Ecotoxicology of Polystyrene Microplastic Fragments: Oxidative Stress Effects in Neonate Versus Adult Daphnia magna

Polystyrene is an inert polymer; however, sublethal effects have been observed in aquatic organisms exposed to microparticles of this plastic. Information on microplastic-related oxidative stress and related antioxidative responses as adverse effects and the underlying mechanisms of toxicity are limited. Daphnids are key contributors in aquatic ecosystems, linking primary producers to consumers and predators, facilitating energy transfer and thus being considered an ideal bioindicator organism to study the adverse effects of polystyrene fragments on the oxidative stress status and subsequent enzymatic antioxidant response. In the present study, neonates (≤ 24 h) and adult daphnids were acutely exposed to polystyrene fragments sieved to three size factions (< 25 µm, 45—63 µm, and 100—500 µm), and adverse effects were evaluated after 24 and 48 h. In adults, larger particles elicited reactive oxygen species by 61%, which was met with elevated superoxide dismutase (17%) and catalase activities (98%), restoring redox homeostasis within 48 h. However, in neonates, the reactive oxygen species increased with exposure to all polystyrene sizes within 24 h, and homeostasis was regained within 48 h without eliciting the enzymatic antioxidant defense. The adverse effects were associated with size fractions that were too large to be consumed, suggesting that leached additives may be involved. Further studies are needed to determine whether nonenzymatic antioxidants were responsible for neutralizing excess reactive oxygen species in neonates.


Introduction
Polystyrene (PS), a polymer composed of aromatic hydrocarbon styrene monomers, is soluble in organic solvents but insoluble in water and resistant to acids, alkalis, salts, mineral oils, organic acids, and alcohols (Schellenberg, 2009).Depending on the manufacturing process and additives used, which may include plasticizers, flame retardants, antioxidants, acid scavengers, light and heat stabilizers, lubricants, pigments, antistatic agents, slip compounds, and thermal stabilizers (Smith & Taylor, 2002), PS may be solid or foamed, translucent or colored.As a durable, solid plastic, PS is often used to manufacture transparent products, such as food packaging, and as a lightweight foam, it is used for thermal insulation.Therefore, the production of PS accounts for approximately 10% of the total non-fiber plastic production globally (Plas-ticsEurope, 2016).However, PS also occurs as one of the most common (micro)plastic debris observed in the environment (Geyer et al., 2017;Scopetani et al., 2019).PS concentrations of up to 0.138 µg/L have been reported in natural waters collected from Port d'Illa de Mar, Spain (Schirinzi et al., 2019).Badylak et al. (2021) detected up to 76,000 PS particles per liter in a coastal lagoon in the Florida Keys.According to Hwang et al. (2020), the average weight of a 3 µm PS particle is 0.015 ng.Assuming this value to convert the PS concentration reported by Badylak et al. (2021), this would equate to a PS concentration of approximately 1.140 µg/L.However, these samples contained mixed plastics therefore, the total concentration of MP may be substantially higher.Nevertheless, it has been predicted that the global plastic waste output into aquatics will increase tenfold by 2050 (Jambeck et al., 2015).
Although PS is considered a chemically inert polymer, toxic effects have been associated with exposure (Lu et al., 2016;Shengchen et al., 2021).However, Gambardella et al. (2017) demonstrated that even though PS beads (0.1 μm diameter, 0.001 to 10 mg/L exposure concentrations) accumulated in the nauplii of the barnacle Amphibalanus amphitrite and larvae of brine shrimp Artemia franciscana, survival was not affected.Nevertheless, mobility and the enzyme activities of cholinesterases and catalase were affected, which the authors associated with oxidative stress.These data indicated sublethal effects related to PS exposure.
The ecotoxicological effects of microplastics (MPs) in Daphnia spp.have recently been reviewed (Samadi et al., 2022), and the lack of data on oxidative stress in Daphnia spp.associated with MP exposure was evident.Eltemsah and Bøhn (2019) found significant differences between juvenile and adult daphnids' responses to PS exposure regarding survival, growth, fecundity, maturation age, and body size of neonates.Nevertheless, knowledge of physiological effects, such as the exposed biota's antioxidative stress responses, may bring insight into the mechanism of PS's toxicity.Daphnia spp.function as keystone species in many aquatic ecosystems due to their central role in many food webs by facilitating energy transfer (Lampert, 1987).Previous studies have proven daphnia, especially Daphnia magna, a reliable bioindicator organism for investigating toxicological influences and effects of several emerging contaminants (Siciliano et al., 2015).Reasons for the wide use of daphnids in ecotoxicological research include sensitivity, economy and ease of culturing in the laboratory, and hatching speed (Samadi et al., 2022;Siciliano et al., 2015).Therefore, D. magna was used as a test organism in the present study to investigate the effect of PS fragments on neonates and adults.
D. magna was shown to ingest PS particles selectively based on size.Rist et al. (2017) reported that daphnids consumed five times more 2 μm PS beads than 0.1 μm beads (1 mg/L exposure concentration) over 24 h.Additionally, feeding rates significantly decreased in the presence of the 0.1 μm PS beads.Furthermore, Liu et al. (2022) demonstrated that the toxicity of polyvinyl chloride microplastics (MPs) is sizedependent.Therefore, the effect of particle size was considered in the present study.Daphnids are filter feeders and typically consume particles in the range of 0.6 to 40 μm (Barnett et al., 2007;Geller & Muller, 1981).Nevertheless, Canniff and Hoang (2018) demonstrated that D. magna ingested MP microbeads ranging from 63 to 75 μm.When exposed to regularly and irregularly shaped MP between 10 and 106 μm in size at concentrations ranging from 0.01 to 10 g/L, D. magna has been shown to ingest up to 50 plastic particles/animal/day (Frydkjaer et al., 2017).Therefore, in the present study, various size fractions of PS fragments were used in acute exposure experiments to investigate the impact of PS on the antioxidative response of adult and neonate daphnids.For neonates, small (< 25 μm) and large (> 100 μm) sieved PS fragments, and for adults, in addition to small and large, also medium (45-63 μm) sieved fragments were used.The small fragments' size range was selected as this size range would undoubtedly be consumed by both the neonates and adults, the medium would be consumed by the adults, and the > 100 μm sieved fragments would be uningestible for both age groups.This size fraction division would allow distinguishing whether the observed effects could be accounted for by ingestion or exposure to leached toxic substances.
Reactive oxygen species (ROS) generation is a common product of normal cell processes during aerobic metabolism.ROS plays a crucial role in various biological systems, such as signaling cascade, gene expression and signaling, or responses associated with diseases or xenobiotic exposure (Lushchak, 2011;Tanabe et al., 2022).Xenobiotic-associated ROS production beyond antioxidant capacity has been related to adverse effects on biological homeostasis in organisms and biomolecule damage.Oxidative stress occurs when the intracellular reactive oxygen species (ROS) concentration increases, which causes DNA and protein damage as well as lipid peroxidation.Thus, elevated ROS has been implicated as a mechanism of pollutant toxicity in aquatic organisms (Livingstone, 2001;Magni et al., 2018) and, therefore, is used as a biomarker for toxicity.The enzymatic and nonenzymatic antioxidative systems commonly counteract the disturbance.Thus, oxidative stress status and antioxidative responses are characteristic toxicity biomarkers, as ROS spikes are associated with exposure to pollutants or inhibition of the antioxidative response (Hook et al., 2014).Typically, the enzymes involved include superoxide dismutase (SOD) as a first line of defense, catalyzing the conversion of ROS to hydrogen peroxide, which is then converted to water and oxygen by catalase (CAT) or peroxidase (Esterhuizen-Londt et al., 2011).Therefore, the study aimed to expose neonate and adult D. magna to PS fragments of different sizes to evaluate the survival, oxidative stress status as the intracellular ROS level, and the enzymatic antioxidant defense as the SOD and CAT activities in response to exposure for 24 and 48 h, respectively.The exposure concentration of 0.5 mg/L was selected based on the highest MP concentrations reported to date (Badylak et al. (2021), considering the predicted future increase of MP pollution (Jambeck et al., 2015) as well as the no observed effect PS level on D. magna in freshwater of 0.1 mg/L (Yang & Nowack, 2020).Acute exposure was selected for the toxicology evaluations, as is typically done in standardized toxicology testing (OECD, 2004).Furthermore, the acute exposure scenario was considered to more accurately depict environmental conditions based on settling velocity studies conducted by Elagami et al. (2022), who reported that for MP particles around 100 μm, the modeled mean residence time in lakes was, on average, less than one day.

Daphnia Cultivation
D. magna ephippia were purchased from Micro-Biotests Inc. (Gent, Belgium) and hatched under 7000 lx light with a 16-h light and 8-h dark cycle at 20.0 ± 1.0 °C in Elendt M4 medium (OECD, 2004).A population of 15 D. magna individuals was maintained in a 2 L glass beaker containing 1.5 L Elendt M4 medium in a controlled climate incubator.The daphnids were fed daily with Chlorella vulgaris (~ 1.5 × 10 8 cells/mL), purchased from the Culture Collection of Algae at Cologne University (Essen, Germany), at a density of 0.1-0.2mg C per individual.The daphnid culture was additionally supplemented with yeast, cerophyl, and trout chow at a concentration of 0.5 μL/mL (v/v) thrice a week.New neonates were removed daily, and cultural media and beakers were renewed thrice weekly.The pH and dissolved oxygen content were checked and maintained according to OECD guidelines (OECD, 2004).Interlaboratory tests were performed using potassium dichromate (Sigma-Aldrich, St. Louis, MO, USA) as a reference toxicant.For the present study, neonates were ≤ 24 h old, and adult daphnids were 10 days old and fully developed.

Consumables
Polystyrene granules (3-5 mm particle size; Product Code: ST31-GL-000111; LS555533; batch C3586) 713 Page 4 of 11 Vol:.( 1234567890) were purchased from Goodfellow (Huntingdon, UK) and ground into smaller fragments employing lowtemperature ball milling on a Retsch Cryomill (Haan, Germany) maintained between -196 °C and -100 °C using liquid nitrogen.A 50 ml cell and a 2 cm diameter steel ball were used for mechanical cryogenic grinding.A third of the cell was filled with the plastic material and ground for approximately 30 min, with 5 min of pre-cooling.The ground plastic fragments were separated according to size using a Vibratory Sieve shaker AS 300 Control (Retsch GmbH, Haan, Germany) via four sieves with mesh sizes of 100, 63, 45, and 25 µm according to ISO 3310-01 (ISO, 2016).Three fractions were retained for the exposures (small ≤ 25 µm, medium 45-63 µm, and large 100-500 µm).All other consumables were purchased from Sigma Aldrich unless stated otherwise and were of analytical grade.
The visualization of the PS fragments was performed using Quanta 250 FEG scanning electron microscopy (Eindhoven, Netherlands) in low vacuum mode (200 Pa) with an LFD sensor.The accelerating voltage was set to 5.0 kV.
Fourier transform infrared (FTIR) spectra were obtained using an IRSpirit FTIR spectrometer (Shimadzu, Kyoto, Japan) equipped with a singlereflection attenuated total reflectance (ATR) accessory (QART™-S, Shimadzu, Kyoto, Japan).The spectra were recorded over the range of 4000-400 cm −1 with 45 scans.The obtained spectra were processed using Origin 2019b (9.6.5.169) software.The spectra were subjected to baseline correction to remove the background signal.Peak smoothing was applied to reduce the noise and enhance the peak resolution.

Exposures and Assessments
Immobilization tests were performed to assess whether the daphnids were physically affected by exposure to PS particles that were consumable (small and medium sized fragments) and fragments too large to be ingested (large fragments).Neonate and adult daphnia were exposed to the three size fractions (sieve sizes: small < 25 µm, medium 45-63 µm, and large > 100 µm) of PS fragments at a concentration of 0.5 mg/L for 24 and 48 h in beakers.Tween® 20 (0.2 µl per 1400 mL media) was added as an emulsifying agent to disperse the PS fraction in the medium.Controls (n = 3), which were run alongside the exposures, showed no effects on adult or neonate daphnids in the presence and absence of Tween® 20 (p = 1).Neonate (45 organisms per replicate, n = 3) and adult (12 organisms per replicate, n = 3) daphnids were exposed to the PS at room temperature (21ºC) with no additional feeding over the exposure period, and the media was not changed during this time.After 24 and 48 h, respectively, immobile daphnids were noted.Live organisms were collected, and the reactive oxygen species concentration was assessed, as well as the antioxidant enzyme activities of catalase and superoxide dismutase as indicators of oxidative stress and the corresponding response to retain cellular homeostasis.
For the ROS determination, as a measure of the oxidative stress status, one-third of the mobile daphnids were snap-frozen in liquid nitrogen and thoroughly homogenized in 1.0 mL phosphate-buffered saline (PBS), followed by centrifugation at 3,000 × g at 4 °C for 10 min.Then, 10 μL of supernatant was used to determine the protein content using a bicinchoninic acid kit (Thermo Fisher Scientific, Waltham, MA, USA).ROS levels were assessed by measuring the oxidation product of 2′,7′-dichlorofluorescein (DCFDA) with excitation and emission spectra at 495 and 529 nm using the DCFDA/H2DCFDA Cellular ROS Assay Kit (catalog no.ab113851; Abcam, Cambridge MA, USA) as per the manufacturer's instructions on a High-performance multi-mode microplate reader (SPARK, TECAN, Switzerland) using 20 μL of supernatant from each sample.The fluorescence intensities were normalized by the protein content of each specimen.
For extracting the S9 enzyme fraction, the other two-thirds of the mobile daphnids were snap-frozen in liquid nitrogen and homogenized with a micropestle in 20 mM potassium phosphate buffer (pH 7) on ice.Cell debris was removed by centrifugation at 13,000 × g for 10 min, and the supernatant was used to assess the enzyme activities.The SOD activity was evaluated using the SOD Assay Kit (19160-1KT-F; Sigma-Aldrich, St. Louis, Missouri, USA).CAT (E.C. 1.11.1.6)activity was assayed using hydrogen peroxide as substrate as per Claiborne (1985) on a Tecan Infinite 200 Pro Infinite M Nano + (Tecan GmbH, Grödig, Austria).Catalase activity was normalized against the protein content, determined as per Page 5 of 11 713 Vol.: ( 0123456789) Bradford (1976), i.e., enzyme activity was calculated as μkat/mg protein.

Statistical Analysis
Statistical analysis was performed using IBM® SPSS® Statistics 28.0.0.0 (190) (2021).Descriptive analysis was performed on all data sets, followed by normality and homogeneity analysis.For data sets meeting the requirements of homogeneity and normality, a one-way analysis of variance (ANOVA) was performed.Tukey posthoc tests were performed with an alpha value of 0.05, indicating significance between treatments and the control.These data sets included the intracellular ROS analysis and SOD enzymatic activity.ROS concentration and SOD enzyme activity served as dependent variables, and exposure treatment (size) as independent variables.The non-parametric Kruskal-Wallis test with pairwise comparisons was used for the catalase activity data set, as requirements for ANOVA were met.CAT activity served as the dependent variable, and the independent variable was the exposure treatment (size).An alpha value of 0.05 was observed as an indicator of statistical significance (Sokal & Rolf, 2013) .

Results and Discussion
The PS fragments generated via cryo-milling were irregularly shaped and sized with jagged edges (Fig. S1).The physicochemical properties of PS were investigated before and after fragmentation via FTIR, which showed no significant changes before and after the physical fragmentation process, as shown in Fig. S2.The samples showed clear peaks at approximately 1450 cm −1 and 2910 cm −1 , corresponding to the stretching and bending vibration of CH 2 groups characteristic of PS.Moreover, the FTIR spectrum exhibited a distinct peak at 690 cm −1 for the phenyl binding vibration.These results indicate that the fragmentation process did not alter the molecular structure, and chemical bonds were retained.
The neonates (Fig. S3A) and adult daphnids (Fig. S3BB), exposed to 0.5 mg/L of each of the sizes of PS fragments, all remained mobile and thus alive for the entire 48-h exposure period (one-way ANOVA p > 0.05).No color change to red or molting was observed as a visible indicator of microbial infection or stress.Even though the exposure concentration used is larger than the higher range of reported environmental concentrations of MPs (Amobonye et al., 2021;Correa-Araneda et al., 2022), the concentrations in nature may rise in the future.The data confirmed that acute exposure to PS fragments at a concentration as high as 0.5 mg/L was not lethal to D. magna, irrespective of the fragment size, in other words, whether they could be consumed or not.Eltemsah and Bøhn (2019) and Schür et al. (2020Schür et al. ( , 2021) ) stated that effects on mobility and survival were observed with chronic exposure; nevertheless, sublethal effects were observed with acute short-term exposure, thus warranting the need to investigate the oxidative status and antioxidative responses and how these responses differ in neonates and adult daphnids.
Exposure of the neonates to small and large PS fragments correlated to a significant rise in the intracellular ROS concentration after 24 h (Fig. 1A).With the small fragments (< 25 µm), ROS increased by 47.1% (Kruskal-Wallis pairwise comparison p = 0.038) and by 61.3% with the large fragments (Kruskal-Wallis pairwise comparison p = 0.004).These data contrast existing findings that smaller MP sizes are more toxic (Jeong et al., 2016(Jeong et al., , 2017)), especially considering that the neonates could have consumed PS fragments in the < 25 µm exposure range and not those in the > 100 µm range.However, after 48 h (Fig. 1B), the ROS concentration in the exposed organisms re-achieved redox homeostasis compared to the control (Kruskal-Wallis p = 0.317).As ROS elevation was the highest with the larger particles and these fragments were too large to be ingested by the daphnids, the adverse effects observed may thus be due to additives being released from the plastic pieces.However, Fig S1 showed that sub-micronsized plastic pieces were electrostatically attached to large fragments, and mechanical vibration may not have been sufficient to induce detachment.Previously, plastic leachates have been shown to cause damaging effects on several plant species (Esterhuizen et al., 2022;Pflugmacher et al., 2020).
In contrast to the neonates, only exposure to the medium fragments caused significantly elevated ROS levels in adults after 24 h of exposure (Fig. 1C), which was a 30.1% rise compared to the control (oneway ANOVA p = 0.042; Tukey posthoc p = 0.027).Despite elevated ROS in all organisms after 48 h (Fig. 1D) compared to 24 h (Fig. 1C), the ROS levels in the exposed organisms were statistically similar to the control (one-way ANOVA p = 0.077), suggesting that homeostasis was regained after 48 h.As reported in several previous studies (Becker et al., 2011;Zhou et al., 2019), the increment of ROS generation in D. magna under stress was regulated by the antioxidant defense system for maintaining physiological homeostasis.The reduced ROS level in adult daphnids compared to neonates indicates that the adults are less sensitive to PS than the neonates, irrespective of whether the fragments are ingestible (small and medium fragments) or not (large fragments).SOD, CAT, glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx), and ascorbate peroxidase (APx) are some of the primary antioxidant enzymes that minimize the harmful effects of ROS (Tanabe et al., 2022).However, as a first line of defense against ROS and damaging hydrogen peroxide generated from ROS, SOD and CAT were considered suitable biomarkers of the antioxidant response in the daphnids.MPs have been shown to disturb the capacity of the antioxidant system, leading to the accrual of intracellular ROS (Shengchen et al., 2021).In the present study, the SOD activity in the neonates was not elevated irrespective of the PS particle size after 24 (Fig. 2A; one-way ANOVA p = 0.283) or 48 h (Fig. 2B; oneway ANOVA p = 0.067) despite elevated intracellular ROS detected after 24 h.As ROS levels returned to those of the control levels in the neonates after 48 h, nonenzymatic antioxidants likely played a role in regaining redox homeostasis.These antioxidants include ascorbic acid, α-tocopherol, glutathione, and other minor antioxidants (Fan et al., 2012).It is therefore suggested that this should be investigated in future studies.
In adult daphnids, the SOD activity was significantly elevated after 24 h with exposure to the larger PS fragments (Fig. 2C); i.e., by 17.7% relative to the control (one-way ANOVA, Tukey posthoc p = 0.047).However, after 48 h (Fig. 2D), the SOD levels stabilized to their normal levels (one-way ANOVA p = 0.081).Elevated SOD may indicate that ROS was increased in adults exposed to large fragments but that SOD reestablished baseline cellular ROS concentrations within 24 h.Jeong et al. (2016) reported increased SOD activities in Brachionus koreanus (rotifer) exposed to 0.05 and 0.5 μm PS microbead at a 10 mg/L exposure concentration.However, the study by Parenti et al. (2020)  As with SOD, CAT activity was not elicited in the D. magna neonates after 24 (Fig. 3A) or 48 (Fig. 3B) hours (one-way ANOVA p = 0.111) of exposure despite elevated ROS.In adults, the CAT activity increased by 98.2% with exposure to medium fragments (Kruskal-Wallis pairwise comparison p = 0.008) and 105.4% with large fragments (Kruskal-Wallis pairwise comparison p = 0.006).After 48 h, the CAT activities returned to levels equal to the control (Kruskal-Wallis p = 0.115).The elevated CAT activities, in conjunction with the increased SOD levels, suggest that the adult daphnids' enzymatic antioxidant response system rapidly responds to elevated ROS reestablishing redox homeostasis within 24 h.Studies investigating the effect of PS MP on CAT activities are limited.Nevertheless, Zhang et al. (2019) reported increased CAT activity in D. magna after 48 h of exposure to 0.1 mg/L PS beads; however, SOD activity was not increased.High CAT activity was also reported in Nelumbo nucifera (Lotus) exposed to PS fragments (Esterhuizen & Kim, 2022).
Based on reviews published by Hu and Palić (2020) and Yin et al. (2023), studies investigating MP-related oxidative stress and related antioxidative responses as adverse outcome pathways for MP toxicity in daphnia are limited.However, these recent reviews indicated that exposure of daphnia to nano/ microplastic led to the acute increment of ROS level and the alteration of antioxidative responses.In the present study, we demonstrate that D. magna neonates and adults physiologically respond differently to exposure to PS fragments.In neonates, ROS significantly increased, proportional to the size of the fragments.However, SOD and CAT activities were neither raised nor inhibited in response to the increased intracellular ROS concentration.In previous studies, low PS exposure concentrations induced substantial ROS production and activated SOD and CAT to combat ROS (Jeong et al., 2016).However, Liang et al. (2017) reported that with high PS concentrations, SOD and CAT activities were inactivated by excess intracellular ROS, leading to oxidative stressrelated damage (Liang et al., 2021).In the present study, despite elevated intracellular ROS in neonates after 24 h, which returned to control ROS levels after 48 h, the measured antioxidant enzymes were not elevated over the 48-h period, suggesting that nonenzymatic mechanisms may be involved in the neonates' response to PS-related ROS.Nevertheless, in adults, increased ROS was met with increased SOD and CAT activities to regain redox homeostasis.In general, exposure to the larger-sized PS fractions caused the most significant ROS production and oxidative stress response in adults.Thus, the adverse effects were not due to consumption but rather exposure to harmful additives released from the particles.
In the study conducted by Jeong et al. (2016), exposure to smaller PS particle sizes prompted more significant oxidative stress in Brachionus koreanus.However, in contrast, the findings presented here agree with Lu et al. (2016), who demonstrated a more substantial antioxidant response with larger PS particles in Danio rerio, suggesting that physiological reactions to PS may be species-specific in addition to the age and life stage of the organism.

Conclusion
The study examined the effects of exposure to different size fractions of polystyrene fragments on oxidative stress and enzymatic antioxidant response in neonate and adult daphnids.The results indicated that larger particles caused ROS in adult daphnids, countered by increased SOD and CAT activities, restoring redox homeostasis within 48 h.However, in neonates, all polystyrene sizes caused increased ROS within 24 h, and homeostasis was restored within 48 h without increased enzymatic antioxidant defense.
Based on the presented data, PS particle-related oxidative stress as an adverse outcome pathway for PS toxicity in D. magna is not plausible due to the reliable antioxidant response.As the PS concentration is higher than currently environmentally reported MP levels, paired with the antioxidant defense in daphnia, PS fragments do not pose an immediate risk to daphnid populations.However, future studies should consider the effects of mixture toxicity, including various MP polymer types, as well as pollutants for which MPs serve as transporters.
Page 9 of 11 713 Vol.: (0123456789) Funding Open Access funding provided by University of Helsinki (including Helsinki University Central Hospital).This work was supported by the South Korean Ministry of Science and ICT(Grant numbers NRF-2017M3A7B6052455).Open Access funding was provided by the University of Helsinki, including Helsinki University Central Hospital.

Data Availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations
Competing Interests The authors have no relevant financial or non-financial interests to disclose.
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