OECD 317: Bioaccumulation in Terrestrial Oligochaetes – Bioaccumulation in Earthworms

The purpose of the study is to determine residues of a test substance in earthworms Eisenia fetida after different intervals up to 21 days exposure (uptake phase) in artificial soil and elimination (post-exposure phase) of residues after different intervals up to another 21 days. By means of bioconcentration and biomagnification processes uptake and elimination parameters as the uptake rate constant and the elimination rate contant as well as the bioaccumulation factor will be calculated.

Study Design

Test setup

The test will be carried out using 14C-labelled test substance or non-labelled test substance. The test will be conducted using artificial soil. Incubation will be performed in boxes with perforated transparent lids to enable exchange of air, to minimise evaporation from soil and to prevent the worms from escaping. The boxes will be filled with approximately 250 g artificial soil (dry weight). The amount of soil corresponds to 50 g dry weight per earthworm. The selected concentration of the test substance should preferably be about 1% of its acute asymptotic LC50.

Test Organisms

For the test soil ingesting invertebrates, such as Eisenia fetida, (Annelida: Oligochaeta) are used. The adult earthworms (age: 2-12 months) with well developed clitellum have a body weight of 250-600 mg and are bred under standardised conditions at IBACON.

Course of the test

Typically, one concentration of the test substance will be used for the test. The earthworms, 5 individuals per replicated test unit, will be exposed to the test substance treated soil for 21 days (uptake phase). The test units will be incubated at 20 °C ± 2 °C and under a controlled 16/8 hours light/dark cycle, preferably 400 to 800 lux.

During the uptake phase at least 6 sampling points will be done. Earthworm and soil samples will be analysed for the test substance. After the end of the uptake phase the earthworms from remaining test units will be transferred to untreated soil and will be incubated for another 21 days (elimination phase). During the elimination phase at least 6 sampling points will be done. Earthworm samples will be analysed for the test substance.

Besides samples containing the test item additional control samples for determining water content of the artificial soil and fat content of the earthworms will be prepared.

Analysis of the test substance and transformation products

In case of the 14C-labelled test substance the analytical methods will base on LSC and HPLC coupled with UV and radio detection. LC-MS/MS can be carried out as additional analytical method. Non-labelled test substances will be determined by LC-MS/MS method.The analytical method and sample preparation will be established to allow a limit of quantification of ≤ 5% of the applied radioactivity or ≤ 10% of the applied concnetration in soil.

Special test setup

  • Depending on the customers needs, artificial soil of different organic carbon content or even natural soil from unpolluted agricultural land can be utilised.


  • Mortality and mass change of the earthworms
  • Calculations of uptake rate constant and elimination rate constant and statistical analysis, Bioaccumulation Factor at Steady State and Kinetic Bioaccumulation Factor

Guidelines and Literature

  • OECD Guideline for the Testing of Chemicals, No. 317, ”Bioaccumulation in Terrestrial Oligochaetes”; adopted July 22, 2010.