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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: screening test, other
Remarks:
Screening test for inherent biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-12-09 to 2012-01-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Performed according to relevant guidelines and compliant to GLP
Qualifier:
according to guideline
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. QA statement)
Remarks:
according to German Chemikaliengesetzt and directive 88/320/EEC
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 the second washing the settled sludge was re-suspended in mineral salts medium. 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 4 days.
- Inoculation: 80 ml/L inoculum (functional control: 32 ml/L) corresponding to the final concentration of 55 mg/L (functional control: 22 mg/L) suspended solids was used to initiate inoculation.
- Initial cell/biomass concentration: 80 mL/L inoculum was used to initiate inoculation
- Water filtered: no
Duration of test (contact time):
28 d
Initial conc.:
30 mg/L
Based on:
test mat.
Initial conc.:
45 mg/L
Based on:
ThOD/L
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 +/- 2°C
- pH:
pH-Value
Start End
Inoculum Control 7.96 1) 7.12
2) 7.14
Functional Control 7.95 1) 6.82
2) 7.15
Test Item 7.85 1) 7.06
2) 7.04
3) 7.05
Blank 6.40 8.04



- 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: 25 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.

Calculation of the theoretical oxygen demand:
Performed according to the following formula: ThOD [mg O2/mg] = 16(2C+0.5*(H - Cl - 3N) + 3S + 2.5*P + 0.5*Na - O)/MW
The biodegradation in % was calculated based on the ThOD of 1.50 mg O2/mg test item.



Reference substance:
aniline
Test performance:
Based on the calculated oxygen demand (ThOD), the test concentration of 30 mg/L corresponded to an oxygen demand of 45.0 mgO2/L in the vessel.
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)

Separate replicates for the test item and blank were prepared for pH measurement at test start:
• 1 incubation vessel for the test item (PpH)
• 1 incubation vessel for the blank (BpH)

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 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 functional control 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.
Key result
Parameter:
% degradation (O2 consumption)
Value:
14
Sampling time:
28 d
Remarks on result:
other: Arithmetic mean of 3 replicates
Details on results:
Colony forming units (CFU) of the inoculum were determined at test start by standard dilution plate count: 5.3 * 10^9 CFU/L.
The concentration of suspended solids of the inoculum at test start was 0.69 g/L corresponding to 55.2 mg/L in the test vessels of the test item and control replicates and 22.1 mg/L in the vessels of the reference item replicates.
All test item replicates reached the 10 % level (beginning of biodegradation) after 5 days. The 1st test item replicate came to a maximum of 14 % after 5 days, the 2nd and the 3rd test item replicate to a maximum of 20 % after 24 and 20 days, respectively. The pass level > 70 % for evidence of inherent, ultimate biodegradability was not reached within 28 days. After 28 days the mean biodegradation rate was 14 %.
Results with reference substance:
The adaptation phase changed to degradation phase after 3 days in both replicates. The functional control reached the pass level of > 65 % after 16 days in the 1st replicate and after 8 days in the 2nd replicate. The mean biodegradation rate on day 14 was 73%. Hence the validity criterion that the degradation should be > 65 % after 14 days was fulfilled. It came to a biodegradation rate of 85 % after 28 days.

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

2011-12-10

1

5.9

2.8

-3.1

1.4

-4.5

-4.2

2011-12-11

2

8.7

5.6

-3.1

4.2

-4.5

-4.2

2011-12-12

3

10.4

22.5

12.1

19.7

9.3

-2.8

2011-12-13

4

12.1

42.3

30.2

42.3

30.2

-2.8

2011-12-14

5

12.7

45.1

32.4

47.9

35.2

0.0

2011-12-15

6

13.5

45.1

31.6

50.7

37.2

0.0

2011-12-16

7

14.4

46.5

32.1

52.1

37.7

1.4

2011-12-17

8

15.5

47.9

32.4

55.0

39.5

1.4

2011-12-18

9

16.1

47.9

31.8

57.8

41.7

1.4

2011-12-19

10

16.4

49.3

32.9

59.2

42.8

1.4

2011-12-20

11

16.9

50.7

33.8

62.0

45.1

1.4

2011-12-21

12

17.2

52.1

34.9

64.8

47.6

1.4

2011-12-22

13

17.5

53.5

36.0

66.2

48.7

1.4

2011-12-23

14

18.3

55.0

36.7

69.0

50.7

2.8

2011-12-24

15

18.3

56.4

38.1

69.0

50.7

1.4

2011-12-25

16

18.6

57.8

39.2

70.5

51.9

2.8

2011-12-26

17

18.9

57.8

38.9

71.9

53.0

2.8

2011-12-27

18

19.2

59.2

40.0

73.3

54.1

2.8

2011-12-28

19

19.7

59.2

39.5

74.7

55.0

2.8

2011-12-29

20

19.4

59.2

39.8

74.7

55.3

2.8

2011-12-30

21

19.7

59.2

39.5

74.7

55.0

2.8

2011-12-31

22

20.0

60.6

40.6

76.1

56.1

2.8

2012-01-01

23

20.6

60.6

40.0

76.1

55.5

2.8

2012-01-02

24

20.6

62.0

41.4

77.5

56.9

2.8

2012-01-03

25

20.9

63.4

42.5

76.1

55.2

2.8

2012-01-04

26

21.1

63.4

42.3

77.5

56.4

2.8

2012-01-05

27

21.1

66.2

45.1

77.5

56.4

2.8

2012-01-06

28

21.4

67.6

46.2

77.5

56.1

2.8

*) Corrected values due to different incoulum concentrations in inoculum control and functional control.

Biological Oxygen Demand (BOD) of the 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

2011-12-10

1

14.8

18.3

3.5

15.5

0.7

14.1

-0.7

2011-12-11

2

21.8

22.5

0.7

22.5

0.7

21.1

-0.7

2011-12-12

3

26.1

28.2

2.1

26.8

0.7

26.8

0.7

2011-12-13

4

30.3

31.0

0.7

31.0

0.7

29.6

-0.7

2011-12-14

5

31.7

38.0

6.3

38.0

6.3

38.0

6.3

2011-12-15

6

33.8

39.5

5.7

40.9

7.1

40.9

7.1

2011-12-16

7

35.9

40.9

5.0

43.7

7.8

42.3

6.4

2011-12-17

8

38.8

42.3

3.6

45.1

6.4

45.1

6.4

2011-12-18

9

40.2

43.7

3.5

47.9

7.7

46.5

6.3

2011-12-19

10

40.9

45.1

4.2

49.3

8.4

47.9

7.0

2011-12-20

11

42.3

45.1

2.8

49.3

7.0

49.3

7.0

2011-12-21

12

43.0

45.1

2.1

50.7

7.7

50.7

7.7

2011-12-22

13

43.7

46.5

2.8

52.1

8.4

52.1

8.4

2011-12-23

14

45.8

47.9

2.1

53.5

7.7

53.5

7.7

2011-12-24

15

45.8

47.9

2.1

53.5

7.7

53.5

7.7

2011-12-25

16

46.5

47.9

1.4

55.0

8.5

55.0

8.5

2011-12-26

17

47.2

49.3

2.1

55.0

7.8

55.0

7.8

2011-12-27

18

47.9

49.3

1.4

55.0

7.1

56.4

8.5

2011-12-28

19

49.3

50.7

1.4

56.4

7.1

56.4

7.1

2011-12-29

20

48.6

50.7

2.1

56.4

7.8

57.8

9.2

2011-12-30

21

49.3

50.7

1.4

57.8

8.5

57.8

8.5

2011-12-31

22

50.0

52.1

2.1

57.8

7.8

59.2

9.2

2012-01-01

23

51.4

52.1

0.7

59.2

7.8

60.6

9.2

2012-01-02

24

51.4

53.5

2.1

60.6

9.2

60.6

9.2

2012-01-03

25

52.2

53.5

1.4

60.6

8.5

60.6

8.5

2012-01-04

26

52.9

53.5

0.6

60.6

7.8

62.0

9.2

2012-01-05

27

52.9

55.0

2.2

60.6

7.8

62.0

9.2

2012-01-06

28

53.6

55.0

1.5

62.0

8.5

62.0

8.5

Validity criteria fulfilled:
yes
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 according to OECD Guideline 302 C. The test item concentration selected as appropriate was 30 mg/L, corresponding to a ThOD of 45.0 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 53.6 mg O2/L on day 28.

In order to check the activity of the test system Anilin was used as functional control. The functional control reached the pass level of > 65 % after 16 days in the 1st replicate and after 8 days in the 2nd replicate. The mean biodegradation rate on day 14 was 73%. It came to a biodegradation rate of 85 % after 28 days.

All test item replicates reached the 10 % level (beginning of biodegradation) after 5 days. The 1st test item replicate came to a maximum of 14 % after 5 days, the 2nd and the 3rd test item replicate to a maximum of 20 % after 24 and 20 days, respectively. The pass level > 70 % for evidence of inherent, ultimate biodegradability was not reached within 28 days. After 28 days the mean biodegradation rate was 14 %.

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

11

5

3

3

2

17

17

19

19

3

14

17

19

19

mv

14

13

14

14

Functional Control
25 mg/L

1

53

61

66

77

2

63

84

91

93

mv

58

73

79

85

Endpoint:
biodegradation in water: screening test, other
Remarks:
Screening test for inherent biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
assessment report
Key result
Parameter:
% degradation (O2 consumption)
Value:
14
Sampling time:
28 d
Remarks on result:
other: Arithmetic mean of 3 replicates
Validity criteria fulfilled:
yes
Interpretation of results:
not inherently biodegradable
Conclusions:
Under our test conditions, the test item was not inherently biodegradable within 28 days.
Executive summary:

The study used as source investigated C.I. Pigment Yellow 1. The study results of the source compound were considered applicable to the target compound and were used for classification and labelling acc. to REGULATION (EC) No 1272/2008. Justification and applicability of the read-across approach (structural analogue) is outlined in the read-across report in section 13 or find a link in cross reference “assessment report”.

Description of key information

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

Key value for chemical safety assessment

Additional information