# Commission Regulation (EC) No 761/2009 of 23 July 2009 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance), Division 1.7.6: Test and control groups

Extent: E+W+S+N.I.

Prior knowledge of the toxicity of the test substance to Lemna, e.g. from a range-finding test, will help in selecting suitable test concentrations. In the definitive toxicity test, there should normally be at least five test concentrations arranged in a geometric series. Preferably the separation factor between test concentrations should not exceed 3,2, but a larger value may be used where the concentration-response curve is flat. Justification should be provided if fewer than five concentrations are used. At least three replicates should be used at each test concentration.
In setting the range of test concentrations (for range-finding and/or for the definitive toxicity test), the following should be considered:
To determine an ECx, test concentrations should bracket the ECx value to ensure an appropriate level of confidence. For example, if estimating the EC50, the highest test concentration should be greater than the EC50 value. If the EC50 value lies outside the range of test concentrations, associated confidence intervals will be large and a proper assessment of the statistical fit of the model may not be possible.
If the aim is to estimate the LOEC/NOEC, the lowest test concentration should be low enough so that growth is not significantly less than that of the control. In addition, the highest test concentration should be high enough so that growth is significantly lower than that in the control. If this is not the case, the test will have to be repeated using a different concentration range (unless the highest concentration is at the limit of solubility or the maximum required limit concentration, e.g. 100 mg·l–1).
Every test should include controls consisting of the same nutrient medium, number of fronds and colonies, environmental conditions and procedures as the test vessels but without the test substance. If an auxiliary solvent or dispersant is used, an additional control treatment with the solvent/dispersant present at the same concentration as that in the vessels with the test substance should be included. The number of replicate control vessels (and solvent vessels, if applicable) should be at least equal to, ideally twice, the number of vessels used for each test concentration.
If determination of NOEC is not required, the test design may be altered to increase the number of concentrations and reduce the number of replicates per concentration. However, the number of control replicates must be at least three.

---
Source: https://www.legislation.gov.uk/eur/2009/761/annex/VI/division/1/division/1.7/division/1.7.6 (the official version)
Page: https://openlegislation.uk/eu-regulation-2009-761/annex-VI/division-1/division-1.7/division-1.7.6
Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives.