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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:
from 2020-03-03 to 2020-03-31
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:
17 July 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
440/2008/EC, 30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
January 1998
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: the sewage plant for domestic sewage in Balatonfüred, Hungary

- Pretreatment: The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 5 g dry material per litre was mixed with test water (mineral medium, see above) and then aerated under test conditions (for 6 days) until use. The pH of the activated sludge inoculum after preparation was 7.39, just before use: 7.30. A pH adjustment of the activated sludge inoculum was not performed.
Duration of test (contact time):
28 d
Initial conc.:
3 mg/L
Based on:
ThOD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Prepared according to OECD guideline
- Test temperature: 20.1 – 20.7 °C (during preparation)
20.3 - 20.7 °C (during incubation)
- pH: 7.37
- pH adjusted: no
- CEC: ~ 10000 – 1000000 cells/L
- Aeration of dilution water: yes
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Winkler bottles (300 mL) with special neck and glass stoppers.
- Number of bottles containing the test item and inoculum: 16 (+ 2 reserve)
- Number of bottles containing procedure control: 16 (+ 2 reserve)
- Number of bottles containing inoculum control: 16 (+ 2 reserve)
- Number of bottles containing toxicity control: 16 (+ 2 reserve)
- Measuring equipment: O2 electrode
- Test performed in closed vessels: yes

- Sampling frequency: Oxygen concentration measurements were performed in all duplicate bottles in the test item, procedure control, inoculum control and toxicity control groups on days 0, 2, 5, 7, 12, 14, 21 and 28. Temperature was measured continuously using a built-in thermometer of the thermostat and noted at least once a day.

CONTROL AND BLANK SYSTEM
- Inoculum control: yes
- Procedure control: yes
- Toxicity control: yes
- Test suspension: yes
Reference substance:
benzoic acid, sodium salt
Test performance:
Based on the theoretical oxygen demand (ThODNH4) of the test item (2.02 mg O2 per mg), at the start of the test a suitable volume (60 mL) of stock solution (nominal concentration of 300 mg/L) of the test item was thoroughly mixed into the respective volume (ad. 6000 mL) of aqueous test medium corresponding to 3.0 mg/L test item. (In 3.0 mg/L test item concentration, the ThODNH4 corresponded to about 3.0 x 2.02 = 6.06 mg O2/L). Based on the theoretical oxygen demand (ThODNH4) of the reference substance, sodium benzoate (1.67 mg O2 per mg), at the start of the test a stock solution of the test item (60 mL) was mixed into the aqueous test medium (ad. 6000 mL, corresponding to 3.0 mg/L reference item, respectively a ThODNH4 of about 5.01 mg O2/L). Only filtered inoculum (12 mL) was added to the aqueous test medium (ad. 6000 mL). 60 mL of test item stock solution (300 mg/L) and 60 mL of reference item stock solution (300 mg/L) were mixed into the aqueous test medium (ad. 6000 mL) corresponding to the test item concentration of 3.0 mg/L [chosen based on the calculated theoretical oxygen demand] and to 3.0 mg/L concentration of the reference item. Microbial inoculum (2.0 mL per litre test medium) was added to each preparation bottle.
Key result
Parameter:
% degradation (O2 consumption)
Value:
8.8
Sampling time:
28 d
Details on results:
Under the test conditions the percentage biodegradation of the test item reached a mean of 8.8 % after 28 days based on the estimated theoretical oxygen demand (ThODNH4). The highest biodegradation value of 8.9 % was obtained on the 7th day of the test. The pass level for ready biodegradability is the removal of 60 % ThODNH4 in a 10-day window. The obtained biodegradation value remained far below the pass level; therefore, the test item was considered to be not readily biodegradable. Details of the results are presented on the table in the section “Any other information on results and tables”.
Results with reference substance:
The reference item, sodium benzoate, was sufficiently degraded to a mean of 75.3 % after 14 days, and to a mean of 77.5 % after 28 days of incubation, based on ThODNH4. The biodegradation reached its plateau on about the 7th day and from this day onwards the observed slight changes were considered as being within the biological variability range of the applied test system. Details of the results are presented on the table in the section “Any other information on results and tables".

Table 1: Dissolved Oxygen Concentrations at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration [mg/L]

Flask No.

mg O2/L after n days of exposure

0

2

5

7

12

14

21

28

Test item

3.0

1a

8.07

7.59

7.19

7.18

6.78

6.50

6.41

6.21

1b

8.11

7.65

7.31

7.16

6.75

6.59

6.36

6.19

mean

8.09

7.62

7.25

7.17

6.77

6.55

6.39

6.20

Reference item

3.0

2a

8.12

6.06

4.53

3.80

3.23

3.54

2.87

2.73

2b

8.12

5.66

4.35

4.29

3.85

2.99

3.17

3.04

mean

8.12

5.86

4.44

4.05

3.54

3.27

3.02

2.89

Inoculum control

3a

8.12

7.84

7.81

7.71

7.43

7.19

6.92

6.81

3b

8.12

7.75

7.67

7.77

7.09

6.87

6.92

6.71

mean

8.12

7.80

7.74

7.74

7.26

7.03

6.92

6.76

Toxicity control

Test item: 3.0 Reference item: 3.0

4a

8.11

5.83

4.29

4.11

3.93

3.17

3.30

2.97

4b

8.10

5.52

3.83

3.80

3.31

3.01

2.93

2.67

mean

8.11

5.68

4.06

3.96

3.62

3.09

3.12

2.82

 

Table 2: Oxygen Depletion at Different Time Intervals during the Exposure Period of 28 Days

 

Treatment

Concentration [mg/L]

Flask No.

mg O2/L after n days of exposure

2

5

7

12

14

21

28

Test item

3.0

1a

0.16

0.50

0.51

0.43

0.48

0.46

0.50

 

1b

0.14

0.42

0.57

0.50

0.43

0.55

0.56

Reference item

3.0

2a

1.74

3.21

3.94

4.03

3.49

4.05

4.03

 

2b

2.14

3.39

3.45

3.41

4.04

3.75

3.72

Toxicity control

Test item: 3.0 Reference item: 3.0

4a

1.96

3.44

3.62

3.32

3.85

3.61

3.78

4b

2.26

3.89

3.92

3.93

4.00

3.97

4.07

 

Table 3: BOD at Different Time Intervals during the Exposure Period of 28 Days

 

Treatment

Concentration [mg/L]

Flask No.

BOD after n days of exposure

2

5

7

12

14

21

28

Test item

3.0

1a

0.05

0.17

0.17

0.14

0.16

0.15

0.17

1b

0.04

0.14

0.19

0.17

0.14

0.18

0.19

Reference item

3.0

2a

0.58

1.07

1.31

1.34

1.16

1.35

1.34

2b

0.71

1.13

1.15

1.14

1.35

1.25

1.24

Toxicity control

Test item: 3.0 Reference item: 3.0

4a

0.33

0.57

0.60

0.55

0.64

0.60

0.63

4b

0.38

0.65

0.65

0.66

0.67

0.66

0.68

 

Table 4: Percentage Biodegradation at Different Time Intervals during the Exposure Period of 28 Days

 

Treatment

Concentration [mg/L]

Flask No.

Percent of biodegradation after n days of exposure

2

5

7

12

14

21

28

Test item

3.0

1a

2.6

8.3

8.4

7.1

7.9

7.6

8.3

1b

2.2

6.9

9.4

8.3

7.1

9.1

9.2

mean

2.4

7.6

8.9

7.7

7.5

8.3

8.8

Reference item

3.0

2a

34.7

64.2

78.8

80.6

69.8

81.0

80.6

2b

42.7

67.8

69.0

68.2

80.8

75.0

74.4

mean

38.7

66.0

73.9

74.4

75.3

78.0

77.5

Toxicity control

Test item: 3.0 Reference item: 3.0

4a

17.7

31.1

32.7

30.0

34.8

32.7

34.2

4b

20.4

35.2

35.5

35.6

36.2

35.9

36.8

mean

19.0

33.2

34.1

32.8

35.5

34.3

35.5

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The percentage biodegradation of the test item reached a mean of 8.8 % after 28 days based on its theoretical oxygen demand (ThODNH4).
Under the conditions of study, the test item is considered to be not readily biodegradable, since the pass level for ready biodegradability is removal of 60 % theoretical oxygen demand (ThODNH4) in a 10-day window.
Executive summary:

The ready biodegradability of the test material was investigated in a Closed Bottle Test conducted in accordance with OECD TG 301D and EU method C.4 under GLP conditions. For this purpose the test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant in completely full and closed bottles in the dark at controlled temperature (22 ± 2 °C) for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure.

The test item was investigated at the concentration of 3.0 mg/L. The test item concentration was chosen based on the theoretical oxygen demand of the test item (ThODNH4 calculated according to equation given in the guidelines, assuming that no nitrification occurs) of 2.02 mg O2/mg. In parallel (under the same conditions as the test item), positive reference item, sodium benzoate at the concentration of 3.0 mg/L (as procedure control), inoculum control and toxicity control were investigated. All validity criteria of the study were met. Under the applied test conditions, no ready biodegradation of this test item was noticed. The percentage biodegradation of the test item reached a mean of 8.8 % after 28 days based on its theoretical oxygen demand (ThODNH4). (The highest biodegradation value of 8.9 % was obtained on the 7th day of the test.).

The concurrently conducted analytical determination of possible nitrite and nitrate development showed slight changes in nitrite concentrations in both parallels of the end (28-day) toxicity control samples; however, the measured dissolved oxygen concentrations did not correspond to the consumed oxygen of ammonium oxidation processes; the relationship between oxygen uptake resulting from a possible ammonium oxidation and oxygen uptake of applied microbial population was equivocal; therefore any correction of the measured dissolved oxygen concentrations was considered as not possible. Most likely technical effects (turbidity and/or discoloration) influenced the nitrite concentration determinations. The biodegradability value of the test item was calculated based on its ThODNH4; any correction, based on the measured nitrite nitrate content was not performed

The reference item, sodium benzoate, was sufficiently degraded to a mean of 75.3 % after 14 days, and to a mean of 77.5 % after 28 days of incubation, based on ThODNH4. (The biodegradation reached its plateau on about the 7th day and from this day onwards the observed slight changes were considered as being within the biological variability range of the applied test system). In the toxicity control containing both, the test item and the reference item, a mean of 35.5 % biodegradation was noted within 14 days and after 28 days of incubation (from about the 5th - 7th days of the test onwards the obtained slight changes in the biodegradability values were considered as being within the biological variability range of the applied test system).

The test item is considered to be not readily biodegradable, since the pass level for ready biodegradability is removal of 60 % theoretical oxygen demand (ThODNH4) in a 10-day window. The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum. According to the test guidelines the test item can be assumed as not inhibitory at the applied concentration level on the activated sludge microorganisms because the degradation in the toxicity control group was higher than 25 % within 14 days.

Description of key information

The percentage biodegradation of the test item reached a mean of 8.8 % after 28 days based on its theoretical oxygen demand (ThODNH4).

Under the conditions of study, the test item is considered to be not readily biodegradable, since the pass level for ready biodegradability is removal of 60 % theoretical oxygen demand (ThODNH4) in a 10-day window.

Key value for chemical safety assessment

Biodegradation in water:
not biodegradable
Type of water:
freshwater

Additional information

The ready biodegradability of the test material was investigated in a Closed Bottle Test conducted in accordance with OECD TG 301D and EU method C.4 under GLP conditions. For this purpose the test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant in completely full and closed bottles in the dark at controlled temperature (22 ± 2 °C) for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure.

The test item was investigated at the concentration of 3.0 mg/L. The test item concentration was chosen based on the theoretical oxygen demand of the test item (ThODNH4 calculated according to equation given in the guidelines, assuming that no nitrification occurs) of 2.02 mg O2/mg. In parallel (under the same conditions as the test item), positive reference item, sodium benzoate at the concentration of 3.0 mg/L (as procedure control), inoculum control and toxicity control were investigated. All validity criteria of the study were met. Under the applied test conditions, no ready biodegradation of this test item was noticed. The percentage biodegradation of the test item reached a mean of 8.8 % after 28 days based on its theoretical oxygen demand (ThODNH4). (The highest biodegradation value of 8.9 % was obtained on the 7th day of the test.).

The concurrently conducted analytical determination of possible nitrite and nitrate development showed slight changes in nitrite concentrations in both parallels of the end (28-day) toxicity control samples; however, the measured dissolved oxygen concentrations did not correspond to the consumed oxygen of ammonium oxidation processes; the relationship between oxygen uptake resulting from a possible ammonium oxidation and oxygen uptake of applied microbial population was equivocal; therefore any correction of the measured dissolved oxygen concentrations was considered as not possible. Most likely technical effects (turbidity and/or discoloration) influenced the nitrite concentration determinations. The biodegradability value of the test item was calculated based on its ThODNH4; any correction, based on the measured nitrite nitrate content was not performed

The reference item, sodium benzoate, was sufficiently degraded to a mean of 75.3 % after 14 days, and to a mean of 77.5 % after 28 days of incubation, based on ThODNH4. (The biodegradation reached its plateau on about the 7th day and from this day onwards the observed slight changes were considered as being within the biological variability range of the applied test system). In the toxicity control containing both, the test item and the reference item, a mean of 35.5 % biodegradation was noted within 14 days and after 28 days of incubation (from about the 5th - 7th days of the test onwards the obtained slight changes in the biodegradability values were considered as being within the biological variability range of the applied test system).

The test item is considered to be not readily biodegradable, since the pass level for ready biodegradability is removal of 60 % theoretical oxygen demand (ThODNH4) in a 10-day window. The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum. According to the test guidelines the test item can be assumed as not inhibitory at the applied concentration level on the activated sludge microorganisms because the degradation in the toxicity control group was higher than 25 % within 14 days.