PERFORMANCE OF THE STUDY

Preparations

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 test substance, 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 25.0 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:

Table Amounts of test substance and positive control in the flasks

   Flask  Positive Control 1  Positive Control 2  Test 1  Test 2  Abiotic Control  Toxicity Control 
   Amount STABILGREEN®K
   (Polyglyceryl-4 Oleyl Ether, Dioleyl
   Phosphate)in mg / L 
—  —  28.1  27.5  27.7  28.0 
   Amount Aniline in mg / L  24.5  24.5  —  —  —  24.5 
   organic C (calculated) in mg / L  20.0  20.0  20.3  19.9  20.1  40.2 

Apparatus

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.

Sampling

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 analyser TOC multi N/C 2100S, Analytik Jena. Each sample was measured at least in duplicate. The carbon analyser 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.

FINDINGS

Tables

IC-Values

In the following tables, the IC values (given in mg/L) which were measured in the samples of the front scrubber flasks are stated.

Table IC Values in mg/L Apparatus Blanks, Controls, front scrubber

Day  Apparatus blank 1  Apparatus blank 2  Control 1  Control 2 
0 0.74  1.20  1.28  1.80 
2 2.70  4.08  10.24  9.09 
4 4.14  5.18  21.03  17.16 
7 4.69  6.07  35.74  29.69 
9 5.79  7.64  42.17  37.43 
11 6.33  8.34  43.62  41.19 
14 7.24  11.36  51.94  50.40 
18 10.24  13.27  59.40  57.75 
23 12.48  17.61  65.40  66.07 
29 16.60  22.49  75.91  76.79 

Table IC Values in mg/L Positive Control, Test Flasks, front scrubber

Day  Positive Control 1  Positive Control 2  Test 1  Test 2  Abiotic Control  Toxicity Control 
0 1.42  1.00  0.63  0.35  0.55  1.86 
2 7.51  11.91  23.60  18.76  5.20  43.74 
4 55.54  109.72  88.12  58.05  5.38  303.68 
7 193.95  251.25  187.58  162.56  6.97  448.58 
9 264.64  278.75  204.48  210.59  8.09  493.20 
11 270.29  282.51  227.77  234.35  8.05  504.15 
14 289.95  288.15  259.95  261.16  10.01  519.19 
18 318.81  322.09  281.70  277.74  12.01  516.20 
23 335.71  338.78  301.60  291.50  13.31  551.95 
29 347.17  353.45  334.16  332.07  16.02  583.85 

In the following tables, the IC values which were measured in the samples of the back scrubber flasks are stated.

Table IC Values in mg/L Controls, Apparatus Blanks, back scrubber

Day  Apparatus blank 1  Apparatus blank 2  Control 1  Control 2 
0 0.86  1.88  0.72  1.29 
29 10.06  12.28  3.22  4.31 

Table 3.1-d IC Values in mg/L Positive Control, Test Flasks, back scrubber

Day  Positive Control 1  Positive Control 2  Test 1  Test 2  Abiotic Control  Toxicity Control 
0 1.19  0.73  1.08  1.67  0.64  2.20 
29 5.29  3.20  4.16  6.57  21.56  10.96 

Net IC

For each flask, the IC value which was measured at the start of the test (d = 0) was subtracted from all following measurements. The net IC was calculated using this corrected measurement value and subtracting the mean IC value of the apparatus blanks of that sampling date.
The net IC values are presented in the following table.

Table Net IC-values in mg/L front scrubber flasks

Day  Control 1  Control 2  Positive Control 1  Positive Control 2  Test 1  Test 2  Abiotic Control  Toxicity Control 
0 1.3  1.8  1.4  1.0  0.6  0.3  0.6  1.9 
2 7.8  6.7  5.1  9.5  21.2  16.3  2.8  41.3 
4 17.3  13.5  51.9  106.0  84.4  54.4  1.7  300.0 
7 31.3  25.3  189.5  246.8  183.2  158.2  2.6  444.2 
9 36.4  31.7  258.9  273.0  198.7  204.8  2.3  487.5 
11 37.3  34.8  263.9  276.1  221.4  228.0  1.7  497.8 
14 43.6  42.1  281.6  279.8  251.6  252.8  1.7  510.9 
18 48.6  47.0  308.0  311.3  270.9  267.0  1.2  505.4 
23 51.3  52.0  321.6  324.7  287.5  277.4  -0.8  537.9 
29 57.3  58.2  328.6  334.9  315.6  313.5  -2.6  565.3 

Table Net IC-values in mg/L back scrubber flasks

Day  Control 1  Control 2  Positive Control 1  Positive Control 2  Test 1  Test 2  Abiotic Control  Toxicity Control 
0 0.7  1.3  1.2  0.7  1.1  1.7  0.6  2.2 
29 -6.6  -5.5  -4.5  -6.6  -5.6  -3.2  11.8  1.2 

Negative values occur, when the apparatus blank was higher than the respective treatment. As the measured values in these blanks as well as in the abiotic control are very low, measurement uncertainties lead to negative degradation values in the abiotic control.

pH

In the following table, the pH at the end of the test (before addition of HCl) is given:

Table pH Test flasks on day 28

Day  Control 1  Control 2  Positive Control 1  Positive Control 2  Test 1  Test 2  Abiotic Control  Toxicity Control 
28 7.4  7.4  7.2  7.2  7.4  7.3  6.8  7.2