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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1999-07-29 to 1999-08-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Collected from secondary effluent of a domestic sewage treatment plant (Kläranlage Odenthal, Germany; date of collection: 1999-07-23)
- Treatment given: Separation of coarse particles by filtration
- Concentration of sludge: 5 mL/L
- Initial cell/biomass concentration: 2.2 mg/L test substance
- Preconditioning: aeration for 6 days
Duration of test (contact time):
28 d
Initial conc.:
2.2 mg/L
Based on:
test mat.
Remarks:
corresponding ThOD: 3288 mg/g
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
20 +- 1 °C
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
19
Sampling time:
28 d
Results with reference substance:
44% biodegradation after 28 days.
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test substance is not readily biodegradable, since a within the test period of 28 days a biodegradation of 19% was determined.
Executive summary:

To assess the ready biodegradability of the test substance, a GLP-complient Closed Bottle Test according to OECD Guideline 301 E and EU Method C.4-E was carried out. The test substance was supended in a mineral medium, inoculated with non-adapted activated sludge from the secondary effluent of a domestic sewage treatment plant and incubated under aerobic conditions in the dark at 20 +- 1 °C. During this period, the biodegradation of the test substance was determined on the basis of the removal of dissolved oxygen. In parallel, a blank control (with inoculum, but without the test substance and the reference substance), a postive control (with inoculum and 2.9 mg/L of the reference substance sodium benzoate) as well as a toxicity control (containing inoculum, 2.2 mg/L test substance and 2.9 mg/L reference substance) was performed. In the toxiciy control, the biodegradation was 39% after 14 days and 44% after 28 days. The result for the positive control (80% biodegradation) confirmed the suitability of the test system. In result, after 28 days the biodegradation of the test substance reached 19%. Thus, the test substance was not readily biodegradable in this test, since the pass level of  70% DOC removal was not reached within a 10-day-window or after after 28 days. The test is regarded as valid.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-11-06 to 2018-01-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
adopted 17.07.1992
Deviations:
yes
Remarks:
Please refer to "Pronciples of method if other than guideline"
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
yes
Remarks:
Please refer to "Pronciples of method if other than guideline"
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
yes
Remarks:
Please refer to "Pronciples of method if other than guideline"
Principles of method if other than guideline:
Due to the high volatility of the test item, the following modifications were made:
- Glass serum bottles, sealed with Teflon lined chlorobutyl septa and aluminium crimp caps were used instead of BOD bottles with glass stoppers. This allowed the injection of the volatile test item without loss directly into the sealed test bottles.
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum: aerobic activated, predominantly domestic sewage sludge from the aeration stage of the sewage treatment plant ARA Birs (Birsfleden / Switzerland)
- Preparation of inoculum for exposure: The aerobic activated sewage sludge was washed three times by centrifugation, decantation of the supernatant liquid phase and resuspension of the solid material in tap water and finally in mineral medium. Aliquots of the homogenized final sludge suspension were weighed, thereafter dries and the dry weight of the suspended solids was determined.

Based on this determination, a calculated amount of wet sludge was suspended in mineral medium to obtain a concentration equivalent to 4 g dry material per liter. During the holding period of four days prior to use, the sludge was aerated at room temperature. Prior to use, the dry weight of the sludge was again determined and the sludge concentration was readjusted to 4 g dry material per liter with mineral medium and filtered through synthetic cotton wool. A volume of 0.5 mL of the filtered sludge was added per 1000 mL test medium as inoculum.
- Pretreatment: No pre-adaption of the inoculum to the test item was done.
- Concentration of sludge: Prior to use, the dry weight of the sludge was again determined and the sludge concentration was readjusted to 4 g dry material per liter with mineral medium and filtered through synthetic cotton wool. A volume of 0.5 mL of the filtered sludge was added per 1000 mL test medium as inoculum.
- Water filtered: Yes.
- Type of filter used: Synthetic cotton wool
Duration of test (contact time):
60 d
Initial conc.:
2.52 mg/L
Based on:
test mat.
Remarks:
corresponding to ThOD = 8.13 mg/L
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: The mineral medium was prepared according to the guidelines and is considered not to contain any contaminant that would affect the integrity or outcome of the study.
- Additional substrate: No
- Solubilising agent: No
- Test temperature: 20 - 21 °C
- pH: 7.4 – 7.5 °C
- pH adjusted: No
- Aeration of dilution water: Shaking at 200 rpm
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: The sealed bottles were placed on an orbital shaker, with a shaking rate of 200 rpm, sufficient to keep the bottle contents well mixed and in suspension. The shaker was placed in a temperature-controlled environment.
- Number of culture flasks/concentration: 49 (including bottles in reserve)
- Method used to create aerobic conditions: Shaking in orbital shaker
- Measuring equipment: For the measurement of the dissolved oxygen (DO) concentration an oxygen meter (F4 from Mettler-Toledo) with DO sensor (LE621 from Mettler-Toledo) was used. The sensor was equipped with a stirring attachment.
- Test performed in closed vessels due to significant volatility of test substance: Yes

SAMPLING
- Sampling frequency: Bottles (in duplicate) were sacrificed for analysis on the day of sampling as follows:
Test item and inoculum control: Exposure days 0, 3, 5, 7, 10, 14, 18, 21, 28, 35, 42, 49, 56 and 60
Procedure control: Exposure days 0, 3, 7, 14, 28, 60
Toxicity control: Exposure days 0, 14, 28 and 60
- Sampling method: The dissolved oxygen (DO) concentration in the test bottles was measured in an airtight system with a DO sensor (under constant stirring at about 500 rpm) connected to an oxygen meter. Two separate test bottles were chosen from each treatment.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 48 bottles
- Procedure control: 22 bottles
- Toxicity control: 18 bottles
Reference substance:
benzoic acid, sodium salt
Preliminary study:
A pre-experiment was performed in order to evaluate if a pH adjustment of the bottle series containing test item will be needed in the test. Therefore, a test bottle filled with 160 mL inoculated mineral medium and sealed, was injected with 0.48 µL test item, mixed and the pH of the dispersion was measured after a few minutes. The pH of the dispersion was 7.5 and therefore, no pH adjustment was necessary.
Test performance:
Oxygen depletion in the inoculum control was 1.2 mg O2/L after 28 days and 1.3 mg O2/L at the end of the test, on day 60 (criterion: should not exceed 1.5 mg O2/L after 28 days).

The residual oxygen concentration in the test item bottles was 5.9 mg O2/L at the end of the test on day 60 (criterion: should not drop below 0.5 mg O2/L at any time). In the reference item bottles, the residual oxygen concentration was 0.6 mg O2/L at the end of the test on day 60. In the toxicity control, by the addition of the same amounts of the test item and reference item as in the test item bottles and reference bottles, respectively, the sum of the ThOD was higher than the availbale dissolved oxygen. As expected, since the test item had no toxic effect, the oxygen was completely depleted.

The degradation rates of the two replicate test bottles containing the test item in inoculated mineral medium deviated by less than 20% at day 28 (normal end of the test) and day 60, i.e. at the end of the test (criterion: should not deviate by more than 20% at the end of the 10-day window, at the time the plateau is reached or the end of the test, as appropriate).

The percentage biodegradation of the reference item sodium benzoate calculated from the oxygen consumption was 91% by day 14 (criterion: should be at least 60% of the ThOD by day 14).
Key result
Parameter:
% degradation (O2 consumption)
Value:
30
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
25
Sampling time:
60 d
Details on results:
The biochemical oxygen demand of the test item in the test media increased from exposure day 3 until exposure day 18, when a mean biodegradation value of 24% was reached. Thereafter, the biodegradation remained more or less constant until the end of the test, on day 60, when a mean value of 25% was obtained. During this phase the calculated mean biodegradation values oscilated between a minimum of 16% on day 49 and a maximum of 30% on day 28.

The pass level for ready biodegradability, i.e. biodegradation of at least 60% of the ThOD in a 10-day window within the 28-day period of the test was not met.
Results with reference substance:
The percent biodegradation of the reference item sodium benzoate was calculated based on the ThOD of 1.67 mg O2/mg. In the procedure control, the reference item was degraded by an average of 75% and 91% by exposure day 3 and 14, respectively, thus conforming the suitability of the activated sludge (>= 60% degradation by exposure day 14). On exposure day 28, the reference item was completely degraded.

Table1: Dissolved Oxygen Concentration in the Test Vessels during the Incubation Period

Time [days]

Dissolved Oxygen Concentration [mg O2/L]

Inoculum Control

Test item

Reference Item

Toxicity Control

Replicate No

Replicate No

Replicate No

Replicate No

0

9.2

9.1

9.1

9.2

9.3

9.2

9.3

9.5

9.4

9.2

9.4

9.3

3

9.3

9.3

9.3

9.5

9.4

9.4

4.1

3.7

3.9

-

-

n.a.

5

9.3

9.2

9.2

8.3

8.3

8.3

-

-

n.a.

-

-

n.a.

7

8.9

9.0

9.0

8.1

8.1

8.1

2.5

2.3

2.4

-

-

n.a.

10

8.8

8.6

8.7

6.4

7.9

7.2

-

-

n.a.

-

-

n.a.

14

8.3

8.1

8.2

7.4

6.0

6.7

1.7

1.4

1.6

0.1

0.1

0.1

18

8.5

8.5

8.5

6.3

6.9

6.6

-

-

n.a.

-

-

n.a.

21

8.3

8.2

8.2

6.6

6.5

6.6

-

-

n.a.

-

-

n.a.

28

8.0

8.0

8.0

5.8

5.6

5.7

0.8

0.7

0.7

0.2

0.2

0.2

35

8.2

8.3

8.2

6.8

6.0

6.4

-

-

n.a.

-

-

n.a.

42

7.7

7.7

7.7

5.9

6.1

6.0

-

-

n.a.

-

-

n.a.

49

7.7

8.0

7.8

7.0

6.3

6.6

-

-

n.a.

-

-

n.a.

60

7.6

8.1

7.8

6.1

5.7

5.9

0.6

0.6

0.6

0.2

0.1

0.1

-: No samples taken

n.a.: Not applicable

 

Table2: Percentage Biodegradation of the Test Item and the Reference Item Sodium Benzoate during the Incubation Period

Time [days]

Test Item

Reference Item

Toxicity Control

 

Replicate No

Replicate No

Replicate No

 

1

2

mean

1

2

mean

1

2

Mean

 

Biodegradation [% of added ThOD]

3

-0.6

0.5

0

72.8

77.3

75

-

-

n.a.

5

12.6

12.3

12.3

-

-

n.a.

-

-

n.a.

7

11.3

11.5

11

88.9

91.3

90

-

-

n.a.

10

28.9

10.4

20

-

-

n.a.

-

-

n.a.

14

11.2

28.1

20

89.8

93.0

91

52.5

52.6

53

18

28.1

20.6

24

-

-

n.a.

-

-

n.a.

21

20.9

22.1

21

-

-

n.a.

-

-

n.a.

28

28.1

31.1

30

98.9

100.2

100

50.8

50.8

51

35

19.2

28.6

24

-

-

n.a.

-

-

n.a.

42

23.5

21.6

23

-

-

n.a.

-

-

n.a.

49

11.8

20.0

16

-

-

n.a.

-

-

n.a.

60

22.5

27.9

25

99.0

99.0

99

50.3

50.3

50

-: No samples taken

n.a.: Not applicable

ThOD: Theoretical oxygen demand in mg O2 per mg test item and/or reference item. For the toxicity control the individual ThOD and the amount of the test item and reference item added were taken into consideration.

Table 3: Biochemical Oxygen Demand [mg O2/mg substance]

Test Conc. Test Item Reference Item Toxicity Control
2.5 mg subst./L 4.5 mg subst./L 7.0 mg subst./L
ThOD 3.23 mg O2/mg subst. 1.67 mg O2/mg subst. 2.23 mg O2/mg subst.
Time [days] Replicate No. Replicate No. Replicate No.
1 2 mean 1 2 mean 1 2 mean
Biochemical Oxygen Demand [mg O2/mg substance]
3 0.0 0.0 0.0 1.2 1.3 1.3 -- -- n.a.
5 0.4 0.4 0.4 -- -- n.a. -- -- n.a.
7 0.4 0.4 0.4 1.5 1.5 1.5 -- -- n.a.
10 0.9 0.3 0.6 -- - n.a. -- -- n.a.
14 0.4 0.9 0.6 1.5 1.6 1.5 1.2 1.2 1.2
18 0.9 0.7 0.8 -- -- n.a. -- -- n.a.
21 0.7 0.7 0.7 -- -- n.a. -- -- n.a.
28 0.9 1.0 1.0 1.7 1.7 1.7 1.1 1.1 1.1
35 0.6 0.9 0.8 -- -- n.a. -- -- n.a.
42 0.8 0.7 0.7 -- -- n.a. -- -- n.a.
49 0.4 0.6 0.5 -- -- n.a. -- -- n.a.
60 0.7 0.9 0.8 1.7 1.7 1.7 1.1 1.1 1.1

--: No samples taken.

n.a.: Not applicable.

ThOD: Theoretical oxygen demand in mg O2 per mg test item and/or reference item. For the toxicity control the individual ThOD and the amount of the test item and reference item added were taken into consideration.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item is not readily biodegradable by OECD criteria (30% in 28 days), but moderately biodegradable under the test conditions chosen.
Executive summary:

The ready biodegradability of the test item was assessed in an GLP-compliant enhanced ready biodegradability test according to OECD Guideline 301 D (1992), EU Method C.4-E and US EPA OPPTS 835.3110 over an extended duration of 60 days. due to the high volatility of the test item the following modification was applied: Instead of BOD bottles with glass stoppers, glass serum bottles, sealed with Teflon lined chlorobutyl septa and aluminium crimp caps were used. This allowed the injection of the volatile test item without loss directly into the sealed test bottles. As inoculum, aerobic activated, predominantly domestic sewage sludge from the aeration stage of a sewage treatment plant was used. The sludge concentration was adjusted to 4 g dry material per liter with mineral medium and filtered through synthetic cotton wool. A volume of 0.5 mL of the filtered sludge was added per 1000 mL test medium as inoculum. The initial test item concentration was 2.52 mg test item/L (based on test material, corresponding to ThOD = 8.13 mg/L; 49 replicates including bottles in reserve). Additionally, an inoculum blank control (48 bottles), a procedure control (with the reference substance sodium benzoate, 22 bottles) and a toxicity control (with the test item and the reference substance, 18 bottles) was performed. The sealed bottles were placed on an orbital shaker, with a shaking rate of 200 rpm, sufficient to keep the bottle contents well mixed and in suspension. The shaker was placed in a temperature-controlled environment. For the measurement of the dissolved oxygen (DO) concentration, an oxygen meter (F4 from Mettler-Toledo) with DO sensor (LE621 from Mettler-Toledo) was used. The sensor was equipped with a stirring attachment. In result, the biochemical oxygen demand of the test item in the test media increased from exposure day 3 until exposure day 18, when a mean biodegradation value of 24% was reached. Thereafter, the biodegradation remained more or less constant until the end of the test, on day 60, when a mean value of 25% was obtained. During this phase the calculated mean biodegradation values oscilated between a minimum of 16% on day 49 and a maximum of 30% on day 28. The pass level for ready biodegradability, i.e. biodegradation of at least 60% of the ThOD in a 10-day window within the 28-day period of the test was not met. The percentage biodegradation of the reference item sodium benzoate calculated from the oxygen consumption was 91% by day 14 (criterion: should be at least 60% of the ThOD by day 14). All vailidity criteria of the guidelines were fulfilled. In conclusion, the test item was not readily biodegradable by OECD criteria (30% in 28 days), but considered to be inherently, primarily biodegradable under the test conditions chosen as the biodegradation rate after 28 and 60 days was above 20%.

Description of key information

The ready biodegradability of the test substance was investigated in an ehanced ready biodegradability test according to the OECD 301 D. The test substance undergoes 30 % biodegradation after 28 days and 25% after 60 days (mean value). Therefore, the test substance is not regarded as readily biodegradable according to OECD criteria, but considered to be inherently, primarily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, fulfilling specific criteria

Additional information

Key information

The ready biodegradability of the test item was assessed in an GLP-compliant enhanced ready biodegradability test according to OECD Guideline 301 D (1992), EU Method C.4-E ans US EPA OPPTS 835.3110 over an extended duration of 60 days (Symrise AG, 2018). Due to the high volatility of the test item the following modification was applied: Instead of BOD bottles with glass stoppers, glass serum bottles, sealed with Teflon lined chlorobutyl septa and aluminium crimp caps were used. This allowed the injection of the volatile test item without loss directly into the sealed test bottles. As inoculum, aerobic activated, predominantly domestic sewage sludge from the aeration stage of a sewage treatment plant was used. The sludge concentration was adjusted to 4 g dry material per liter with mineral medium and filtered through synthetic cotton wool. A volume of 0.5 mL of the filtered sludge was added per 1000 mL test medium as inoculum. The initial test item concentration was 2.52 mg test item/L (based on test material, corresponding to ThOD = 8.13 mg/L; 49 replicates including bottles in reserve). Additionally, an inoculum blank control (48 bottles), a procedure control (with the reference substance sodium benzoate, 22 bottles) and a toxicity control (with the test item and the reference substance, 18 bottles) was performed. In result, the biochemical oxygen demand of the test item in the test media increased from exposure day 3 until exposure day 18, when a mean biodegradation value of 24% was reached. Thereafter, the biodegradation remained more or less constant until the end of the test, on day 60, when a mean value of 25% was obtained. During this phase the calculated mean biodegradation values oscilated between a minimum of 16% on day 49 and a maximum of 30% on day 28. All vailidity criteria of the guidelines were fulfilled. In conclusion, the test item was not readily biodegradable by OECD criteria (30% in 28 days), but considered to be inherently, primarily biodegradable under the test conditions chosen as the biodegradation rate after 28 and 60 days was above 20%.

Supporting information

To assess the ready biodegradability of the test substance, a GLP-complient Closed Bottle Test according to OECD Guideline 301 E and EU Method C.4-E was carried out (Symrise AG, 2000). The test substance was supended in a mineral medium, inoculated with non-adapted activated sludge from the secondary effluent of a domestic sewage treatment plant and incubated under aerobic conditions in the dark at 20 +- 1 °C. During this period, the biodegradation of the test substance was determined on the basis of the removal of dissolved oxygen. In parallel, a blank control (with inoculum, but without the test substance and the reference substance), a postive control (with inoculum and the reference substance sodium benzoate) as well as a toxicity control (containing inoculum, test substance and reference substance) was performed. In result, after 28 days the biodegradation of the test substance reached 19%. Thus, the test substance was not readily biodegradable in this test, since the pass level of  70% DOC removal was not reached within a 10-day-window or after after 28 days. In the toxiciy control, the biodegradation was about 39% after 14 days and 44% after 28 days. Based on this result it can be concluded that no inhibition of the microbial activity occured at a substance concentration of 2.2 mg/L. The result for the positive control (80% biodegradation) confirmed the suitability of the test system. The test is regarded as valid.

Conclusion

Based on the most current and reliable information (Symrise AG, 2018), the test item is considered to be not readily biodegradable by OECD criteria, since only max. 30% biodegradation in 28 days was reached and the pass level for ready biodegrability, i.e. biodegradation of at least 60% of the ThOD in a 10-day window within a 28-day period was not passed. This result is supported by an older ready biodegradability study (Symrise AG, 2000), as the test item was not readily biodegradable according to OECD criteria (19% biodegradation after 28 days). But the test item is considered to be inherently, primarily biodegradable because the max. biodegradation was 30 % on day 28 (mean value) and 25 % (mean value) after 60 days.