Preparation of microplastic particles
The polypropylene bags were purchased from the local market (51-micron thickness; density-0.85 – 0.92 g/cm3) Karaikudi, Tamil Nadu. Polypropylene bags were cut into small pieces and they were sterilized using Tween 20 solution followed by 70% ethanol. Then it was boiled with xylene at 60 °C and obtained solute was air-dried, washed with distilled water and kept in a hot air oven at 60 °C up to evaporation of water (Das and Kumar 2015). Finally, this powdered polypropylene microplastic was filtered through a 50µm pore size filter.
Preparation of microplastic particles for toxicity assessment
Microplastic particles were immersed in 4% crystal violet and vortexed well (Supplementary figure 1) and elimination of extra stain by frequently washing with sterile water and dried in a hot air oven. Then 1000mg /L concentration stock suspension was prepared and this stock suspension was sonicated for 20 minutes and further, this solution was stirred at 600rpm for 20 minutes.
Physiochemical characterization of Polypropylene microplastic samples
Polypropylene plastic was identified by Attenuated Total Reflection-Fourier transform infrared spectroscopy (ATR- FTIR) for commercially available polypropylene film and Fourier transforms infrared spectroscopy (FTIR) for powdered polypropylene microplastic.10 milligrams of microplastic particles were mixed with potassium bromide and make into a pellet. The pellet was kept in a sample holder and scanned with 4500cm-1 – 400 cm-1. Field Emission - Scanning Electron Microscope (FE-SEM) was used to determine the polypropylene microplastic's surface morphology and their particle size. FTIR and SEM analysis characterized powdered polypropylene microplastic samples.
Culture conditions of Daphnia similis
D. similis was used as test organisms for evaluating the polypropylene microplastic toxic effects. In the present study, D. similis stock culture was maintained in our laboratory for approximately three months before the study. Culture organisms alternative feed with blue-green algae and yeast for maintained at 22 ±2°C and pH – 7.2 – 7.8 in a photoperiod of 16:8 (light: dark). Water renewed twice for a week. The D. similis culture was maintained by following the previous method of Vijayakumar et al. (2018).
Acute toxicity experiment
Acute toxicity test was conducted according to OECD test guidelines 202 (2004). Newborn neonates of D. similis less than 24 hours old were selected and was transferred to a glass test container with microplastic particles. Controls contained only test medium without polypropylene microplastics. Three repetitions per treatment were done. In each container, add 20 animals and were exposed to different concentrations (0, 12.5, 25, 50, 100 mg/L) of microplastic added to the test medium. Acute toxicity study was conducted for up to 24 hours. (Previously, we experimented on polypropylene microplastic exposure for up to 48 hours. As results show that (Data not shown) animals were destroyed. Ingestion, accumulation and adherence of polypropylene microplastic particles can be visualized under the stereomicroscope.
Biochemical parameter analysis
After 24 hours of exposure, experimental water was filtered and collect the animals. Collected animals were rinsed with deionized water and ice-cold 0.1M phosphate buffer saline (PBS - pH7.4) to obliterate adherent microplastic particles. Then, these animals were homogenized using 1.5 mL of ice-cold PBS (pH 7.4) for 10 minutes and centrifuged at 10,000 rpm for 15 minutes (Galhano et al. 2020). The supernatant was collected and stored at -20°C used for further enzyme analysis.
The biochemical markers associated with antioxidant enzyme activity (Catalase, CAT; Glutathione peroxidase, GPx; Superoxide dismutase, SOD), biotransformation (Glutathione S- transferase, GST), non-enzyme antioxidant activity (Reduced glutathione, GSH) and neurotransmission (acetylcholinesterase, AChE), oxidative stress (Lipid peroxidation, LPO; Carbonyl protein, CP), were determined. The protein content of the D. similis was determined by the dye-binding method of Bradford (1976) using bovine serum albumin as standard.
Antioxidant and Non- antioxidant enzyme activity
SOD activity was done by following the method of Suzuki (2000). Absorbance was measured at 560nm and expressed as U/mg of protein. Enzyme CAT activity was determined by the following method of Cohen et al. (1970). Catalase enzyme can decompose substrate hydrogen peroxide into hydrogen and water and monitor at 240nm in ELISA reader and expressed in U/mg of protein. GPx activity determines the NADPH oxidation presence of H2O2 and reduced glutathione (GSH) following the procedure Habig et al. (1974) and measured at 340nm. This activity was represented as U/mg of protein. GST activity was determined by Habig et al. (1974) method using the substrate as 1-chloro 2,4- dinitrobenzene (CDNB) for 96 well microtiter plate and measured at 340nm. Unit of GST activity denoted in U/mg of protein. Reduced glutathione (GSH) was measured by adding DTNB formed yellow colour and absorbed at 412nm. This procedure followed Jollow et al. (1974) and their concentration expressed at µmol GSH mg-1.
Oxidative stress effects on protein and lipid
LPO was determined by measure the amount of thiobarbituric acid and malondialdehyde complex, from which thiobarbituric acid was used as a substance, followed by Buege and Aust (1978). The spectrophotometrically was measured at 532nm and expressed at µmol MDA mg-1 protein. Carbonyl protein (CP) quantification was measured at 360nm in ELISA reader following by the method of Reznick and Packer (1994) and oxidation of CP expressed by µmol carbonyl mg-L protein.
Acetylcholine esterase activity
AChE activity was measured by the following procedure of Ellman et al. (1961) and acetylcholine iodide used as the substrate, spectrophotometrically measured at 412nm and their unit expressed as U/mg of protein.
All assays were done in Triplicates. Data were processed with Microsoft excel and one way of variance (ANOVA) followed by Tukey's HSD test. Results were expressed standard error, and the adapted level of significance is p < 0.05.