A microalga called Scenedesmus sp. employed in this investigation was extracted from pond water samples that were taken (3°05′24.00″ North, 80°16′12.00″ East). A 0.1-milliliter water sample was introduced into Petri plates containing sterilized BG-11 agar medium. Sodium nitrate (NaNO₃): 1.5 grams; Dipotassium hydrogen phosphate (K₂HPO₄.3H₂O): 0.04 grams; Magnesium sulfate (MgSO₄.7H₂O): 0.075 grams; Calcium chloride (CaCl₂.2H₂O): 0.036 grams; Citric acid: 0.0006 grams; Ferric ammonium citrate (C₆H₁₁FeNO₇³⁺): 0.006 grams; EDTA (Disodium magnesium salt): 0.001 grams; Sodium carbonate (Na₂CO₃): 0.01 grams; A5 solution: 1.0 milliliter which contains Boric acid (H₃BO₃): 2.86 grams; Manganese(II) chloride (MnCl₂.4H₂O): 1.81 grams; Zinc sulfate (ZnSO₄.7H₂O): 0.222 grams; Sodium molybdate (Na₂MoO₄.2H₂O): 0.39 grams; Copper sulfate (CuSO₄.5H₂O): 0.79 grams; Cobalt nitrate (Co (NO₃)₂. 6H₂O: 0.0494 g and pH: 8.0) [29]. The water was then dispersed over the surface of the medium using a sterile glass rod. The medium was incubated for seven days at a temperature of 28 ± 2°C. The cultures were exposed to 2500 Lux of light intensity at the plate surface using a tubular fluorescent lamp (PHILIPS Master 85 W/840). Individual colonies were isolated from the medium and re-cultivated on fresh medium under the previously described conditions. To purify the chosen microalgal species many sub-culturing was carried out using fresh medium. Also, to prevent bacterial contamination the subculturing was carried out with the addition of an antibiotic mixture (streptomycin 30 ppm and 30 ppm tetracycline 30 ppm) [30].

The polymers (polyethylene and polypropylene) utilized for the study were obtained from the Central Institute of Petrochemicals Engineering and Technology (CIPET) in Chennai, India. The procured MPs were sorted out into sizes greater than 500 μm and less than 500 μm using a standard sieve of the size of 500 μm and the microalgal Scenedesmus sp. was exposed to different concentrations of MPs: 50 mg L⁻¹, 100 mg L⁻¹, and 150 mg L⁻¹. The varying concentrations of MPs were prepared weighing specific amounts of MPs (50 mg, 100 mg, and 150 mg) and added to 1 L of distilled water. The suspensions were thoroughly mixed using a magnetic stirrer and then sonicated for 5 min for better dispersion. The experiment consisted of the following treatments: T1: PP 150 mg L⁻¹ (>500 μm); T2: PP 150 mg L⁻¹ (<500 μm); T3: PE 150 mg L⁻¹ (>500 μm); T4: PE 150 mg L⁻¹ (<500 μm); T5: PP+PE 150 mg L⁻¹ (>500 μm); T6: PP+PE 150 mg L⁻¹ (<500 μm); T7: PP 100 mg L⁻¹ (>500 μm); T8: PP 100 mg L⁻¹ (<500 μm); T9: PE 100 mg L⁻¹ (>500 μm); T10: PE 100 mg L⁻¹ (<500 μm); T11: PP+PE 100 mg L⁻¹ (>500 μm); T12: PP+PE 100 mg L⁻¹ (<500 μm); T13: PP 50 mg L⁻¹ (>500 μm); T14: PP 50 mg L⁻¹ (<500 μm); T15: PE 50 mg L⁻¹ (>500 μm); Tl6: PE 50 mg L⁻¹ (<500 μm); T17: PP+PE 50 mg L⁻¹ (>500 μm); T18: PP+PE 50 mg L⁻¹ (<500 μm). For treatments T5, T6, T11, T12, T17, and T18 the PE+PP mixture was prepared in the ratio of 1:1. The ratio was designed to depict the variation in MPS concentration in the aquatic environment. Subsequently, the MPs of various concentrations were added to the medium (BG-11) and kept for 7 days.

A 150 mL volume of BG-11 medium was prepared in 250 mL Erlenmeyer flasks made of borosilicate glass. The flasks were then subjected to autoclaving at a temperature of 121°C and a pressure of 15 lbs for 15 minutes. Subsequently, one milliliter of Scenedesmus sp. was transferred into the flask and incubated under light. The cells were harvested using a centrifuge (Remi RM-02 Plus Mini Centrifuge, India) and resuspended in sterile distilled water after seven days. Each flask containing BG-11 medium with MPs previously added was supplemented with 1 milliliter of cell suspension, containing 1 × 10⁸ Colony Forming Units (CFU). The flasks were thereafter placed in an incubator and subjected to light for 72 hours. The incubator was equipped with a rotary shaker operating at a speed of 100 revolutions per minute. The temperature within the incubator was maintained at 23 ± 2°C. For each treatment, three replications were maintained. Also, adequate control without any inoculation was kept. Following 72 hours, samples from each treatment were serially diluted and inoculated on a solid BG-11 medium. The samples were then incubated under illumination for 7 days. After 7 days, the proliferation of cells, which indicates a measurable quantity, was seen and recorded.

To investigate the impact of various concentrations of MPs on photosynthetic pigments, the MPs were introduced into BG-11 broth and inoculated with microalga Scenedesmus sp. Subsequently, the flasks were incubated under the light in a rotary shaker. Further, a control was maintained without adding MPs. After 20 days, a volume of 5 mL was extracted from each treatment and subjected to centrifugation at a speed of 12,000 rpm for 2 minutes using a microcentrifuge (Remi RM-02 Plus Mini Centrifuge, India). The liquid portion was removed, and the solid portion was mixed with 5 mL of pure acetone. Subsequently, the pellet was suspended in a liquid and subjected to sonication using an ultrasonicator (QSonica, USA) for 5 minutes at room temperature. This was then followed by incubation at a temperature of 4°C for 24 hours without light. Afterward, the samples were centrifuged in an incubator shaker (Remi RM-02 Plus Mini Centrifuge (India)) at 12,000 rpm for 2 minutes to separate the liquid portion above. The supernatant was analyzed for absorbance at wavelengths of 470.0 (A₄₇₀) nm, 644.8 (A_644.8) nm, and 661.6 (A_661.6) nm to determine the concentrations of chlorophyll-a, chlorophyll-b, and carotenoids, respectively using a UV/VIS spectrophotometer (UV 3200, Labindia Analytical). The chlorophyll-a, chlorophyll-b, total chlorophyll, and carotenoids were calculated using Eq. (1)., Eq. (2)., Eq. (3)., and Eq. (4) respectively [31].

The quantity of phthalates released from MPs in the growing media was quantified. BG-11 medium was supplemented with 150 mg L⁻¹ of PP (>500 μm) and PE (>500 μm) and inoculated with Scenedesmus sp. After 7 days of incubation, one milliliter of the growth medium was withdrawn and extracted the phthalates using a mixture of 20 milliliters of hexane and dichloromethane in equal proportions (1:1, volume to volume). This extraction process was carried out in an ultrasonic bath operating at a frequency of 40 kHz and a power of 1100 W, at a temperature of 40°C for 20 minutes. Phthalates were measured using a gas chromatograph/mass spectrometer (Agilent 7890 B-7000 C, Agilent Technologies, USA) with electron ionization in the specified ion monitoring mode [32].

The Scenedesmus sp. was inoculated into BG-11 medium, which included PP (150 mg L⁻¹) and PE (150 mg L⁻¹) with a size of more than 500 μm. The mixture was then left to incubate for a period of 20 days. After 20 days, the MPs were retrieved using centrifugation at 1,500 rpm for 10 minutes. They were then delicately washed with 0.5 mL of phosphate-buffered saline (PBS). The retrieved MPs were then placed back into the same solution (0.5 mL of PBS) along with a solution of Nile red (9-(Diethylamino)-5H benzo [α] phenoxazin-5-one) (0.1 mg mL⁻¹ in acetone). The stained microalgal cells attached to MPs were examined using a fluorescent microscope (Olympus BX40) with a color CCD digital camera (DP12, Olympus). The microscope was set with a 450–490 nm excitation filter, a 505-nm diachronic mirror, and a 520-nm barrier filter. The examination was performed using a 40× objective lens. The digitized color images were transformed into grayscale illustrations using computer software.

An analysis of variance (ANOVA) was employed to examine the effect of different types and concentrations of MPs on growth (CFU) and production of photosynthetic pigments (chlorophyll and carotenoids) of microalgal species Scenedesmus sp. The ANOVA and correlation analysis was performed using XL-Stat Pro Software. The Principal Component Analysis (PCA) illustrates the relationship between the variable chlorophyll (Chlorophyll a, Chlorophyll b, Total Chlorophyll), total carotenoids, and CFU and the different treatments. The blue-colored arrows represent the variables. Each arrow points in the direction where that variable has a stronger influence.

The growth of Scenedesmus sp. was assessed using different sizes (<500 μm and >500 μm) and concentrations (50, 100, and 150 mg L⁻¹) of PP and PE. At a concentration of 50 mg L⁻¹, a maximum population of 34 × 10³ CFU mL⁻¹ was observed when the size of PP was <500 μm. Similarly, when the size of PP was >500 μm, the algal population was maximum when the concentration was 50 mg L⁻¹. The highest algal population of 27 × 10³ CFU mL⁻¹ and 23 × 10³ was noted when the PP sizes were <500 μm and >500 μm respectively at 50 mg L⁻¹. Also, combining PP and PE (1:1) the growth of algal population was found to be maximum at lower concentrations. The algal population at 50 mg L⁻¹ concentration was 30 CFU mL⁻¹ and 30 CFU mL⁻¹ when the size was <500 μm and >500 μm respectively. However, the reduction in growth was variable and showed no stability among various treatments. Similarly, the growth at higher concentrations (100, 150 mg L⁻¹) did not show a significant reduction compared to lower concentrations (50 mg L⁻¹) (Fig. 1).

Microalgae are abundant and vital in freshwater ecosystems, playing crucial roles in food webs, primary production, and nutrient cycling [16]. These indicators are important for assessing environmental health and water quality because of the complex interplay between living and non-living factors that influence their distribution in ecosystems [33]. In the present study, the reduction in the growth of microalga Scenedesmus sp. was observed when exposed to MPs agrees with previous studies [34–37] Studies have shown that polyethylene and polyvinyl chloride microplastics reduce growth due to physical blockage and chemical stress Ansari et al. [38]. In the present study, even at higher concentrations of MPs, microalgal growth inhibition was minimal compared to lower concentrations. The growth of the microalgae population in the presence of MPs can be attributed to their use of MPs as a substrate [39]. Additionally, microalgal species exhibit adaptive mechanisms such as the production of exopolysaccharides, lipids, and pigments when exposed to stressors like salt, heavy metals, hydrocarbons, and microplastics, as explained by Zhao et al. [40].

In the present study, the effect of particle size, concentration, and polymer type on the total chlorophyll and total carotenoid levels was investigated and the results are presented in Table S1 and Fig. 2. Considering the polymer type PP, PE, and mixture of PP and PE (1:1), the chlorophyll level decreased consistently for each polymer type with an increase in concentration from 50 mg L⁻¹ to 150 mg L⁻¹ for both particle sizes (<500 μm and >500 μm) and the total carotenoid level was relatively same for concentrations 50 mg L⁻¹ and 100 mg L⁻¹. However, the carotenoid level was decreased at 150 mg L⁻¹ for both particle sizes (<500 μm and >500 μm). The observed stability in carotenoid levels of Scenedesmus sp. at concentrations of 50 mg L⁻¹ and 100 mg L⁻¹ across the MP size ranges of > 500 μm and < 500 μm suggests that these concentrations are not enough to induce significant changes in carotenoid production. However, at 150 mg L⁻¹, the reduction in total carotenoid levels indicates that higher concentrations lead to inhibited synthesis of carotenoids. Considering the sizes, the total chlorophyll and carotenoid level was less for particle size <500 μm than the particle size >500 μm. The findings indicate that the decrease in total chlorophyll and carotenoid concentration in microalgae varies significantly depending on size. Compared to particles >500 μm, microplastic particles <500 μm exhibited a higher percentage reduction in these pigments. These results indicate that a combination of particle size and concentration affects the quantity of carotenoid and chlorophyll. However, it is important to note that the total chlorophyll (3.075 mg L⁻¹) and total carotenoids (0.704 mg L⁻¹) of control when compared with microalgae Scenedesmus sp. exposed to microplastics, showed considerable reduction in their levels. For chlorophyll, the highest percentage reduction of 88% was seen in PE (size < 500 μm) at a concentration of 150 mg L^−1, and the lowest percentage reduction of 16.9% was seen in PP+PE at size > 500 μm (concentration: 50 mg L⁻¹). Considering carotenoids, the highest percentage reduction was seen in PE (size < 500 μm) at a concentration of 150 mg L⁻¹ (88%), and the lowest concentration of 15.4% was seen in PP >500 μm at a concentration of 50 mg L⁻¹. This indicates that regardless of the size of MPs and concentration, microplastic exposure negatively impacted these essential photosynthetic pigments.

Microalgae primarily carry out photosynthesis by using chlorophyll [41]. Photosynthesis is a photochemical process that utilizes light energy to drive a redox reaction, converting carbon dioxide into carbohydrates and simultaneously releasing oxygen [42]. Pollutants such as MPs primarily affect photosynthesis and ribosomes [43]. The reduction in total chlorophyll and carotenoid in this study could be due to the microplastics that can adhere to the algal cell surface causing a reduction in light absorption leading to lower photosynthetic activity and consequently reduction in chlorophyll synthesis. According to Li et al. [44], the microalgae Chlamydomonas reinhardttii exposed to polystyrene microplastics reduced photosynthetic activities and according to Tunali et al. [36] higher concentrations of polystyrene microplastics of size 0.5μm reduced the chlorophyll content of C. vulgaris. According to Ansari et al. [38] the photosynthetic efficiency of microalga Acutodesmus obliquus reduced as the microplastic concentration (HDPE, PP, and PVC) increased from 0 mg L⁻¹ to 250 mg L⁻¹ during the early log phase. Accordingly, the present study also shows that the chlorophyll content decreases with an increase in the microplastic concentration. MPs can stick to and accumulate on the outer surface of microalgae. Physical contact can hinder the transfer of gasses and the absorption of vital nutrients, which might affect the metabolic processes of the algae, including the synthesis of carotenoids [19].

The addition of MPs with Scenedesmus sp., several phthalates were detected, including dibutyl phthalate (389.38 ng mL⁻¹) benzyl butyl phthalate (0.81 ng mL⁻¹) and diisobutyl phthalate (3.92 ng mL⁻¹) (Fig. S1). Dibutyl phthalate had the highest concentration among all the phthalates, with diethyl phthalate ranking second. Small chemical compounds called phthalates are sporadically incorporated into plastic polymers by van der Waals forces or hydrogen bonds as opposed to covalent interactions [45]. This makes it possible for them to be readily absorbed by the environment during the processing and deterioration of plastic [46]. Additionally, phthalates and other plasticizers with low molecular weights (300–600 g mol⁻¹) may be easily released from plastics into the environment [47]. Furthermore, it was discovered that plastic additives enter the environment through a process called diffusion, which is impacted by the polymers’ porosity and thickness, the additives’ molecular weight and hydrophobicity, the matrices’ surrounding characteristics, weathering, and aging processes [48]. Additionally, as plastics age, their polymer chains break, which promotes the leaching of additives from the parent materials [48]. Phthalates and other plasticizers are liberated from their parent plastics and end up as plastic waste in the environment. They continue to exist as persistent and stable organic contaminants in this form [49]. Fricks’ law governs all of the processes involved in additives leaching from the parent material, including sorption, desorption, diffusion, and adsorption/dispersion [45]. According to Li et al. [50] the combined effect of polystyrene microplastics and dibutyl phthalate induced little toxicity on the microalga Chlorella pyrenoidosa. According to Liao et al. [51] the accumulation of di(2-ethylhexyl) phthalate in C. Pyrenoidosa in freshwater amplified through trophic transfer and had negative impacts on the organism. The predominant phthalates released from the PE bags were di-isobutyl phthalate (DiBP) and di-n-butyl phthalate (DnBP). The amounts of the substances were discovered to be 83.4±12.5 and 120.1±18.0 ng g⁻¹ of plastic, respectively [46]. A study found that exposure to PVC reduced the growth of the microalgae Dunaliella tertiderta due to plastic additives (namely phthalates) emitted by MPs. Li et al. [52] stated that phthalate esters (PE) have a low affinity for plastics and polymers, resulting in their susceptibility to leaching into the environment. Cunha et al. [53] demonstrated that DBP had the most significant influence on Scenedesmus sp. The growth rate within the initial 48 hours of exposure was determined to be 41.88 μg L⁻¹, as shown by the EC₅₀ value. On the contrary, a comprehensive investigation conducted over an extended time showed that the interaction between HDPE (250 mg L⁻¹) and PP (100 mg L⁻¹) had no discernible effect on the growth and development of microalgae [44].

The findings revealed that there were no statistically significant differences in the levels of chlorophyll content (p-value = 0.99) or colony-forming units (CFU) (p-value = 0.99) across varied concentrations of MPs. Despite the study’s lack of sufficient statistical proof, it nonetheless contributed to our comprehension of the effects of MPs on Scenedesmus sp. The findings revealed that the examined concentration range of MPs did not have a substantial effect on the chlorophyll content or growth of Scenedesmus sp. The axis in Fig. 3 representing CFU extends to the right side of the image. Treatments 4 and 11 significantly influence elevated CFU values, facilitating microbial growth in reaction to increased microplastic concentrations. The arrows for chlorophyll a, chlorophyll b, and total chlorophyll point to the left, indicating that treatments 3, 8, and 16 on the left side have higher chlorophyll values, while the arrow for total carotenoids points towards treatment 5, which has more carotenoids level indicating that these treatments have less impact on pigment synthesis. The responses of treatments close to the origin, like 1, 9, and 10, to the variables (chlorophyll and carotenoids) were average or neutral. This means that multiple factors had an equal effect on the treatments. Also, treatments 2, 7, 14, 15, and 17 cluster together, indicating they have similar profiles across the variables analyzed equally impacting microbial growth and pigment production. Overall, treatment 4 (size < 500μm) is strongly associated with higher microbial growth whereas treatment 5 (size > 500μm) is more associated with high total carotenoids. Overall, treatment 4 is strongly associated with CFU, whereas treatment 5 is more associated with total carotenoids. Nevertheless, this work highlights the need for more investigation to determine the enduring and indirect effects of MPs on microalgae.

Fig. 4 displays the photomicrograph and fluorescence micrographs of Scenedesmus sp. and Scenedesmus sp. colonized on polyethylene (PE) and polypropylene (PP). The photomicrographs revealed that the chloroplasts of algal cells exhibited a green color when examined under an optical microscope and a red color when observed under a fluorescent microscope.

Multiple studies have shown that MPs may be colonized by a wide range of species through the biofouling process [16, 54, 55]. The polymer type is a critical determinant as it directly influences the pace of development, the concentration of photosynthetic pigments, and the extent of oxidative stress [55]. Lagarde et al. [56] discovered that the initial interaction between microalgae and MPs bigger than 400 μm did not directly impact the development of microalgae. Microalgae synthesize and excrete high molecular weight polymers known as extracellular polymeric substances (EPS) [53], which can enhance the proliferation of adhered microalgae [57, 58]. The colonization of microalgae on microplastics is influenced by surface texture since smooth-surfaced microplastics are less prone to colonization compared to those with rough surfaces [59]. In the current investigation, Scenedesmus sp. colonization on rough-surfaced PP was more common than on smooth-surfaced PE. Moreover, these coarse surfaces often provide ideal conditions for the formation of biofilms [60]. The gaps created by MPs favor the availability of nutrients and light thus improving the colonization of microalgae [57]. The adherence of microalgae to MPs increases the density of the colonized polymer, hence affecting the vertical displacement of plastics [61]. Furthermore, the process of biofilm growth can lead to the deterioration of the plastic surface, hence accelerating the release of chemicals associated with plastic production [62]. MPs and chemicals produced by plastic might potentially accumulate in the biofilms that develop on plastic waste, serving as a temporary reservoir for these substances. Kumar et al. [63] found that this can contaminate the food supply for grazing creatures at the bottom of aquatic food chains.