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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 2013-02-27 to 2013-04-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study was performed according to the OECD Guideline 301 D. All validity criteria were fulfilled.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
. Minor deviation: ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound).
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
The Netherlands GLP compliance programme (inspected on August 14 to 16, 2012 / signed on September 18, 2012)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
Activated sludge was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This activated sludge plant treats predominantly domestic wastewater. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 400 mg Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted in the BOD bottles (van Ginkel and Stroo, 1992).
Duration of test (contact time):
60 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 litre of deionized water; 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.3 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 nitrification.
- Solubilising agent (type and concentration if used): Dichloromethane (DCM)
- Test temperature: 22-24 °C
- CEC (meq/100 g): No data
- Aeration of dilution water: No data
- Suspended solids concentration: 2 mg/L (dry weight)
- Continuous darkness: Yes
- Other: None

TEST SYSTEM
- Culturing apparatus: Test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.
- Number of culture flasks/concentration: 10 bottles containing only inoculum, 6 bottles containing inoculum and sodium acetate, 10 bottles containing inoculum and test substance, and 10 bottles treated with DCM (added and evaporated) only containing inoculum.
- Method used to create aerobic conditions: No data
- Method used to create anaerobic conditions: Not applicable
- Measuring equipment: The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode (WTW TrioXmatic EO 200) and meter (WTW OXI 530) (Retsch, Ochten, The Netherlands). The pH was measured using a Eutech Cyberscan pH11 pH meter (Eutech Instruments, Nijkerk, The Netherlands). The temperature was measured and recorded with a sensor connected to a data logger.
- Test performed in closed vessels: Yes
- Test performed in open system: No

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Procedure control: Yes with the reference substance
- Toxicity control: Yes

OTHERS:
- Duration: 7, 14, 21, 28, 42 and 60 days
- The dry weight (DW) of the inoculum was determined by filtrating 50 mL of the activated sludge over a pre-weighed 12 µm cellulose nitrate filter. This filter was dried for 1.5 hours at 104 ± 2 °C and weighed after cooling. DW was calculated by subtracting the weighed filters and by dividing this difference by the filtered volume.
Reference substance:
acetic acid, sodium salt
Remarks:
6.7 mg/L
Preliminary study:
None
Test performance:
The oxygen consumption of each flask is recorded and correct temperature is checked. At the end of the test period, the pH of each flask is measured again.
Key result
Parameter:
% degradation (O2 consumption)
Value:
32
Sampling time:
28 d
Remarks on result:
other: Closed Bottle test
Key result
Parameter:
% degradation (O2 consumption)
Value:
62
Sampling time:
60 d
Remarks on result:
other: Prolonged Closed Bottle test
Details on results:
Toxicity: Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test was not determined because possible toxicity of test item to microorganisms degrading acetate is not relevant. At day 7, a slight inhibition of the endogenous respiration was detected. Retardation of the onset of biodegradation due to the "high" initial concentration of the test substance can therefore not be excluded.
Results with reference substance:
Degradation of sodium acetate is 65 and 74 % after 7 and 14 days, respectively: the activity of the inoculum is thus verified.

Validity of the test

The validity of the test is demonstrated by an endogenous respiration of 1.4 mg/L at day 28. Furthermore, the differences of the replicate values at day 28 were less than 20%.

The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 74.

Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Table 5.2.1/1: Oxygen consumption (mg/L) and the percentages biodegradation of the test substance and sodium acetate (BOD/ThOD) in the Closed Bottle test.

 

Time (days)

Oxygen consumption (mg/L)

Biodegradation (%)

Test

substance

Sodium acetate

Test

substance

Sodium acetate

0

0.0

0.0

0

0

7

-0.1

3.5

0

65

14

0.6

4.0

10

74

21

1.5

 

25

 

28

1.9

 

32

 

42

2.6

 

43

 

60

3.7

 

62

 

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test substance is biodegraded by 32 % at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test this compound was biodegraded by 62 % at day 60. Although test substance is not readily biodegradable, the biodegradation reached at the last day of the test demonstrates that test substance should be classified as not persistent.
Executive summary:

This study was performed according to the OECD TG Guideline 301 with GLP statement, to determine the biodegradability of the test substance by the Closed Bottle Test.

The test material was exposed to activated sewage sludge micro-organisms at a concentration of 2 mg/L with culture medium in closed bottles in the dark at 22-24 °C for 7, 14, 21, 28, 42 and 60 days. The degradation of the test material was assessed by the measurement of oxygen consumption.

Test substance was biodegraded by 32 % at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test this substance was biodegraded by 62 % at day 60. Test substance is not readily biodegradable. The biodegradation reached at the last day of the test, however, demonstrates that test substance should be classified as not persistent.

 

The test is valid as shown by an endogenous respiration of 1.4 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 74% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period.  

Description of key information

OECD Guideline 301D, GLP, key study, validity 1:

32% and 62% biodegradation after 28 and 60 days, respectively.

Not readily biodegradable but not persistent in the environment.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable

Additional information

To assess the biodegradation of the registered substance, one valid study is available.

This biodegradation study (Akzo Nobel, 2013) was assessed as the key study and was performed on the registered substance according to the OECD Guideline 301D in compliance with GLP. The test substance was exposed to activated sewage sludge micro-organisms at a concentration of 2 mg/L with culture medium in closed bottles in the dark at 22-24 °C for 7, 14, 21, 28, 42 and 60 days. The degradation of the test substance was assessed by the measurement of oxygen consumption. Test substance was biodegraded by 32 % at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test this substance was biodegraded by 62 % at day 60. In conclusion, the test substance is not readily biodegradable. However, the biodegradation reached at the last day of the test demonstrates that test substance should be classified as not persistent in the environment.

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