Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 March 1997 - 17 May 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in accordance with OECD, EC, and US EPA test guidelines, and in compliance with GLP.
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
Note that the version of this guideline cited in the study report was dated January 1998 (after the testing period but prior to the report issue date).
Deviations:
no
Principles of method if other than guideline:
A preliminary test using the the closed bottle method was undertaken prior to the definitive test which followed the modified Sturm procedure.
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Oakley STW, collected on the day before the test.
- Storage conditions: Not specified.
- Storage length: Not more than one day (refer to above comment)
- Preparation of inoculum for exposure: The solids content of the sludge was determined by filtering the sludge through pre-weighed filters, then drying and re-weighing the filters. The appropriate volume of inoculum was added to the control and test vessels to give a final suspended solids concentration of 30 mg/L.
- Initial cell/biomass concentration: 30 mg/L.
- Water filtered: yes - Ultrapure water was used.
- Type and size of filter used, if any: Whatman GFC filter paper
Duration of test (contact time):
29 d
Initial conc.:
8.9 other: mgC/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Remarks:
for modified Sturm
Parameter followed for biodegradation estimation:
O2 consumption
Remarks:
for preliminary closed bottle assessment
Details on study design:
For definitive modified Sturm test

TEST CONDITIONS
- Composition of medium: Consistent with the medium described in OECD test guideline 301B
- Solubilising agent (type and concentration if used): Solution in acetone was prepared, but the acetone was removed by evaporation under a stream of nitrogen.
- Test temperature: 21.9 - 23.5°C
- pH: Between 7.5 and 7.6 at the start, 7.3 and 7.5 at the end of the test.
- pH adjusted: no
- Aeration of dilution water: Air flow rate was between 33 to 46 mL/minute
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Glass bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Continuous flushing with treated air through an air inlet tube reaching approximately 10 cm under the liquid surface.
- Measuring equipment: CO2 evolution measured by trap system described below.
- Test performed in open system: An air outlet was located just below the stopper of each test flask.
- Details of trap for CO2 and volatile organics if used: Three Drechsel bottles in series, each containing 100 mL of 0.025N Barium hydroxide.

SAMPLING
- Sampling frequency: Samples taken on days 2, 3, 4, 6, 10, 12, 14, 18, 24, and 28. A Sample was taken on day 29, in the abiotic phase of the test (test and control mixtures were acidified on day 28 of the test to kill the microbial inoculum and to drive off any residual carbon dioxide).
- Sampling method: The nearest of the three dechsel bottles was removed and the second connected in its place. The residual barium hydroxide concentration in the removed bottle was determined by duplicate titrations with hydrochloric acid (0.05N) using phenolphthalein indicator.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes (performed in duplicate)
- Abiotic sterile control: No
- Toxicity control: No
Reference substance:
benzoic acid, sodium salt
Preliminary study:
Sodium benzoate was degraded to 54% of its ThOD (Theoretical Oxygen Demand) after five days of incubation which indicates that the inoculum was viable and exerting normal biodegradive activity. In the preence of the test material at 8.9 mg C/L, degradation of sodium benzoate was completely inhibited.
Oxygen consumption by mixtures containing Benzoflex 2-45 alone at 8.9 mg C/L was equivalent to 8% of its ThOD which indicated that it was not readily biodegradable under the conditions of this preliminary test.
The level of oxygen consumption in bottles containing inoculated mineral salts medium alone at five days (0.3 mgO2/L) was considered to be acceptable for this assay system.
Parameter:
% degradation (CO2 evolution)
Value:
17
Sampling time:
2 d
Parameter:
% degradation (CO2 evolution)
Value:
71
Sampling time:
10 d
Parameter:
% degradation (CO2 evolution)
Value:
93
Sampling time:
28 d
Parameter:
% degradation (CO2 evolution)
Value:
93
Sampling time:
29 d
Remarks on result:
other: Includes residual CO2 released following acidification of medium.
Parameter:
BOD5
Value:
0.17 g O2/g test mat.
Results with reference substance:
The results obtained for the degradation of sodiurn benzoate (65% of its TCO2 after 6 days and 85% after 29 days) and for cumulative CO2 production by the control mixtures (69.0 and 65.1 mg) fulfil the validity criteria for this test.
Although DEGDB appeared to be inhibitory to the inoculum in the preliminary test, it did not inhibit the biodegradation of benzoic acid in the definitive test (modified Sturm)
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Substances are considered to be readily degradable in the modified Sturm test if CO2 production is equal to or greater than 60% of the theoretical value within ten days of the level achieving 10%. DEGDB met these criteria, so may be considered to be readily biodegradable.
Although DEGDB appeared to be inhibitory to the inoculum in the preliminary test, it did not inhibit the biodegradation of benzoic acid in the definitive test (modified Sturm)
Executive summary:

A ready biodegradability test was performed to determine the biodegradability of the test material DEGDB. The study was conducted according to EC, OECD, and US EPA test guidelines, and in compliance with GLP.

In a modified Sturm test DEGDB was found to have degraded by 17% after 2 days, 71% after 10 days, and by 93% at the end of the 28 -day biotic phase of the test. The postive control substance, sodium benzoate, which was analysed contemporaneously degraded rapidly (65% degradation after 6 days), and confirmed that the inoculum was viable and that the test was valid.

Substances are considered to be readily degradable in this test if CO2 production is equal to or greater than 60% of the theoretical value within ten days of the level achieving 10%. In the Modified Sturm test, DEGDB met these criteria, so may be considered to be readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 April 1997 - 17 May 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in accordance with OECD, EC, and US EPA test guidelines, and in compliance with GLP.
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
Note that the version of this guideline cited in the study report was dated January 1998 (after the testing period but prior to the report issue date).
Deviations:
no
Principles of method if other than guideline:
A preliminary test using the closed bottle method was undertaken prior to the definitive test which followed the modified Sturm procedure.
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A sample of activated sludge was collected on the day before the test from Oakley STW a sewage treatment works that treats predominantly domestic waste.
- Storage conditions: Not specified.
- Storage length: Not more than one day (refer to above comment)
- Preparation of inoculum for exposure: The solids content of the sludge was determined by filtering the sludge through pre-weighed filters, then drying and re-weighing the filters. The appropriate volume of inoculum was added to the control and test vessels to give a final suspended solids concentration of 30 mg/L.
- Initial cell/biomass concentration: 30 mg/L.
- Water filtered: yes - Ultrapure water was used.
- Type and size of filter used, if any: Whatman GFC filter paper
Duration of test (contact time):
29 d
Initial conc.:
10 other: mgC/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Remarks:
for modified Sturm
Parameter followed for biodegradation estimation:
O2 consumption
Remarks:
for preliminary closed bottle assessment
Details on study design:
For definitive modified Sturm test

TEST CONDITIONS
- Composition of medium: Consistent with the medium described in OECD test guideline 301B
- Solubilising agent (type and concentration if used): Solution in acetone was prepared, but the acetone was removed by evaporation under a stream of nitrogen.
- Test temperature: 21.1 - 22.9°C
- pH: 7.6 at the start, 7.3 and 7.5 at the end of the test.
- pH adjusted: no
- Aeration of dilution water: Air flow rate was between 30 to 46 mL/minute
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Glass bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Continuous flushing with treated air through an air inlet tube reaching approximately 10 cm under the liquid surface.
- Measuring equipment: CO2 evolution measured by trap system described below.
- Test performed in open system: An air outlet was located just below the stopper of each test flask.
- Details of trap for CO2 and volatile organics if used: Three Drechsel bottles in series, each containing 100 mL of 0.025N Barium hydroxide.

SAMPLING
- Sampling frequency: Samples taken on days 2, 3, 4, 6, 10, 12, 14, 18, 24, and 28. A Sample was taken on day 29, in the abiotic phase of the test (test and control mixtures were acidified on day 28 of the test to kill the microbial inoculum and to drive off any residual carbon dioxide).
- Sampling method: The nearest of the three dechsel bottles was removed and the second connected in its place. The residual barium hydroxide concentration in the removed bottle was determined by duplicate titrations with hydrochloric acid (0.05N) using phenolphthalein indicator.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes (performed in duplicate)
- Abiotic sterile control: No
- Toxicity control: No
Reference substance:
benzoic acid, sodium salt
Remarks:
Both both preliminary and definitive tests
Preliminary study:
Sodium benzoate was degraded to 54% of its ThOD (Theoretical Oxygen Demand) after five days of incubation which indicates that the inoculum was viable and exerting normal biodegradive activity. In the presence of the test material at 10 mg C/L, degradation of sodium benzoate was reduced to 10% of its ThOD
inhibited. This indicated that DPGDB was inhibitory to the inoculum under the conditions of this test.
Oxygen consumption by mixtures containing DPGDB alone at 10 mg C/L was equivalent to 6% of its ThOD which indicated that it was not readily biodegradable under the conditions of this preliminary test.
The level of oxygen consumption in bottles containing inoculated mineral salts medium alone at five days (0.3 mgO2/L) was considered to be acceptable for this assay system.
Parameter:
% degradation (CO2 evolution)
Value:
6
Sampling time:
2 d
Parameter:
% degradation (CO2 evolution)
Value:
62
Sampling time:
12 d
Parameter:
% degradation (CO2 evolution)
Value:
85
Sampling time:
28 d
Parameter:
% degradation (CO2 evolution)
Value:
87
Sampling time:
29 d
Remarks on result:
other: Includes residual CO2 released following acidification of medium.
Parameter:
BOD5
Value:
0.12 g O2/g test mat.
Results with reference substance:
The results obtained for the degradation of sodium benzoate (63% of its TCO2 after 6 days and 79% after 29 days) and for cumulative CO2 production by the control mixtures (69.9 and 65.7 mg) fulfil the validity criteria for this test.
Although DPGDB appeared to be inhibitory to the inoculum in the preliminary test, it did not inhibit the biodegradation of benzoic acid in the definitive test (modified Sturm).
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Substances are considered to be readily degradable in the modifed Sturm test if CO2 production is equal to or greater than 60% of the theoretical value within ten days of the level achieving 10%. DPGDB met these criteria, so may be considered to be readily biodegradable.
Although DPGDB appeared to be inhibitory to the inoculum in the preliminary test, it did not inhibit the biodegradation of benzoic acid in the definitive test (modified Sturm)
Executive summary:

A ready biodegradability test was performed to determine the biodegradability of the test material DPGDB. The study was conducted according to EC, OECD, and US EPA test guidelines, and in compliance with GLP.

The modified Sturm test DPGDB was found to have degraded by 6% after 2 days, 62% after 12 days, and by 85% at the end of the 28 -day biotic phase of the test. The postive control substance, sodium benzoate, which was analysed contemporaneously degraded rapidly (63% degradation after 6 days), and confirmed that the inoculum was viable and that the test was valid.

Substances are considered to be readily degradable in this test if CO2 production is equal to or greater than 60% of the theoretical value within ten days of the level achieving 10%. In the Modified Sturm test, DPGDB met these criteria, so may be considered to be readily biodegradable.

Description of key information

DEGDB:

Biodegradability:

- Key study, reliability 1, OECD 301B - Modified stturm test - readily biodegradable

- Supporting study, reliability 1, OECD 301D - Closed Bottle Test - not readily biodegradable

- Supporting study, reliability 1, ISO Method 11734 - Anaerobix Biodegradability of Organic Chemicals - Ultimately biodegradable under anaerobic conditions

- Supporting study, reliability 2, OECD 301A - Ready Biodegradability: DOC Die Away Test - readily biodegradable

DPGDB:

Biodegradability:

- Key study, reliability 1, OECD 301B - Modified sturm test - readily biodegradable

- Supporting study, reliability 1, OECD 301D - Closed Bottle Test - not readily biodegradable

- Supporting study, reliability 1, ISO Method 11734 - Anaerobix Biodegradability of Organic Chemicals - Ultimately biodegradable under anaerobic conditions

- Supporting study, reliability 2, OECD 301A - Ready Biodegradability: DOC Die Away Test - readily biodegradable

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

This substance is a reaction mass of dipropylene glycol dibenzoate (DPGDB) and diethylene glycol dibenzoate (DEGDB). No testing has been performed on the reaction mass itself, but data are available for DPGDB and DEGDB. Both DPGDB and DEGDB were demonstrated to be readily biodegradable in a Modified Strum Ready Biodegradability test. DPGDB achieved 62% degradation by day 12, meeting the 10 -day window criterion and 85% by test day 28. In addition, DEGDB achieved 71% after 10 days and 93% by test termination. Based on these data, the reaction mass of DPGDB and DEGDB is expected to be readily biodegradable.