Sampling Earthworms

Literally, flip logs & rocks, strip off bark, root around, under and through litter and see what you find. This sampling emphasizes epigeic and some endogeic species, you may occasionally get a nightcrawler (anecic) if they occupy the site.
Types of data you can collect using this method:

• A list of the species and/or ecological groups present in your site

Types of data you cannot collect with this method:

• Relative abundance of different species or ecological group
• Density of earthworms on an area basis (i.e. number of earthworms per m²)
• Biomass of earthworms on an area basis (i.e. grams of earthworm biomass per m²)
• Relative depth of different earthworm species in the upper soil horizons

Take a shovel or hand spade and dig up some soil. Sift through it with your fingers looking for worms. To get a good sample, be sure to dig to a depth of at least 15 cm, 30 cm is even better, especially if you are interested in getting any endogeic species that might be present. It’s handy to have a piece of plastic or tarp to pile the excavated soil on. Pile the shovels full of soil on one end, grab handfuls of soil and sift it through your fingers. You should not have to sift the soil through a screen, simply by grabbing handfuls and sifting it through your fingers with reasonable care you will find most of the earthworms present, but be sure to break up clumps of soil, they are often in those clumps. If there is a lot of surface litter, the very small epigeic earthworms can hide very well (especially on the undersides of wet leaves) and it can be very labor intensive to search by hand.  In addition, you will almost never get nightcrawlers (Lumbricus terrestris) using hand sampling since they feel the vibrations of the digging and rapidly move deep into the soil. Therefore, if you are interested in generating a full species list for your site or relative abundance of all species present, this method must be done in conjunction with at least midden counts so that Lumbricus terrestris is not missed. If you are only interested in generating a species list for your site, then you can choose to sample here and there across your site. However, if you want to measure relative abundance of different earthworms for your site then use a plot sampling scheme (see study design).

Types of data you can collect using this method:

• Relative abundance of different species or ecological groups (except the anecic species, Lumbricus terrestris)
• Density of earthworms on an area basis (i.e. number of earthworms per m²)
• Biomass on an area basis (i.e. grams of earthworm biomass per m²)
• Relative depth of different earthworm species in the upper soil horizons

Types of data you cannot collect with this method alone:

• A complete list of the species and/or ecological groups present

In this method you pour a solution of mustard water on the soil allowing it to percolate down. The mustard solution irritates the skin of earthworms and they come to the surface to avoid it, where they can be collected, preserved and identified. To make the solution, mix 4 liters (~1 gallon) of water with 40 grams (~1/3rd cup) ground yellow mustard seed. This is the same powdered yellow mustard you will find in the spice rack in any grocery store. However, if you plant on doing this a lot it is MUCH cheaper to buy it in bulk at the local food coop.

A 4L jug of mustard solution is enough to sample a 35cm x 35 cm (1 square foot) sample plot. Clear away the dry surface litter in your sample area (if the litter is wet you may have to search it by hand for errant earthworms). Slowly pour about half of the jug of mustard solution over the area allowing it to soak in as much as possible. If worms are present and active they should begin coming up almost immediately. If they don’t come up immediately, wait 2 minutes or so and then pour again. A forceps is handy for picking the earthworms up off the surface. Have a collection tray to put them in until you’re done, since they can come up in rapid succession and if you are collecting and preserving your earthworms, put some alcohol (isopropyl or rubbing alcohol) in the collection tray to anesthetize the earthworms as they surface. When picking up the surfacing earthworms, WAIT until they come all the way up and out of the soil before you grab them or they will try to go back down and you will most often get an earthworm piece, rather than a whole earthworm. After a few minutes, and the initial flush of earthworms slows down, pour more of the solution over the sample area. You will probably get another flush of earthworms coming to the surface. Continue this until the whole gallon is used up (2 or 3 pourings). Deeper dwelling species take longer to surface than those close to the surface to don’t be too impatient. Total sample time is usually 5-10 minutes.

This technique works well for all species of earthworms but only when the earthworms are active. If it has been very dry, very hot or very cold in the week(s) prior to sampling they may not respond as very well since they may be in aestivation (earthworm version of hibernation). In contrast, if air temperatures have been moderate and it has rained recently they are likely to be active and respond well to the liquid extraction. AN EXCEPTION – if the soil is very compacted and/or has a poor structure (heavy clay, fields, roads, etc.) the extractant doesn’t move well through the soil and the earthworms will not respond because the liquid doesn’t reach them. BUT, in most hardwood forests of our region the liquid extraction method works very well. Try it, it’s fun!

If you are only interested in generating a species list for your site, then you can choose to sample here and there across your site. However, if you want to measure relative abundance of different earthworms for your site then use a plot sampling scheme (see study design).

Types of data you can collect using this method:

• A complete list of the species and/or ecological groups present
• Relative abundance of different species or ecological groups
• Density of earthworms on an area basis (i.e. number of earthworms per m²)
• Biomass on an area basis (i.e. grams of earthworm biomass per m²)

Types of data you cannot collect with this method alone:

• Relative depth of different earthworm species in the upper soil horizon

The anecic species, Lumbricus terrestris, also known as the nightcrawlers, form distinctive piles of cast material (earthworm poop) around the openings to their deep vertical burrows. The pile, called a midden, is usually about 2-5cm in diameter and 1-2cm in height with a burrow hole, 2-4mm in diameter, near the center. The cast material is dark brown to black in color and has a distinctive smooth-globular texture, rather than the angular or crumb structure you would see in native soils. Because nightcrawlers drag whole leaves into their burrows, the opening of the midden will often be covered with a mat of leaf petioles (the leaf stem) and other fragments of leaves. Watch for these telltale signs and once you develop an eye for them, you will find that middens are readily identifiable. If you simply want to know if nightcrawlers are present, then look for these distinctive middens. Lumbricus terrestris is the only earthworm in our region that makes middens. If you want to estimate the relative abundance of nightcrawlers, use a plot sampling scheme (see study design) to determine the average number of middens per unit area. There is one nightcrawlers per burrow, so the presence and number of middens is a good estimate of the population of this species. If you are attempting to do a very thorough assessment of species relative abundance, it is a good idea to do midden counts in conjunction with another sampling method, since Lumbricus terrestris is not always sampled fully using the other methods.

Types of data you can collect using this method:

• Relative abundance of Lumbricus terrestris (anecic ecological group)
• Density of Lumbricus terrestris on an area basis (i.e. number of Lumbricus terrestris per m²)

Types of data you cannot collect with this method alone:

• A complete list of the species and/or ecological groups present at a site
• Biomass on an area basis (i.e. grams of earthworm biomass per m²)
• Relative depth of different earthworm species in the upper soil horizons

Once you have sampled your earthworm populations, what do you do with them?

• Find out how to preserve your earthworms (next tab) .
• Find out how to identify your earthworms.
• Find out how to calculate earthworm biomass.

Once you have earthworms, what do you do with them? If you are documenting an earthworm occurrence or want to save your specimens for identification later, then you need to preserve them.

1. Kill the earthworms in alcohol (isopropyl – rubbing alcohol works great)

1. Get as much dirt off the earthworms as possible
2. Put the earthworms in the alcohol one at a time (rather than a whole handful all at once) so they don’t get tangled up into a big mess of earthworms. After a few seconds, the earthworm will become anesthetized and relax into a mostly extended position.
3. Place the anesthetized earthworms into a vial. You can keep the earthworms in alcohol for up to 24 hours (keeping them cool and out of direct sunlight). But don’t wait to long, after a day or so in alcohol they start to get mushy.

2. Within 24 hours you need to put the earthworms in Formalin

1. Place the earthworms in a leak proof vial and cover them in Formalin which is a cellular fixative that will prepare the specimens for long-term storage.
2. Once the earthworms have been in formalin for at least 24 hours, they can be placed back into alcohol (70-100% isopropyl, NOT ethyl which bleachs any pigmentation) for long-term storage. This allows you to re-use the formalin over and over again.

CAUTION: Formalin is a dangerous chemical and safety precautions must be taken. It should be used only in a well ventilated area. It should not be breathed, swallowed or come in contact with bare skin or eyes. If external contact does occur wash the area with large amounts of water. If ingested contact a poison control center immediately. Formalin has been shown to cause cancer in laboratory animals.

If you do not have access to specimen vials and formalin, we can provide them. Contact us [email protected]