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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 2019-06-27 to 2019-09-12
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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
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: domestic wastewater treatment plant, Bensheim, Germany
- Preparation of inoculum for exposure: The activated sludge was deposited for 15 min, 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. 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.
Duration of test (contact time):
28 d
Initial conc.:
102 mg/L
Based on:
test mat.
Initial conc.:
131.7 mg/L
Based on:
ThOD
Remarks:
NH4
Initial conc.:
207.1 mg/L
Based on:
ThOD
Remarks:
NO3
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: prepared according to OECD 301, pH adjusted to 7.5 with 1M HCL solution
- Test temperature: 22 ± 1 °C
- pH: 7.2 - 7.6
- pH adjusted: no
- Aeration of dilution water: no
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Manometric Test System with test flasks, volume 500 mL
- Number of culture flasks/concentration: 2
- Measuring equipment: BSB/BOD-Sensor-System
- Test performed in closed vessels: The test flasks were closed gas-tight by a measuring head
- Test performed in open system: no
- Details of trap for CO2 and volatile organics: with Potassium hydroxide solution (45 %)

SAMPLING
- Sampling frequency: every day of the exposure period

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 replicates
- Abiotic sterile control: 1 replicate
- Toxicity control: 1 replicate
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Results with reference substance:
The reference item was sufficiently degraded to 82 % after 14 days and to 85 % after 28 days of incubation, thus, confirming the suitability of the aerobic activated sludge inoculum used.

Validity Criteria

- Inoculum Control: The oxygen demand of the inoculum control (medium and inoculum) was 0 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 7.3 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 item and a reference item less than 25 % biodegradation (based on ThODNH4) occurred within 14 days, the test item can be assumed to be inhibitory. The biodegradation was 48 % at day 14; the test item was not inhibitory.

Biodegradation of the Test Item

Nitrogen is part of the molecular structure of the test item; therefore nitrification (ThODNO3) was considered for the evaluation of the results. The test item never reached 10 % biodegradation. The mean biodegradation percentage at the end of the 28-day exposure period was 0 % (ThODNH4, ThODNO3).

Biodegradation of the Toxicity Control

In the toxicity control containing both, the test item and the reference item, 48 % (ThODNH4) biodegradation was noted within 14 days and 50 % (ThODNH4) biodegradation after 28 days of incubation (39 % and 40 % based on ThODNO3). 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.

Oxygen Demand in the Abiotic Control

The oxygen demand in the abiotic control was 0 mg/L during the test duration. There was no need to correct the degradation of the test item and toxicity control.

Table 1. Percentage Biodegradation of Test Item, Sodium Benzoate and Toxicity Control based on ThOD NH4

Time

(Days)

Percentage Biodegradation

Test item*

Sodium Benzoate**

Toxicity
control*,**

Flask 1 [%]

Flask 2 [%]

Flask 5 [%]

Flask 7 [%]

l

0

0

21

8

2

0

0

47

22

3

0

0

62

32

4

0

0

65

38

5

0

0

71

42

6

0

0

74

43

7

0

0

79

45

8

0

0

79

45

9

0

0

79

45

10

0

0

79

45

11

0

0

82

47

12

0

0

82

47

13

0

0

82

47

14

0

0

82

48

15

0

0

85

48

16

0

0

85

50

17

0

0

85

50

18

0

0

85

50

19

0

0

85

50

20

0

0

85

50

21

0

0

85

50

22

0

0

85

50

23

0

0

85

50

24

0

0

85

50

25

0

0

85

50

26

0

0

85

50

27

0

0

85

50

28

0

0

85

50

* ThOD NH4 of the test item: 1.291 mg O2/mg test item
** ThOD NH4 of sodium benzoate: 1.666 mg O2/mg reference item

Table 2. Percentage Biodegradation of Test Item and Toxicity Control based on ThOD NO3 and of Sodium Benzoate based on ThOD NH4

Time

(Days)

Percentage Biodegradation

Test item*

Sodium Benzoate**

Toxicity
control*,**

Flask 1 [%]

Flask 2 [%]

Flask 5 [%]

Flask 7 [%]

l

0

0

21

7

2

0

0

47

17

3

0

0

62

25

4

0

0

65

31

5

0

0

71

33

6

0

0

74

35

7

0

0

79

36

8

0

0

79

36

9

0

0

79

36

10

0

0

79

36

11

0

0

82

37

12

0

0

82

37

13

0

0

82

37

14

0

0

82

39

15

0

0

85

39

16

0

0

85

40

17

0

0

85

40

18

0

0

85

40

19

0

0

85

40

20

0

0

85

40

21

0

0

85

40

22

0

0

85

40

23

0

0

85

40

24

0

0

85

40

25

0

0

85

40

26

0

0

85

40

27

0

0

85

40

28

0

0

85

40

* ThOD NO3 of the test item: 2.029 mg O2/mg test item
** ThOD NH4 of sodium benzoate: 1.666 mg O2/mg reference item

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The mean biodegradation after 28 days was 0 %. Therefore, the test item is considered to be not readily biodegradable based on ThOD(NH4) and ThOD(NO3).
Executive summary:

The ready biodegradability of the test item was determined with a aerobic activated sludge from a domestic wastewater treatment plant over a test period of 28 days in theManometric Respirometry Testaccording to OECD 301F and Regulation (EC) No. 440/2008 Method C.4-D under GLP. The test item was tested at a concentration of 102 mg/L in a duplicate, corresponding to ThOD NH4 of 131.7 mg/L and to ThOD NO3 of 207.1 mg/L. The oxygen concentration was measured usingBSB/BOD-Sensor-System.The test flasks were closed gas-tight by a measuring head. Potassium hydroxide solution (45 %) was used for trapping the produced carbon dioxide. The amount of O2 consumed by the activated sludge was calculated from the decrease of pressure in the reaction vessel.The biodegradation of the reference item was not inhibited by the test item in the toxicity control. The reference item was biodegraded to 85 % after 28 days of incubation. The biodegradation of the test item was 0 % after 28 days. In conclusion, the test item is not readily biodegradable as not biodegradation at all was observed.

Description of key information

The mean biodegradation of the test item after 28 days incubation was 0 %. Therefore, the test item is considered to be not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

The ready biodegradability of the test item was determined with a aerobic activated sludge from a domestic wastewater treatment plant over a test period of 28 days in the Manometric Respirometry Test according to OECD 301F and Regulation (EC) No. 440/2008 Method C.4-D under GLP. The test item was tested at a concentration of 102 mg/L in a duplicate, corresponding to ThOD NH4 of 131.7 mg/L and to ThOD NO3 of 207.1 mg/L. The oxygen concentration was measured using BSB/BOD-Sensor-System. The test flasks were closed gas-tight by a measuring head. Potassium hydroxide solution (45 %) was used for trapping the produced carbon dioxide. The amount of O2 consumed by the activated sludge was calculated from the decrease of pressure in the reaction vessel. The biodegradation of the reference item was not inhibited by the test item in the toxicity control. The reference item was biodegraded to 85 % after 28 days of incubation. The biodegradation of the test item was 0 % after 28 days. In conclusion, the test item is not readily biodegradable as not biodegradation at all was observed.