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.

实验第28天时，随机选取4个生物学重复组，再从每个重复组中随机选取4条蚯蚓以提取其肠道内容物。本实验设置如下组别：对照组（control，未处理土壤）、1%浓度MP35暴露组（MP35L）、1%浓度MP125暴露组（MP125L）、1%浓度MP500暴露组（MP500L）、10%浓度MP35暴露组（MP35H）、10%浓度MP125暴露组（MP125H）、10%浓度MP500暴露组（MP500H）；盐对照组（Scontrol，仅氯化钠（NaCl）暴露），以及氯化钠-MP联合暴露组，包括氯化钠+1%MP35组（SMP35L）、氯化钠+1%MP125组（SMP125L）、氯化钠+1%MP500组（SMP500L）、氯化钠+10%MP35组（SMP35H）、氯化钠+10%MP125组（SMP125H）、氯化钠+10%MP500组（SMP500H）。首先将蚯蚓安乐处死，并用70%乙醇进行表面灭菌。为暴露肠道，在蚯蚓生殖环带（clitellum）后方处剪开。随后用灭菌镊子小心挤出肠道内容物，收集至1.5 mL艾本德（Eppendorf）离心管中。后续采用DNeasy PowerSoil Pro试剂盒（Qiagen，德国），按照制造商提供的实验规程从肠道内容物中提取脱氧核糖核酸（DNA），最终共获取56份DNA样本。使用纳米滴分光光度计（Nanodrop Spectrophotometer）评估DNA质量。将DNA样本保存于-80℃，随后送至诺禾致源股份有限公司（Novogene Company Limited，英国剑桥），开展细菌16S核糖体RNA（rRNA）基因的高通量测序。采用正向引物341F与反向引物806R扩增V3-V4可变区。