Combined ecotoxicological effects of different sizes and concentrations of polyethene microplastics and soil salinization on the earthworm Dendrobaena veneta

On Day 28 of the experiments, four replicates were selected at random and four earthworms were selected at random from each replicate to extract gut contents. The experimental setting included a control group (control, untreated soil), MP exposure groups with 1 percent MP35 (MP35L), 1 percent MP125 (MP125L), 1 percent MP500 (MP500L), 10 percent MP35 (MP35H), 10 percent MP125 (MP125H), and 10 percent MP500 (MP500H), a salt control (Scontrol, NaCl exposure only), and combined NaCl-MPs exposure groups, including NaCl plus 1 percent MP35 (SMP35L), NaCl plus 1 percent MP125 (SMP125L), NaCl plus 1 percent MP500 (SMP500L), NaCl plus 10 percent MP35 (SMP35H), NaCl plus 10 percent MP125 (SMP125H), and NaCl plus 10 percent MP500 (SMP500H). The earthworms were first euthanized and surface-sterilized using 70 percent ethanol. To expose the gut, the earthworms were cut just behind the clitellum. The intestinal contents were carefully extruded with sterilized tweezers and collected in 1.5 mL Eppendorf tubes. DNA was subsequently extracted from the gut contents using DNeasy PowerSoil Pro Kits (Qiagen, Germany) following the manufacturer's protocol, resulting in 56 DNA samples in total. DNA quality was assessed with a Nanodrop Spectrophotometer. The DNA samples were stored at minus 80 degrees Celsius before submission to Novogene Company Limited (Cambridge, UK) for high-throughput sequencing of bacterial 16S rRNA gene. The V3-V4 region was amplified using forward primer 341F and reverse primer 806R.