Division 1.4 PRINCIPLE OF THE TEST

The test is performed in batch by incubating the test substance with either surface water only (pelagic test) or surface water amended with suspended solids/sediment of 0,01 to 1 g/L dry weight (suspended sediment test) to simulate a water body with suspended solids or re-suspended sediment. The suspended solids/sediment concentration in the lower range of this interval is typical for most surface waters. The test flasks are incubated in darkness at an environmental temperature under aerobic conditions and agitation. At least two different concentrations of test substance should be used in order to determine the degradation kinetics. The concentrations should differ from each other by a factor of 5 to 10 and should represent the expected range of concentrations in the environment. The maximum concentration of the test substance should not exceed 100 µg/L, but maximum test concentrations below 10 µg/L or less are preferred to ensure that the biodegradation follows first order kinetics. The lowest concentration should not exceed 10 µg/L, but lowest test concentrations of 1-2 μg/L or less than 1 μg/L are preferred. Normally an adequate analysis of such low concentration can be achieved by use of commercially available ¹⁴C-labelled substances. Because of analytical limitations, it is frequently impossible to measure the concentration of the test substance with the required accuracy, if the test substance is applied at a concentration ≤ 100 µg/L (see second paragraph in section 1.7.2). Higher concentrations of test substance (> 100 µg/L and sometimes > 1 mg/L) may be used for the identification and quantification of major transformation products or if a specific analysis method with a low detection limit is not available. If high concentrations of test substance are tested, it may not be possible to use the results to estimate the first order degradation constant and half-life, as the degradation will probably not follow first order kinetics.

Degradation is followed at appropriate time intervals, by measuring either the residual ¹⁴C or the residual concentration of test substance when specific chemical analysis is used. ¹⁴C labelling of the most stable part of the molecule ensures the determination of the total mineralisation, while ¹⁴C labelling of a less stable part of the molecule, as well as the use of specific analysis, enable the assessment of only primary biodegradation. However, the most stable part does not necessarily include the relevant functional moiety of the molecule (that can be related to a specific property such as toxicity, bioaccumulation, etc.). If this is the case, it may be appropriate to use a test substance, which is ¹⁴C-labelled, in the functional part in order to follow the elimination of the specific property.