Test No. 222: Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei)

4.The following information relating to the test chemical should be available to assist in the design of appropriate test procedures:-water solubility; -log Kow; -vapour pressure; -and information on fate and behaviour in the environment, where possible (e.g.rate of photolysis and rate of hydrolysis where relevant to application patterns). 5.This Guideline is applicable to all test chemicals irrespective of their water solubility.The method may not be applicable to substances for which the air/soil partition coefficient is greater than one, or to substances with vapour pressure exceeding 300 Pa at 25 o C. Other factors such as high water solubility or high adsorption to soil limiting the volatilisation potential should be taken into account when deciding whether or not the test chemical can be tested.For volatile, unstable or readily degrading substances (e.g.data generated from a TG 307 study may be considered), or where there is otherwise uncertainty in maintaining the nominal soil concentration, analytical measurements of the exposure concentrations at the beginning, during and at the end of the test should be considered. 6.Before use of the Test Guideline for the testing of a mixture intended for a regulatory purpose, it should be considered whether, and if so why, it may provide adequate results for that purpose.Such considerations are not needed, when there is a regulatory requirement for testing of the mixture. REFERENCE SUBSTANCE 7.The NOEC and/or the EC x of a reference substance must be determined to provide assurance that the laboratory test conditions are adequate and to verify that the response of the test organisms does not change statistically over time.It is advisable to test a reference substance at least once a year or, when testing is carried out at a lower frequency, in parallel to the determination of the toxicity of a test chemical.Carbendazim or benomyl are suitable reference substances that have been shown to affect reproduction (3).Significant effects should be observed between (a) 1 and 5 mg active ingredient (a.i.)/kg dry mass or (b) 250-500 g/ha or 25-50 mg/m 2 .If a positive toxic standard is included in the test series, one concentration is used and the number of replicates should be the same as that in the controls.