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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 1996-01-30 to 1996-03-29
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Although a guideline study (OECD 301B & 301D and EU Methods C4-C & C4-E.) conducted according to the principles of Good Laboratory Practice, the purity and expiry date of the test substance was not supplied and its stability under the test conditions was not determined.
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
Due to a failure in the control system on Day 27, the temperature of the test area dropped to 14.8°C which falls outside the minimum recommended for this assay (20°C) it is not thought to be significant or to have affected the integrity of the test.
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
yes
Remarks:
Due to a failure in the control system on Day 27, the temperature of the test area dropped to 14.8°C which falls outside the minimum recommended for this assay (20°C) it is not thought to be significant or to have affected the integrity of the test.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch No.: + 19951208
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: Oakley STW
- Method of cultivation: Five test vessels (five-litre brown glass carboys) each containing mineral salts medium, and the bacterial inoculum at a concentration of 1% were aerated overnight with air that had been passed through cylinders containing calcium chloride and Carbosorb AS followed by two bottles containing 0.025N barium hydroxide to remove CO2.
- Preparation of inoculum for exposure: The sample of activated sludge was aerated in the laboratory for four hours.
- Pretreatment: A sample of the mixed liquor was homogenised in a mechanical blender and allowed to settle for 50 minutes. An aliquot of the supernatant was used as the inoculum for the test.
- Concentration of sludge: not stated
- Initial cell/biomass concentration: 1% (innoculm)
- Water filtered: through reverse osmosis unit
- Type and size of filter used, if any: Elga Ltd, Prima 4 reverse osmosis unit
Duration of test (contact time):
28 d
Initial conc.:
396 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral salts medium (MSMS)
- Additional substrate: none
- Solubilising agent (type and concentration if used): The test material was added as ultrasound-treated suspensions
- Test temperature: 21.2°C to 23.9°C
- pH: The pH of test mixtures ranged between 7.4 and 7.5 at the start of the test and between 7.4 and 7.5 at the end.
- pH adjusted: no
- CEC (meq/100 g): not stated
- Aeration of dilution water: yes
- Suspended solids concentration: not stated
- Continuous darkness: not stated

TEST SYSTEM
- Culturing apparatus: five-litre brown glass carboys
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Each vessel was then fitted with a stopper holding an air inlet tube reaching approximately 15 cm below the liquid surface and an air outlet just below the stopper. The vessels contents were continuously flushed for 29 days with treated air.
- Measuring equipment: duplicate titration of 20 mL samples with hydrochloric acid (0.05N), using phenolphthalein indicator.
- Test performed in closed vessels due to significant volatility of test substance: No
- Test performed in open system: yes
- Details of trap for CO2 and volatile organics if used: The air outlet from each vessel was connected to three Drechsel bottles in series, each containing 0.025N barium hydroxide (100 mL).

SAMPLING
- Sampling frequency: 2, 3, 4, 5, 6, 8, 15, 18, 25, 28, 29 d
- Sampling method: not stated

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: N/A
- Toxicity control: sodium benzoate 1.72 g/L

STATISTICAL METHODS: none used
Reference substance:
benzoic acid, sodium salt
Preliminary study:
A five-day bacterial inhibition test was performed under the conditions of the Closed Bottle Test (EC Procedure C.4-E, OECD Procedure 301D). This showed that the test item at a nominal concentration of 10 mgC/L did not significantly inhibit degradation of the reference material, sodium benzoate. In this preliminary test, the test item showed no evidence of biodegradation.
Test performance:
Sodium benzoate was degraded by 60% after six days and 88% after 29 days.
Cumulative CO2 production in the controls after 29 days (30.5 and 25.1 mg CO2) was within the acceptable range for this assay system (recommended maximum = 120 mgCO2)
These results confirm that the inoculum was viable and that the test was valid.
Parameter:
% degradation (CO2 evolution)
Value:
2
Sampling time:
28 d
Details on results:
In the Modified Sturm Test, mean cumulative CO2 production by mixtures containing test item at 10 mgC/L was equivalent to 2% of the TCO2.
Results with reference substance:
The degradation of sodium benzoate was rapid and achieved 60% of its TCO2 after six days and 88% after 29 days.

Modified Sturm test - blank-corrected cumulative CO2 production and degradation as a percentage of TCO2 in reference and test mixtures

Day

Reference

Test item (10 mgC/L)

Culture 1

Culture 2

CO2 (mg)

%TCO2

CO2 (mg)

%TCO2

CO2 (mg)

%TCO2

2

8.5

8

0

0

0

0

3

29.1

27

0

0

0

0

4

46.1

43

0

0

0

0

5

56.3

53

0

0

0.3

0

6

64.2

60

0.2

0

0.5

0

8

73.2

69

0.2

0

0.7

1

15

84.5

79

0.5

0

1.0

1

18

88.6

83

0.7

1

1.8

2

25

91.6

86

0.7

1

1.8

2

28

92.7

87

1.3

1

1.8

2

29

93.2

88

1.3

1

1.8

2

The rate of air flow during the test ranged from 38 to 43 mL/minute. Temperatures of the test area ranged from 21.2°C to 23.9°C.

Because of the stringency of this type of test, substances that fail to show degradation are not necessarily non-biodegradable under environmental conditions. Conditions more favorable to degradation, such as a larger microbial inoculum and a longer incubation time, may be necessary for biodegradation to occur.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the conditions of the test, the test item was not regarded as be readily degradable.
Executive summary:

The ready biodegradability of test item was assessed in the CO2 Evolution Test (Modified Sturm Test, Procedure C.4-C of the Annex to Directive 92/69/EEC; OECD Procedure 301B). A five-day bacterial inhibition test was performed under the conditions of the Closed Bottle Test (EC Procedure C.4-E, OECD Procedure 301D). This showed that test item at a nominal concentration of 10 mgC/L did not significantly inhibit degradation of the reference material sodium benzoate. In this preliminary test, test item showed no evidence of biodegradation. In the Modified Sturm Test, test item was added to two vessels containing inoculated mineral salts medium, to give a nominal test concentration of 10 mgC/L. Control vessels comprised two containing inoculated mineral salts medium alone and one containing inoculated mineral salts medium plus the reference material sodium benzoate (10 mgC/L). Test and control vessels were aerated for 29 days with air that had been treated to remove carbon dioxide (CO2). The CO2 produced by each culture was trapped in a series of Drechsel bottles containing barium hydroxide which were connected to the outlet from each test vessel. Sodium benzoate was degraded by 60% after six days and 88% after 29 days. Cumulative CO2 production in the controls after 29 days (30.5 and 25.1 mgCO2) was within the acceptable range for this assay system (recommended maximum =120 mgCO2). These results confirm that the inoculum was viable and that the test was valid. Mean cumulative CO2 production by the mixtures containing test item at 10 mgC/L was equivalent to 2% of the theoretical value (TCO2, 106.4 mgCO2). 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%. The test item cannot therefore be considered to be readily degradable.

Description of key information

The ready biodegradability of test item was assessed in the CO2 Evolution Test (Modified Sturm Test, Procedure C.4-C of the Annex to Directive 92/69/EEC; OECD Procedure 301B). Mean cumulative CO2 production by the mixtures containing test item at 10 mgC/L was equivalent to 2% of the theoretical value (TCO2, 106.4 mgCO2). 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%. The test item cannot therefore be considered to be readily degradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information