The medium was prepared from the stock solutions. The inoculum was taken from its source, washed, aerated and the dry matter was determined.

The test vessels were filled with medium and inoculum. Then all flasks were aerated for 72 hours with purified, CO2-free, moistened air to purge the system of CO2.

Experimental Parameters

Flask volume 1500 mL
Apparatus blanks 2, containing mineral medium only
Controls 2, containing mineral medium and inoculum
Positive control flasks 2, containing positive control, mineral medium and inoculum
Test flasks 2, containing positive control, mineral medium and inoculum
Abiotic control 1, containing test substance, mineral medium and HgCl2
Toxicity control  1, containing test substance, positive control, mineral medium and inoculum
Inoculum concentration: 25 mg/L
Temperature 19.2 – 21.4 °C
Duration 28 days

The test was performed with a nominal start concentration of 20 mg organic carbon/L.

The following amounts of test substance and positive control were added to the flasks:


Positive Control 1

Positive Control 2

Test 1

Test 2

Abiotic Control

Toxicity Control

in mg / L





Amount Aniline in mg / L




organic C (calculated) in mg / L








The test vessels were aerated with purified (by activated charcoal), CO2-scrubbed, moistened air. The scrubbing of carbon dioxide was achieved by bubbling the purified air through a flask containing 1.5 m-NaOH. To control the absence of CO2, the air was then led through a flask containing a solution of Ba(OH)2 before reaching the test vessels.

Magnetic stirrers were used to prevent deposition of inoculum.

The emitted CO2 was trapped in 0.25-m-NaOH. Two scrubbers containing 100 mL each were connected in series to the test vessels. The initial IC value of the 0.25 m-NaOH was separately determined in each flask.


From each front scrubber flask, ten samples were taken in order to determine the emitted CO2. (on days 0, 2, 4, 7, 9, 11, 14, 18, 23 and 29). The sample volume was 1 mL. The resulting change in the volume of the front flask was considered in the calculation of emitted CO2.

On day 28, 5 mL HCl 2-m. were added to each test flask in order to drive off dissolved CO2. On day 29, samples from both scrubber flasks were taken.

CO2 Determination

Analyses of the emitted CO2 were made by IC measurement using the carbon analyzer TOC multi N/C 2100S, Analytik Jena. Each sample was measured at least in duplicate. The carbon analyzer was calibrated with freshly prepared reference solutions containing potassium hydrogen phthalate and sodium carbonate once a week. After every start, quality control samples were measured.