Division 1.8.11 Measurements and analytical determinations

The algal biomass in each flask is determined at least daily during the test period. If measurements are made on small volumes removed from the test solution by pipette, these should not be replaced.

Measurement of biomass is done by manual cell counting by microscope or an electronic particle counter (by cell counts and/or biovolume). Alternative techniques, e.g. flow cytometry, in vitro or in vivo chlorophyll fluorescence (6)(7), or optical density can be used providing a satisfactory correlation with biomass can be demonstrated over the range of biomass occurring in the test.

The pH of the solutions shall be measured at the beginning and at the end of the test.

Provided an analytical procedure for determination of the test substance in the concentration range used is available, the test solutions should be analysed to verify the initial concentrations and maintenance of the exposure concentrations during the test.

Analysis of the concentration of the test substance at the start and end of the test of a low and high test concentration, and a concentration around the expected EC₅₀ may be sufficient where it is likely that exposure concentrations will vary less than 20 % from nominal values during the test. Analysis of all test concentrations at the start and end of the test is recommended where concentrations are unlikely to remain within 80-120 % of nominal. For volatile, unstable or strongly adsorbing test substances, additional sampling for analysis at 24 hour intervals during the exposure period is recommended in order to better define loss of the test substance. For these substances, extra replicates will be needed. In all cases, determination of test substance concentrations need only be performed on one replicate vessel at each test concentration (or on the contents of the vessels pooled by replicate).

Test media prepared specifically for analysis of exposure concentrations during the test should be treated identically to those used for testing, i.e. they should be inoculated with algae and incubated under identical conditions. If analysis of the dissolved test substance concentration is required, it may be necessary to separate algae from the medium. Separation should preferably be made by centrifugation at a low g-force, sufficient to settle the algae.

If there is evidence that the concentration of the substance being tested has been satisfactorily maintained within ±20 % of the nominal or measured initial concentration throughout the test, analysis of the results can be based on nominal or measured initial values. If the deviation from the nominal or measured initial concentration is greater than ±20 %, analysis of the results should be based on geometric mean concentration during exposure or on models describing the decline of the concentration of test substance (3)(8).

The alga growth inhibition test is a more dynamic test system than most other short-term aquatic toxicity tests. As a consequence, the actual exposure concentrations may be difficult to define, especially for adsorbing substances tested at low concentrations. In such cases, disappearance of the substance from solution by adsorption to the increasing algal biomass does not mean that it is lost from the test system. When the result of the test is analysed, it should be checked whether a decrease in concentration of the test substance in the course of the test is accompanied by a decrease in growth inhibition. If this is the case, application of a suitable model describing the decline of the concentration of test substance (8) may be considered. If not, it may be appropriate to base the analysis of the results on the initial (nominal or measured) concentrations.