Earthworms can be extracted from the soil by pouring an irritant solution on the soil surface as formalin, mustard or allyl isothiocyanate, which flows into the soil pores and earthworm burrows. Once it meets an earthworm, it irritates the skin and brings the earthworm to the soil surface. However, earthworms can hide in niches, where the irritant solution cannot go to, therefore excavate and screen the soil for the hidden individuals by hand is often necessary for a precise recording. Depending on the preferred location of earthworms and the type of burrows, irritant is aimed primarily towards anecic species, and no hand sorting method carries the risk of underestimating endogeic. The combination of the two methods is considered as the gold standard in detecting abundance and diversity of earthworms.

Extraction with an irritant solution drives a large part of the worms to the surface, where the individuals can be collected easily. The use of mustard solution is less efficient as compared to formol or allyl isothiocyanate, abbreviated AITC (Pelosi et al., 2009), but in combination with hand sorting, nearly all earthworms can be found. Another difficulty is the standardization of the mustard concentration. The advantage of mustard is its availability and harmlessness to the users. The extraction efficiency of the irritating solutions differs.

Fründ and Jordan (2003) give the following ranking: table mustard < mustard‐flour ≈ mustard seeds < formalin.

It is possible to combine the earthworm extraction with the spade test by hand sorting the excavated soil first and then use the mustard extraction in the hole. However, the tremor produced by the digging and taking the spade sample may chase some earthworms to remote areas, where they can no longer be reached. This approach may therefore lead to a lower or more variable numbers.

Research question and experimental conditions

Occurrence and activity of earthworms vary greatly depending on weather, season and cultivation. The following conditions need to be considered for earthworm sampling:

• The ideal time to do the earthworm counting is their time period of maximum activity (March‐April and September‐October; Central Europe).
• Since the site is disturbed, be sure not to sample again on the same place. Sampling before farm practices as soil tillage.
• In dependence of soil heterogeneity and due to a general high variability, 4 to 6 replicates are recommended per field.
• The sampling design should be adapted to the particular research question. 

Under the following conditions, studies on earthworm popu lation are more difficult or impossible:

• Dry, wet or waterlogged soil
• Hot weather and temperatures below zero
• In heavy clay soils, the extraction is difficult. In sandy soils earthworms occur in low numbers.