Nortriptyline (purity≥99.9%) purchased from Aladdin Co., Ltd. (Shanghai, China). Nortriptyline stock solution (10.0 mg/mL) was prepared by dissolving nortriptyline in ultrapure water. PVC microplastic particles (C, 76.03%; O, 12.77%; Cl, 11.20%; O/C, 0.17) with a particle size of approximately 1 μm were purchased from Jinheng Plastics Co., Ltd. (Dongguan, China). Their physical properties included a relative density ranging from 1.35 to 1.46 and a refractive index of 1.544 measured at 20°C.

C. vulgaris was obtained from Yuanquan Biotechnology Co., Ltd. (Hainan, China) and cultivated in 250 mL conical flasks with sterile BG-11 medium. The half-maximal effective concentration (EC₅₀) of nortriptyline for C. vulgaris was determined as 22.32 mg/L via preliminary experiments. Accordingly, 5 and 25 mg/L were chosen as the nortriptyline exposure concentrations in this study. Additionally, in the co-exposure group, 5 mg/L of PVC microplastics was added alongside nortriptyline. Each treatment group had three replicates, and cultures were maintained at 28 ± 1°C with a light intensity of 3000 lux and a 16 h/8 h light/dark cycle.

The experimental group consisted of C. vulgaris cultured in medium containing 10 mg/L PVC and varying concentrations of nortriptyline. The growth kinetics of C. vulgaris were monitored by measuring optical density (OD) at 680 nm using a Shimadzu UV-2700i spectrophotometer, The OD values were calibrated against dry cell weight based on a standard curve established in our previous study [10]. Photosynthetic pigments, including chlorophyll-a, chlorophyll-b, and carotenoids, were quantified using a methanol extraction method followed by spectrophotometric analysis [11]. The measurement of fluorescence parameters was performed on 3 mL algal suspensions from each group after a 15-min dark adaptation period using a PHYTO-PAM analyzer (Walz, Germany) for the assessment of photosynthetic activity [12].

A 5 mL aliquot of the algal suspension was centrifuged at 5000 rpm for 10 min. The pellet was resuspended in 3 mL of phosphate buffer (PBS, pH 7.2) and subjected to cell disruption via magnetic bead oscillation. Following homogenization, the samples were assayed for catalase (CAT) activity, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, China).

The pellet was collected after initial centrifugation (5000 rpm, 10 min) of 15 mL of algal suspension, then re-suspended in homogenization medium (0.1 mol/L) at a 1:9 (w/v) ratio (pellet:medium) and mechanically homogenized in an ice bath. The resulting mixture was centrifuged at 2500 rpm for 10 min. The supernatant was then collected, mixed with Coomassie Brilliant Blue dye solution, and allowed to stand for 10 min; subsequently, the OD values of each group were measured at 595 nm using a UV-Vis spectrophotometer.

The removal of nortriptyline through biological and non-biological removal was quantified according to our previously established methodology [10]. 10 mL of the C. vulgaris culture exposed to PVC microplastics and nortriptyline was transferred to a centrifuge tube and centrifuged at 9000 rpm for 10 min. The supernatant was collected for measurement of the residual nortriptyline concentration to determine the total removal amount (A_t). Abiotic degradation controls were conducted in the absence of microalgae to account for non-biological removal amount (A_p), specifically including photodegradation and adsorption by PVC microplastics. The biological removal amount (A_b) was calculated according to Eq. (1):

The concentration of nortriptyline is detected by high-performance liquid chromatography [13]. Separation was achieved using an Agilent C18 column (250 mm × 4.6 mm, 5 μm). The mobile phase, composed of acetonitrile and 0.2% triethylamine (40:60, v/v), was adjusted to pH 3.0 with phosphoric acid. Detection was performed at 210 nm.

Data analysis was conducted using Origin 2024, RStudio, and SPSS (version 19.0). Statistical significance across groups was assessed by one-way analysis of variance (ANOVA) with Tukey-Kramer post hoc tests. Differences were considered significant at p < 0.05 and highly significant at p < 0.01.

The effects of varying concentration of nortriptyline and PVC on the dry weight of C. vulgaris during a 7-day cultivation period were shown in Fig. 1(a). The addition of solely 5 mg/L PVC produced a mild stimulatory effect on the growth of C. vulgaris. However, the inhibition rate of C. vulgaris exposed to 5 mg/L nortriptyline for 7 days was 39.2%. Notably, the inhibition rate decreased to 29.6% with the addition of 5 mg/L PVC microplastics, as shown in Fig. 1(b). This detoxification effect of PVC microplastics on nortriptyline was also observed under 25 mg/L nortriptyline exposure conditions, where the inhibition rate of C. vulgaris decreased from 58% to 34% upon addition of 5 mg/L PVC microplastics.

The contents of chlorophyll a, chlorophyll b, and carotenoids are widely used as key indicators to evaluate photosynthetic performance of microalgae [14,15]. Exposure to nortriptyline led to a substantial decrease in the contents of chlorophyll a, chlorophyll b, and carotenoids in C. vulgaris, and this reduction exhibited a concentration of-dependent relationship (p<0.05). Among these photosynthetic pigments, chlorophyll a exhibited the most pronounced decrease, declining from 0.18 to 0.02 mg/g as the nortriptyline concentration of increased from 5 to 25 mg/L (Fig. 2).

The contents of all three photosynthetic pigments were increased by the addition of 5 mg/L PVC microplastics, with more significant increases observed under the high nortriptyline exposure concentration (25 mg/L) than under the low concentration (5 mg/L). After a six-day exposure period, chlorophyll a content decreased to 0.17 mg/g and 0.01 mg/g when exposed to 5 mg/L and 25 mg/L nortriptyline alone, but recovered to 0.22 mg/g and 0.02 mg/g in the corresponding PVC-nortriptyline co-exposure groups.

Similarly, as the concentration of nortriptyline increased from 5 to 25 mg/L, carotenoids levels decreased from 0.079 to 0.029 mg/g. However, in the presence of PVC microplastics, carotenoids contents increased to 0.08 and 0.059 mg/g, respectively. These findings demonstrate that nortriptyline inhibits carotenoid biosynthesis, whereas co-exposure with PVC microplastics enhances and maintains carotenoid production, potentially mitigating nortriptyline-induced stress and improving cellular physiological regulation in C. vulgaris.

Zhou et al. [16] report that the values of Fv/Fm and photosystem II (ΦPSII) can effectively assess the relationship between algal cells and stress factors. As illustrated in Fig. 3(a), the Fv/Fm and ΦPSII values of C. vulgaris in the control group maintained relative stability throughout the 5-day cultivation period. However, Exposure to nortriptyline induced concentration-dependent and time-dependent decreases in these two parameters. Notably, the addition of PVC microplastics significantly amplified these photosynthetic parameters, with the mitigating effects becoming more pronounced over time. Specifically, in groups co-exposed to 5 mg/L nortriptyline and PVC microplastics, the Fv/Fm ratio recovered from 0.3 to 0.6 by day 5, closely approaching the control level of 0.7. As shown in Fig. 3(b), even more strikingly, the ΦPSII value in the 25 mg/L nortriptyline group was restored from a severely inhibited level of 0.05 (15% of the control) to 0.25 upon co-exposure with PVC microplastics, representing a fivefold increase relative to its inhibited state. These results demonstrates that PVC microplastics potentiate the photosynthetic efficiency of PSII by mitigating the phytotoxic effects of nortriptyline on the microalgal photosynthetic machinery.

The photochemical parameters of C. vulgaris photosynthesis after six days of exposure are shown in Fig. 4. Exposure to nortriptyline significantly decreased the values of Ik, α, and ETRmax in a concentration-dependent manner. In contrast, the co-exposure of PVC microplastics and nortriptyline resulted in a discernible increase in all three parameters. Specifically, upon addition of PVC microplastics, the Ik, α, and ETRmax values in the group exposed to 5 mg/L nortriptyline increased by 6.5%, 48.7%, and 63.9%, respectively. Correspondingly, the increases were 13.4%, 36.7%, and 55.2%, respectively, at a higher nortriptyline exposure concentration (25 mg/L). These results indicate that PVC microplastics at low concentrations can effectively alleviate the nortriptyline induced inhibition and enhance the photosynthetic efficiency of C. vulgaris.

Fig.5(a) illustrates the temporal variations in total protein (TP) content of C. vulgaris under different treatment conditions. Compared to the control group, exposure to 5 mg/L nortriptyline resulted in a significant reduction in TP, with a 73.1% decrease observed on day 2 and a further 32.0% decline by day 6. However, the co-exposure of nortriptyline with PVC microplastics partially reversed this trend, leading to a 44.8% increase in TP on day 2 and a 13.0% increase on day 6. In contrast, exposure to 25 mg/L nortriptyline induced a more pronounced reduction in TP. Notably, the addition of PVC microplastics under high concentration of nortriptyline conditions significantly restored TP levels, with increases of 77.5% on day 2 and 78.2% on day 6. These findings demonstrate that nortriptyline exerts a concentration dependent inhibitory effect on the TP content of C. vulgaris. Moreover, the presence of low concentration of PVC microplastics appears to mitigate the detrimental effects of nortriptyline on TP.

As shown in Fig. 5(b) and 5(c), SOD and CAT are two crucial enzymes for cells to mitigate oxidative damage caused by ROS [17]. On the second day of exposure, SOD activity is increased with higher nortriptyline concentrations, indicating that ROS-mediated cellular damage intensifies with elevated nortriptyline concentrations. on days 2, 4, and 6, respectively. However, following the addition of PVC microplastics, SOD activity decreased by 0.5%, 2.4%, and 12.7% on the same days. In the 25 mg/L nortriptyline exposure group, SOD activity rose by 44.4% on day 2 and 23.4% on day 4, but showed a sharp decline of 58.7% on day 6 after PVC microplastics addition. The observed decrease in SOD activity may be attributed to structural modification of the enzyme by excessive ROS, leading to impaired enzymatic function. Consistent with this explanation, the addition of PVC microplastics further reduced SOD activity by 8.3%, 15.0%, and 15.1% on days 2, 4, and 6, respectively. Taken together, these findings indicate that while nortriptyline exposure significantly induces SOD activity as part of the oxidative stress response, the co-presence of PVC microplastics appears to attenuate this response, possibly by mitigating nortriptyline induced ROS generation or through direct interaction with the enzymatic defense system. Moreover, the presence of PVC microplastics further suppressed SOD activity, suggesting that PVC may alleviate the oxidative stress induced by nortriptyline, thereby reducing the demand for enzymatic antioxidant defense. As shown in Fig. 5c, both concentrations of nortriptyline significantly elevated CAT activity in C. vulgaris. Compared with the control group, CAT activity was increased by 19.9%, 12.8%, and 11.3% on days 2, 4, and 6, respectively, following exposure to 5 mg/L nortriptyline. In contrast, for cells exposed to 25 mg/L of nortriptyline, the CAT activity increased by 23.5%, 16.1%, and 13.5% on the corresponding days. Notably, the CAT activity in the groups co-exposed to both PVC and nortriptyline was lower than that in the groups exposed to nortriptyline alone. Throughout the experimental period, relative to the control group, the CAT activity in the group treated with a combination of 5 mg/L PVC and 5 mg/L nortriptyline increased by 11.9%, 9.5%, and 10.0% on the 2nd, 4th, and 6th days, respectively. Similarly, in the group co-exposed to 25 mg/L nortriptyline and 5 mg/L PVC, the CAT activity increased by 19.0%, 8.9%, and 3.1% on these days.

MDA is the final product of membrane peroxidation and serves as an indicator of oxidative and lipid damage induced by ROS [18]. As shown in Fig. 5(d), the effects of nortriptyline and PVC microplastics on the MDA content of C. vulgaris were evaluated. The results indicate that nortriptyline exposure significantly increased the MDA content in C. vulgaris cells, suggesting elevated oxidative stress. By contrast, the addition of PVC microplastics effectively reduced the intracellular MDA content, implying a protective effect against nortriptyline-induced membrane damage. In comparison with the control group, the MDA content in cells exposed to 5 mg/L of nortriptyline exhibited increases of 10.6%, 20.4%, and 33.1% on the 2nd, 4th, and 6th days respectively. Similarly, exposure to 25 mg/L nortriptyline resulted in increased MDA content by 17.4%, 27.0%, and 34.9% on days 2, 4, and 6, respectively. In comparison to the control, cells co-exposed to 5 mg/L nortriptyline and 5 mg/L PVC showed lower increases in MDA content: 7.2%, 23.1%, and 21.0% on days 2, 4, and 6, respectively. Likewise, under co-exposure to 25 mg/L nortriptyline and 5 mg/L PVC, the MDA content rose by 16.8%, 19.0%, and 43.1% over the same period.

The study investigates the effects of low concentration PVC microplastics on the bioremediation of nortriptyline by C. vulgaris, focusing on algal growth characteristics, removal mechanisms, and kinetic processes. Results show that nortriptyline exposure significantly inhibits growth and photosynthetic activity of C. vulgaris while inducing cellular oxidative stress, thereby impairing its bioremediation efficiency for nortriptyline. The results of this study are consistent with previous findings, which indicate that a low concentration of PVC microplastics not only promotes the growth of C. vulgaris but also mitigates the toxic effects induced by nortriptyline [19,20]. As illustrated in Fig. 6, nortriptyline removal efficiency positively correlates with microalgae dry weight and photosynthetic fluorescence parameters, but negatively correlates with intracellular MDA content and CAT activity. Furthermore, a positive correlation was observed between the level of growth inhibition induced by nortriptyline and both CAT activity and MDA content. In contrast, inhibition was negatively correlated with photosynthetic efficiency, as shown by significant reductions in photosynthetic pigment (chlorophyll a, b, and carotenoid) content and a marked decline in fluorescence parameters. These findings indicate that nortriptyline exposure negatively impacts the growth and photosynthesis performance of C. vulgaris, with the severity of the suppression increasing in a dose-dependent manner as the nortriptyline exposure concentration increases. The addition of PVC microplastics not only mitigates the negative effects of nortriptyline exposure but also enhances the growth and photosynthesis performance of C. vulgaris by increasing chlorophyll a and b, and carotenoid levels, this enhancement results in a significant improvement in fluorescence parameters. Yang et al. [21] reported that the maximum growth promotion rates of C.pyrenoidosa were 40.51% in the presence of tire microplastics. The addition of PVC enhances the photosynthetic efficiency of C. vulgaris by promoting the activity of the light reactions and increasing hydrogen production. Additionally, the elevation of chlorophyll a and b, along with carotenoid levels, further optimizes the electron transfer processes in the light reactions and facilitates the transfer of these electrons to the Calvin cycle in the dark reactions, thereby improving overall photosynthetic performance. Zhao et al. [22] reported that growth and photosynthesis-related indicators suggested that polystyrene microplastics exerted a hormetic effect on Haematococcus pluvialis,with short-term exposure stimulating photosynthetic activity and growth. Low concentration of PVC microplastics enhances chloroplast pigments synthesis conditions by improving intracellular material transport and energy metabolism, thereby alleviating nortriptyline-induced inhibition [23].

The alteration in C. vulgaris’ total protein content under stress is a holistic cellular response, encompassing both defense mechanism activation (e.g., boosted stress-related protein synthesis) and metabolic impairment [24–26]. An inhibitory effect on the total protein content of C. vulgaris was observed in response to nortriptyline exposure, demonstrating a clear concentration-dependent manner, with more pronounced suppression at the higher exposure concentration of 25 mg/L. However, this adverse impact was significantly alleviated by the co-presence of PVC microplastics, which consistently reversed the decline in total protein content during both early and late exposure periods. These findings suggest that the toxicity of nortriptyline to algal cells may be mitigated through interaction processes involving microplastics.

Plant cells activate their internal antioxidant defense mechanisms to mitigate the damage caused by ROS under stress conditions. Cellular homeostasis is disrupted under nortriptyline exposure, leading to the accumulation of reactive ROS and subsequent oxidative damage [27–29]. SOD and CAT are two of the most critical enzymes in this antioxidant system. The primary function of SOD is to catalyze the conversion of superoxide anions (O₂⁻) into hydrogen peroxide (H₂O₂) and oxygen (O₂), thereby reducing the damage inflicted by this reactive oxygen species on cellular structures. On the other hand, CAT is responsible for catalyzing the decomposition of H₂O₂ into H₂O and O₂, thereby neutralizing hydrogen peroxide and protecting cells from oxidative damage. The activities of these two enzymes typically increase in response to elevated ROS levels under stress conditions, which helps maintain the intracellular redox balance and mitigate oxidative stress [30]. When C. vulgaris is exposed to nortriptyline, the activities of SOD and CAT in cells increase. Nonetheless, upon further prolonging the nortriptyline exposure, SOD activity in cells exposed to high concentration of nortriptyline declines. This likely occurs because high concentration of nortriptyline exposure generates excessive ROS, which attack the molecular structure of SOD, altering its active center or spatial conformation and thus impairing its activity. As exposure time increased, the ROS induced structural changes in SOD become more marked, leading to a continuous decrease in SOD activity. Similar observations have been reported in studies such as the one by He et al., who investigated the effects of long-term exposure to high concentrations of microplastics on antioxidant enzyme activity in sweet pepper [31]. Moreover, the oxidative stress induced by microplastics stimulates the production of more antioxidant enzymes and other protective compounds in the root system, thereby enhancing its metabolic activity and overall resilience [32].

PVC microplastics might induce a stress response in C. vulgaris, leading to an upregulation of SOD synthesis. When exposed to PVC-induced environmental changes, C. vulgaris may activate defense mechanisms, including enhanced production of antioxidant enzymes to mitigate potential oxidative damage. Moreover, PVC can bind to intracellular receptors and trigger signal transduction pathways that boost the transcription and translation of the CAT or SOD gene, thus augmenting the synthesis and activity [24,33]. Additionally, the formation of complexes between PVC microplastics and metal ions may impair enzymatic activity by altering the availability of metal cofactors essential for SOD function. Furthermore, microplastics can adsorb onto the enzyme surface, potentially inducing conformational changes that lead to further inhibition of enzyme activity [34].

The results of this study demonstrate that the removal efficiency of nortriptyline from water by C. vulgaris decreases with increasing nortriptyline exposure concentration. This phenomenon can be attributed to the strong inhibitory effect of high nortriptyline concentrations on the metabolic activity of C. vulgaris. Higher exposure concentrations cause severe disruption of cellular functions, which in extreme cases may even lead to cell death. The impairment of essential physiological functions, including nutrient uptake, enzyme-mediated biochemical reactions, and cellular respiration processes, effectively obstructs the mechanisms through which C. vulgaris enables the removal of nortriptyline. Consequently, the reaction rate constant experiences a reduction, and in tandem, the half-life of the reaction is correspondingly prolonged [35].

The kinetic process of nortriptyline removal from water by C. vulgaris under various conditions was effectively described by a pseudo-first-order kinetic model. When the drug concentration of in the nortriptyline alone treatment group increases, the reaction rate constant k1 decreases and the half-life increases. In contrast, compared with the nortriptyline alone treatment group, the k1 value of in C. vulgaris co-exposure to the PVC microplastics and nortriptyline increases and the half-life of pollutants decreases, indicating that low - concentration of PVC helps to improve the efficiency of C. vulgaris in removing nortriptyline [36].

Evidently, low concentration PVC microplastics can effectively mitigate the toxicity of nortriptyline towards C. vulgaris, and the adsorption of nortriptyline by PVC microplastics may be one of the reasons for this mitigation. In recent years, research on adsorptive removal technologies for organic micropollutants in aquatic environments has increasingly utilized comparative studies of multiple adsorbents and evaluations under realistic water conditions. Significant progress has been made in the development of efficient and low cost adsorbent materials, as demonstrated in studies on per and polyfluoroalkyl substances adsorption [37], where materials such as activated carbon, ion exchange resins, and modified clays are commonly employed. However, the development of efficient and environmentally friendly water purification technologies for emerging contaminants remains a challenge. Although advanced materials like radiation synthesized hydrogels show unique advantages in adsorbing and removing pollutants [38].

In the present study, the acute toxic effect of nortriptyline on C. vulgaris was partially alleviated in the presence of PVC microplastics. It must be emphasized, however, that this mitigating effect was observed only under the specific conditions of low concentration and larger particle size applied in this experiment. The observed mitigating effect under specific conditions cannot be generalized to imply that microplastics typically reduce the ecological risks of environmental pollutants [39]. In natural aquatic systems, microplastics concentrations are highly heterogeneous, and microplastics themselves remain persistent and multi-faceted environmental hazards. The apparent reduction in toxicity reported here was only evident in this specific co-exposure system under strictly controlled laboratory conditions, and does not contradict the well-documented adverse ecological impacts of microplastics at broader spatiotemporal scales, including long-term ecosystem-level risks and bioaccumulation potential [40–41]. Therefore, the overall environmental risks associated with co-exposure to microplastics and specific antidepressant compounds still require more in-depth and systematic investigation.