jeyavani et al., 2022 ). Future studies should focus on chronic toxicity testing methods, which are expected to be more sensitive to toxic effects, and consider the use of aMP of other materials to evaluate the presence or absence of MP effects. Declarations Funding: This study was supported in part by a grant-in-aid from the Fisheries Agency of Japan (2BC413). Please note that the views expressed in this study are not necessarily those of the Ministry of Agriculture, Forestry and Fisheries of Japan. Declaration of interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contributions: All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Nobuyuki Ohkubo, Mana Ito, Chisato Kataoka, and Toshimitsu Onduka. The first draft of the manuscript was written by Nobuyuki Ohkubo and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Acknowledgements We are grateful to Dr. Rei Yamashita (Atmosphere and Ocean Research Institute, The University of Tokyo) and Dr. Alam Md Khorshed (National Research Institute of Fisheries Technology) for their useful advice, and to Mses. Miki Shoda, Miki Ishimoto, Mika Momosaki, Mr. Hideki Nagano (National Research Institute of Fisheries Technology) for their kind assistance. Data Availability: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References Abreu PC, Ballester ELC, Odebrecht C, Wasielesky W, Cavalli RO, Granéli W, Anesio AM (2007) Importance of biofilm as food source for shrimp ( Farfantepenaeus paulensis ) evaluated by stable isotopes (δ13C and δ15N). 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Environ Sci Technol 47(13):7137–7146 Supplementary Files 260224Ohkubosupplementarytable.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 19 Apr, 2026 Editor assigned by journal 19 Mar, 2026 First submitted to journal 18 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9144095","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":609007942,"identity":"87edc7a1-c650-4cca-b0c6-485f73f4fdbf","order_by":0,"name":"Nobuyuki Ohkubo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABE0lEQVRIiWNgGAWjYDACHgY2MG0AxIwNBjVy/MwHIDKMDcRpOWYs2ZZAkhYG5sQNxxLwu4uf54zZgw8Md+TMJXLMJGcUsCVuPsb78APDLxsG5tnYrZHs7TE3nMHwzNhyBlDLBgMZ423H2I0lGPvSGBjnHMCqxeA8j5k0D8PhxA03gFoeGLDJbrvfxiDB2HOYgXEGdheia2Fm3NzGxvwDr5azPUhaNhgwK25gY2OTYPiBW4tkz7FyoF8OG1v2PCu2nAEMZIljbGwWiQ1pPLj8ws+TvA0YYoflzNmTN97s+QOMSqDDbnz4YyNniCPEwIDxH4jkMECIJLYx8AAtJwTYHyBx/jAwyEsQ1DIKRsEoGAUjAwAAQBZcENldJpwAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-9476-3620","institution":"Japan Fisheries Research and Education Agency: Kokuritsu Kenkyu Kaihatsu Hojin Suisan Kenkyu Kyoiku Kiko","correspondingAuthor":true,"prefix":"","firstName":"Nobuyuki","middleName":"","lastName":"Ohkubo","suffix":""},{"id":609007943,"identity":"8ec06af2-6d05-4140-85e0-747dc8776ccf","order_by":1,"name":"Mana Ito","email":"","orcid":"","institution":"Japan Fisheries Research and Education Agency: Kokuritsu Kenkyu Kaihatsu Hojin Suisan Kenkyu Kyoiku Kiko","correspondingAuthor":false,"prefix":"","firstName":"Mana","middleName":"","lastName":"Ito","suffix":""},{"id":609007944,"identity":"6926963d-667a-4d10-bc26-c263ede8fc39","order_by":2,"name":"Chisato Kataoka","email":"","orcid":"","institution":"Japan Fisheries Research and Education Agency: Kokuritsu Kenkyu Kaihatsu Hojin Suisan Kenkyu Kyoiku Kiko","correspondingAuthor":false,"prefix":"","firstName":"Chisato","middleName":"","lastName":"Kataoka","suffix":""},{"id":609007945,"identity":"6a13d380-a066-4f6c-97c0-cb68dde0e31b","order_by":3,"name":"Toshimitsu Onduka","email":"","orcid":"","institution":"Japan Fisheries Research and Education Agency: Kokuritsu Kenkyu Kaihatsu Hojin Suisan Kenkyu Kyoiku Kiko","correspondingAuthor":false,"prefix":"","firstName":"Toshimitsu","middleName":"","lastName":"Onduka","suffix":""},{"id":609007946,"identity":"42d507f2-26bf-4271-9fa0-3eab54949e2b","order_by":4,"name":"Takeshi Hano","email":"","orcid":"","institution":"Japan Fisheries Research and Education Agency: Kokuritsu Kenkyu Kaihatsu Hojin Suisan Kenkyu Kyoiku Kiko","correspondingAuthor":false,"prefix":"","firstName":"Takeshi","middleName":"","lastName":"Hano","suffix":""}],"badges":[],"createdAt":"2026-03-17 05:01:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9144095/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9144095/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105283791,"identity":"909321bd-de1b-40ee-bd3e-c7da6de394fa","added_by":"auto","created_at":"2026-03-24 10:44:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":446890,"visible":true,"origin":"","legend":"\u003cp\u003eImages of 0.2-mm- and 10-mm-filtered aged microplastic (aMP) examined with fluorescence microscopy (A, B) and crystal violet staining (C, D). Scale bars indicate 200 mm in (A, B) and 2 mm in (C, D). Arrowheads show examples of stained aMP (C, D).\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-9144095/v1/1346f5f1a9ea2c0faefab98e.png"},{"id":105283792,"identity":"465afd5a-7cd1-40b7-a6c7-c63143df9613","added_by":"auto","created_at":"2026-03-24 10:44:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":807046,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of 16S rRNA gene copies of the bacterial communities attached to each microplastic (MP) type. Initial, before aging treatment; 0.2-mm-filtered, 0.2-mm-filtered aged MP (aMP); 10-mm-filtered, 10-mm-filtered aMP. Taxa shown with a hatched pattern have been observed in MP collected from the marine environment (Zettler et al., 2013).\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-9144095/v1/65d5d5e9b7b57b681d09648c.png"},{"id":105564690,"identity":"f18568b8-1661-4bdb-bd32-a01b08c16122","added_by":"auto","created_at":"2026-03-27 12:50:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2051918,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9144095/v1/11120621-3f9e-4a21-ae24-af0280733e47.pdf"},{"id":105283793,"identity":"d42b33a1-6811-4cff-b463-2d608f98cf11","added_by":"auto","created_at":"2026-03-24 10:44:51","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19738,"visible":true,"origin":"","legend":"","description":"","filename":"260224Ohkubosupplementarytable.docx","url":"https://assets-eu.researchsquare.com/files/rs-9144095/v1/edecfadba655050d8008e408.docx"}],"financialInterests":"","formattedTitle":"Subchronic exposure to pristine and aged microplastics causes no impairment of survival or growth in a marine mysid (Americamysis bahia)","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePlastic waste of various sizes affects organisms in different ways (Koelmans et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The necessity of controlling marine plastic waste is gaining increasing international attention (e.g., G20 Implementation Framework for Actions against Marine Plastic Litter, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). There is an urgent need to understand the biological impacts of marine plastic waste (Isobe et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) as a basis for determining what action should be taken. Microplastics (MPs), which are variously defined as plastic fragments smaller than 5 or 1 mm, are a relatively new threat to marine organisms and ecosystems (e.g., Andrady \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Chae and An, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Galloway and Lewis, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). They are mainly formed when plastic waste breaks down into small pieces and becomes scattered throughout the ocean (e.g., Morales-Caselles et al., 2021). Presently, ocean monitoring efforts primarily focus on MPs larger than 300 \u0026micro;m (Isobe et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, recent reports have detected finer MPs (20\u0026ndash;300 \u0026micro;m, \u0026ldquo;micro-MP\u0026rdquo;), with numbers reported to be hundreds to thousands of times greater than that of larger MPs (Song et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ueda et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Yang. et al., 2025). In particular, Jacob et al., (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) reports that micro-MPs smaller than 50 \u0026micro;m are more toxic to fish. There are concerns that micro-MP is ingested by marine organisms that feed on small prey, such as plankton (Gao et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Jeyavani et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe available information on the biological effects of MP is compromised by biases in the materials studied (e.g., Koelmans et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Paul-Pont et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; de Ruijter et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). For example, polystyrene (PS) is rarely found in the ocean (e.g., Yang et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), yet it is the material most often used in biological toxicity assessments (Paul-Pont et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The effects of MP itself on marine organisms are still unclear. MPs have a variety of shapes (spherical, fragmented, fibrous, etc.), contain a variety of materials (e.g., PS; polyethylene, PE; polypropylene, PP; polyethylene terephthalate), and occur in various sizes (e.g., Auta et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Moreover, once released into the marine environment, MPs inevitably experience aging processes (e.g., UV radiation, biofouling, and hydrolysis) (Alimi et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Auta et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). MPs are sometimes covered in a biofilm (Carson et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zettler et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), which affects their properties (Harrison et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Tu et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), including buoyancy, that could affect their interaction with various organisms (Gaylarde et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Harrison et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Oberbeckmann et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). For these reasons, it is likely insufficient to assess only the effects of MPs of various sizes and shapes. To assess effects of MP in a more environmentally relevant manner, researchers have proposed conducting exposure tests with MP collected from the ocean (e.g., de Ruijter et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, MP collected from environment can carry attached hazardous chemicals or pathogens; because of this, exposure testing using field-collected MP runs the risk of overestimating the effect of the MP itself (Cormier et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Feng et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Oberbeckmann et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Pannetier et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Rochman et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), making it challenging to identify the source of any biological effects and ensure the reproducibility of test results.\u003c/p\u003e \u003cp\u003eIn this study, we examined the biological effects of aged MP (aMP) by stimulating biofouling of micro-MP in a laboratory that closely resembles that found in the natural environment. By preparing aMP modeled on natural MP and conducting exposure tests, we can evaluate the biological effects of the MP itself in a biologically relevant manner. Importantly, the adhesion of harmful chemicals can be minimized by performing MP aging treatment in the laboratory. A microbial community analysis was conducted to determine the type of biofilm formed during the aging process, and the results were then compared to MP collected from the ocean. Furthermore, to investigate the biological effects based on the presence or absence of bacterial flora adhering to the MP, MP samples were aged in seawater containing bacteria and in seawater from which bacteria had been removed by 0.2-\u0026micro;m filtration. In addition, a separate exposure test was conducted using pristine MP for comparison. Polyethylene was selected as the material because it is one of the most frequently detected plastics in the ocean (e.g., Yang et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), yet it has been the focus of only a minority of impact assessment studies (Paul-Pont et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Additionally, the exposure concentration was set at 10 times the highest anticipated marine MP concentration in 2060 (1 mg/L, as reported by Isobe et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and exposure was conducted at this elevated level.\u003c/p\u003e \u003cp\u003eFor the evaluation, the juvenile stage of crustaceans, which has been reported to be affected in some cases (e.g., Jeyavani et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), was used. Our study evaluated the toxic effects of aMP on a marine mysid (\u003cem\u003eAmericamysis bahia\u003c/em\u003e), because it is a typical marine crustacean with a crucial role in marine food webs as a link between producers and high-trophic-level consumers (e.g., fish) with high ecological and economic value (Verslycke et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Despite its important role in marine ecosystems (Oliveira et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), this species has only been included in a few MP impact studies to date (Biefel et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. MP preparation for the aging process\u003c/h2\u003e \u003cp\u003eWe used fluorescent yellow PE microspheres of diameter 45\u0026ndash;53 \u0026micro;m. The MPs had a density of 1.00 g/cm\u003csup\u003e3\u003c/sup\u003e, and were purchased from Cospheric (Santa Barbara, CA, USA).\u003c/p\u003e \u003cp\u003eSeawater collected on 26 August 2024 in our laboratory was sand- and then active-carbon filtered at 20\u0026deg;C, then pre-filled into bottles and allowed to stand at room temperature for two days to allow any suspended sand particles to settle out. The decanted filtered seawater (2 L) was then filtered through a 10-\u0026micro;m mesh filter. One liter of the seawater was filtered again through a 0.2-\u0026micro;m bottle-top filter (Thermo Fisher; Nalgene Rapid-Flow sterile disposable bottle top filters with polyethersulfone membranes, Mexico).\u003c/p\u003e \u003cp\u003eAged MP was prepared by adding MP at a concentration of 20 mg/L (converted value: 6.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e particle/L) to the 10-\u0026micro;m and 0.2-\u0026micro;m mesh-filtered seawater (to create 10-\u0026micro;m-filtered and 0.2-\u0026micro;m-filtered aMP, respectively). The mixture was stirred continuously for 28 days in an incubator (LH-241PFDT-SP, NKsystem, Osaka, Japan,) under constant temperature (20.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u0026deg;C) and light cycle (12-h light:12-h dark). The 0.2-\u0026micro;m-filtered aMP served as a negative control for biofilm formation. The aMP was collected using a 47-mm-diameter PTFE filter (Omnipore 5.0 \u0026micro;m PTFE membrane; Merk Millipore, Ireland), placed in 4 L of artificial seawater (Marine Art SF-1, Osakayakken. Co. Ltd, Japan) adjusted to a salinity of 25 and pH of 8.0 and containing 0.5 ppm of Tween 80, and resuspended by processing with an ultrasonic agitator for 2 min. The shape and size of MP particles were determined by fluorescence microscopy (IX73; Olympus, Japan) with an Olympus model DP73 Color Fidelity Microscope Digital Camera. To stain the peptidoglycan layer of any bacteria, crystal violet staining was performed by slightly modifying established protocols (Merritt et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), as follows. The aged MP was stained with a 0.1% crystal violet (FUJIFILM Wako Pure Chemical Corporation, Japan) solution for 30 s. Then, it was washed with distilled water and examined under a stereomicroscope (SZX7; Olympus) equipped with an Olympus model DP21 Color Fidelity Microscope Digital Camera.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. DNA extraction and MiSeq sequencing\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo evaluate the microbial community formed on the aMP surface, 10-mL samples of both the 10-\u0026micro;m-filtered and 0.2-\u0026micro;m filtered aMP solutions (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2 each) were filtered onto the PTFE membrane (Merck Millipore) at the end of the aging process. DNA was extracted from the MP before and after aging with a DNeasy PowerSoil Pro kit (QIAGEN, Hilden, Germany). The hypervariable V3\u0026ndash;V4 region of bacterial 16S rRNA was amplified on an Illumina Miseq platform. Obtained sequences were clustered into OTUs (97% similarity) against the Greengenes database (ver. 13_8) using QIIME2 software (ver. 2024.10). Quantitative PCR (qPCR) assays targeting genes encoding 16S rRNA (337F-797R) were carried out with a SsoAdvanced universal SYBR green supermix (Bio-Rad, United Kingdom) on a CPX96 Real-Time System (Bio-Rad).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Subchronic toxicity test\u003c/h2\u003e \u003cp\u003eMysids (\u003cem\u003eA. bahia\u003c/em\u003e) were purchased from the National Institute for Environmental Studies (Tsukuba, Japan). The culture has been maintained in our laboratory for more than 5 years. The culture medium was prepared using sand-filtered seawater, diluted with aerated deionized tap water to a final salinity of 25. A 16-h-light:8-h-dark photoperiod was used during culturing and the water temperature was maintained at 25\u0026deg;C. The mysids were fed every two days with \u003cem\u003eArtemia\u003c/em\u003e sp. nauplii (24 h after hatching) ad libitum.\u003c/p\u003e \u003cp\u003eSurvival and growth tests were performed using \u003cem\u003eA. bahia\u003c/em\u003e by following a procedure from the US EPA on 7-day-old juveniles (US EPA, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) with minor modifications. For each test, 150\u0026ndash;250-mL samples of artificial seawater adjusted to a salinity of 25 and pH of 8.0 were placed in 300-mL glass beakers, and each treatment consisted of 5 individuals per beaker with triplicate (aMP exposure, 15 mysids total per treatment) or quadruplicate (pristine MP exposure, 20 mysids total) trials. The larvae were exposed to MP for 7 days in an incubator at 25\u0026deg;C with a 16-h-light:8-h-dark photoperiod. During the test, the artificial seawater was completely replaced every 2\u0026ndash;3 days with a semi-static exposure method. The main exposure concentration was 10 mg/L, which is 10\u0026times; the predicted maximum concentration of MP in the ocean in 2060 (1 mg/L; Isobe et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Additionally, 3-mg/L and 1-mg/L treatments were established in the pristine MP exposure test. The number of MPs in the exposure medium was counted on days 0 and 2 of the experiment with a fluorescence microscope (BZ-X800; Keyence Corp., Osaka, Japan). The solvent control group was exposed only to the solvent carrier (Tween80, 0.5 ppm) in the artificial seawater. We confirmed in a separate experiment that this concentration of Tween 80 does not exhibit toxicity to marine mysids. Approximately 200 \u003cem\u003eArtemia\u003c/em\u003e nauplii were provided to each beaker on each day of the test except the fourth. The total number of molts (i.e., shedding of the old exoskeleton [exuvia]) and mortality in each treatment group were counted daily. At the end of the test, test specimens were fixed in 70% ethanol (v/v) for 24 h, and measured for total length under a stereomicroscope equipped with a digital camera and for body weight with an electronic balance (CPA224S; Sartorius AG, Germany). The number of individuals showing evidence of MP uptake (MP uptake frequency) was then counted under a fluorescence microscope (IX73).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Statistical analysis\u003c/h2\u003e \u003cp\u003eWe used the Cochran\u0026ndash;Armitage test to determine the significance of differences in survival rate among treatments. Total length, body weight, and total number of molts at the end of the test were subjected to one-way analysis of variance (ANOVA) to identify significant differences among treatments, and a Bonferroni correction was applied for multiple tests. If the data were heteroscedastic, a Kruskal\u0026ndash;Wallis test and a post-hoc Steel\u0026rsquo;s test were performed. Differences were regarded as significant if the type I error rate (\u003cem\u003ep\u003c/em\u003e) was less than 0.05. The Microsoft Excel add-in software Excel Tokuei for Windows (SSRI Inc., Tokyo, Japan) was used for all statistical analyses.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Characteristics of aMP\u003c/h2\u003e \u003cp\u003eStirring caused the aMP to fragment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B), resulting in an average long diameter of 25 \u0026micro;m and a short diameter of 16 \u0026micro;m in the 0.2-\u0026micro;m-filtered aMP and an average long diameter of 24 \u0026micro;m and a short diameter of 15 \u0026micro;m in the 10-\u0026micro;m-filtered aMP. The 10-\u0026micro;m-filtered aMP stained more intensely in crystal violet stain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) compared to the 0.2-\u0026micro;m-filtered aMP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), indicating that more biofilm was attached. These results suggest that the ageing treatment produced micro-MPs that were fragmented and coated with biofilm. The copy number of bacterial 16S rRNA genes was higher in the 10-\u0026micro;m-filtered aMP (3.3 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e copies/mg MP) than in the 0.2-\u0026micro;m-filtered aMP (1.9 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e copies/mg MP) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), showing that a large number of bacteria proliferated on the MP surface in the 10-\u0026micro;m-filtered treatments. The composition and distribution of biofilm on the surface of MPs tends to differ from those in the surrounding seawater, and are enriched with certain bacteria (Feng et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Rhodobacteraceae (Elifantz et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and Flavobacteriaceae (Feng et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), which are major biofilm components in various regions, were detected from the 10-\u0026micro;m-filtered aMP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), and have also been associated with MP (both PP and PE) collected from actual marine environments (Zettler et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This suggests that we were able to produce aged MP that successfully simulated that produced under natural conditions. Of the bacterial sequences obtained from the 10-\u0026micro;m-filtered aMP, 67% were homologous to bacteria detected in MP from natural marine environments. Previous studies have artificially formed biofilms on MP and evaluated their biological effects (Amariei et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Vroom et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The present study is the first to analyze the microbial community and verify its similarity to natural conditions. Our technique for artificially aging MP is likely to be important for evaluating the impact of MP on marine organisms. Furthermore, the technique could prove useful for experimentally simulating the adsorption of chemicals onto the plastic particles and their subsequent uptake by marine organisms, which is another major issue concerning marine plastic debris (e.g., Carson et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Wang et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Subchronic toxicity of aMP\u003c/h2\u003e \u003cp\u003eThe results of the pristine MP exposure test are shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The Cochran\u0026ndash;Armitage test revealed no significant difference in survival rates between the solvent control and pristine MP exposure groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The MP uptake frequency was higher at higher MP concentrations; however, no significant differences were observed. A one-way ANOVA showed no significant differences in the total number of molts, body weight, or total length (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Measurements of the number of pristine MP particles in solutions of 1, 3, and 10 mg/L yielded values 77%, 230%, and 40%, respectively, of the predicted number of particles calculated from the nominal weight concentration, average MP size (50 \u0026micro;m), and specific gravity. The difficulty of controlling the concentration of MP in water has been well established (Sun and Wu, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), resulting in variations between the actual and nominal concentrations.\u003c/p\u003e \u003cp\u003eThe results of the aMP exposure test are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The Cochran\u0026ndash;Armitage test revealed no significant difference in survival rates between the solvent control and MP exposure groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The count of aMP particles in solution was slightly higher for the 10-\u0026micro;m-filtered aMP, however, the two were almost identical. Most mysid individuals had ingested MP. However, a one-way ANOVA showed no significant differences in the total number of molts or body weights (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Although the 10-\u0026micro;m-filtered aMP exposure group had significantly greater total length (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02) than the solvent control group at the end of the experiment (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), the difference was slight (about 10%). The 10-\u0026micro;m-filtered aMP had more attached biofilm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), and crustaceans use aquatic biofilms as an important nutrient source (Abreu et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Tang et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), suggesting that this may have slightly promoted growth in the 10-\u0026micro;m-filtered group, as has been reported for \u003cem\u003eD. magna\u003c/em\u003e (Amariei et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In addition, an exposure study using naturally derived MP has also reported positive effects on survival (de Ruijter et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and these data suggested that even if MP exposure has negative effects on growth due to the inhibition of food assimilation, such effects can be counterbalanced by the ingestion of attached biofilms in the natural environment.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSurvival rate, total number of molts, microplastic (MP) uptake frequency, total length, and body weight of mysids at the end of the aged MP (aMP) exposure test\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExperimental group\u003c/p\u003e \u003cp\u003e(mean measured MP concentration)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSurvival rate (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal number of molts\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMP uptake frequency (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTotal length(mm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBody weight(mg)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSolvent control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e93\u003c/p\u003e \u003cp\u003e(14/15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.2-\u0026micro;m-filtered aMP (9.5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e particle/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100 (15/15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12/15 (80)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10-\u0026micro;m-filtered aMP (1.6 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e particle /L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e93\u003c/p\u003e \u003cp\u003e(14/15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14/14 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e6.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e-, n.d.; *, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05 compared to solvent control\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eOur results show no adverse effects on mysid larvae from pristine or aMP exposure for the tested sizes (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\le\\:\\)\u003c/span\u003e\u003c/span\u003e53 \u0026micro;m), materials (PE), and concentrations (10 mg/L). As we previously mentioned, the biological effects of pristine MP exposure can differ from those of exposure to MP in the marine environment (Carson et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, we prepared an aMP sample that was closer to the MPs that are found in the field and conducted exposure tests at a concentration 10\u0026times; higher (10 mg/L) than that predicted for 2060 (Isobe et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). As also mentioned above, planktonic crustaceans are the taxonomic group most frequently reported to be adversely affected by MP (e.g., Jeyavani et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The lack of observed harmful effects on the mysid larvae suggests that the biological impact on marine planktonic crustaceans will be minimal at current levels of MP concentration, and that the small magnitude of any potential impact will make detection difficult in subchronic toxicity tests.\u003c/p\u003e \u003cp\u003eOn the other hand, there are reports of adverse effects on the survival of other crustaceans, particularly juveniles, following prolonged exposure at higher concentrations of 1- or 10-\u0026micro;m PS (Lee et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), or 10\u0026ndash;45-\u0026micro;m PP (Jeyavani et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Future studies should focus on chronic toxicity testing methods, which are expected to be more sensitive to toxic effects, and consider the use of aMP of other materials to evaluate the presence or absence of MP effects.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis study was supported in part by a grant-in-aid from the Fisheries Agency of Japan (2BC413). Please note that the views expressed in this study are not necessarily those of the Ministry of Agriculture, Forestry and Fisheries of Japan.\u003c/p\u003e \u003cp\u003eDeclaration of interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003ch2\u003eAuthor Contributions:\u003c/h2\u003e \u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Nobuyuki Ohkubo, Mana Ito, Chisato Kataoka, and Toshimitsu Onduka. The first draft of the manuscript was written by Nobuyuki Ohkubo and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe are grateful to Dr. Rei Yamashita (Atmosphere and Ocean Research Institute, The University of Tokyo) and Dr. Alam Md Khorshed (National Research Institute of Fisheries Technology) for their useful advice, and to Mses. Miki Shoda, Miki Ishimoto, Mika Momosaki, Mr. Hideki Nagano (National Research Institute of Fisheries Technology) for their kind assistance.\u003c/p\u003e\u003ch2\u003eData Availability:\u003c/h2\u003e \u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbreu PC, Ballester ELC, Odebrecht C, Wasielesky W, Cavalli RO, Gran\u0026eacute;li W, Anesio AM (2007) Importance of biofilm as food source for shrimp (\u003cem\u003eFarfantepenaeus paulensis\u003c/em\u003e) evaluated by stable isotopes (δ13C and δ15N). J Exp Mar Biol Ecol 347(1):88\u0026ndash;96\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlimi OS, Farner Budarz J, Hernandez LM, Tufenkji N (2018) Microplastics and nanoplastics in aquatic environments: aggregation, deposition, and enhanced contaminant transport. 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Mar Pollut Bull 212:117538\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZettler ER, Mincer TJ, Amaral-Zettler LA (2013) Life in the Plastisphere: Microbial Communities on Plastic Marine Debris. Environ Sci Technol 47(13):7137\u0026ndash;7146\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-environmental-contamination-and-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aect","sideBox":"Learn more about [Archives of Environmental Contamination and Toxicology](https://www.springer.com/journal/244)","snPcode":"244","submissionUrl":"https://submission.nature.com/new-submission/244/3","title":"Archives of Environmental Contamination and Toxicology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Polyethylene, Aged MP, Biofouling, Marine mysid, Subchronic toxicity","lastPublishedDoi":"10.21203/rs.3.rs-9144095/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9144095/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThere are increasing concerns that \u0026micro;icroplastics (MPs) will be ingested by marine organisms that feed on small prey, such as plankton. In this study, we examined the biological effects (survival and growth) of exposure to pristine polyethylene MP and aged MP (aMP; length\u0026thinsp;\u0026le;\u0026thinsp;53 \u0026micro;m) prepared by maintaining the MP under laboratory conditions at 20\u0026deg;C for 28 days to create biofouling that closely resembles that found in the natural environment. To determine the type of biofilm formed during the aging process, microbial community analysis of aMP was conducted. \u003cem\u003eRhodobacteraceae\u003c/em\u003e and \u003cem\u003eFlavobacteriaceae\u003c/em\u003e were detected from the aMP, and these same bacterial members were associated with MP collected from actual marine environments, suggesting that we successfully produced aMP under conditions that simulated those in the field. We then evaluated the subchronic toxicity (7 d) of MP by using marine mysid (\u003cem\u003eAmericamysis bahia\u003c/em\u003e) larvae. After 10 mg/L pristine and aMP exposure, no significant differences in survival rate or body weight were observed between the solvent control group and MP exposure groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), though aMP exposure slightly increased total length (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02). Overall, no remarkable adverse effects on marine mysid larvae were observed after pristine MP and aMP exposure at the tested sizes, materials, and concentrations. This suggests that the biological impact of aMP on marine planktonic crustaceans will be minimal at current levels of MP contamination, and that the small impact will make detection difficult in subchronic toxicity tests.\u003c/p\u003e","manuscriptTitle":"Subchronic exposure to pristine and aged microplastics causes no impairment of survival or growth in a marine mysid (Americamysis bahia)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-24 10:44:46","doi":"10.21203/rs.3.rs-9144095/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-04-19T17:52:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-19T15:56:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Environmental Contamination and Toxicology","date":"2026-03-18T21:20:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-environmental-contamination-and-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aect","sideBox":"Learn more about [Archives of Environmental Contamination and Toxicology](https://www.springer.com/journal/244)","snPcode":"244","submissionUrl":"https://submission.nature.com/new-submission/244/3","title":"Archives of Environmental Contamination and Toxicology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a5dbe650-272f-4c9d-853f-8ff88327ff4b","owner":[],"postedDate":"March 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-24T10:44:46+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-24 10:44:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9144095","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9144095","identity":"rs-9144095","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}