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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:
15-03-2018 - 14-04-2018
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:
1992
Deviations:
yes
Remarks:
Minor deviation: Ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification. This omission did not result in nitrogen limitation as was shown by the biodegradation of the reference compound.
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 10707 Water quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method by analysis of biochemical oxygen demand (closed bottle test)
Version / remarks:
1994
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
September 2017
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Secondary activated sludge (09-03-2018) was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge plant treating predominantly domestic wastewater.
- Preparation of inoculum for exposure: The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 0.40 g Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted in the bottles to 2.0 mg/L (van Ginkel and Stroo, 1992).
- Adaptation: The inoculum was not pre-adapted to the test substance.
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: The nutrient medium of the Closed Bottle test contained per liter of deionized water; 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4·2H2O, 22.5 mg MgSO4·7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrifi-cation.
- Addition of test material: Accurate administering of the test substance was accomplished by preparing a solid stock of 3.0 mg of the test substance per g of silica gel in a 50-mL serum flask. Only part of the top layer of the silica gel was brought into contact with the test substance. The serum flask was closed with a screw top after having flushed the headspce with nitrogen gas. The content was then mixed vigorously. Subsequently, 0.20 g of silica gel with the test substance was added to the test bottles. Silica gel (0.20 g) was added to the control bottles. Next the bottles were filled with nutrient medium with inoculum and closed. Sodium acetate was added to the bottles using a stock solution of 1.0 g/L.
- Test temperature: 22.6 to 23.0°C
- pH: at start of the test: 7.3; at day 28: 7.3 (controls) and 7.2 (test).
- pH adjusted: no
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 0.30 L BOD (biological oxygen demand) bottles with glass stoppers
- Number of culture flasks/concentration: 10 bottles containing only inoculum, 10 bottles containing silica gel and inoculum, 10 bottles containing inoculum, silica gel and test substance, 6 bottles containing sodium acetate and inoculum.
- Other: The concentrations of the test substance, and sodium acetate in the bottles were 2.0 and 6.7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles.

SAMPLING
- The zero time bottles were immediately analyzed after preparation for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28.

Reference substance:
acetic acid, sodium salt
Key result
Parameter:
% degradation (DOC removal)
Value:
65
Sampling time:
28 d
Details on results:
Theoretical oxygen demand (ThOD) of the test substance: 2.7 g O2/g substance. The ThOD of sodium acetate: 0.8 g O2/g substance.

Toxicity: inhibition of endogenous respiration of the inoculum by the test substance at day 7 was not detected. Therefore, no inhibition of the biodegradation due to the "high" initial test substance concentration is expected.

Biodegradation: The biodegradation of a substance consisting of many constituents is therefore an addition of different biodegradation curves. It is thus possible that individual compounds meet the time window criterion whereas the biodegradability curve of the multi-constituent suggests that the test substance is not readily biodegradable. The time window applied as a validity criterion to Closed Bottle tests is therefore considered not applicable (OECD 2006). The test substance should as a consequence be classified as readily biodegradable based only on 65% biodegradation reached at day 28.
Results with reference substance:
The reference compound sodium acetate was biodegraded for 91% at day 14.

Table I: Dissolved oxygen concentrations (mg/L) in the closed bottles.

Time (days)

Oxygen concentration (mg/L)

O(cs)

O(t)

O(c)

O(a)

0

8.7

8.7

8.7

8.7

0

8.7

8.7

8.7

8.7

Mean (M)

8.7

8.7

8.7

8.7

7

8.3

7.1

8.2

3.4

7

8.3

6.9

8.2

3.6

Mean (M)

8.3

7.0

8.2

3.5

14

8.1

4.9

8.0

3.1

14

8.0

5.2

7.9

3.0

Mean (M)

8.1

5.1

8.0

3.1

21

7.9

4.7

8.0

 

21

8.0

4.5

8.0

 

Mean (M)

8.0

4.6

8.0

 

28

7.7

4.3

7.8

 

28

7.8

4.2

7.8

 

Mean (M)

7.8

4.3

7.8

 

O(cs): Mineral nutrient solution with inoculum silica gel.

O(t): Mineral nutrient solution with inoculum, silica gel and test material (2.0 mg/L)

O(c): Mineral nutrient solution with only inoculum.

O(a): Mineral nutrient solution with inoculum and sodium acetate (6.7 mg/L)

Table II Oxygen consumption (mg/L) and the percentages biodegradation of the test substance, Muguet Aldehyde (multi-constituent) (BOD/ThOD) and sodium acetate (BOD/ThOD) in the Closed Bottle test.

Time (days)

Oxygen consumption (mg/L)

Biodegradation (%)

Test substance

Acetate

Test substance

Acetate

0

0.0

0.0

0

0

7

1.3

4.7

24

87

14

3.0

4.9

56

91

21

3.4

 

63

 

28

3.5

 

65

 

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' for details on validity criteria.
Interpretation of results:
readily biodegradable
Conclusions:
The substance was biodegraded by 65% at day 28 in the OECD 301D Closed Bottle test. The multi-constituent substance should therefore be classified as readily biodegradable.
Executive summary:

The biodegradation potential of the test substance was examined in a study according to OECD TG 301D (Closed Bottle test), ISO and EU Test Guidelines, and in compliance with GLP.

The test substance (2.0 mg/L) was incubated for 28 days in the dark with inoculum (activated sludge). Two duplicate bottles were samples for analysis of the dissolved oxygen concentration at day 0, 7, 14, 21 and 28 days and biodegradation percentage was calculated. The substance did not cause a reduction in the endogenous respiration at day 7, and is therefore considered non-inhibitory to the inoculum. The test substance was biodegraded by 65% at day 28. The time window applied as a validity criterion to Closed Bottle tests is considered not applicable (OECD 2006) to multi-constituent substances. Therefore, the test substance should be regarded as readily biodegradable based only on 65% biodegradation reached at day 28. The test met all validity criteria and is considered reliable without restriction.

Description of key information

The substance was biodegraded by 65% at day 28 in the OECD 301D Closed Bottle test. The multi-constituent substance should therefore be classified as readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

The biodegradation potential of the test substance was examined in a study according to OECD TG 301D (Closed Bottle test), ISO and EU Test Guidelines, and in compliance with GLP.

The test substance (2.0 mg/L) was incubated for 28 days in the dark with inoculum (activated sludge). Two duplicate bottles were samples for analysis of the dissolved oxygen concentration at day 0, 7, 14, 21 and 28 days and biodegradation percentage was calculated. The substance did not cause a reduction in the endogenous respiration at day 7, and is therefore considered non-inhibitory to the inoculum. The test substance was biodegraded by 65% at day 28. The time window applied as a validity criterion to Closed Bottle tests is considered not applicable (OECD 2006) to multi-constituent substances. Therefore, the test substance should be regarded as readily biodegradable based only on 65% biodegradation reached at day 28. The test met all validity criteria and is considered reliable without restriction.

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