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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 19-10-2011 to 29-01-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to slightly modified OECD, EU and ISO Test Guidelines with GLP statement. All validity criteria were fulfilled.
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
. Minor deviations were introduced: a) ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification, and b) river water instead of an effluent/extract/mixture was used as inoculum.
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: ISO 10707 (1994) 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).
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
2010-03-18
Oxygen conditions:
aerobic
Inoculum or test system:
other: River water
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): River water was sampled from the Rhine near Heveadorp, The Netherlands (14-10-2011). The nearest plant (Arnhem-Zuid) treating domestic wastewater biologically was 3 km upstream.
- Preparation of inoculum for exposure: The river water was aerated for 7 days before use to reduce the endogenous respiration. River water without particles was used as inoculum. The particles were removed by sedimentation after 1 day while moderately aerating.
- Pre-treatment: The river water used in the Closed Bottle test was spiked per litre of water with 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. Sodium acetate was added to the bottles using a stock solution of 1.0 g/L.
Duration of test (contact time):
100 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: Deionized water containing no more than 0.01 mg/L Cu was prepared in a water purified system.
- Additional substrate: none.
- Solubilising agent (type and concentration if used): none.
- Test temperature: 22-24°C
- pH: 8.2
- pH adjusted: no
- Suspended solids concentration: no data
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 0.30 L BOD bottles with glass stoppers
- Number of culture flasks/concentration: 10 bottles containing only inoculum, 10 bottles containing inoculum and silica gel, 10 bottles containing inoculum and silica gel dosed with test substance (at 2 mg/L), and 6 bottles containing sodium acetate (at 6.7 mg/L) and inoculum.
- Method used to create aerobic conditions: no data
- Method used to create anaerobic conditions: not applicable
- Measuring equipment: Oxygen electrode (WTW TrioXmatic EO 200).
- Test performed in closed vessels: yes
- Test performed in open system: no
- Other: All bottles were completely filled without air bubbles.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: No
- Toxicity control: No
See "Test system".
Reference substance:
acetic acid, sodium salt
Preliminary study:
None
Test performance:
The Theoretical Oxygen Demand (ThOD) of the test substance used to calculate the biodegradation percentages is 3.0 mg/mg. The ThOD of sodium acetate is 0.8 mg/mg.
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
21
Sampling time:
60 d
Parameter:
% degradation (O2 consumption)
Value:
28
Sampling time:
100 d
Details on results:
The test substance was not biodegraded at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test, the test substance was biodegraded by 21% at day 60 and 28% at day 100.
See tables in "Any other information on results incl. tables".
Results with reference substance:
The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 83.

Table 5.2.1/1: Dissolved oxygen concentrations (mg/L) in the closed bottles

Time (days)

Oxygen concentration (mg/L)

Blank control (with silica gel)

Test substance

Blank control (without silica gel)

Reference substance

0

 

Mean

8.9

8.9

8.9

8.9

8.9

8.9

8.9

8.9

8.9

8.9

8.9

8.9

7

 

Mean

8.2

8.2

8.2

8.4

8.3

8.4

8.1

8.3

8.2

4.4

4.4

4.4

14

 

Mean

7.8

7.9

7.9

7.8

7.9

7.9

8.0

7.9

8.0

3.5

3.4

3.5

21

 

Mean

7.5

7.7

7.6

7.7

7.6

7.7

7.6

7.6

7.6

 

28

 

Mean

7.4

7.5

7.5

7.6

7.5

7.6

7.4

7.4

7.4

 

42

 

Mean

7.3

7.1

7.2

5.7

5.8

5.8

 

 

60

 

Mean

6.9

6.7

6.8

5.3

5.6

5.5

 

 

100

 

Mean

6.4

6.2

6.3

4.8

4.4

4.6

 

 

Blank control (with silica gel): Mineral nutrient solution without test substance but with inoculum and silica gel.

Test substance: Mineral nutrient solution with test material (2.0 mg/L), silica gel and inoculum.

Blank control (without silica gel): Mineral nutrient solution with only inoculum.

Reference substance: Mineral nutrient solution with sodium acetate (6.7 mg/L) and with inoculum.

Table 5.2.1/2: Oxygen consumption (mg/L) and the percentages biodegradation of the test substance and sodium acetate in the Closed Bottle test.

Time (days)

Oxygen consumption (mg/L)

Biodegradation (%)

Test substance

Reference substance

Test substance

Reference substance

0

7

14

21

28

42

60

100

0.0

-0.1

0.0

-0.1

-0.1

1.4

1.3

1.7

0.0

3.8

4.5

 

 

 

 

 

0

0

0

0

0

23

21

28

0

70

83

 

 

 

 

 

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Remarks:
but inherently biodegradable
Conclusions:
The test substance was not biodegraded at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test, the test substance was biodegraded by 21% at day 60 ahd 28% at day 100. The biodegradation found in the prolonged test demonstrates that this test substance should be classified as Inherently biodegradable. The biodegradation percentage of 21 reached at day 60 strongly indicates that the test substance is partially biodegradable.
Executive summary:

This study was performed according to slightly modified OECD 301D, EU Method C.4 -E and ISO 10707 Test Guidelines with GLP statement, to assess the biodegradability of the test substance using a ready biodegradability test.

In this ready biodegradability test, microorganisms are inoculated into a chemically defined liquid medium containing the test substance as sole carbon and energy source. The test substance is exposed to a relatively low numbers of microorganisms present in river water, under aerobic conditions for a period of 100 days.

The test was performed in 0.30 L BOD Bottles with glass stoppers. Use was made of 10 bottles containing only inoculum, 10 bottles containing inoculum and silica gel, 10 bottles containing inoculum and silica gel dosed with test substance (at 2 mg/L), and 6 bottles containing sodium acetate (at 6.7 mg/L) and inoculum. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The zero time bottles were immediately analysed 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, 28, 42, 60 and 100.

The pH of the media was 8.2 at the start of the test. The pH of the medium at day 28 was also 8.2 (control, control with silica gel, test). Temperatures were within the prescribed temperature range of 22 to 24 °C.

The test substance was not biodegraded at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test, the test substance was biodegraded by 21% at day 60 and 28% at day 100. The biodegradation found in the prolonged test demonstrates that this test substance should be classified as Inherently biodegradable. The biodegradation percentage of 21 reached at day 60 strongly indicates that the test substance is partially biodegradable.

Possible inhibition of biodegradation by the test substance may be detected prior to the onset of the biodegradation through suppression of the oxygen consumption in the presence of a test substance. The endogenous respiration was slightly inhibited during the first four weeks of the test. Inhibition of the biodegradation due to the "high" initial concentration of the test substance can therefore not be excluded.

The test is valid as shown by an endogenous respiration of 1.5 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 83% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentration >0.5 mg/L in all bottles during the test period. However, a toxicity test containing both the test substance and a reference compound would have been interesting to perform, to verify if the test substance inhibits or not the micro-organisms activity.

Description of key information

OECD Guideline 301D, EU Method C.4-E, ISO Guideline 10707, GLP, key study, validity 1:

0% biodegradation within 28 days. The substance is not readily biodegradable.

However, there is unequivocal evidence of primary biodegradation at 60 days (21% biodegradation). Therefore, the substance is inherently biodegradable and probably completely primarily biodegraded (higher biodegradation observed at day 100: 28% biodegradation).

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable
Type of water:
other: river water

Additional information

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

This biodegradation study (Akzo Nobel, 2012) was assessed as the key study and was performed on the registered substance according to the OECD Guideline 301D, EU Method C.4 -E and ISO Guideline 10707 in compliance with GLP. In this ready biodegradability test, micro-organisms were inoculated into a chemically defined liquid medium containing the test substance as sole carbon and energy source. The test substance was exposed to a relatively low numbers of micro-organisms present in river water, under aerobic conditions for a period of 100 days. The dissolved oxygen concentraton was measured at days 7, 14, 21, 28, 42, 60 and 100. The test substance was not biodegraded at day 28 in the Closed Bottle test. Therefore, the registered substance should not be classified as readily biodegradable. However, in the prolonged Closed Bottle test, the test substance was biodegraded by 21% at day 60 and 28% at day 100. The biodegradation found in the prolonged test demonstrates that the registered substance should be classified as inherently biodegradable. The biodegradation percentage of 21 reached at day 60 strongly indicates that the test substance is partially biodegradable, and probably completely primarily biodegraded (higher biodegradation observed at day 100).