Commission Regulation (EC) No 761/2009Show full title

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 (F_B). 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.