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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
other: ready biodegradability test OECD 301B : CO2 Evolution (Modified Sturm Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
The medium was prepared from the stock solutions. The stock solution of the positive control was prepared and its TOC was measured. 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
Positive control flasks: 2, containing positive control, mineral medium and inoculum
Toxicity control: 1, containing test item, positive control, mineral medium and inoculum
Inoculum concentration: 24.9 mg/L
Temperature 20.0 – 21.6 °C
Duration 28 days

The test was performed with a nominal start concentration of 20 mg organic carbon/L.
In the positive control flasks, 25.9 mg/L of Aniline were added.
In the toxicity control flask, 25.9 mg/L of Aniline and 27.3 mg/L of phosphorous acid, tri-C12-C14-alkyl esters were added
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Activated sludge from a biologic sewage treatment plant was used. The chosen plant is treating mostly domestic sewage.
Source: The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, NW-Lachen-Speyerdorf.
Date of collection: 30. Mar 2012, batch no: 20120330.
Pre-Treatment: The sludge was filtrated, washed with tap water twice, then washed with and re-suspended in test medium. It was then aerated for  12 hours. The dry matter was determined with 3740 mg suspended solids/litre
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
28 d
Test temperature:
20.0 – 21.6 °C
pH:
7.41
Nominal and measured concentrations:
Nominal concentration: 27.3 mg/L
Details on test conditions:
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.
Analyses of the emitted CO2 were made by IC measurement using the carbon analyser TOC multi N/C 2100S, Analytik Jena.
Reference substance (positive control):
yes
Remarks:
Aniline
Duration:
29 d
Dose descriptor:
NOEC
Effect conc.:
ca. 27.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
The degradation observed in the toxicity flask was 66.4% at the end of the test (29 days). This degradation is very closed to the degradation percentage observed in the positive controls (68.7% at day 29) containing Aniline alone, without test substance. Therefore, it can be stated that the test item is not toxic towards the inoculum in a concentration of 27.3 mg/L.

In the following table, the calculated emitted carbon (in mg/L) is presented:


Day

Control 1

Control 2

Toxicity Control

2

0.42

0.57

0.86

4

0.98

1.28

8.74

7

1.50

1.80

12.54

9

1.98

2.05

21.10

11

2.34

2.66

25.42

14

2.56

3.01

30.16

18

2.63

3.11

28.41

23

3.04

3.65

32.50

29

3.14

3.95

30.33

In the following table, the percentage biodegradation is presented:

Day

Positive Control 1

Positive Control 2

Positive Control Mean

Toxicity Control

0

0.0

0.0

0.0

0.0

2

-0.2

1.1

0.5

0.9

4

16.6

10.0

13.3

18.9

7

49.8

50.5

50.1

27.0

9

60.8

61.8

61.3

47.3

11

68.5

68.5

68.5

56.8

14

77.3

74.7

76.0

67.9

18

75.9

73.4

74.7

63.4

23

79.3

81.3

80.3

72.3

29

68.7

68.3

68.5

66.4

Because of a poor calibration, the calculated degradation of the positive control and in the toxicity flask on day 23 were slightly higher than on day 29. This can be stated as uncritical for the outcome of the study.

No observations were made which might cause doubts concerning the validity of the study outcome.

The result of the test can be considered valid.

Validity criteria fulfilled:
yes
Conclusions:
NOEC (29 d) = 27.3 mg/L (highest and only dose tested)
Executive summary:

The toxicity toward the microorganisms of the phosphorous acid, triC12-C14 alkylesters was investigated in a GLP-compliant ready biodegradability screening study performed in accordance with OECD Guideline No. 301B via the toxicity control flask. The phosphorous acid, triC12-C14 alkylesters was not found to be toxic to microorganisms under the test conditions of the readily biodegradation test.

The NOEC (29 d) was 27.3 mg/L corresponding to the highest and only dose tested.

Description of key information

The toxicity toward the microorganisms of the phosphorous acid, triC12-C14 alkylesters was investigated in a GLP-compliant ready biodegradability screening study performed in accordance with OECD Guideline No. 301B via the toxicity control flask. The phosphorous acid, triC12-C14 alkylesters was not found to be toxic to microorganisms under the test conditions of the readily biodegradation test.

The NOEC (29 d) was 27.3 mg/L corresponding to the highest and only dose tested.

Key value for chemical safety assessment

Additional information