OECD 207/222 Earthworm Acute Toxicity and Reproduction Test

Earthworms play an important role in the soil biocenosis since they enhance soil characteristics such as the soil function of litter breakdown, soil fertility and soil microstructure. Therefore, earthworms are used as a standard test species to investigate the impact of a substance that is applied on soil or when a contamination of soil is possible. Usually, is in a first step the acute toxicity tested. Persistent substances or substances with several applications per season cause a longer exposure for the earthworm community. In these cases the performance of a study on sublethal effects is required.

Study Design

Test Organisms

Eisenia fetida (Savigny 1826) belongs to the Annelida, Oligochaeta. The test organisms originate from an internal synchronous laboratory breeding culture. Adult hermaphrodites are used for testing when they are between two and twelve months old


Course of the Test

Earthworm tests are performed with an artificial soil (according to OECD requirements) consisting of quartz sand, sphagnum peat, kaolin clay and calcium carbonate. Testing is performed in a climatic chamber with defined temperature, light regime and intensity.


OECD 207: Earthworm, Acute Toxicity Tests

On the basis of the results of a non-GLP pre-test, the applied concentrations for the main test are selected. The test item is mixed into the artificial soil.

Usually 5 concentrations in a geometric row with 4 replicates is performed. In total 40 earthworms are exposed to the test item per treatment group i.e. ten individuals per replicate. Each test comprises a negative control. A test with a toxic reference substance is performed once a year to proofs the sensitivity of the test system. After 7 and 14 days of exposure the mortality of earthworms is assessed.
As sublethal parameters the mean body weight and behavioural abnormalities are recorded.


OECD 222: Earthworm Reproduction Test

Usually 5 test substance concentrations (8 concentrations for ECx-design) with four replicates each are tested (control 8 replicates). The application can be performed by mixing the substance into the artificial soil or by spraying onto the soil surface. For plant protection products the main focus of this study is to show that the no observed effect concentration (NOEC) of the product is less than the 5 fold predicted environmental concentration (PEC), which is set as the acceptability threshold. Otherwise, an earthworm field study is the option to prove environmental safety.

10 adult worms (not older than 1 year) are exposed for 4 weeks. Mortality and weight changes are assessed after this time. The offspring is exposed another 4 weeks until a total exposure period of 8 weeks is reached.



Acute Toxicity:

Derivation of a concentration-response curve for mortality. The LC50 and if possible a NOEC are determined statistically.

Reproduction Test:

As the focus of this study lies on reproduction, the tested concentration range mostly results in slight or no mortality. Reproductive success of the worms. NOEC and/or ECx are determined statistically. Additionally other sublethal effects like body weight changes and feeding activity are recorded.


Guidelines and Literature

  • OECD-Guideline for the testing of chemicals No. 207 “Earthworm, Acute Toxicity Test” (adopted April 4, 1984).
  • ISO-Guideline 11268-1:1993 “Soil quality – Effects of pollutants on earthworms (Eisenia fetida) - Part 1: Determination of acute toxicity using artificial soil substrate”.
  • OECD, Guideline for the testing of chemicals Nr. 222 "Earthworm, Reproduction Test" (adopted April 13, 2004).
  • ISO-Guideline 11268-2, “Soil quality - Effects of pollutants on earthworm (Eisenia fetida) - Part 2: „Determination of effects on reproduction“ (1998).