# 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), Appendix 2: Semi-continuous procedure

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

Prolonged incubation for up to several months may be required in order to achieve a sufficient degradation of recalcitrant substances. The duration of the test should normally not exceed 60 days unless the characteristics of the original water sample are maintained by renewal of the test suspension. However, the test period may be extended to a maximum of 90 days without renewal of the test suspension, if the degradation of the test substance has started within the first 60 days.
During incubation for long periods, the diversity of the microbial community may be reduced due to various loss mechanisms and due to possible depletion of the water sample of essential nutrients and primary carbon substrates. It is therefore recommended that a semi-continuous test is used to adequately determine the degradation rate of slowly degrading substances. The test should be initiated by use of the semi-continuous procedure if, based on previous experience, an incubation period of three months is expected to be necessary to achieve 20 % degradation of the substance. Alternatively, the normal batch test may be changed into a semi-continuous test, if no degradation of the test substance has been achieved during approximately 60 days of testing using the batch procedure. The semi-continuous procedure may be stopped and the test continued as a batch experiment, when a substantial degradation has been recorded (e.g. > 20 %).
In the semi-continuous test, every two weeks, about one third of the volume of the test suspension is replaced by freshly collected water with the test substance added to the initial concentration. Sediment is likewise added to the replacement water to the initial concentration (between 0,01 and 1 g/l), if the optional suspended sediment test is performed. Carrying out the test with suspended sediment solids, it is important that a fully suspended system is maintained also during water renewal, and that the residence time is identical for solids and water, as otherwise the intended similarity to a homogenous aqueous system with no fixed phases can be lost. For these reasons, an initial concentration of suspended sediments in the lower range of the specified interval is preferred, when the semi-continuous procedure is used.
The prescribed addition of test substance implies that the initial concentration of test substance is not exceeded by the partial renewal of the test suspension and, hence, the adaptation, which is frequently seen with high concentrations of a test substance, is avoided. As the procedure comprises both a re-inoculation and a compensation of depleted nutrients and primary substrates, the original microbial diversity is restored, and the duration of the test can be extended to infinity in principle. When the semi-continuous procedure is used, it is important to note that the residual concentration of the test substance must be corrected for the amounts of test substance added and removed at each renewal procedure. The total and the dissolved test substance concentration can be used interchangeably for compounds that sorb little. Sorption is insignificant (< 5 %) under the specified conditions (0,1-1 g solids/l) for substances of log Kow < 3 (valid for neutral, lipophilic compounds). This is illustrated by the following calculation example. 0,1 g/l of solids roughly corresponds to 10 mg of carbon per litre (fraction of carbon, fC = 0,01). Assuming that:
Log Kow (of the test substance) = 3
Koc = 0,42 × Kow
Partition coefficient, Kd = fC × Koc
then, the dissolved fraction of the total concentration (C-water (Cw)/C-total (Ct) is:
Cw/Ct = 1/(1 + Kd × SS) = 1(1 + Koc × fC × SS) = 1/(1 + 0,42 × 103 × 0,01 × 0,1 × 10–3) = 0,999

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Source: https://www.legislation.gov.uk/eur/2009/761/annex/V/appendix/2 (the official version)
Page: https://openlegislation.uk/eu-regulation-2009-761/annex-V/appendix-2
Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives.