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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:
1 November 2005 to 26 January 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Carbon content: 50.2%
- Description: amber coloured paste
- Storage: room temperature, in the dark, under nitrogen
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: activated sewage sludge micro-organisms obtained on 31 October 2005 from the aeration stage of the Severn Trent Water Plc sewage treatment plan (Loughborough, Leicestershre, UK), which treats predominately domestic sewage.
- Method of cultivation: washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC)
- Storage conditions: maintained on continuous aeration in the laboratory at a temperature of approximately 21°C and used on the day of collection
- Concentration of sludge: The suspended solids concentration was equal to 1.9 g/l prior to use. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper (rinsed three times with 20 ml deionised reverse osmosis water prior to drying in an oven) using a Buchner filter for 3 minutes after rinsing the filter three successive times with 10 ml of deionised reverse osmosis water. The filter paper was then dried in an oven at approximately 105°C for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained.
Duration of test (contact time):
28 d
Initial conc.:
10 other: mg C/L
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: The aqueous mineral salts medium used for testing was prepared according to OECD Guideline 301B.
- Test temperature: 21˚C
- pH: 7.5 to 7.6
- Aeration of dilution water: purged with CO2-free air
- Suspended solids concentration: 30 mg solids/L (inoculum)

TEST SYSTEM
- Culture apparatus: 5-L glass bottle, each containing 3 L, each connected to two CO2 absorber traps (500 mL Dreschel bottles) containing 300 mL of 0.05M sodium hydroxide (NaOH) prepare using purified de-gassed water.
- Number of culture flasks/concentration: Six; test substance (2); inoculum blanks (2), sodium benzoate procedural control (1), and toxicity control (1).
- Method of test substance administration: Dispensed onto a Whatman GF/A filter paper (21 mm) prior to dispersal in inoculated culture medium and total volume adjusted to achieve a final concentration of 10 mg C/L. The control blank, procedural control and toxicity control vessels were prepared in the same manner with filter paper for consistency.
- Method used to create aerobic conditions: continuous aeration (40 mL/min); CO2 free air was pumped under positive pressure through a hydration flask before entering the test system.

SAMPLING
- Sampling frequency: Days 0, 2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, 28 and 29
- Sampling method: samples were removed from the first absorber flask on each test system and analyzed for CO2 evolution.
- Terminal sampling: On Day 28, 1 mL of concentrated hydrochloric acid was added to each test vessel following terminal pH measurements. After overnight aeration to drive any residual inorganic carbon from the test vessels, samples for analysis of CO2 evolution were removed from the first and second absorber flasks.
- Sterility check: none
- Sample storage before analysis: all but the Day 12 and 18 samples were analyzed immediately; Day 12 and 18 samples were stored at approximately -20˚C and analysis of these samples was considered unnecessary since the results from the previously and subsequent analyses showed no significant increase in degradation.

CONTROL AND BLANK SYSTEM
- Inoculum blank: two flasks containing mineral medium, inoculum and Whatman GF/A filter paper (21 mm)
- Procedure control: one flask containing mineral medium, inoculum, reference substance (10 mg C/L) and Whatman GF/A filter paper (21 mm)
- Toxicity control: one flask containing mineral medium, inoculum, test material (added using Whatman GF/A filter paper, 21 mm) and reference substance (10 mg C/L of each)
- Abiotic sterile control: none
Reference substance:
benzoic acid, sodium salt
Remarks:
10 mg C/L
Key result
Parameter:
% degradation (CO2 evolution)
Value:
20
Sampling time:
28 d
Details on results:
The total CO2 production in the control vessels on Day 28 was 24.56 mg/L.

The ratio of inorganic to total carbon (IC/TC) of the test material suspension at the start of the test was below 5%. The mean cumulative CO2 production in the test material vessels by Day 28 was 24.56 mg/L.
The cumulative net percent CO2 production (blank control values subtracted) for the test substance was determined to be 20%. The difference between replicate values for CO2 production at the end of the test was less than 20%.

The mean cumulative CO2 production in the toxicity control vessel was 40% by Day 14 and 45% by Day 28.
Results with reference substance:
The mean cumulative CO2 production in the procedural control vessels was 95% by Day 14 and 93% by Day 28.
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test material attained 20% degradation after 28 days and the test substance therefore cannot be classified as readily biodegradable by the criteria set forth in OECD Guideline 301B.
Executive summary:

This study was performed to determine the ready biodegradability of the test material in an aerobic aqueous medium by the carbon dioxide evolution method following OECD Guideline 301B, EC Test Method C.4-C, and U.S. EPA Guideline OPPTS 835.3110. The amount of carbon dioxide (CO2) released upon biodegradation of the test substance (10 mg C/L, with inoculum) was measured over 28 days in the dark at 21˚C. Due to the low solubility of the test material, the test substance was adsorbed to an inert support (Whatman GF/A filter paper, 21 mm) prior to addition to the test medium to aid in dispersion of the test material in the test medium and increase the surface area of the test material exposed to the test organisms. Two blank controls (containing inoculum), a procedural control (containing inoculum and reference substance, 10 mg C/L), and a toxicity control (containing inoculum, and test and reference substance, 10 mg C/L each) were test concurrently to account for background CO2 production, viability of the inoculum, and the toxicity of the test substance, respectively. Preparation of these control vessels also included the filter paper for consistency.

The total CO2 production in the control vessels on Day 28 was 24.56 mg/L. The ratio of inorganic to total carbon (IC/TC) of the test material suspension at the start of the test was below 5%. The mean cumulative CO2 production in the test material vessels by Day 28 was 24.56 mg/L. The cumulative net percent CO2 production (blank control values subtracted) for the test substance was determined to be 20%. The difference between replicate values for CO2 production at the end of the test was less than 20%. The mean cumulative CO2 production in the toxicity control vessel was 40% by Day 14 and 45% by Day 28. The mean cumulative CO2 production in the procedural control vessels was 95% by Day 14 and 93% by Day 28.

The test material attained 20% degradation after 28 days and the test substance therefore cannot be classified as readily biodegradable by the criteria set forth in OECD Guideline 301B. 

Description of key information

The test material attained 20% degradation after 28 days and the test substance therefore cannot be classified as readily biodegradable by the criteria set forth in OECD Guideline 301B. 

Key value for chemical safety assessment

Additional information