OECD 232: Collembolan Reproduction Test in Soil

The reproduction of Folsomia candida will be determined in a laboratory experiment. Therefore the collembolans are exposed to soil that is treated with the test substance and compared to a control and to a reference item. The Directive 1107/2009 requires assessment of effects on soil organisms for all test compounds. For substances applied directly to soil testing on Folsomia candida is mandatory, as they are considered to be a relevant representative of soil fauna. The reproduction provides information about inhibition of the specific population growth rate

Study Design

Test Organisms

The springtail Folsomia candida is a soil inhabiting species of worldwide distribution. A short life cycle and easy breeding qualifies this species for laboratory testing. They are very common and frequent soil arthropods. Collembolans are feeding on organic matter, soil fungi, bacteria and pollen. Springtails play a central role in the soil food chain since they generate organic matter for the bacterial mineralization process and they serve as prey for predatory soil animals. Furthermore they control the growth of soil fungi and contribute thereby to plant growth. The test organisms used are bred in our own laboratory culture.


Course of the Test (OECD 232)

The studies are performed in controlled environmental rooms with defined temperature. Illumination follows a night and day rhythm.

If there is no information about the toxicity of the test substance, a range finding pre-test helps to identify the suitable concentration range. The number of test concentrations and replicates depends on the experimental design.

Usually 5 test item concentrations (8 concentrations for ECx-design) with four replicates and a negative control comprising eight replicates are tested. A toxic reference substance is tested in a dose response design at least once per year.

The test substance is mixed into an artificial soil consisting of quartz sand, sphagnum peat, kaolin clay and calcium carbonate. Moisture and pH are adjusted.

Ten springtails between 9 and 12 days old are introduced into each test vessels and exposed for 28 days. The exposure route is via dermal and alimentary uptake. During the test the springtails are fed with dry yeast.

At the end of the test the surviving adult springtails and the number of offspring produced are counted. For the assessment the complete soil is mixed with water and ink. Thus the springtails are drifting on the soil surface and can be counted under the binocular.



As the focus of this study lies on reproduction, the tested concentration range mostly results in slight or no mortality. The number of surviving springtails and the offspring of the springtails of the test item groups is compared to the numbers of the control group.

Results on reproduction are used to determine a NOEC (no observed effect concentration) or to calculate an EC10 and EC20.


Guidelines and Literature

  • OECD Guidelines for the Testing of Chemicals Test No. 232: OECD 232: Collembolan Reproduction Test in Soil, adopted guideline 7 September 2009.
  • ISO 11267 ISO Soil Quality – Inhibition of reproduction of Collembola (Folsomia candida) by soil pollutants, 1999.