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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-09-04 to 2017-11-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
adopted July 17, 1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
May 30, 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: the sewage treatment plant Bensheim, Germany
- Preparation of inoculum for exposure: The aerobic activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in test water and centrifuged again. This procedure was done three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined.
- Pretreatment: Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water and aerated overnight. This suspension was used for the experiment.
- Water filtered: no, decanted
Duration of test (contact time):
28 d
Initial conc.:
103.1 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: reconstituted water
- Test temperature: 22 ± 1 °C
- pH: 7.6 (measured at the start of the test), 7.5 to 7.6 (measured at the end of the test)
- pH adjusted: yes
- Surrounding Type: Climatised chamber
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Manometric Test System with test flasks containing a volume of approximately 500 mL.
- Number of culture flasks/concentration: 2
- Method used to create anaerobic conditions: The test flasks were closed gas-tight by a measuring head.
- Measuring equipment: BSB/BOD-Sensor-System
- Test performed in closed vessels due to significant volatility of test substance: yes

CONTROL AND BLANK SYSTEM
- Test suspension: 2
- Inoculum blank: 2
- Abiotic sterile control: 1
- Toxicity control: 1
- Procedure control: 1
Reference substance:
benzoic acid, sodium salt
Test performance:
The amounts of test item and reference item were directly weighed into the test flasks. No emulsifiers or solvents were used. The closed test flasks were incubated in a climatised chamber under continuous stirring for 28-days. The consumption of oxygen was determined by measuring the change of pressure in the flasks. Evolved carbon dioxide was absorbed in an aqueous solution (45 %) of potassium hydroxide. The change of pressure in the test flasks was measured by means of a manometric method each day.
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Details on results:
The mean biodegradation of the test item at test end after 28 days was 0% (ThOD-NH4).
Results with reference substance:
The reference item sodium benzoate was sufficiently degraded to 80 % after 14 days and to 83 % after 28 days of incubation. The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.

Table 1: Preparation of the Test Solutions

Treatment

Flask

Test Item

mg per flask

Reference Item1

mg per flask

CuSO4

 mL

Activated

Sludge2

mL

Test

Water

mL

Final Volume

mL

Test Item

1

25.2

2

242

244

2

25.1

2

242

244

Inoculum Control3

3

2

242

244

4

2

242

244

Procedure Control4

5

24.7

2

242

244

Abiotic Control5

6

25.2

5

239

244

Toxicity Control

7

25.1

24.8

-

2

242

244

 

1Reference item: sodium benzoate

2Stock suspension of 3.5 g/L on dry matter base (final sludge concentration in test flasks: 28.7 mg sludge/L)

3The inoculum control was also used for other projects which ran in parallel

4The procedure control was also used for other projects which ran in parallel

5Poisoned with CuSO4 (stock solution of 1 g/L) —: Not applicable

Table 2: Cumulative Biochemical Oxygen Demand (mg O2/L) in Test Flasks during the Test Period of 28 Days

Time (days)

Flask No.

1

2

3

4

5

6

7

1

0

0

0

5

50

0

45

2

0

5

5

5

80

0

75

3

5

5

5

10

110

0

105

4

5

5

10

10

120

0

115

5

5

5

10

10

125

0

120

6

5

5

10

10

130

0

125

7

10

5

15

15

135

0

130

8

10

10

15

15

140

0

135

9

10

10

15

15

145

0

140

10

10

10

15

20

150

0

140

11

10

10

15

20

150

0

145

12

10

10

20

20

150

0

145

13

15

10

20

20

155

0

150

14

15

10

20

20

155

0

150

15

15

10

20

20

155

0

150

16

15

10

20

20

155

0

150

17

15

10

20

20

160

0

155

18

20

15

20

25

160

0

155

19

20

15

20

25

160

0

155

20

20

15

20

25

160

0

155

21

20

15

20

25

160

0

155

22

25

20

20

25

160

0

160

23

25

20

20

25

165

0

160

24

25

20

20

25

165

0

165

25

25

20

20

25

165

0

165

26

25

20

20

25

165

0

165

27

25

20

20

30

165

0

165

28

25

20

20

30

165

0

165

Flasks 1 and 2: test item

Flasks 3 and 4: inoculum control

Flask 5: reference (procedure control)

Flask 6: abiotic control

Flask 7: toxicity control

Table 3: Percentage Biodegradation of Test Item, of Sodium Benzoate and of the Toxicity Control based on ThODNH4

Time

(Days)

Percentage Biodegradation1

 

Test Item1

Sodium Benzoate2

Toxicity control1,2

Flask 1 [%]

Flask 2 [%]

Flask 5 [%]

Flask 7 [%]

 

1

0

0

28

10

2

0

0

44

16

3

0

0

61

22

4

0

0

65

24

5

0

0

68

25

6

0

0

71

26

7

0

0

71

26

8

0

0

74

27

9

0

0

77

29

10

0

0

79

28

11

0

0

79

29

12

0

0

77

29

13

0

0

80

30

14

0

0

80

30

15

0

0

80

30

16

0

0

80

30

17

0

0

83

31

18

0

0

82

30

19

0

0

82

30

20

0

0

82

30

21

0

0

82

30

22

1

0

82

31

23

1

0

84

31

24

1

0

84

33

25

1

0

84

33

26

1

0

84

33

27

0

0

83

32

28

0

0

83

32

1 ThODNH4 of test item: 2.611 mg O2/mg test item

2 ThODNH4 of sodium benzoate: 1.666 mg O2/mg reference item

 

 

Validity Criteria of the Study

Inoculum Control: The oxygen demand of the inoculum control (medium and inoculum) was 25 mg O2/L and thus not greater than 60 mg O2/L within 28 days as required by the test guideline.

pH-Value: The pH-value of the test item flasks at the end of the test was 7.5 and therefore within the range of pH 6.0 to 8.5 as required by the test guideline.

Reference Item: The percentage degradation of the reference item should reach the level for ready biodegradability (>60 %) within 14 days as required by the test guideline. The reference item sodium benzoate was degraded to more than 60 % after 3 days of incubation.

Test Item: The difference of duplicate values for the degradation of the test item at the plateau, at the end of the test and at the end of the 10-day window was less than 20 %. The difference of duplicate values at day 28 differed by 0 %. The validity criterion was fulfilled.

Toxicity Control: If in a toxicity test, containing both the test substance and a reference compound less than 25 % biodegradation (based on total ThOD) occurred within 14 days, the test substance can be assumed to be inhibitory. The biodegradation was 30 % at day 14; the test item was not inhibitory.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The degradation rate of the test item did not reach 60 % within the 10-day window or after 28 days. Therefore, the test item is considered to be not readily biodegradable.
Executive summary:

The Ready biodegradability of the test item was assessed in a Manometric Respirometry Test according to OECD Guideline for Testing of Chemicals No. 301 F over a period of 28 days at 22 °C ± 1 °C in darkness. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item, sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. The test item loading rate was corresponding to an oxygen demand of about 269.2 mg/L (ThODNH4) and reference item loading rate was 101.2 mg/L corresponding to an oxygen demand of about 168.6 mg/L (ThODNH4). Degradation rate of test item calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 28 days of incubation. The reference item sodium benzoate was sufficiently degraded to 80 % after 14 days and to 83% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used. In the toxicity control containing both, the test item and the reference item sodium benzoate, 30 % biodegradation was noted within 14 days and 32 % biodegradation after 28 days of incubation. According to the test guidelines, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25 % within 14 days. The test item did not reach a mean biodegradation of 10 % during the incubation time of 28 days. The mean biodegradation at test end after 28 days was 0 % (ThODNH4). Therefore, the test item is considered to be not readily biodegradable based on ThODNH4.

Description of key information

The mean biodegradation at test end after 28 days was 0 % (ThODNH4). Therefore, the test item is considered to be not readily biodegradable based on ThODNH4.

Key value for chemical safety assessment

Biodegradation in water:
not biodegradable
Type of water:
freshwater

Additional information

The Ready biodegradability of the test item was assessed in a Manometric Respirometry Test according to OECD Guideline for Testing of Chemicals No. 301 F over a period of 28 days at 22 °C ± 1 °C in darkness. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item, sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. The test item loading rate was corresponding to an oxygen demand of about 269.2 mg/L (ThODNH4) and reference item loading rate was 101.2 mg/L corresponding to an oxygen demand of about 168.6 mg/L (ThODNH4). Degradation rate of test item calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 28 days of incubation. The reference item sodium benzoate was sufficiently degraded to 80 % after 14 days and to 83 % after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used. In the toxicity control containing both, the test item and the reference item sodium benzoate, 30 % biodegradation was noted within 14 days and 32 % biodegradation after 28 days of incubation. According to the test guidelines, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25 % within 14 days. The test item did not reach a mean biodegradation of 10 % during the incubation time of 28 days. The mean biodegradation at test end after 28 days was 0% (ThODNH4). Therefore, the test item is considered to be not readily biodegradable based on ThODNH4.