# 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 3: Determination of 14CO2

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

Indirect 14CO2 determination
For routine measurements, the indirect method is normally the least time-consuming and most precise method if the test substance is non-volatile and is not transformed into volatile transformation products. Simply transfer unfiltered samples e.g. 5 ml size to scintillation vials. A suitable activity in samples is 5 000 dpm-10 000 dpm (80-170 Bq) initially, and a minimum initial activity is about 1 000 dpm. The CO2 should be stripped off after acidifying to pH 2-3 with 1-2 drops of concentrated H3PO4 or HCl. The CO2 stripping can be performed by bubbling with air for about 1/2-1 hour. Alternatively, vials can be shaken vigorously for 1-2 hours (for instance on a microplate shaker) or with more gentle shaking be left overnight. The efficiency of the CO2 stripping procedure must be checked (by prolonging the aeration or shaking period). A scintillation liquid, suitable for counting aqueous samples should then be added, the sample homogenised on a whirling mixer and the radioactivity determined by liquid scintillation counting, subtracting the background activity found in the test blanks (FB). Unless the test water is very coloured or contains a high concentration of particles, the samples will normally show uniform quenching and it will be sufficient to perform quench corrections using an external standard. If the test water is highly coloured, quench correction by means of internal standard addition may be necessary. If the concentration of particles is high it may not be possible to obtain a homogeneous solution or gel, or the quench variation between samples may be large. In that case the counting method described below for test slurries can be used. If the test is carried out as a suspended sediment test, the 14CO2 measurement could be done indirectly by taking a homogeneous 10-ml sample of the test water/suspension and separating the phases by centrifugation at a suitable speed (e.g. at 40 000 m/s2 for 15 min.). The aqueous phase should then be then treated as described above. The 14C activity in the particulate phase (POA) should be determined by re-suspending the sediment into a small volume of distilled water, transferring to scintillation vials, and adding scintillation liquid to form a gel (special scintillation liquids are available for that purpose). Depending on the nature of particles (e.g. their content of organic material), it may be feasible to digest the sample overnight with a tissue solubiliser and then homogenise on a whirling mixer prior to the addition of scintillation liquid. Alternatively, the POA can be determined by combustion in excess of oxygen by use of a sample oxidiser. When counting, internal standards should always be included, and it may be necessary to perform quench corrections using internal standard addition for each individual sample.
Direct 14CO2 determination
If the evolved 14CO2 is measured directly, it should be done by setting up more flasks at the start of the test, harvesting the test flasks at each measuring point by acidifying the test flasks to pH 2-3 and collecting the 14CO2 in an internal (placed in each test flask at the start of the test) or external absorber. As absorbing medium either alkali (e.g. 1 N NaOH solution, or a NaOH pellet), ethanolamine or an ethanolamine-based, and commercially available absorbers can be used. For direct measurement of the 14CO2, the flasks should be closed with e.g. butyl rubber septa.
Figure 1a
Example of arithmetic plot of data (residual activity versus time)
Figure 1b
Example of semi-logarithmic plot of data (ln to residual activity versus time)

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Source: https://www.legislation.gov.uk/eur/2009/761/annex/V/appendix/3 (the official version)
Page: https://openlegislation.uk/eu-regulation-2009-761/annex-V/appendix-3
Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives.