Earthworm Field Study (ISO 11268-3)

Advanced risk assessment on earthworm populations in natural environment

Environmental risk assessments of plant protection products and in specific cases also of veterinary medicinal products take into account the effects of substances on soil organisms like e.g. earthworm species. If prior laboratory studies are not sufficient to exclude unacceptable effects on earthworms it may be necessary to start with a higher tier approach by performing earthworm field studies under realistic conditions as given by GAP (good agricultural practice) to receive further data for the decision makers.

Study Design

according to ISO 11268-3

Test Organisms

Earthworms, living in natural populations. Adults are identified to species level, juveniles are assigned to the two morphological groups: tanylobous and epilobous.

Design of the Test Field

The basic study design comprises three treatment groups: the test item treatment, the water treated control and the toxic reference item treated with 6 to 10 kg/ha carbendazim. Each treatment has four replicates (plots) arranged in a randomized plot design (plot size 10 m x 10m). Distance between the plots is at least 3 m and approximately 10 m distance to the fields next to the test site. Sampling plots are chosen from grassland or arable field sites with homogeneous conditions.

Application of Test Substances

The application of the test substances are carried out with a calibrated movable plot sprayer or granule applicator. The number of test item applications and application rates follows the principles of GAP taking into account realistic conditions of application, product persistence and possible interception by vegetation.

Analytical Verification of the Test Item Application

According to ISO 11268-3 the application of the test item has to be analytically verified. ibacon offers three methods for verfication:

  • analysis of spray liquid samples taken before test item application
  • determination of the a.i. content in soil samples
  • analysis of spray residues from Petri dishes placed on the test item treated plots during application

Link to Chemistry

Sampling Procedure
Formaldehyde extraction combined with hand sorting.

In a first step the upper soil layer (circa 0-20 cm) of a sample place (size 0.125 to 1.0 m²) is excavated and the soil and vegetation layer are carefully searched for earthworms. For the investigation of deeper soil layers 5 to 10 liters of an aqueous solution of 0,2% formaldehyde is uniformly applied to the remaining surfaces of the trial plots. After a few minutes the earthworms come to the soil surface and are collected into a preservation liquid. Four samples are taken per plot (= 16 per treatment group).

Recording of environmental Conditions

Soil parameter and climate data are taken throughout the study.

Soil characterisation: soil profile, pH, C-content, water holding capacity, particle size composition, vegetation observation

Microclimate: soil and air temperature, soil humidity, precipitation


Course of the Study

Prior to the test item application a pre-sampling on the testing site verifies the required earthworm abundance and presence of important ecological groups (anecic and endogeic species). After test item application artificial overhead irrigation can be necessary to ensure exposure of earthworms if insufficient rainfall (under 10 mm) occurs within three days after application. Three additional earthworm samplings are performed within the next 12 months after the test item application.

Sampling dates: pre-application sampling within 14 days before application, three post application samplings approx. 1, 4-6 and 12 months after last test item application

Duration: 1 year



Abundance and biomass of adult and juvenile earthworms are monitored over a time period of one year. The data of the most abundant species are analysed in detail. The results are evaluated for possible differences of the control compared with the test and reference item treatment.


Guidelines and Literature

ISO 11268-3 / 2014/ Soil quality - Effects of pollutants on earthworms - Part 3: Guidance on the determination of effects in field situations", 2nd edition.
Kula et al. 2006: Technical recommend-dations for the update of the ISO Earthworm Field Test Guideline (ISO-11268-3). Journal of Soils and Sediments 6: 182-186.
ISO 23611-1 / 2006 / Soil quality – sampling of soil invertebrates – Part 1: Hand-sorting and formalin extraction of earthworms.