Analysis of Earthworm Populations in Soil

1. Preparation of Mustard Concentrate Solution

1. Turn on balance, place a weigh boat on top, and zero the balance.
2. Weigh out 38.1 g of ground oriental mustard into weigh boat and transfer to a plastic container with cap.
3. Measure 100 mL of tap water in a graduated cylinder and add to the plastic container with mustard.
4. Secure cap on container and shake vigorously until all mustard is mixed off the bottom of the plastic container and dissolved into the tap water.
5. Let solution sit for 24 h for maximum capsaicin extraction from mustard.
6. Fill two 8 L water carriers halfway with tap water (approximately 4 L of water into each carrier).
7. Shake mustard concentrate several times to mix then transfer mustard concentrate solution to water carrier.
8. Transfer a small amount of the solution from the water carrier back into the concentrate container and shake vigorously. Pour back into the water carrier to transfer all of concentrate into the diluted solution.
9. Seal the water carrier cap, ensure cap valve is in “OFF” position, and invert water carrier three times to mix evenly.

2. Extraction of Earthworms

1. Label three sample cups for each collection site.
2. Proceed to sampling site with one quadrat, labeled sampling cups with lids, and water carrier with diluted mustard solution.
3. At the sampling site, clear away brush, leaves, or mulch as much as possible to clearly expose the ground.
4. Place quadrat randomly on the ground in a cleared spot.
5. Invert one water carrier three more times to mix.
6. Turn the water carrier cap valve to the “ON” position and pour approximately a third (1.3 L) of the diluted mustard solution within the quadrat, concentrating on the center of the quadrat area. If soil becomes saturated and solution pools, stop pouring and wait until pooled solution infiltrates into soil before continuing to pour.
7. Observe quadrat area closely for earthworm appearance for five minutes, including the area directly under sides of quadrat.
8. Use forceps to collect all earthworms that appear in quadrate area by waiting for worms to completely emerge from ground before transferring to first sample cup. After five minutes lid the sample cup and proceed to next sampling site.
9. Repeat collection steps for all sampling sites, with three replicates per collection site (6 replicates total)

3. Comparing Earthworm Population Density Between Collection Sites

1. Count the number of earthworms collected for each sample and calculate the mean and standard deviation for each collection site.
2. To compare earthworm densities between collection sites, create a bar graph from the means and use the standard deviations to create error bars on the graph.