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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 March 1998 - 12 November 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study; well documented study report

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
Activated sludge was collected from the Prospect Bay Wastewater Treatment Facility in April 1998. The sludge was sieved using a 2 mm screen and then aerated for approximately 4 hours. The sludge was homogenized in a blender at medium speed for approximately 2 minutes and then was allowed to settle for approximately 30 minutes. The supernatant was used as the inoculum for this study and was used the same data that it was prepared. A total suspended solids measurement and standard plate count were performed on the inoculum using procedures based on Standard Methods. The average measured total suspended solids concentration of the inoculum was 78.6 mg/L. The plates were incubated at 20 degrees C for approximately 48 hours and then the number of colony forming units was enumerated. The result of the standard plate count performed on the inoculum was 1.37x10^6 CFU/mL. The standard plate count result indicated that the inoculum used in this study contained an acceptable number of viable microbes according to the guideline.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
other: 10 mg carbon/liter Carbon determined by elemental analysis
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: The test medium was a modified biochemical oxygen demand test dilution water. The test medium contained the following standard reagent solutions per liter of high quality Nanopure water: 1mL ammonium sulfate solution 4%; 1 mL of calcium chloride solution (2.75%); 4 mL of ferric chloride solution (0.025%); 1 mL of magnesium sulfate solution (2.25%); 2 ml of phosphate buffer solution (pH 7.2).

- Test temperature: 20C
- pH: The pH values ranged from 5.2 to 6.9.
- pH adjusted: yes/no

- Suspended solids concentration: The solids concentration of the test solution was 0.786 mg/L


TEST SYSTEM
- Culturing apparatus: The test chambers were ambered 4 liter bottles. The air entering the chambers was passed through Drierite to remove ambient moisture and then through Ascarite to produce CO2 free air. The air exiting the test chambers was passed through a series of three gas washing bottles each containing KOH to trap the CO2 that had evolved within the chamber. All chambers were aerated with CO2 free air for 24 hours at a rate of 50 to 100 mL per minute to purge the system of CO2. After the aeration period, the flow of CO2 was stopped and three CO2 traps each containing KOH were connected to the exit air lines of each chamber. An amount of test substance necessary to deliver 10 mg C/L was added to the treatment group test chambers by direct weight addition. An amount of reference substance necessary to deliver 10 mg C/L was added to the reference group test chambers by direct weight addition. The biodegradation was started by bubbling CO2 free air through each of the test chambers at a rate of 50 to 100 mL per minute. The CO2 produced from the degradation of organic carbon sources within the test chamber was trapped as K2CO3 in the KOH solution and the amount of inorganic carbon in the trapping solution was measured at various intervals during the study using a Shimadzu TOC 5000 carbon analyzer.

- Number of culture flasks/concentration: 3 Replicates


SAMPLING
The CO2 traps were removed for analysis on days 1, 4, 8, 11, 14, 19, 21, 25, and 29. The CO2 trap nearest the chamber was removed and analyzed for inorganic carbon. The two remaining traps were placed one position closer to the test chamber and a new trap was placed on the end of the series. On the 28h day of the test, an aliquot of the contents of each test chamber was removed and the pH determined. The contents of each chamber then were acidified by the addition of 3 mL of concentrated hydrochloric acid to drive off inorganic carbonate. The chambers were aerated overnight after which the trapping solutions closest to the test chambers were analyzed for inorganic carbon.

CONTROL AND BLANK SYSTEM
The test contained a blank control group, a reference group, and a treatment group. The blank control, reference, and treatment groups each contained three replicate test chambers
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
1.5
Sampling time:
28 d
Details on results:
The mean percent theoretical CO2 evolved for the test substance was 1.5% (range 0.6 to 2.0%). The control chambers evolved an average of 6.5 milligrams of CO2 over the test period. The mean percent theoretical CO2 evolved for the reference substances was 99%. The temperature range recorded during the test was 18 to 20C. The average measured total suspended solids concentration of the inoculum was 78.6 mg/L. The result of the standard plate count performed on the inoculum was 1.37 x 10^6 CFU/mL. The measured pH values ranged from 5.2 to 6.9. The control chambers evolved an average of 6.5 milligrams of CO2 over the test period.

BOD5 / COD results

Results with reference substance:
The mean percent theoretical carbon dioxide evolved for sodium benzoate was 99%

Any other information on results incl. tables

Cumulative Percent of Theoretical Carbon Dioxide Evolved

Day

Benzoate

Replicate values

OS13961BG

Replicate values

1

0.0; 0.0; 0.0

0.0; 0.1; 0.0

4

13.1; 34.8; 50.0

1.2; 0.5; 0.0

8

64.4; 46.8; 73.2

1.1; 0.5; 0.0

11

85.6; 94.9; 94.5

1.3; 0.5; 0.1

14

91.1; 100.4; 98.4

1.2; 1.0; 1.8

19

92.9; 101.5; 100.4

1.8; 0.6; 1.9

21

92.7; 102.1; 100.9

2.0; 0.8; 2.0

25

94.1; 103.4; 101.2

2.2; 0.8; 2.2

29

94.6; 103.0; 100.4

2.0; 0.6; 1.9

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
other: Not readily biodegradable under test conditions
Conclusions:
The mean percent theoretical CO2 evolved for the test substance was 1.5% (range 0.6 to 2.0%). The control chambers evolved an average of 6.5 milligrams of CO2 over the test period. The mean percent theoretical CO2 evolved for the reference substances was 99%.
Executive summary:

The objective of the study was to measure the amount of carbon dioxide produced from the biodegradation of the test substance under the conditions tested. The test contained a blank control group, a reference group, and a treatment group, and each contained three replicate test chambers. The study was conducted according to procedures specified by the OECD Guideline for Testing of Chemicals, Method 301B and EEC Method C.4-C. The test substance was administered to the treatment group test chambers by direct weight addition at a concentration of 10 mg Carbon per liter. After 28 days the cumulative percent of theoretical carbon dioxide evolved for the test material was 1.5%. The solids concentration of the test solution was within the acceptable limit specified I the test guideline. The standard plate count result indicated that the inoculum used in this study contained an acceptable number of viable microbes according to the guidelines