Increased bio-toxicity of leachates from polyvinyl chloride microplastics during the photo-aging process in the presence of dissolved organic matter

Microplastics (MPs), consisting of plastic particles or fibers with sizes below 5 mm, have emerged as a global concern due to their potential toxicity. A significant proportion of the millions of tons of plastic produced annually enters the environment and undergoes natural weathering processes, resulting in the formation of MPs and nanoplastics (Wu et al. 2020). These small plastic particles can persist in the environment for extended periods, potentially reaching hundreds of years (Zhou et al. 2021). The harmful effects of MPs on various aquatic organisms, including zooplankton (Botterell et al. 2019), marine mammals (Moore et al. 2020), microalgae (Gao et al. 2023), and benthic foraminifera (Langlet et al. 2020), have been reported. These impacts are not solely attributed to the physical presence of MPs but also to the release of chemicals from the particles. Aging processes can alter the properties of MPs, such as particle size, specific surface area, roughness, morphology, functional groups, and the release of chemicals, thereby influencing the ecological risks associated with these particles (Luo et al. 2022).

Photo-aging is a prevalent degradation process that occurs in plastic materials within aquatic environments. Previous studies have documented the changes in the surface structure that accompany the aging of MPs, including increased surface roughness and the formation of specific functional groups, such as vinyl (C–C) and oxygen-containing groups (–OH, –COOH, and –CHO) (Wang et al. 2023). Additionally, photo-irradiation can lead to the release of organic substances from MPs (Wang et al. 2020; Du et al. 2023). The photo-aging process of MPs can be categorized into direct and indirect photo-aging. The former involves the breakage of intermolecular bonds on the surface of MPs upon exposure to ultraviolet light with wavelengths ranging from 290 to 400 nm (Fan et al. 2021; Zhang et al. 2021). The latter refers to free radical-sensitized photo-aging, which is mediated by chromophores present in the environment or impurities within the polymer (Yang et al. 2018; Wang et al. 2020). Previous research by Wang et al. (2020) indicated that low-molecular-weight organic acids, such as oxalate and citrate, can promote the aging process of polyvinyl chloride MPss (PVC-MPs) and enhance the release of organic chemicals from PVC-MP by generating hydroxyl radicals. Dissolved organic matter (DOM), which is widespread in aquatic environments, often acts as an important photosensitizer. The photochemical degradation of organic pollutants through photolysis sensitized by DOM has been extensively studied in recent years. During this process, reactive oxygen species (ROS) such as hydroxyl radicals (^·OH), singlet oxygen (¹O₂), and triplet states of DOM are formed under sunlight exposure (Chen et al. 2016). These ROS also play a crucial role in the degradation and aging of MPss (Duan et al. 2022; Liu et al. 2022).

Despite the ubiquitous presence of DOM in surface water, limited research has been conducted on its potential influence on the photo-aging behavior of MPs and the resultant ecological risks. The objective of this study was to investigate the impact of humic acid (HA), a common type of DOM, on the photo-aging process of PVC-MPs and the resulting toxicity of leachates. PVC was selected as the target material due to its widespread usage and presence in water bodies, as well as its greater susceptibility to embrittlement and decomposition compared to other MPs (Browne et al. 2010; Wu et al. 2019). Considering the complex composition and low concentration of chemicals leaching from PVC-MP, the potential risk of the leachate during the photo-aging process was evaluated by the bioassay. The results of this study will enhance our understanding of the photo-aging process of MPs in natural waters and the associated ecological risks.

PVC particles with a diameter of 75 μm were obtained from Jiaquan Plastic Co., Ltd (Dongguan, China). HA bought from Sigma-Aldrich (USA) was dissolved in ultrapure water and filtered through a 0.45-μm membrane to prepare a stock solution. The concentration of the HA stock solution, measured in mg C/L, was determined using a carbon analyzer (Shimadzu, Tokyo, Japan). Ultrapure water with a resistivity of 18.2 MΩ⋅cm was generated using a Milli-Q water purification system (Millipore).

The photo-aging experiments were performed using 30 mL quartz glass tubes as reaction vessels. The reaction solution consisted of PVC-MP at a concentration of 100 mg/L, with the option of incorporating either 0 or 5 mg/L HA, along with ultrapure water. The initial pH of each solution was adjusted to 7.0 using hydrochloric acid or sodium hydroxide. Subsequently, the reaction tubes were placed within a photochemical reactor (YM-GHX-V, Yuming Instrument Company, Shanghai, China) equipped with a 500 W central xenon lamp in the center. The reaction solutions were subjected to magnetic stirring, while a constant temperature of 25 °C was maintained throughout the experiment using a recycled cooling water system. Control experiments were also conducted concurrently, employing tubes covered with aluminum foil within the reactor. After an irradiation period of 72 h, the aged PVC-MPs were collected by centrifugation at 6,000 rpm for 10 min and subsequently freeze-dried. Fourier transform infrared spectroscopy (FTIR) analysis was then conducted, utilizing a Nicolet iS 10 instrument (USA), to identify and evaluate the functional groups within the 4,000–500 cm⁻¹ range, employing KBr pellet technology. The supernatants obtained from the centrifugation step were collected for subsequent bioassay tests.

The acute toxicity assessment was conducted using the bioluminescence inhibition assay, as described by Villa et al. (2014) and Chen & Wang (2018). Ultrapure water served as the blank control in the experiment. Prior to the test, the freeze-dried bacteria Vibrio qingaiensis sp. nov. (Q67) was reconstituted. In each quartz tube, 0.1 mL of the test solution was mixed with 0.9 mL of the cultivated bacterial suspension. Subsequently, the luminator (Berthold, Germany) was employed to measure the relative light unit of each test medium and the blank control after a 20-min exposure. The degree of light emission inhibition, which was calculated by comparing the relative light unit (RLU) of each test medium to that of the blank control, was used to determine the acute toxicity of the test solutions.

The genetic toxicity of each test solution was assessed using the Vicia faba roots tip micronucleus assay, following the methodology described by Chen & Wang (2018). Dry broad bean (V. faba) seeds, which were obtained from the Central China Normal University and harvested from an unpolluted area, were utilized for the assay. The seeds were soaked and germinated at 25 °C until roots measuring 2–3 cm in length emerged. For each test solution and the blank control (ultrapure water), five germinated seeds with uniform root growth were exposed for a duration of 6 h, followed by cultivation at 25 °C for 24 h. Subsequently, the root tips of the beans were excised, fixed using Carnoy liquid (a mixture of acetic acid and ethanol in a ratio of 1:3), stained using the Feulgen staining technique, and finally mounted on slides. The number of micronuclei observed on each slide was counted under a microscope. The genetic toxicity was evaluated based on the frequency of micronuclei, which was represented as the average number of micronuclei per 1,000 cells in each root tip.

Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) version 16.0 (USA) for Windows. To identify significant differences, a one-way analysis of variance (ANOVA) was employed. Statistical significance was considered when p < 0.05.

Moreover, the intensities of both C = O and O–H were observed to be higher in the presence of HA, suggesting that the presence of HA enhanced the photo-aging process of PVC-MP. Only a few studies have explored the influence of environmental factors such as salinity, low-molecular-weight organic acids, and clay minerals on the photo-aging of MPs (Cai et al. 2018; Wang et al. 2020; Ding et al. 2022). However, the impact of DOM in surface water has received relatively little attention. Wang et al. (2020) demonstrated that the presence of low-molecular-weight organic acids could accelerate the photo-aging process of PVC-MP under both simulated and natural sunlight irradiation, primarily due to the formation of •OH radicals. Similarly, HA, being a type of DOM, contains various chromophores that can absorb light and generate reactive oxygen species (ROS), including ³HA*, •OH, and ¹O₂, which might contribute to the enhanced photo-aging of PVC-MPs.

Photo-aging plays an important role in the degradation process of MPs in the natural environment, which usually involves free radical-mediated reactions initiated by solar irradiation (Zhang et al. 2021). Specifically, when exposed to light irradiation, PVC experiences rapid dehydrochlorination, leading to the formation of short sequences of conjugated unsaturation within the polymer. These unsaturated sequences are susceptible to further photodegradation (Ainali et al. 2021). In cases where PVC contains chromophores due to impurities or low-molecular-weight organic acids, photosensitized photo-aging may occur, resulting in the formation of polyene structures. These structures can then be oxidized to ketones and alcohols by O₂ and •OH radicals (Wang et al. 2020). Given the ubiquitous presence of HA in the natural aquatic environments, it is likely that HA promotes the photo-aging process of PVC-MP by generating ROS, thereby influencing the fate and ecological risk associated with PVC-MP.

Previous studies have demonstrated the toxic effects of leachates from MPs on various organisms, including the sea urchin Lytechinus variegatus, blue mussel Mytilus edulis, benthic foraminifera Haynesina germanica, and so on (Nobre et al. 2015; Langlet et al. 2020; Uguen et al. 2022). The observed toxicity of these leachates was primarily attributed to the presence of chemical additives in the plastic debris. These additives are compounds added to enhance the performance and functionality of plastic products, and they can readily leach from the polymer matrix into aquatic environments when plastic waste enters such ecosystems (Oliviero et al. 2019). Certain organic additives, including plasticizers, flame retardants, stabilizers, antioxidants, and pigments, are known to have carcinogenic, mutagenic, or endocrine-disrupting properties toward aquatic organisms (Groh et al. 2019; Do et al. 2022). Therefore, it can be inferred that the observed toxicity of PVC-MP leachates in the absence of light is likely caused by the release of the additives present in the plastic particles.

The bio-toxicity of leachate derived from MPs exhibited a significant increase after exposure to light irradiation, in comparison to the dark condition. Previous research has consistently reported an escalation in toxicity for leachates derived from aged MPs. Rummel et al. (2019) conducted cell-based bioassays and observed an increased induction of oxidative stress response when exposed to leachates derived from UV-weathered MPs. However, Bejgarn et al. (2015) reported different findings that leachate from four plastics became more toxic after light irradiation, while two became less toxic and two did not change significantly, depending on the types of plastic products. The increased toxicity was mainly attributed to the enhanced release of plastic additives under irradiation (Bejgarn et al. 2015; Luo et al. 2020). Besides, during the photo-aging process of PVC-MP, the degradation of the polymer can result in the release of organic substances. Recent studies have demonstrated the generation of microplastic-derived DOM during the weathering processes of MPs, including photo-aging and biodegradation (Miri et al. 2022; Ouyang et al. 2023). The MP-derived DOM primarily consists of monomers, oligomers, and oxygenated intermediates derived from MPs, which have been identified as emerging environmental hazards and toxic to organisms (Abolghasemi-Fakhri et al. 2019; Xu et al. 2022).

In the presence of HA, the toxic effects of the leachates were found to be significantly intensified. Light irradiation can lead to the production of ROS such as ³HA*, •OH, and ¹O₂. It has been established that ROS could promote the aging of MPs and facilitate the release of microplastic-derived DOM (Lee et al. 2020; Xu et al. 2022), thereby increasing the bio-toxicity of the leachate. However, to date, no research has specifically investigated the impact of DOM on the ecological risk associated with MPs.

The process of photo-aging plays a crucial role in the aging of MPs within aquatic environments; thus, it is necessary to address the ecological risks associated with this phenomenon. Given the intricate composition and low concentrations of leachates, bioassays offer a cost-effective approach for evaluating the ecological risks involved. In comparison to the blank controls, PVC-MP leachates exhibited statistically significant acute and genetic toxicity under both dark and light irradiation conditions. Notably, the toxicity of the leachates was considerably higher under light irradiation, suggesting a greater release of toxic chemicals during the photo-aging process. Furthermore, the presence of HA was found to enhance the aging process and intensify the ecological risk associated with the leachates. Considering the ubiquitous presence of DOM in surface water, it is imperative to account for its potential to promote microplastic photo-aging and the resulting increase in bio-toxicity.

This work was supported by the National Natural Science Foundation of China (grant no. 42077323) and the College Students' Innovation and Entrepreneurship Training Program of Jiangsu Province (grant no. 202210298026Z).

L.C. conceptualized the work, wrote and reviewed the edited manuscript, and supervised the work. H.Q. wrote the preparation of original draft and visualized the work. K.Y. performed data curation and did software techniques. B.G. investigated the work, analyzed the methodology, and validated the work.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.