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Environmental fate & pathways

Biodegradation in water: screening tests

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Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between May 14, 2003 and June 17, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to guideline under GLP conditions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: ASTM D-5864-95
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report):
- Physical state: pale yellow liquid
- Storage condition of test material: stored at ambient room temperature.
- Other:
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable.

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): activated sludge was collected from the Denton Wastewater Treatment Facility in Denton Maryland (USA).

- Preparation of inoculum for exposure: Activated sludge was sieved through a 2mm screen, homogenized in a blender at medium speed for 2 minutes and allowed to settle for approx. 30 min. One liter aliquot of the supernatant above the settled solids was removed and supplemented with 25 mg vitamin-free casamino acids and 25 mg yeast extract.

-Pretreatment: The inoculum was adapted for 14 days. 100 mL of supplemented inoculum was combined with 900 mL of mineral medium in a 2-L Erlenmeyer flask. An amount of test substance to give the equivalent of 4 mg carbon/L was added by the direct addition method. The mixture was aerated with CO2-free air for a total of 14 days. Two additional increments of test substance were made on day 7 and day 11, each to give the equivalentconcentration of 8 mg carbon/L.  On day 14, the inoculum was filtered through glass wool and homogenized for 2 min.

- Concentration of sludge: 30 mL/3000 mL

- Initial cell/biomass concentration: The result of a standard plate count performed on the inoculum after adaption was 7600 CFU/mL
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
other: 10 mg carbon/L
Based on:
other: carbon content of test material
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
inorg. C analysis
Details on study design:
TEST CONDITIONS
- Composition of medium:

2919 mL Nanopure water
3 mL ammonium sulfate solution (4.0%)
3 mL calcium chloride solution (2.75%)
12 mL ferric chloride solution (0.025%)
3 mL magnesium sulfate solution (2.25%)
30 mL phosphate buffer (pH 7.2)
30 mL adapted inoculum

- Test temperature: 20- 22 C

- pH: test material pH 7.1; reference substance pH 6.3


TEST SYSTEM

- Culturing apparatus: Test chambers were 4-liter amber glass bottles aerated with moisture-free and CO2-free compressed air.

- Number of culture flasks/concentration:

Test substance (3 replicates):  The carbon content of the test substance determined by elemental analysis was 55.69%.  Dosing was based on the carbon content of the test material.  An amount of test substance equivalent to 10 mg carbon/L was added by direct addition.

Reference substance (3 replicates):  An amount of CriscoTM canola oil  equivalent to 10 mg carbon/L was added to the test chambers by direct addition.

Control (3 replicates):  Control chambers contained only the inoculum in mineral medium.  No additions were made to the control.
The final volume in all chambers was 3000 mL.

- Method used to create aerobic conditions: Air entering the test chambers was scrubbed using Drierite and Ascarite absorbants. All chambers were aerated with CO2-free air for approx. 24 hours prior to addition of test and reference materials to purge the systems from CO2. After addition of test material and reference substance to test chambers, solutions were continuously aerated with CO2-free air.

- Measuring equipment: A carbon analyzer was used to measure the evolved CO2 as inorganic carbon.

- Details of trap for CO2 and volatile organics if used: Three CO2 traps containing approx. 100 mL of 0.5 N KOH were connected to the exit air lines of each test chamber to trap CO2 producted from degradation of organic carbon sources as K2CO3


SAMPLING

- Sampling frequency: CO2 traps were removed for analysis on days 1, 5, 8, 13, 15, 19, 21 and 26.

- Sampling method: The CO2 trap nearest the test chamber was removed and analyzed for inorganic carbon. The two remaining traps were moved one position closer to the test chamber and a new trap was placed at the end of the series.

- Other: On day 28, the test was terminated. An aliquot of the contents of each chamber was removed and the pH measured. The remaining contents of each chamber were acidified with 1 mL of concentrated hydrochloric acid. The chambers were aerated overnight and the trapping solutions closest to the test chambers was analyzed for inorganic carbon.


CONTROL AND BLANK SYSTEM

- Inoculum blank: Control chambers (3 replicates) contained only the inoculum in mineral medium.  No additions were made to the control.

- Other: Reference substance (3 replicates):  An amount of CriscoTM canola oil  equivalent to 10 mg Carbon/L was added to the test chambers containing mineral medium by direct addition.



STATISTICAL METHODS:
-Calculations:

(1) Inorganic carbon was converted to mg CO2 using the equation:

mg CO2 = mg carbon/L x volume KOH x 3.67 mg CO2/mg carbon

(2) The cumulative mg CO2 for the test and reference substances were corrected for the amount of CO2 evolved by the control using the equation:

cumulative mg CO2 evolved = Σ mg CO2 test – mean Σ mg CO2 control

(3) The % theoretical CO2 (%ThCO2) evolved was determined using the equation:

%ThCO2 = mg/CO2 produced / (mg carbon in test) x (3.67 mg CO2/mg carbon) x 100
Reference substance
Reference substance:
other: canola oil (Crisco brand name)

Results and discussion

Test performance:
No unusual conditions were noted that would affect the study.
% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
3.6
St. dev.:
4.5
Sampling time:
28 d
Details on results:
The inorganic carbon content, cumulative mg CO2 and %ThCO2 at time points during the study are shown in Tables 1, 2 and 3, respectively

BOD5 / COD results

Results with reference substance:
Results with the reference substance were valid. The % biodegradation of the canola oil reference was 78.8% in 28 days.

Any other information on results incl. tables

Table 1. Inorganic Carbon (mg carbon/L)

Day

Control A

Control B

Control C

Ref A

Ref B

Ref C

Test A

Test B

Test C

1

5.0

5.0

5.0

5.3

6.6

5.5

10.2

4.5

5.1

5

10.6

8.9

12.1

61.7

64.1

67.6

9.6

11.6

9.5

8

11.3

9.3

8.3

86.4

91.4

85.7

9.5

9.7

8.2

13

13.8

6.1

8.6

75.1

72.6

70.8

9.6

13.0

5.6

15

13.1

8.7

9.7

24.3

15.8

26.5

18.1

13.0

9.5

19

15.0

7.0

10.6

28.0

7.7

27.6

12.4

15.4

9.8

21

16.2

8.9

9.1

15.4

28.6

15.7

12.9

10.6

11.8

26

18.3

8.1

10.6

18.7

28.5

13.3

16.2

8.8

15.2

29

15.0

9.0

11.3

13.1

13.6

13.8

13.7

21.3

12.3

 

 

Table 2. Cumulative mg CO2 Evolved

Day

Control A

Control B

Control C

Ref A

Ref B

Ref C

Test A

Test B

Test C

1

1.8

1.8

1.8

0.1

0.6

0.2

1.9

-0.2

0.0

5

5.7

5.1

0.3

18.9

20.2

21.1

1.6

0.2

-0.3

8

9.9

8.5

9.3

47.1

50.3

49.0

1.5

0.2

-0.9

13

14.9

10.8

12.5

71.1

73.4

71.5

1.6

1.5

-2.3

15

19.7

13.9

16.0

76.2

75.4

77.4

4.3

2.4

-2.7

19

25.2

16.5

19.9

82.5

74.2

83.6

4.9

4.1

-3.1

21

31.2

19.8

23.3

84.0

80.5

85.1

5.5

3.8

-2.9

26

37.9

22.8

27.2

86.3

86.4

85.5

6.9

2.5

-1.9

29

43.4

26.1

31.3

86.8

87.1

86.2

7.6

6.0

-1.7

Avg.

33.6

86.7

4.0

  

Table 3. Cumulative % ThCO2

Day

Ref A

Ref B

Ref C

Test A

Test B

Test C

1

0.1

0.5

0.2

1.7

-0.2

0

5

17.2

18.4

19.2

1.4

0.2

-0.3

8

42.7

45.6

44.5

1.4

0.2

-0.8

13

64.6

66.7

65

1.4

1.4

-2.1

15

69.2

68.4

70.3

3.9

2.2

-2.4

19

74.9

67.4

75.9

4.5

3.7

-2.8

21

76.3

73.1

77.3

5

3.5

-2.6

26

78.4

78.5

75.9

6.2

2.3

-1.7

29

78.8

79.1

78.3

6.9

5.5

-1.5

average

78.7

3.6

Std dev.

0.4

4.5

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test substance is not biodegradable.
Executive summary:

The % biodegradation of the test material was determined according to guideline ASTM D-5864 -95.

Activated sludge obtained from a domestic water treatment facility was adapted to the test material for 14 days under aerobic conditions. Following the acclimation, inoculated mineral medium was dosed with an amount of test substance equivalent to 10 mg carbon/L as the nominal sole source of organic carbon and aerated with CO2-free air. The CO2 produced within the test chambers was trapped as K2CO3 in KOH trapping solution.  At intervals between day 0 and day 28, a carbon analyzer was used to measure the amount of carbon in the trapping solution which was then mathematically converted to %ThCO2 (percent theoretical CO2).

The results of the 28 day study indicate that the test substance is not readily biodegradable. The %ThCO2 of the test material was 3.6% ±4.5 after 28 days. The %ThCO2 of the canola oil reference was 78.8% ± 0.4 after 28 days which validated the test.