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Environmental fate & pathways

Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2011-08-15 to 2011-09-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to relevant guidelines and compliant to GLP

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))
Deviations:
yes
Remarks:
Instead of the test system generated of a mixed inoculum from different sources, inoculum of the aqueous phase of non-adapted activated sludge was used.
GLP compliance:
yes (incl. certificate)
Remarks:
according to German Chemikaliengesetzt and directive 88/320/EEC

Test material

Reference
Name:
Unnamed
Type:
Constituent

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): Inoculum of the aqueous phase of non adapted activated sludge. Municipal sewage treatment plant, D-31137 Hildesheim. Activated sludge from the sewage plant at Hildesheim is well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste.
- Pretreatment: The activated sludge was washed twice with autoclaved tap water. After washing, the supernatant was decanted and the settled sludge was maintained in an aerobic condition by aeration with CO2 free air until test start.
- Inoculation: 10 ml/L inoculum (functional control: 4 ml/L) corresponding to the final concentration of 100 mg/L (functional control: 40 mg/L) suspended solids was used to initiate inoculation.
- Initial cell/biomass concentration: 10 mL/L inoculum was used to initiate inoculation
- Water filtered: no
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
30 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 302C
- Test temperature: 25.0 - 25.5 °C
- pH:
pH-Values in the Test Vessels at Test Start and Test End
pH-Value
Start End
Inoculum Control - 1) 6,42
2) 6,24
Functional Control - 1) 6,40
2) 6,58
Test Item 7,28 1) 6,40
2) 6,36
3) 6,36
Blank - 8,23

- pH adjusted: no
- Aeration of dilution water: Continuous stirring
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: Brown glass bottles, volume 500 mL
- Number of culture flasks/concentration: Triplicates
- Method used to create aerobic conditions: Continuous stirring
- Measuring equipment: The oxygen consumption was determined in the incubation vessels by the OxiTop measuring system at 360 measuring points during the 28 d incubation period.
- Details of trap for CO2 and volatile organics if used: A rubber sleeve with soda lime was hung into the opening of the bottles. The bottles were closed with OxiTop measuring heads and the measuring system was activated.


SAMPLING
- Sampling frequency: The temperature in the incubator was documented continuously throughout the test with a hygrothermograph.
The colony forming units and concentration of suspended solids of the inoculum were determined at test start.
The pH of the test item solution was determined in a separate replicate at test start. No adjustment of pH was necessary.
The pH of the test solutions was determined at test end.
The oxygen consumption was determined in the incubation vessels by the OxiTop measuring system at 360 measuring points during the 28 d incubation period.


CONTROL AND BLANK SYSTEM
- Inoculum blank: 30 mg/L test item in aqua demin.
- Abiotic sterile control: No
- Functional control: Anilin, 25 mg/L
- Inoculum control: Test medium and inoculum without test and reference item.



Reference substance
Reference substance:
aniline

Results and discussion

Test performance:
Based on the calculated oxygen demand (ThOD), the test concentration of 30 mg/L corresponding to an oxygen demand of
38.1 mgO2/L in the vessel was selected.

The test solutions were prepared in measuring flasks and given into brown glass bottles as incubation vessels (inoculum control, functional control) or were prepared directly in brown glass
bottles (test item, blank):
• 3 incubation vessels for the test item (P1, P2, P3)
• 2 incubation vessels for the functional control (R1, R2)
• 1 incubation vessel for the blank (B1)
• 2 incubation vessels for the inoculum control (C1, C2)

A separate test item replicate was prepared for pH measurement at test start:
• 1 incubation vessel for the test item (PpH).

The test item was weighed out and was transferred directly into the brown glass bottles with 250 mL test medium (consisting of the appropriate volumes of mineral medium stock solutions, de-mineralised water and inoculum) for the test item replicates and with 250 mL demineralised water for the blank, respectively.

For the functional control the reference item was weighed out and was transferred into a 500 mL measuring flask with demineralised water. The appropriate volumes of mineral medium stock solutions and inoculum were added, then the flask was filled up with demineralised water.

The inoculum control, consisting of mineral medium stock solutions, demineralised water and inoculum, was prepared in a 500 mL measuring flask.
The test solutions for reference item and inoculum control were divided using 250 mL measuring flasks before being filled into the brown glass bottles.
A rubber sleeve with soda lime was hung into the opening of the bottles. The bottles were closed with OxiTop measuring heads and the measuring system was activated.
% Degradation
Parameter:
% degradation (O2 consumption)
Value:
10
Sampling time:
28 d
Details on results:
Colony forming units (CFU) of the inoculum were determined at test start by standard dilution plate count: 2.0 x 109 CFU/L.
The concentration of suspended solids of the inoculum at test start was 9.79 g/L corresponding to 97.9 mg/L in the test vessels of the test item and control replicates and 39.2 mg/L in the vessels of the reference item replicates.

BOD5 / COD results

Results with reference substance:
The functional control reached the pass level of > 65 % after 4 days in the 1st replicate and after 10 days in the 2nd replicate. It came to a biodegradation rate of 100 % after 28 days. Hence the validity criterion that the degradation should be > 65 % after 14 days was fulfilled.

Any other information on results incl. tables

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

BOD [mg O2/L]

Inoculum
Control

Functional Control
25 mg/L

Blank
30 mg/L

R1

R2

B1

Date

[d]

mean*

gross

Net

gross

net

gross

16.08.2011

1

2.2

0.0

-2.2

0.0

-2.2

-5.6

17.08.2011

2

4.8

0.0

-4.8

0.0

-4.8

-1.4

18.08.2011

3

7.3

40.9

33.6

11.3

4.0

-4.2

19.08.2011

4

9.3

49.3

40.0

39.5

30.2

0.0

20.08.2011

5

11.8

59.2

47.4

42.3

30.5

0.0

21.08.2011

6

13.2

63.4

50.2

45.1

31.9

0.0

22.08.2011

7

14.9

69.0

54.1

46.5

31.6

0.0

23.08.2011

8

16.1

74.7

58.6

47.9

31.8

0.0

24.08.2011

9

16.6

77.5

60.9

50.7

34.1

1.4

25.08.2011

10

17.5

80.3

62.8

53.5

36.0

0.0

26.08.2011

11

18.0

83.1

65.1

56.4

38.4

-1.4

27.08.2011

12

18.3

84.5

66.2

56.4

38.1

-2.8

28.08.2011

13

19.4

87.4

68.0

60.6

41.2

-1.4

29.08.2011

14

20.3

90.2

69.9

63.4

43.1

0.0

30.08.2011

15

21.1

91.6

70.5

66.2

45.1

-1.4

31.08.2011

16

21.4

91.6

70.2

66.2

44.8

-1.4

01.09.2011

17

23.1

94.4

71.3

69.0

45.9

1.4

02.09.2011

18

24.0

94.4

70.4

69.0

45.0

0.0

03.09.2011

19

24.5

95.8

71.3

69.0

44.5

1.4

04.09.2011

20

26.2

97.2

71.0

70.5

44.3

2.8

05.09.2011

21

26.8

98.6

71.8

70.5

43.7

2.8

06.09.2011

22

28.2

98.6

70.4

73.3

45.1

1.4

07.09.2011

23

28.2

98.6

70.4

71.9

43.7

1.4

08.09.2011

24

29.3

98.6

69.3

77.5

48.2

0.0

09.09.2011

25

29.3

100.0

70.7

77.5

48.2

1.4

10.09.2011

26

30.4

101.0

70.6

87.4

57.0

-1.4

11.09.2011

27

31.3

103.0

71.7

90.2

58.9

2.8

12.09.2011

28

31.6

104.0

72.4

91.6

60.0

2.8

   *) corrected values due to different inoculum concentration in inoculum control and functional control

Biological Oxygen Demand (BOD) of Test Item

BOD [mg O2/L]

Inoculum
Control

Test Item
30 mg/L

P1

P2

P3

Date

[d]

mean

gross

net

gross

net

gross

net

16.08.2011

1

5.6

7.0

1.4

5.6

0.0

4.2

-1.4

17.08.2011

2

12.0

16.9

4.9

15.5

3.5

15.5

3.5

18.08.2011

3

18.3

18.3

0.0

16.9

-1.4

16.9

-1.4

19.08.2011

4

23.3

28.2

5.0

28.2

5.0

26.8

3.6

20.08.2011

5

29.6

33.8

4.2

33.8

4.2

32.4

2.8

21.08.2011

6

33.1

38.0

4.9

38.0

4.9

36.6

3.5

22.08.2011

7

37.3

42.3

5.0

42.3

5.0

40.9

3.6

23.08.2011

8

40.2

45.1

4.9

45.1

4.9

43.7

3.5

24.08.2011

9

41.6

49.3

7.7

49.3

7.7

46.5

4.9

25.08.2011

10

43.7

49.3

5.6

49.3

5.6

46.5

2.8

26.08.2011

11

45.1

49.3

4.2

50.7

5.6

47.9

2.8

27.08.2011

12

45.8

46.5

0.7

47.9

2.1

46.5

0.7

28.08.2011

13

48.6

50.7

2.1

52.1

3.5

50.7

2.1

29.08.2011

14

50.7

52.1

1.4

53.5

2.8

52.1

1.4

30.08.2011

15

52.9

53.5

0.6

55.0

2.2

53.5

0.6

31.08.2011

16

53.6

53.5

0.0

56.4

2.9

55.0

1.5

01.09.2011

17

57.8

59.2

1.4

62.0

4.2

60.6

2.8

02.09.2011

18

59.9

60.6

0.7

63.4

3.5

63.4

3.5

03.09.2011

19

61.3

63.4

2.1

67.6

6.3

67.6

6.3

04.09.2011

20

65.6

64.8

-0.8

70.5

5.0

70.5

5.0

05.09.2011

21

67.0

66.2

-0.8

73.3

6.3

73.3

6.3

06.09.2011

22

70.5

66.2

-4.2

74.7

4.3

74.7

4.3

07.09.2011

23

70.5

67.6

-2.8

76.1

5.7

76.1

5.7

08.09.2011

24

73.3

67.6

-5.7

77.5

4.3

78.9

5.7

09.09.2011

25

73.3

69.0

-4.3

80.3

7.1

80.3

7.1

10.09.2011

26

76.1

67.6

-8.5

78.9

2.9

78.9

2.9

11.09.2011

27

78.2

73.3

-4.9

84.5

6.3

84.5

6.3

12.09.2011

28

78.9

74.7

-4.2

84.5

5.6

84.5

5.6

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The percentage degradation of the functional control reached the pass level > 65 % after 14 days. The difference of extremes of replicate values of biodegradation of the test item at the end of the test was less than 20 %.
Interpretation of results:
not inherently biodegradable
Conclusions:
Under our test conditions, the test item was not inherently biodegradable within 28 days.
Executive summary:

The inherent biodegradability of the test item was determined in the Modified MITI Test (II) with non adapted activated sludge for a period of 28 days. The study was conducted from 2011-08-15 to 2011-09-12 according to OECD Guideline 302 C. The test item concentration selected as appropriate was 30 mg/L, corresponding to a ThOD of 38.1 mgO2/L. The oxygen was depleted by respiration of bacteria and the degradation was followed by measuring the oxygen concentration. The biodegradation is therefore expressed as the percentage BOD depletion and was calculated for each study day.

The mean oxygen depletion in the inoculum control was 78.9 mg O2/L on day 28.

In order to check the activity of the test system aniline was used as functional control. The functional control reached the pass level of > 65 % after 4 days in the 1st replicate and after 10 days in the 2nd replicate. It came to a biodegradation rate of 100 % after 28 days.

The 1st test item replicate reached the 10 % level (beginning of biodegradation) after 2 days and came to a maximum of 20 % after 9 days. The 2ndtest item replicate reached the 10 % level after 4 days and came to a maximum of 20 % after 9 days. The biodegradation of the 3rd test item replicate reached the 10 % level after 9 days and reached 15 % on day 28. The pass level > 70 % for evidence of inherent, ultimate biodegradability was not reached within 28 days. After 28 days the mean biodegradation rate was 10 %.

The mean oxygen depletion in the abiotic control was 2.8 mg O2/L on day 28.

The validity criteria of the guideline are fulfilled.

Under our test conditions, the test item was not inherently biodegradable within 28 days.

Biodegradation of the Test Item in Comparison to the
                   Functional Control

Biodegradation [%]

Study Day [d]

Replicate

7

14

21

28

Test Item
30 mg/L

1

13

4

0

0

2

13

7

17

15

3

9

4

17

15

mv

12

5

11

10

Functional Control
25 mg/L

1

90

100

100

100

2

52

72

73

100

mv

71

86

87

100

                            mv = mean value