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Environmental fate & pathways

Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Duration of test (contact time):
29 d
Initial conc.:
20.4 mg/L
Based on:
test mat.
Initial conc.:
20.3 mg/L
Based on:
test mat.
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (CO2 evolution)
Value:
-11
Sampling time:
28 d
Details on results:
Degradation was followed over the total test duration of 29 days after 0, 2, 5, 9, 14, 19, 22, 28 and 29 days via TIC analysis of the bound CO2.

On the 28th day, the residual dissolved CO2 was expelled by acidification of the test batches. No more biodegradation takes place since the micro-organisms are destroyed by the acidification. The degradation values determined on the 29th day (end of incubation) are therefore assigned to the 28th day.

The test item 3 -Dodecyl-1 -(2,2,6,6 -tetramethyl-4 -piperidinyl)-pyrrolidin-2,5 -dion studied reached a degree of degradation of -11 % in the course of 28 days (mean of the test item batches). The details of the test evaluations are presented in Appendix No. 1 -5. The negative value of the degree of degradation is due to mathematical calculations. Probably, the test item had toxic effects on the inoculum used. The additionally performed determination of the number of colony forming at the end of the test did not give significant indications for toxicity.

Validity criteria fulfilled:
yes
Conclusions:
The substance showed no degradation (-11 % degradation) within 28 days.
Executive summary:

A Modified Sturm test (OECD Guideline 301 B) was performed compliant with GLP (RL 1; Infracor, 2002). Non-adapted activated sludge from a municipal sewage treatment plant was used as inoculum. The inoculum had a bacterial count of 114 * 10^4 CFU/mL (colony forming units/mL). Approximately 61 mg test item was used per 3-L vessle (n=2). Further, 2 replicate inoculum controls and one test flask with reference item sodium benzoate were included in the test. Degradation extent was claculated based on CO2 evolution during the test (TIC analysis).

As a result, the substance showed no degradation (- 11 % degradation) within 28 days. While inoculum toxicity cannot be excluded as a reason for this (no toxicity control included), an additionally performed determination of the number of colony forming units at the end of the test did not give significant indications for toxicity (CFU/ml: 59x10^5; reference item: 27x10^3; blank 34x10^2).

Endpoint:
biodegradation in water: screening tests
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-08-12 to 2015-09-10
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))
Deviations:
yes
Remarks:
Non-adapted activated sludge used as inoculum instead of mixed inoculum from different sources (OECD 302 C)
Principles of method if other than guideline:
Instead of the test system generated of a mixed inoculum from different sources, inoculum of non adapted activated sludge was used.
GLP compliance:
yes
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
- Receipt: 2015-08-07
- Preparation of inoculum for exposure: The activated sludge was washed twice with chlorine free tap water. After the second washing the settled sludge was resuspended in tap water and maintained in an aerobic condition by aeration with CO2-free air for 5 days.
- Inoculation: 15.7 mL/L inoculum corresponding to the final concentration of 100 mg/L suspended solids was used to initiate inoculation (Functional control replicates: 4.7 mL/L inoculum corresponding to 30 mg/L suspended solids).

Duration of test (contact time):
28 d
Initial conc.:
30 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral salts medium acc. to OECD 302 C
- Additional substrate: No
- Test temperature: Nominal: 25, ± 2 °C; Actual: 24.3 – 26.3 °C

ThOD 2.63 mg O2/mg test item
ThODNO3 2.94 mg O2/mg (with nitrification)
Test concentration 30 mg/L (corresponding to 150 µL stock solution / 250 mL test solu-tion)
Stock solution 50 g/L
Solvent Dichloromethane PESTINORM (Purity: 99.8 %, VWR CHEMICALS)
ThOD in the test vessel 78.9 mg O2/L
ThODNO3 in the test vessel 88.2 mg O2/L (with nitrification)
Replicates Triplicates

- pH:
pH-Values in the Test Vessels at Test Start and Test End

pH-Value
Start End
Inoculum Control 7.48 C1) 6.65
C2) 6.72
Cs) 7.72
Functional Control
Sodium Benzoate 7.51 1) 7.67
2) 7.63
Test Item 7.44 1) 7.72
2) 7.66
3) 7.74
Blank 5.99 9.19
1) - 3) = replicate number

- pH adjusted: no
- Dispersion treatment: Continuous stirring
- Continuous darkness: Dark, in an incubator


TEST SYSTEM
- Culturing apparatus: Incubator
- Number of culture flasks/concentration: 3
- 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 in the incubation vessels by the 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 Control: Test medium and inoculum without test and/or reference item
- Solvent Control: Test medium, inoculum and 150 µL dichlormethane without test and /or reference item
- Blank: Test item in ultrapure water



STATISTICAL METHODS:
Calculation of the theoretical oxygen demand (ThOD) of the test item and reference item, respectively, according to formula (1):

16  ( 2c + 1/2  ( h - cl - 3n ) + 3s + 5/2p + 1/2na - o )
ThOD =  (1)
MW
MW = Molecular weight of test item / reference item

As described in 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 item and reference item was
calculated from the difference of the test replicates and inoculum control replicates

The biodegradation of the test item and referenceitem was calculated out of the BOD in relation to the ThOD.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
No preliminary study
Test performance:
For information see: Any other information on materials and methods incl. tables
Parameter:
% degradation (O2 consumption)
Value:
3
Sampling time:
28 d
Details on results:
Theoretical Oxygen DemandThe biodegradation in % was calculated based on the ThOD of 2.63 mg O2 /mg test item.
No degradation was observed, therefore, the alternative ThOD with nitrification was not taken into account.

The concentration of suspended solids of the inoculum after test system preparation was 6.38 g/L. 15.7 mL/L inoculum corresponding to the final concentration of 100 mg/L suspended solids was used to initiate inoculation (Functional control replicates: 4.7 mL/L inoculum corresponding to 30 mg/L sus-pended solids).

Results of the Blank:
In the blank, no abiotic degradation was observed (0.0 mg O2/L on day 28).

Course and Stages of Inherent Biodegradation of the Test Item:
The biodegradation of the test item is shown graphically in comparison to the inherent degradable functional control. The test item replicates did not reach the 10 % level (beginning of biodegradation) within 28 days. The pass level > 70 % for evidence of inherent, ultimate biodegrability was not reached within 28 days.

Under the test conditions, the test item Hostavin 3055, was not inherently biodegradable within 28 days.


Results with reference substance:
Results of the Functional Control:
The adaption phase changed to degradation phase after 1 day in both replicates. The functional control Sodium Benzoate reached the pass level of > 65 % after 2 days in the mean of both replicates. The mean biodegradation rate was 100 % on day 7 and remained on that level until day 28.

Biological Oxygen Demand (BOD) of Inoculum Control and Solvent Control

 

 

BOD [mg O2/L]

 

 

Inoculum
Control

Solvent
Control

 

Date

[d]

C1

C2

CS

mean

2015-08-13

1

11.3

11.3

5.6

9.4

2015-08-14

2

19.7

15.5

9.9

15.0

2015-08-15

3

31.0

26.8

21.1

26.3

2015-08-16

4

40.9

33.8

25.4

33.4

2015-08-17

5

47.9

39.5

28.2

38.5

2015-08-18

6

55.0

46.5

32.4

44.6

2015-08-19

7

63.4

55.0

39.5

52.6

2015-08-20

8

69.0

59.2

45.1

57.8

2015-08-21

9

73.3

63.4

46.5

61.1

2015-08-22

10

76.1

67.6

49.3

64.3

2015-08-23

11

77.5

69.0

50.7

65.7

2015-08-24

12

77.5

67.6

47.9

64.3

2015-08-25

13

81.7

73.3

52.1

69.0

2015-08-26

14

84.5

74.7

55.0

71.4

2015-08-27

15

87.4

77.5

55.0

73.3

2015-08-28

16

88.8

78.9

57.8

75.2

2015-08-29

17

90.2

77.5

55.0

74.2

2015-08-30

18

91.6

80.3

56.4

76.1

2015-08-31

19

94.4

81.7

56.4

77.5

2015-09-01

20

98.6

86.0

60.6

81.7

2015-09-02

21

97.2

83.1

56.4

78.9

2015-09-03

22

101.0

88.8

62.0

83.9

2015-09-04

23

101.0

87.4

59.2

82.5

2015-09-05

24

104.0

90.2

60.6

84.9

2015-09-06

25

107.0

91.6

62.0

86.9

2015-09-07

26

106.0

90.2

59.2

85.1

2015-09-08

27

107.0

91.6

60.6

86.4

2015-09-09

28

108.0

93.0

62.0

87.7

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

 

 

 

BOD [mg O2/L]

Inoculum
Control

Functional Control
Sodium Benzoate 45 mg/L

Blank
30 mg/L

 

R1

R2

B1

Date

[d]

mean*

gross

net

gross

net

gross

2015-08-13

1

2.8

49.3

46.5

43.7

40.9

-1.4

2015-08-14

2

4.6

60.6

56.0

59.2

54.6

-2.8

2015-08-15

3

8.0

74.7

66.7

71.9

63.9

0.0

2015-08-16

4

10.1

80.3

70.2

78.9

68.8

0.0

2015-08-17

5

11.7

81.7

70.0

83.1

71.4

-1.4

2015-08-18

6

13.5

86.0

72.5

87.4

73.9

-2.8

2015-08-19

7

15.9

93.0

77.1

93.0

77.1

0.0

2015-08-20

8

17.5

97.2

79.7

97.2

79.7

0.0

2015-08-21

9

18.5

98.6

80.1

98.6

80.1

0.0

2015-08-22

10

19.5

101.0

81.5

101.0

81.5

0.0

2015-08-23

11

19.9

103.0

83.1

101.0

81.1

0.0

2015-08-24

12

19.5

97.2

77.7

101.0

81.5

-2.8

2015-08-25

13

20.9

103.0

82.1

104.0

83.1

-1.4

2015-08-26

14

21.6

106.0

84.4

106.0

84.4

0.0

2015-08-27

15

22.2

106.0

83.8

106.0

83.8

0.0

2015-08-28

16

22.8

108.0

85.2

107.0

84.2

-1.4

2015-08-29

17

22.5

104.0

81.5

107.0

84.5

-1.4

2015-08-30

18

23.1

107.0

83.9

107.0

83.9

-1.4

2015-08-31

19

23.5

107.0

83.5

108.0

84.5

-1.4

2015-09-01

20

24.8

111.0

86.2

111.0

86.2

0.0

2015-09-02

21

23.9

107.0

83.1

110.0

86.1

-1.4

2015-09-03

22

25.4

113.0

87.6

113.0

87.6

0.0

2015-09-04

23

25.0

110.0

85.0

113.0

88.0

0.0

2015-09-05

24

25.7

111.0

85.3

114.0

88.3

0.0

2015-09-06

25

26.3

114.0

87.7

116.0

89.7

1.4

2015-09-07

26

25.8

111.0

85.2

114.0

88.2

-1.4

2015-09-08

27

26.2

111.0

84.8

116.0

89.8

-1.4

2015-09-09

28

26.6

114.0

87.4

116.0

89.4

0.0

*) mean value of C1, C2and Cs, divided by 3.3 in order to take the lower inoculum concentration of the reference item replicates into account

Validity criteria fulfilled:
yes
Interpretation of results:
not inherently biodegradable
Conclusions:
Biodegradation [%] of the Test Item in Comparison to the Functional Control


Biodegradation [%]
Functional Control
Sodium Benzoate Test Item
Date [d] R1NaB R2NaB mv P1 P2 P3 mv
2015-08-13 1 62 54 58 0 1 4 2
2015-08-14 2 75 73 74 0 0 0 0
2015-08-15 3 89 85 87 0 0 0 0
2015-08-16 4 93 91 92 0 0 0 0
2015-08-17 5 93 95 94 0 0 0 0
2015-08-18 6 96 98 97 0 0 0 0
2015-08-19 7 100 100 100 0 0 0 0
2015-08-20 8 100 100 100 0 0 0 0
2015-08-21 9 100 100 100 0 0 0 0
2015-08-22 10 100 100 100 0 0 0 0
2015-08-23 11 100 100 100 0 0 0 0
2015-08-24 12 100 100 100 0 0 0 0
2015-08-25 13 100 100 100 0 0 0 0
2015-08-26 14 100 100 100 0 0 0 0
2015-08-27 15 100 100 100 0 0 0 0
2015-08-28 16 100 100 100 0 0 0 0
2015-08-29 17 100 100 100 0 0 0 0
2015-08-30 18 100 100 100 0 0 0 0
2015-08-31 19 100 100 100 0 0 0 0
2015-09-01 20 100 100 100 0 0 0 0
2015-09-02 21 100 100 100 0 0 2 1
2015-09-03 22 100 100 100 0 0 3 1
2015-09-04 23 100 100 100 0 0 2 1
2015-09-05 24 100 100 100 0 0 3 1
2015-09-06 25 100 100 100 0 0 6 2
2015-09-07 26 100 100 100 0 0 5 2
2015-09-08 27 100 100 100 0 0 7 2
2015-09-09 28 100 100 100 0 1 9 3
mv = mean value

Under test conditions, the test item was not inherently biodegradable within 28 days.
Executive summary:

The inherent, ultimate 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 2015-08-12 to 2015-09-10 according to OECD Guideline 302 C at Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany. The test item concentration selected as appropriate was 30 mg/L, corresponding to a ThOD of 78.9 mg O2/L (88.2 mg O2/L with nitrification). 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 87.7 mg O2/L on day 28.

 

In order to check the activity of the test system Sodium Benzoate was used as functional control in separate replicates. The functional control Sodium Benzoate reached the pass level of > 65 % after 2 days in the mean of both replicates. The mean biodegradation rate was 100 % on day 7 and remained on that level until day 28.

 

The biodegradation of the test item is shown graphically indicated in comparison to the inherent degradable functional control. The test item replicates did not reach the 10 % level (beginning of biodegradation) within 28 days. The pass level > 70 % for evidence of inherent, ultimate biodegradability was not reached within 28 days.

 

The validity criteria of the guideline are fulfilled.

Under 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

1

0

0

0

0

2

0

0

0

1

3

0

0

2

9

 

mv

0

0

1

3

Functional Control

Sodium Benzoate

1

100

100

100

100

2

100

100

100

100

mv

100

100

100

100

mv = mean value

Description of key information

In screening tests no relevant ultimate biodegradation observed.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

Biodegradability of the test item was assessed based on two valid screening tests:

A Modified Sturm test (OECD Guideline 301 B) was performed compliant with GLP (RL 1; Infracor, 2002). Non-adapted activated sludge from a municipal sewage treatment plant was used as inoculum. The inoculum had a bacterial count of 114 * 10^4 CFU/mL (colony forming units/mL). Approximately 61 mg test item was used per 3-L vessel (n=2). Further, 2 replicate inoculum controls and one test flask with reference item sodium benzoate were included in the test. Degradation extent was calculated based on CO2 evolution during the test (TIC analysis).

As a result, the substance showed no degradation (- 11 % degradation) within 28 days. While inoculum toxicity cannot be excluded as a reason for this (no toxicity control included), an additionally performed determination of the number of colony forming units at the end of the test did not give significant indications for toxicity (CFU/ml: 59x10^5; reference item: 27x10^3; blank 34x10^2).    

Therefore, a test on inherent ultimate biodegradability of the test item was performed with non-adapted activated sludge for a period of 28 days. The study was conducted according to OECD Guideline 302 C compliant with GLP (RL 1; Noack, 2015). The test item concentration selected as appropriate was 30 mg/L, corresponding to a ThOD of 78.9 mg O2/L (88.2 mg O2/L with nitrification). 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. All validity criteria were fulfilled.

The test item showed no relevant degradation within 28 days (arithmetic mean value: 3%; n=3). As a functional control sodium Benzoate was used: the pass level of > 65 % was reached after 2 days (mean of two replicates), the mean biodegradation rate was 100 % on day 7 and remained on that level until day 28. This confirmed activity of the test system.

Based on the results of the sewage treatment plant microorganism toxicity test (OECD 209), the concentration applied in OECD 301B would correspond 10% inhibition of respiration (EC10 21.3 mg/L), the respective concentration from OECD 302C equivalent to 20% inhibition (EC20 30.5 mg/L). Thus, inhibition of certain more sensitive microorganisms possibly decisive for biodegradation cannot be excluded.