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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 17th 2009 to December 17th 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 301F with GLP statement. All validity criteria were fulfilled and no deviation was observed.
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
2008-09-30
Specific details on test material used for the study:
Storage condition of test material: stored at approximately 4°C (refrigerator) in the dark under N2 conditons.
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained on November 18th, 2009 from the secondary treatment stage of the sewage treatment plant at Villette (STEP Villette, av. de Thônex 105, 1226 THONEX (Geneva, Switzerland)), which treats predominantly domestric sewage.

Preparation of inoculum:
The sample of activated sewage sludge was maintained on continuous aeration upon receipt. A sample of the activated sewage sludge was washed three times by settlement (centrifuge: Heittich rotenta 460 RS) and suspension in culture medium. To remove any excessive amounts of dissolved organic carbon (DOC) that may have been present, the solution was stirred and maintained on with pure oxygen at room temperature. Determination of dry weight is made to inoculate final solution with 30 mg/L dry weight activated sludge.
Duration of test (contact time):
28 d
Initial conc.:
99.85 mg/L
Based on:
test mat.
Initial conc.:
100.05 mg/L
Based on:
test mat.
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: the culture medium used in this study was that recommended in the OECD Guideline 301F. See details on "Any other information on materials and methods incl. tables".
- Additional substrate: none
- Solubilising agent (type and concentration if used): none
- Test temperature: the test was conducted at a temperature of 22°C +/- 1°C.
- pH: on day 28, all vessels were sampled for pH. The pH varied between 7.27 to 7.69.
- pH adjusted: no
- CEC (meq/100 g): no data
- Aeration of dilution water: no
- Continuous darkness: no. The test was conducted in diffuse light
- Other: none

TEST SYSTEM
The system consists of a sample flask sealed by a sensor head/CO2 trap in a temperature controlled incubator. The samples were stirred for the duration of the study with a magnetically coupled stirrer. As biodegradation progresses, the micro-organisms convert oxygen to carbon dioxide which is absorbed into NaOH causing a net reduction in gas pressure within the sample flask. WTW oxitopC calculate automatically the consumption of oxygen.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes. A control in duplicate was performed, consisting of inoculated culture medium.
- Abiotic sterile control: yes. The crushed test material was dispersed directly in culture medium. An amount of test material (19.97 mg) was dispersed in 200 mL of culture medium inoculated to give the test concentration of 99.85mg/L with abiotic agent (NaN3).
- Toxicity control: yes. The test material (100.15 mg/L) and the standard material (100.10 mg/L) in inoculated culture medium.
- Other: A reference control with standard material (sodium benzoate) was performed.

STATISTICAL METHODS:
None
Reference substance:
benzoic acid, sodium salt
Preliminary study:
No data
Test performance:
3 tests were performed on the ST 10 C 08.
Key result
Parameter:
% degradation (O2 consumption)
Value:
83
Sampling time:
28 d
Remarks on result:
other: test 1
Key result
Parameter:
% degradation (O2 consumption)
Value:
93
Sampling time:
28 d
Remarks on result:
other: test 2
Key result
Parameter:
% degradation (O2 consumption)
Value:
80
Sampling time:
28 d
Remarks on result:
other: test 3
Details on results:
See in "Attached background material".
The test material (test 1) attained 83% debradation after 28 days and satisfied the 10-day window validation criterion.
The test material (test 2) attained 93% debradation after 28 days and satisfied the 10-day window validation criterion.
The test material (test 3) attained 80% debradation after 28 days and satisfied the 10-day window validation criterion.
The toxicity test attained 84% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study.
Parameter:
BOD5
Value:
2.51 other: mg O2/mg test material (test 1)
Parameter:
BOD5
Value:
2.82 other: mg O2/mg test material (test 2)
Parameter:
BOD5
Value:
2.45 other: mg O2/mg test material (test 3)
Results with reference substance:
Sodium benzoate attained 98% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

See tables and figures in "Attached background material".

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test material (test 1, test 2, test 3) attained respectively 83%, 93% and 80% degradation after 28 days and satisfied the 10-day window validation criterion. Therefore, the test material can be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline 301F.
Executive summary:

The study was performed to assess the ready biodegradability of the test material in an aerobic aqueous media. The method followed that described in the OECD Guideline 301F with GLP statement.

The test material, at a concentration of 99.85 mg/L in test 1, 100.05 mg/L in test 2 and 100.00 mg/L in test 3 was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in diffused light at 22°C +/- 1°C for 28 days.

The degradation of the test material was assessed by the measurement of daily oxygen consumption on days 0 and 28. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.

The test material (test 1, test 2, test 3) attained respectively 83%, 93% and 80% degradation after 28 days and satisfied the 10-day window validation criterion. Therefore, the test material can be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline 301F.

The toxicity test attained 84% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study.

Sodium benzoate attained 98% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From September 21st 2009 to October 23rd 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 301F with GLP statement. Due to a connection problem, the automatic record of daily temperature was not recorded. Nevertheless, the temperature indicated by the shaker was checked daily and no significant changes of temperature were observed. Furthermore, the automatic temperature control was certified during the annual control of the shaker. Therefore, it has been concluded that the protocol deviation had no effect on the results of study.
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
yes
Remarks:
, due to a connection problem, the automatic record of daily temperature was not recorded.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
2008-09-30
Specific details on test material used for the study:
Storage condition of test material: stored at approximately 4°C (refrigerator) in the dark under N2 conditons.
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained on September 23rd, 2009 from the secondary treatment stage of the sewage treatment plant at Villette (STEP Villette, av. de Thônex 105, 1226 THONEX (Geneva, Switzerland)), which treats predominantly domestric sewage.

Preparation of inoculum:
The sample of activated sewage sludge was maintained on continuous aeration upon receipt. A sample of the activated sewage sludge was washed three times by settlement (centrifuge: Heittich rotenta 460 RS) and suspension in culture medium. To remove any excessive amounts of dissolved organic carbon (DOC) that may have been present, the solution was stirred and maintained on with pure oxygen at room temperature. Determination of dry weight is made to inoculate final solution with 30 mg/L dry weight activated sludge.
Duration of test (contact time):
28 d
Initial conc.:
100.1 mg/L
Based on:
test mat.
Initial conc.:
100.3 mg/L
Based on:
test mat.
Initial conc.:
99.85 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: the culture medium used in this study was that recommended in the OECD Guideline 301F. See details on "Any other information on materials and methods incl. tables".
- Additional substrate: none
- Solubilising agent (type and concentration if used): none
- Test temperature: the test was conducted at a temperature of 22°C +/- 1°C. However, due to a connection problem, the automatic record of daily temperature was not recorded.
- pH: on day 28, all vessels were sampled for pH. The pH varied between 7.23 to 7.67.
- pH adjusted: no
- CEC (meq/100 g): no data
- Aeration of dilution water: no
- Suspended solids concentration:
- Continuous darkness: no. The test was conducted in diffuse light
- Other: none

TEST SYSTEM
The system consists of a sample flask sealed by a sensor head/CO2 trap in a temperature controlled incubator. The samples were stirred for the duration of the study with a magnetically coupled stirrer. As biodegradation progresses, the micro-organisms convert oxygen to carbon dioxide which is absorbed into NaOH causing a net reduction in gas pressure within the sample flask. WTW oxitopC calculate automatically the consumption of oxygen.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes. A control in duplicate was performed, consisting of inoculated culture medium.
- Abiotic sterile control: yes. The crushed test material was dispersed directly in culture medium. An amount of test material (20.08 mg) was dispersed in 200 mL of culture medium inoculated to give the test concentration of 100.4 mg/L with abiotic agent (NaN3).
- Toxicity control: yes. The test material (99.85 mg/L) and the standard material (100.2 mg/L) in inoculated culture medium.
- Other: A reference control with standard material (sodium benzoate) was performed.

STATISTICAL METHODS:
None
Reference substance:
benzoic acid, sodium salt
Preliminary study:
None
Test performance:
3 tests were performed on the ST 10 C 08.
Key result
Parameter:
% degradation (O2 consumption)
Value:
81
Sampling time:
28 d
Remarks on result:
other: test 1
Key result
Parameter:
% degradation (O2 consumption)
Value:
85
Sampling time:
28 d
Remarks on result:
other: test 2
Key result
Parameter:
% degradation (O2 consumption)
Value:
83
Sampling time:
28 d
Remarks on result:
other: test 3
Details on results:
See "Attached background material".
The test material (test 1) attained 81% debradation after 28 days and satisfied the 10-day window validation criterion.
The test material (test 2) attained 85% debradation after 28 days and satisfied the 10-day window validation criterion.
The test material (test 3) attained 83% debradation after 28 days and satisfied the 10-day window validation criterion.
The toxicity test attained 82% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study.
Parameter:
BOD5
Value:
2.48 other: mg O2/mg test material (test 1)
Parameter:
BOD5
Value:
2.59 other: mg O2/mg test material (test 2)
Parameter:
BOD5
Value:
2.53 other: mg O2/mg test material (test 3)
Results with reference substance:
Sodium benzoate attained 93% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

None

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test material (test 1, test 2, test 3) attained respectively 81%, 85% and 83% degradation after 28 days and satisfied the 10-day window validation criterion. Therefore, the test material can be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline 301F.
Executive summary:

The study was performed to assess the ready biodegradability of the test material in an aerobic aqueous media. The method followed that described in the OECD Guideline 301F with GLP statement.

The test material, at a concentration of 100.1 mg/L in test 1, 100.3 mg/L in test 2 and 99.85 mg/L in test 3 was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in diffused light at 22°C +/- 1°C for 28 days. Due to a connection problem, the automatic record of daily temperature was not recorded. Nevertheless, the temperature indicated by the shaker was checked daily by the study technician throughout the test and no significant changes of temperature were observed.

The degradation of the test material was assessed by the measurement of daily oxygen consumption on days 0 and 28. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.

The test material (test 1, test 2, test 3) attained respectively 81%, 85% and 83% degradation after 28 days and satisfied the 10-day window validation criterion. Therefore, the test material can be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline 301F.

The toxicity test attained 82% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study.

Sodium benzoate attained 93% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Description of key information

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

83, 93 and 80% degradation after 28 days, within the 10-day window .

Readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Two valid studies are available, according to OECD guideline 301F with GLP statement, to determine the readily biodegradation of the substance.

In the first study, assessed as a key study, the substance attained 83%, 93% and 80% degradation after 28 days (corresponding to test 1, test 2 and test 3) within the 10-day window.

In the second study, assessed as a supporting study, the substance attained 81%, 85% and 83% degradation after 28 days (corresponding to test 1, test 2 and test 3) within the 10-day window.

Therefore, based on these studies, the substance can be considered to be readily biodegradable.