Extraction time and mustard concentration for extraction of aquatic oligochaetes by using wet funnel

<p dir="ltr">Supplemental data 1: Exp. S1 </p><p dir="ltr">Relationship between the number of extracted individuals and extraction time in the Baermann apparatus </p><p dir="ltr">Two experiments (Exp. S1.1-1.2) were performed to determine the extraction period, the number of extracted individuals was recorded at 0.5, 1, 1.5, 2, 2.5, 3, 4, and 5 h in Exp. S1.1, and the percentage of extracted individuals was compared with those of the 2 h and 24 h extraction periods in Exp. S1.2. Soil from the Tottori Agricultural Experiment Station was used. The experimental procedure was the same as for Exp. 1 of the article up to the point where the residue was transferred to the stainless-steel mesh screen. In Exp. S1.1, the core soil sample was used for the experiment. After 5 h of extraction, the residue was moved into the plastic tray, and the remaining individuals in the residue were counted by hand-sorting. In Exp. S1.2, a fresh volume of approximately 100 cc of stock soil which had been collected from the same station was used because the original stock soil sample had already been depleted at that point. The method used for calculating the extraction efficiency was the same as in the Exp. 1 of the article. In Exp. S1.1, the individuals collected in each time frame and those remaining in the residue after extraction were summed, and the percentage extracted in each time frame was calculated. In Exp. S1.2, the oligochaetes were extracted over 2 h (the data from Exp. 1 of the article were used for this) and 24 h, those remaining in the residue after extraction were added to this number, and the percentage extracted at each time was again computed. Exp. S1.1 was replicated seven times, and the 24 h extraction in Exp. S1.2 was replicated six times. Extraction efficiency was analyzed using generalized linear models with a quasibinomial error structure and a logit link-function for Exp. S1.2. The extraction time was the explanatory variable and extraction efficiency was the response variable. Statistical calculations were performed in R version 4.2.2 (R Development Core Team, 2022), and the significance of the explanatory variable was assessed with F-tests using package car. Extraction efficiency in Exp. S1.1 increased with extraction time, reaching over 80% at 1.5 h, after which the rate of increase slowed. In Exp.S1.1, under continuous extraction, efficiency increased from 2 h to 5 h but was greater over 2 h than over 24 h using different soil samples in Exp. S1.2 (Table S1). This indicates that extraction efficiency beyond 2 h could be considered essentially stable.</p><p dir="ltr">Supplemental data 2: Exp. S2</p><p dir="ltr"><b>Determining mustard concentration for extraction</b></p><p dir="ltr">To determine the mustard concentration required for extraction, the effects of varying concentrations on oligochaetes were investigated in Exp. S2.1-S2.3. As no information about the response of aquatic oligochaetes to mustard was available, a wide range of mustard concentrations were tested in Exp. S2.1. Aquatic oligochaetes extracted from a Tottori station soil were visually divided into three categories: large, medium, and small specimens. Four mustard concentrations of 0.05, 0.2, 0.5, and 2 g L⁻¹ were prepared, and 10 oligochaetes comprising three large, four medium, and three small individuals were submerged into each concentration at 15°C, then observed for 2 h. It was found that all tested concentrations resulted in the gashing, death, and disintegration of the oligochaetes (Table S2). Sensitivity of aquatic oligochaetes to mustard was high. While a rapid movement response, such as moving down the funnel, was expected at high concentrations, I did not wish to injure the oligochaetes. To determine a suitable non-lethal concentration to induce movement, tap water was first filled into a funnel and then a high concentration of mustard solution was added to the residue, creating a mustard concentration gradient in the funnel. I investigated concentrations that did not kill aquatic oligochaetes in the long term in Exp. S2.2, and concentrations that did not kill them in the short term in Exp. S2.3. For the former experiment, lower concentrations of 0.005, 0.02, 0.05, and 0.1 g L⁻¹ were prepared, and 10 individuals were again introduced at 15°C in the same manner as in Exp. S2.1. Oligochaete body condition and dead/alive status in each concentration were recorded at 2 h, 4 h, 1 d, and 4 d after introduction. For the latter experiment, the mustard concentration was set at 0.02, 0.05, 0.1, 0.2, and 0.5 g L⁻¹. These oligochaetes were transferred into distilled water after 2.5 h, as the intended extraction time was 2 h. Their status was recorded after 5 h in the distilled water. The number of surviving oligochaetes decreased from ten to nine after 2 h at the lowest concentration (0.005 g L⁻¹): this number then remained constant until the end of the experiment (Table S3). It is however possible that this reflects an error in determining the starting number of individuals rather than actual mortality. Individuals over 0.02 g L-1 survived for 4 h, but after 1 d the mortality increased with increasing concentration (Table S3). These outcomes suggested the use of a mustard concentration of 0.01 g L⁻¹ throughout the funnel. In Exp. S2.3 under submergence for 2 h, oligochaetes survived in medium concentrations up to 0.2 g L⁻¹ (Table S4). Since adding mustard to the tap water-filled funnel added a substantial further dilution factor, I eventually decided on using a concentration of 0.25g L^-1.</p>