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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-07-20 to 2011-09-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well performed OECD study with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2,3-Quinoxalinedione, 6-amino-1,4-dihydro-7-methoxy-
EC Number:
615-027-9
Cas Number:
69904-10-5
Molecular formula:
C9H9N3O3
IUPAC Name:
2,3-Quinoxalinedione, 6-amino-1,4-dihydro-7-methoxy-
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Amino-C-Dion trocken
- Molecular formula: C9H9N3O3
- Molecular weight: 207.19 g/mol
- Physical state: Solid, yellow powder
- Analytical purity: 93.8 % (w/w)
- Storage condition of test material: Room temperature, protected from light, in original container

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge from the municipal sewage treatment plant, 31137 Hildesheim, Germany
- Preparation of inoculum for exposure: The activated sludge was washed twice with autoclaved tap water. After the second washing the settled sludge was filled up with mineral salts medium and was maintained in an aerobic condition by aeration for 4 hours. Thereafter the sludge was homogenized with a blender. The supernatant was decanted and maintained in an aerobic condition by aeration with CO2-free air for 2 days.
10 mL/L were used to initiate inoculation.
- Initial cell/biomass concentration: 1.4 x 10e10 CFU/L
- Water filtered: no
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
50 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral salts medium acc. to OECD 301F
- Additional substrate: No
- Test temperature: 21-22 °C
- pH:
pH-Values at Test Start and Test End

pH-Value
Start End
Inoculum Control 7.72 1) 7.74
2) 7.75
Functional Control 7.73 1) 7.93
2) 7.91
Test Item 7.71 1) 7.70
2) 7.72
Toxicity Control 7.71 7.89

- pH adjusted: no
- Dispersion treatment: Continuous stirring
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Incubator
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Closed bottles with a rubber sleeve with soda lime
- Measuring equipment: The bottles were closed with OxiTOP measuring heads.
The oxygen consumption was determined continuously from the pressure in the incubation vessels by OxiTop measuring system at 360 measuring points (every 112 minutes) during the 28 d incubation period.

SAMPLING
- Sampling frequency: 360 measuring points (every 112 minutes) during the 28 d incubation period.
- Sampling method: Pressure measurements directly from the test vessels

CONTROL AND BLANK SYSTEM
- Inoculum blank: Test medium without test and reference item
- Abiotic sterile control: No
- Toxicity control: Test item and reference item in test concentrations

STATISTICAL METHODS:
Calculation of the theoretical oxygen demand (ThOD) of the test item and functional control.
Concerning the OECD-guideline 301 the index of the atoms of the item CcHhClclNnNanaOoPpSs was used for the calculation of the ThOD.
The oxygen depletion of the test and reference item was calculated out of the difference of the test and inoculum control replicates.
The biodegradation of the test and reference item was calculated out of the BOD in relation to the ThOD.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Preliminary study:
No preliminary study
Test performance:
Based on the calculated oxygen demand, the test concentration of 50 mg/L corresponding to an oxygen demand of 58.0 mg O2/L in the vessel was selected.
The test solutions were prepared in measuring flasks and placed in brown glass bottles as incubation vessels (inoculum control, functional control) or were prepared directly in brown glass bottles (test item, toxicity control):

• two for the test item (P1, P2)
• two for the functional control (R1, R2)
• one for the toxicity control (T1)
• two for the inoculum control (C1, C2)

Separate replicates of test item and toxicity control were prepared for measurement of the pH-value at test start:

• one for the test item (P1)
• one for the toxicity control (T1)

The test medium, consisting of the required volumes of mineral medium stock solutions, demineralised water and 10 mL/L inoculum, was prepared in a measuring flask. 250 mL of this solution were filled in the brown glass bottles of the inoculum control, using a 250 mL measuring flask.
The test item was weighed out and transferred directly into the brown glass bottle with 250 mL test medium.
Accordingly, the test and reference item for the toxicity control were weighed out and transferred directly into the brown glass bottle.
For the functional control the reference item was weighed out and transferred into a 250 mL measuring flask with demineralised water. The required volumes of mineral medium stock solutions and 10 mL/L inoculum were added, then the flask was filled up with demineralised water. This test solution was placed in the brown glass bottle.

A rubber sleeve with soda lime was hung into the opening of the bottles to absorb evolved CO2. The bottles were closed with OxiTop measuring heads and the measuring system was activated.
% Degradation
Parameter:
% degradation (O2 consumption)
Value:
7
Sampling time:
28 d
Details on results:
In case of the functional control, the adaptation phase changed to degradation phase after
1 day (degradation > 10 %). The pass level > 60 % was reached after 2 days. The biodegradation rate reached a plateau phase with 67 % degradation in the 1st replicate and 90 % in the 2nd replicate on day 13.

The 1st test item replicate reached the 10 % level (beginning of biodegradation) after 16 days, the 2nd test item replicate did not reach the 10 % level. The 60 % pass level was not reached within 28 days. After 28 days the mean biodegradation was 7 %.

The test item is classified as not readily biodegradable in the 10-d-window and after 28 days.
The pass level for ready biodegradation (> 60 % degradation) was reached after 2 days. The validity criterion that the degradation should be > 60 % after 14 d was fulfilled.

BOD5 / COD results

Results with reference substance:
In the toxicity control the biodegradation achieved 55 % after 14 days. After 28 days the biodegradation came to 62 %. The validity criterion that the test item should not inhibit the biodegradation of the reference item was fulfilled.

Any other information on results incl. tables

Biological Oxygen Demand (BOD) of Test Item Amino-C-Dion trocken

BOD [mg O2/L]

Date

[d]

Test Item
50 mg/L

P1

P2

gross

net

gross

net

2011-08-19

1

1.4

0.7

1.4

0.7

2011-08-20

2

1.4

0.0

1.4

0.0

2011-08-21

3

2.8

1.4

1.4

0.0

2011-08-22

4

4.2

2.1

2.8

0.7

2011-08-23

5

4.2

2.1

2.8

0.7

2011-08-24

6

1.4

0.0

0.0

-1.4

2011-08-25

7

5.6

2.8

4.2

1.4

2011-08-26

8

7.0

4.2

4.2

1.4

2011-08-27

9

7.0

4.2

4.2

1.4

2011-08-28

10

7.0

3.5

5.6

2.1

2011-08-29

11

5.6

2.8

5.6

2.8

2011-08-30

12

5.6

2.8

4.2

1.4

2011-09-01

13

5.6

2.8

5.6

2.8

2011-09-02

14

11.3

4.3

2.8

-4.3

2011-09-03

15

11.3

4.3

4.2

-2.9

2011-09-04

16

12.7

5.7

4.2

-2.9

2011-09-05

17

12.7

5.0

4.2

-3.6

2011-09-06

18

12.7

5.0

4.2

-3.6

2011-09-07

19

12.7

5.0

5.6

-2.2

2011-09-08

20

12.7

5.0

5.6

-2.2

2011-09-09

21

14.1

6.4

5.6

-2.2

2011-09-10

22

14.1

5.7

7.0

-1.5

2011-09-11

23

15.5

7.1

7.0

-1.5

2011-09-12

24

15.5

7.1

7.0

-1.5

2011-09-13

25

14.1

5.7

5.6

-2.9

2011-09-14

26

15.5

7.1

7.0

-1.5

2011-09-15

27

15.5

7.1

7.0

-1.5

2011-09-16

28

16.9

7.8

7.0

-2.2

Biological Oxygen Demand (BOD) of Inoculum Control, Functional Control,
and Toxicity Control

BOD [mg O2/L]

Date

[d]

Inoculum
Control

Functional
Control
45 mg/L

Toxicity Control
50 mg/L Test
Item+ 45 mg/L
Reference Item

R1

R2

T1

mean

gross

net

gross

net

gross

net

2011-08-19

1

0.7

36.6

35.9

39.5

38.8

36.6

35.9

2011-08-20

2

1.4

50.7

49.3

55.0

53.6

52.1

50.7

2011-08-21

3

1.4

55.0

53.6

59.2

57.8

57.8

56.4

2011-08-22

4

2.1

56.4

54.3

62.0

59.9

60.6

58.5

2011-08-23

5

2.1

56.4

54.3

63.4

61.3

62.0

59.9

2011-08-24

6

1.4

53.5

52.1

63.4

62.0

60.6

59.2

2011-08-25

7

2.8

57.8

55.0

67.6

64.8

66.2

63.4

2011-08-26

8

2.8

57.8

55.0

69.0

66.2

66.2

63.4

2011-08-27

9

2.8

57.8

55.0

70.5

67.7

67.6

64.8

2011-08-28

10

3.5

57.8

54.3

73.3

69.8

69.0

65.5

2011-08-29

11

2.8

56.4

53.6

70.5

67.7

67.6

64.8

2011-08-30

12

2.8

55.0

52.2

70.5

67.7

67.6

64.8

2011-09-01

13

2.8

53.5

50.7

70.5

67.7

69.0

66.2

2011-09-02

14

7.1

47.9

40.9

84.5

77.5

80.3

73.3

2011-09-03

15

7.1

47.9

40.9

86.0

79.0

81.7

74.7

2011-09-04

16

7.1

47.9

40.9

86.0

79.0

83.1

76.1

2011-09-05

17

7.8

47.9

40.2

87.4

79.7

83.1

75.4

2011-09-06

18

7.8

47.9

40.2

87.4

79.7

84.5

76.8

2011-09-07

19

7.8

46.5

38.8

88.8

81.1

86.0

78.3

2011-09-08

20

7.8

46.5

38.8

88.8

81.1

86.0

78.3

2011-09-09

21

7.8

46.5

38.8

88.8

81.1

86.0

78.3

2011-09-10

22

8.5

47.9

39.5

90.2

81.8

87.4

79.0

2011-09-11

23

8.5

46.5

38.1

90.2

81.8

87.4

79.0

2011-09-12

24

8.5

45.1

36.7

91.6

83.2

88.8

80.4

2011-09-13

25

8.5

45.1

36.7

91.6

83.2

88.8

80.4

2011-09-14

26

8.5

45.1

36.7

93.0

84.6

90.2

81.8

2011-09-15

27

8.5

43.7

35.3

94.4

86.0

90.2

81.8

2011-09-16

28

9.2

43.7

34.6

95.8

86.7

91.6

82.5

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test item is classified as not readily biodegradable in the 10-d-window and after 28 days.
Executive summary:

The ready biodegradability of the test item Amino-C-Dion trocken (batch number: 241) was determined with non adapted activated sludge in the Manometric Respirometry Test for a period of 28 days. The study was conducted from 2011-07-20 to 2011-09-16 with the definitive exposure phase from 2011-08-19 to 2011-09-16 according to OECD guideline 301 F at Dr.U.Noack-Laboratorien, D-31157 Sarstedt. The test item concentration selected as appropriate was 50 mg/L, corresponding to a ThOD of 58.0mg O2/L per test vessel. The oxygen was depleted by the respiration of bacteria and the degradation was followed by measuring the oxygen concentration. The biodegradation rate is therefore expressed as the percentage BOD (biological oxygen demand) and was calculated for each study day.

The mean oxygen depletion in the inoculum control was 9.2 mg O2/L on day 28.

In order to check the activity of the test system sodium benzoate was used as functional control. The pass level > 60 % was reached after 2 days. The biodegradation rate reached a plateau phase with 67 % degradation in the 1streplicate and 90 % in the 2nd replicate on day 13.

In the toxicity control containing both test and reference item 55 % degradation occurred within 14 days. After 28 days the biodegradation came to 62 %. The degradation of the reference item was not inhibited by the test item.

The 1st test item replicate reached the 10 % level (beginning of biodegradation) after 16 days, the 2nd test item replicate did not reach the 10 % level. The 60 % pass level was not reached within 28 days. After 28 days the mean biodegradation was 7 %.

The validity criteria of the guideline are fulfilled.

The test item is classified as

not readily biodegradable

in the 10-d-window and after 28 days.