BAP

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 July 2008 to 04 February 2009

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Description: Dark amber paste

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- The activated sewage sludge sample was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC).
- The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21°C and used on the day of collection.
- 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 preweighed GF/A filter paper (rinsed three times with 20mL deionised reverse osmosis water prior to drying in an oven) using a Buchner filter.
- Filtration was continued 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.
- The suspended solids concentration was equal to 1.9 g/L prior to use.

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
Approximately 24 hours prior to addition of the test and standard materials the vessels were filled with 2400 mL of culture medium and 47.4 mL of inoculum and aerated overnight. On Day 0 the test and standard materials were added and the volume in all the vessels adjusted to 3 litres by the addition of culture medium.
The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of approximately 40 mL/minute and stirred continuously by magnetic stirrer. The CO2- free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.
- Test temperature: 21 °C
- pH: The pH of the test preparations was determined on Day 28, prior to acidification with
hydrochloric acid, using a WTW pH/Oxi 3401 pH and dissolved oxygen meter.
- pH adjusted: No

TEST SYSTEM
The following test preparations were prepared and inoculated in 5 litre glass culture vessels each containing 3 litres of solution:
a) A control, in duplicate, consisting of inoculated culture medium plus a glass microscope slide.
b) The standard material (sodium benzoate), in duplicate, in inoculated culture medium plus a glass microscope slide to give a final concentration of 10 mg carbon/L.
c) The test material, in duplicate, in inoculated culture medium plus a glass microscope slide to give a final concentration of 10 mg carbon/L.
d) The test material plus the standard material m inoculated culture medium plus a glass microscope slide to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).
A glass microscope slide was added to the control and standard material vessels in order to maintain consistency between these vessels and the test material vessels. Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L.

CONTROL AND BLANK SYSTEM
- Control vessels: prepared containing a glass microscope slide per 3 litres of inoculated culture medium in order to maintain consistency between the control and test material vessels.
- Toxicity control: containing test material and sodium benzoate; 46.2 mg of test material was smeared onto a glass microscope slide prior to addition to inoculated culture medium. An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3 litres to give a final concentration of 15.4 mg test material/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.

STATISTICAL METHODS:
Statistical analysis of the Day 29 IC values for the control and test material vessels was carried out using a Student's t-test to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001 ).

Key result

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

28 d

Results with reference substance:

Sodium benzoate attained 62% degradation after 14 days and 83% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test material attained 0% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Executive summary:

A study was performed to the standardized guideline OECD 301B, under GLP conditions to assess the ready biodegradability of the test material in an aerobic aqueous medium.

 

The test material, at a concentration of 10 mg Carbon/L, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21 °C for 28 days. Following the recommendations of the International Standards Organisation (ISO 1995) the test material was smeared onto a glass microscope slide prior to addition to the test medium.

 

The degradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.

 

The test material attained 0% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of the test.

Description of key information

A study was performed to the standardized guideline OECD 301B, under GLP conditions to assess the ready biodegradability of the test material in an aerobic aqueous medium. The test material attained 0% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of the test (Harlan Laboratories, Ltd., 2009).

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater