Earthworm and Collembolan Field Study

A field study to assess effects of plant protection products on earthworms and collembolans

Application of plant protection products should not significantly affect non target soil organisms. Collembolans and earthworms are ecologically important and contribute substantially to the decomposition of organic matter for bacterial mineralization. A field study that combines earthworms and collembolan samplings under Good Agricultural Practice (GAP) is advised if laboratory studies show considerably high effects on mortality and reproduction of those organisms. A field study allows determining short and long term effects of plant protection products on earthworms and collembolans populations under realistic conditions.

Study Design

meets the requirements of ISO 11268-3


Cost reductions compared with two separate field studies by generating syngeries, two separate final reports can be provided if requested.

Test Organisms

Earthworms and Collembolans, living in natural populations
Adult earthworms are identified to species level. Juveniles are separated into tanylobous and epilobous earthworms.
Collembolans can be identified to suborder, family or species level depending on customers needs

Design of the Test Field

The basic study design comprises three treatment groups: test item treatment, water treated control and toxic reference item treated with carbendazim and chlorpyrifos in a tankmix. Multiple test item dose rates are possible. Alternatively both toxic references can be established a separate treatment groups. Each treatment group has four replicates (plots) arranged in a randomized plot design (plot size 10 m x 15m). Compared with an earthworm field study the plot is enlarged by a centraol collembola sampling area og five m width. The 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 Methods

Earthworms are collected by a combination of hand sorting and formaldehyde extraction. Collembolan soil dwellers are extracted with a McFadyen heat extractor from soil cores and collembolans living in the litter- and top soil layer are caught with pitfall traps. For details see earthworm and collembolan field study.

Recording of environmental Conditions

Soil parameter (soil profile, pH, C-content, water holding capacity, particle size composition) and climate data (soil and air temperature, soil humidity, precipitation) are taken throughout the study.


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.

Collembolans are sampled before the application of the test item and usally four times after application. Duration: 1 year for the earthworm part of the study, 6-12 months for the collembola part.



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.

The collembola field study accepts initial test item effects and usually focuses on the timespan which is necessary to demonstrate recovery. Recovery is considered to have occurred when soil organism numbers are not significantly lower than the control on two consecutive sampling occasions.


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.
  • ISO 23611-2 (2007). Soil quality - Sampling of soil invertebrates - Part 2: Sampling and extraction of micro-arthropods (Collembola and Acarina).
  • Kula C. et al. (2006) Technical recommendations for the update of the ISO Earthworm Test Guideline (ISO 11268-3). Journal of Soils and Sediments 6: 182-186.
  • de Jong F.M.W. et al. (2010) Guidance for summarising and evaluation field studies with non-target arthropods. A guidance document of Dutch Platform for the Assessment of Higher Tier Studies Test Organisms. RIVM, Bilthoven, Netherlands.