Plastic pollution is a pressing environmental issue that is garnering merited attention. The growing global distribution of plastic waste in the environment stems from its widespread use and improper disposal (Dahlbo et al., 2018; van Velzen et al., 2019). While studies on the ecotoxicology of plastic and its breakdown products, such as micro- and nanoplastics, are increasing, and the impacts are being better understood (Esterhuizen and Kim, 2022; Samadi et al., 2022), another aspect of this problem, namely the toxicity of leaching chemicals, is being recognised (Lithner et al., 2009; Lithner et al., 2012; Pflugmacher et al., 2020).

Plastic items can leach harmful chemicals into their surroundings (Do et al., 2022). Additives which are not covalently bound to the polymer matrix and residual monomers could migrate into the environment from virgin plastics (Crompton, 1979; Araújo et al., 2002; Lithner et al., 2011; Kwan and Takada, 2016). Over time, when exposed to environmental factors like sunlight and water, plastics can break down and release a cocktail of toxic compounds, including phthalates, bisphenol A (BPA), and polychlorinated biphenyls (PCBs). These compounds are known as additives and are applied during the manufacture of plastics depending on the intended use and properties of the final product (Wiesinger et al., 2021; Do et al., 2022). They play a crucial role in tailoring plastics for specific applications by imparting desirable properties or allowing the retaining of the original plastic properties during moulding (OECD, 2009; Hahladakis et al., 2018). Regularly used additives include plasticisers, flame retardants, thermal stabilisers, photostabilisers, antioxidants, and pigments (Stevens, 1990; Andrady and Rajapakse, 2016).

Various media have been shown to facilitate leaching, including sea- and freshwater (Bridson et al., 2021). Release of additives from plastics was studied by Do et al. (2022). They found that polymer type, surface characteristics, the environment, and time are the main factors influencing leaching. Furthermore, biodegradation, thermal and photodegradation influenced the liberation of additives.

Some additives are known carcinogens, mutagens, and endocrine disruptors, and thus potentially hazardous to many organisms (EBC, 2008; Groh et al., 2019). As plastic and microplastic (MP) pollution has been found globally (Scopetani et al., 2019; Mammo et al., 2020; Patti et al., 2020; Prabhu et al., 2022), these leached chemicals can contaminate terrestrial and aquatic ecosystems and even enter the food chain, with potentially detrimental effects on the health of both wildlife and humans. Understanding and addressing this facet of plastic pollution is essential for mitigating its far-reaching consequences, especially concerning the toxicity of leachates to native biota.

In addition to intrinsic toxicants that may be liberated, MPs can adsorb and concentrate persistent organic pollutants (POPs, e.g., PCBs and organochlorine pesticides), heavy metals (lead, cadmium, and mercury), and non-plastic pollutants (e.g., polycyclic aromatic hydrocarbons (PAHs)) from the surrounding water (Caruso, 2019). Chemicals that adhere to MP surfaces have been shown to bioaccumulate in organisms that ingest the fragments (Caruso, 2019). Furthermore, microbial biofilms can develop on MPs in the environment, releasing metabolites, enzymes, and other chemicals that have been found to contribute to plastic degradation and, thus, facilitate leaching (Han et al., 2020; Sooriyakumar et al., 2022), which may contribute to toxicity.

Even though several studies have considered the ecotoxicological effects of leachates from plastics (reviewed by Gunaalan et al., 2020), many have used artificial leaching methods and have not considered the effect of factors such as the environment, climate fluctuations, and naturally occurring microbes. Therefore, the present study aimed to investigate the toxicological effects of natural leachate from polystyrene (PS), one of the most detected plastics in the environment (Scopetani et al., 2019; Tian et al., 2023). PS was leached in pure water (MilliQ), freshwater, and seawater for six months outside under environmental conditions and the ecotoxicological effects of the leachate, and dilutions thereof, were investigated in terms of immobility, oxidative stress, and enzymatic antioxidative defence response in Daphnia magna (pure and freshwater) and Artemia salina (seawater) with acute exposure. Many previous studies regarding MP and leachates were conducted with D. magna (Samadi et al., 2022), a recognised model bioindicator organism for evaluating ecotoxicology (Reilly et al., 2023). Likewise, A. salina has been suggested as a model species for ecotoxicity assessments (Kalčíková et al., 2012; Albarano et al., 2022). The three media were selected to assess how the properties of a natural media can influence the toxicity of the leachate, where both fresh- and seawater contained natural microbes and minerals compared to sterile MilliQ water which is both deionized and demineralized. This approach allows the direct comparison of the toxicities of the obtained PS leachates in the various media on keystone bioindicator organisms which have not previously been assessed. Thus, the selection of three distinct media aimed to investigate the impact of natural media properties on leachate toxicity. Both fresh- and seawater, rich in natural microbes and minerals, were compared to sterile MilliQ water, known for its deionized and demineralized composition. This method facilitates a direct comparison of the toxicities exhibited by the obtained PS leachates across different media. The chosen bioindicator organisms, considered keystone species, have not been previously evaluated under these conditions.

D. magna neonates were exposed for 48 h to leachate prepared by incubating PS fragments in freshwater and pure (MilliQ) water, respectively, for 6 months in nature. At the same time, A. salina were exposed to leachate prepared by incubating PS-MP in seawater for six months. For neonate D. magna, exposed to the PS leachate in freshwater ( Figure 1A ), 53.3% fewer neonates were mobile after 48 h of exposure to the undiluted leachate compared to the control (p = 1.000). At the same time, exposure to the 10%, 25%, and 50% concentrations of the leachate did not reduce daphnid mobility (p = 0.001). However, for neonates exposed to PS-leached in pure water ( Figure 1B ), mobility was not significantly affected with any of the concentrations of leachate over the 48 h exposure period (p = 0.596).

A. salina ( Figure 1C ) mobility was the most severely affected by the undiluted seawater leachate, with 80.0% of the organisms remaining mobile after 48 h compared to the control (p < 0.001). With the 50% and 25% concentrations, the mobility was 53.3% (p < 0.001) and 33.3% (p = 0.001), respectively. However, with 10% leachate, mobility was not affected (p = 1.000).

To evaluate physiological adverse effects in live, healthy organisms, the lowest leachate concentration that exhibited sublethal adverse effects was chosen for further assessment. Specifically, a concentration of 50% leachate was selected for D. magna, while for A. salina, a concentration of 10% leachate was deemed appropriate. The selection was based on the data obtained in Figure 1 .

The cellular ROS concentration relative to the protein content was not elevated after 48 h of exposure to the 50% dilution of the freshwater PS-leachate (p = 0.116; Figure 2A ). The ROS concentration in the neonates exposed to the 50% diluted pure water PS-leachate for 48 h was increased by 24.2% (p = 0.022; Figure 2A ). As for A. salina after 48 h of exposure to the 10% seawater PS-leached ( Figure 2B ), the cellular ROS level was statistically equivalent compared to the control (p = 0.064).

The SOD activity was significantly elevated in neonate daphnids after 48 h of exposure to both freshwater PS-leachate as well as pure water PS-leachate compared to the control ( Figure 3A ). With exposure to the freshwater PS-leachate, the SOD activity was increased 104.6% (p = 0.011) and 100.3% with exposure to the pure water PS leachate (p = 0.0140). The SOD activity in A. salina ( Figure 3B ) was statistically equal to that of the control after 48 h of exposure to the seawater PS leachate (p = 0.479).

The CAT activities in neonate daphnids were unchanged relative to the control CAT activity after 48 h of exposure to freshwater as well as pure water PS-leachate (p = 0.897; Figure 4A ). However, in A. salina ( Figure 4B ), CAT activity was significantly increased by 127.9% (p = 0.002).

The goal of this study was to compare the toxicities of PS leachates prepared in three distinct media, seawater, freshwater, and sterile, pure water (MilliQ). These media were selected to represent the properties of natural water bodies in terms of pH, salinity, conductivity, and turbidity, compared to sterile, deionized, and demineralized water. The leachates produced using the various media resulted in significantly different toxic responses in the bioindicator organisms tested. Notably, PS leachate in MilliQ water was significantly less toxic to D. magna compared to PS leachate in freshwater as is evidenced by the immobilization over time. The immobile organisms were not swimming after 15 seconds of agitation. They had all sunken to the bottom of the exposure vessel and their antennae or feeding apparatus showed no movement, which could indicate physiological toxicity leading to death rather than a physical impairment. Additives, which are mostly bound to the MPs by weak dispersion forces (Zhang and Chen, 2014), can leach from plastic materials with fragmentation and degradation accelerating the release of chemicals (Engler, 2012). Since all microplastic fragments were removed before exposure, only leached chemicals could have contributed to the observed immobilization, an established indicator of acute toxicity (OECD, 2004). The additives used in the manufacture of the PS could not be disclosed by the supplier. Nevertheless, several common plastic additives (Smith and Taylor, 2002; Samadi et al., 2022) have been identified as toxic to D. magna and other aquatic organisms (Thaysen et al., 2018; Schiavo et al., 2021; Song et al., 2021; Blinova et al., 2023).

In general, D. magna was less susceptible to the PS-leachate. Only the undiluted PS-leachate in freshwater caused significant changes in mobility. Lithner et al. (2012) leached plastics from old electronic products in water for three days at the same concentration as used in the present study (100 g/L). They found that after acute exposure (24 h and 48 h) the leachate was non-toxic to D. magna. Furthermore, in 2009, Lithner (2011) reported that leachate prepared from PS (100 g/L) did not cause immobilisation of D. magna. However, as deionized water was used, this result concurs with our immobilization findings of daphnids exposed to PS leachate prepared in MilliQ water.

A previous study reported an LD₅₀ of 67 mg/L for 1 μm PS beads in D. magna (Miloloža et al., 2021). Nevertheless, the PS concentration used to prepare the leachate in the present study (100 g/L) far exceeds the highest environmental concentrations reported for PS (Schirinzi et al., 2019; Scopetani et al., 2019; Badylak et al., 2021) and MP in general. Up to 4650 particles/m³ have been detected in freshwater and 102,000 particles/m³ in coastal waters (Wong et al., 2020; Zhang et al., 2020). A conversion from particles per volume to weight per volume was proposed by Leusch and Ziajahromi (2021). However, this accounts for the specific uniform size of reference particles and the density of a specific polymer. Assuming an average size of 1 µm and the density of PS (1.05 g/cm³), the environmental concentrations reported may be equivalent to several orders of magnitude less than the PS concentration used in the present study to prepare the leachate. Thus, leachates from the current level of MP pollution in the environment should not pose a significant threat to D. magna. Nevertheless, MP hotspots may form due to water currents, with localized concentrations exceeding millions of particles per litre (Kane et al., 2020), which should be considered in the comprehensive risk assessment of MPs and their leachates in the environment.

A. salina was significantly more affected by the leachate compared to the D. magna. This may indicate an overall higher susceptibility, or it could be related to the seawater and associated microbiota facilitating a larger concentration of toxicants to be released from the MP. However, in studies comparing the sensitivities of common bioassay organisms to toxicants, A. salina was significantly less susceptible compared to D. magna (Minguez et al., 2014). Furthermore, the study by Wang et al. (2019) showed that Artemia parthenogenetica was not significantly affected by exposure to various concentrations of PS-MP in terms of survival, growth, or development, irrespective of acute or chronic exposure. In support of this, the study by Sait et al. (2021) demonstrated that salinity facilitates additive leaching.

Due to the mobility and distribution of MP, it has been distributed ubiquitously throughout the global marine environment (Cai et al., 2023; Li et al., 2023; Tian et al., 2023). Furthermore, in a review by Wagner et al. (2014), they stated that due to specific oceanic hydrology, plastic pollution accumulates in large oceanic gyres (garbage patches), resulting in more MP than zooplankton. Considering this concentration of plastic pollution in oceanic hotspots, together with the significant impact of the leachate observed on A. salina, the risk of leachate from pollution in marine environments to zooplankton is of concern and poses a threat to marine ecosystems.

Microbes, including bacteria, algae, and fungi, are recognized for their role in facilitating the degradation of plastics through both enzymatic and nonenzymatic hydrolysis of polymers (Devi et al., 2016; Amobonye et al., 2021). Even though the collected waters were filtered through a 0.22 µm filter, removing the majority of microbes, some may have passed into the leaching media (Hahn, 2004). Consequently, the increased toxicity leading to immobilization observed with the leachates in fresh- and seawater, compared to the pure water, could, to some degree, be attributed to microbial action facilitating degradation and allowing more toxicants to be liberated from the plastic material. Furthermore, the potential impact of microbial metabolites, such as exopolysaccharides, on oxidative stress and, consequently, overall toxicity should not be underestimated (Kavitake et al., 2022).

Furthermore, POPs, antibiotics, and heavy metals sorb to plastic debris from the surrounding environment (Liu et al., 2023). Therefore, as the fresh and seawater were collected from natural sources, POPs may have been present and could have contributed to increased toxicity in addition to leached chemicals. Thus, when assessing the risk of MP and leachate, it is essential to these chemical toxicants and their potential additive or synergistic effects on the overall toxicity.

One of the most common non-lethal endpoints observed as a measure of toxicity is oxidative stress and the corresponding antioxidant responses (consisting of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), glutathione reductase(GR), glutathione-S-transferase (GST) and other low molecular weight scavengers such as glutathione, ascorbate, vitamin E, β-carotene and proteins) which combat cellular damage by maintaining the homeostasis of ROS (Yu, 1994). Elevated ROS, an indication of oxidative stress, was only observed in daphnids when exposed to the leachate prepared in pure water. However, this was accompanied by a corresponding increase in the SOD activity, which catalysis the conversion of ROS to hydrogen peroxide. Despite CAT not being activated, this daphnid treatment had the lowest corresponding immortality. Thus, hydrogen peroxide generated from SOD may have been made innocuous by other antioxidative enzymes such as peroxidase, glutathione peroxidase or ascorbate peroxidase (Yu, 1994). In neonates exposed to the leachate prepared in freshwater, ROS homeostasis was maintained as is evident by the still lingering elevated SOD activity. A previous study investigating the effects of PS leachate (50 mg/L) on neonate and adult daphnia found that in neonates ROS was not elevated and SOD activity was not affected but CAT activity was suppressed, with no associated mortality. The authors speculated that the non-enzymatic oxidative stress system may have been involved in maintaining homeostasis. In contrast to D. magna’s antioxidant response, the A. salina’s catalase activity was elevated after 48 h of exposure indicating its involvement in maintaining homeostasis and ensuring mobility and survival. This illustrates the vast differences in species’ response to toxicants.

In conclusion, the physiological data shows that both indicator organisms’ antioxidant response systems could maintain homeostasis at low concentrations of the leachate and considering the currently reported average MP concentrations, leachates from plastic do not pose a severe threat to these organisms. Nevertheless, the potential formation of plastic accumulation points, commonly known as “garbage patches,” raises concerns, as these circumstances may lead to elevated concentrations of leachates, potentially resulting in severe adverse effects on native biota, particularly in saline environments.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The manuscript presents research on animals that do not require ethical approval for their study.

ME: Conceptualization, Data curation, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. S-AL: Conceptualization, Formal analysis, Methodology, Resources, Supervision, Writing – review & editing. YK: Conceptualization, Investigation, Methodology, Supervision, Validation, Writing – review & editing. RJ: Formal analysis, Investigation, Methodology, Writing – review & editing. YK: Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review & editing.