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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-01-08 to 1999-02-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 301 B with GLP statement. All validity criteria were fulfilled and no deviations were observed.
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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Remarks:
Statement signed by the Study Director on 1999-03-24.
Specific details on test material used for the study:
- Theoretical carbon content: 81%
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge was collected from the Prospect Bay Wastewater Treatment Facility on January 07, 1999.
- The sludge was sieved using a 2 mm screen and then aerated for four hours. After the aeration period 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 day 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 plates were incubated at 20 ± 3 °C for approximately 48 h and then the number of colony forming units was enumerated.
Duration of test (contact time):
28 d
Initial conc.:
10 other: mg C/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Test medium was modified biochemical oxygen demand (BOD) test dilution water and was prepared using NANO® pure water.
- Preparation of Test Chambers: The following was added to each chamber:
1) 2943 mL of NANO® pure water
2) 3 mL of ammonium sulfate solution; 3 mL calcium chloride solution; 12 mL ferric chloride solution; 3 mL of magnesium sulfate solution; 6 mL of phosphate buffer
3) 30 mL of the activated sludge inoculum
- All chambers were aerated with CO2 free air for approximately 24 h at a rate of 50 to 100 mL per minute to purge the systems of CO2,. After the aeration period, the flow of CO2 free air was stopped, and three CO2 traps each containing approximately 100 mL of 0.5 N KOH were connected to the exit air lines of each chamber. Amounts of test and reference substance stock solutions necessary to deliver 10 mg C/L were added to the appropriate test chambers.
- Test temperature: 19-22 °C
- pH: 5.04-6.02
- Average measured total suspended solids (TSS) concentration of the inoculum was 209 mg/L; calculated solids concentration of the test solution was 2.09 mg/L.

TEST SYSTEM
- Culturing apparatus: The test chambers were ambered 4-liter gas washing 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 approximately 100 mL of 0.5 N KOH to trap the CO2 that had evolved within the chamber. An additional set of gas washing bottles that were not connected to a chamber were maintained concurrently with the traps connected to the chambers. These bottles contained approximately 100 mL of 0.5 N KOH and the amount of CO2 detected in the KOH solution was subtracted from the CO2 in the blank control traps to determine the amount of CO2 produced by the inoculum in the blank control. Magnetic stir bars and stir plates were used to mix the contents of the test chambers. Stir plate motors were cycled on and off approximately every 15 minutes to prevent the heating of the stirrer motors. The final volume within all chambers was brought up to 3000 mL by the addition of NANO® pure water.
- Number of culture flasks/concentration: 2

SAMPLING
- Sample Collection and Analysis: The CO2 traps were removed for analysis on days 3, 6, 10, 13, 17,20, 24 and 27. 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.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Toxicity control: The toxicity control was dosed with a volume of test substance and reference stock solution necessary to deliver 10 mg C/L as test substance and 10 mg C/L as reference substance.
- Other: The killed control was dosed with a volume of test substance stock solution necessary to deliver 10 mg C/L and adequate mercuric chloride (HgCI2) to achieve 50 mg/L, The mercuric chloride was added by direct weight addition.

BIODEGRADATION TEST INITIATION
The biodegradation test 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.

TEST TERMINATION
On the 28th 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 a sample from each chamber was removed for dissolved organic carbon (DOC) analysis. The trapping solutions closest to the test chambers were analyzed for inorganic carbon.
Reference substance:
benzoic acid, sodium salt
Remarks:
10 mg C/L
Preliminary study:
None
Test performance:
No data
Key result
Parameter:
% degradation (CO2 evolution)
Value:
83.9
Sampling time:
28 d
Details on results:
- The average cumulative percent of theoretical carbon dioxide produced by the test substance at 10 mg C/L was 83.9%.
- The killed control did not evolve a measurable amount of CO2.
- The toxicity control evolved approximately 85.8 % TCO2 indicating that the test substance was not toxic to the inoculum.
- Result of the standard plate count performed on the inoculum was 5.8 x 10^5 CFU/mL.
- The control chambers evolved an average of approximately 12 milligrams of CO2, over the test period. This value has been corrected for the amount of CO2 in the trapping solution since potassium hydroxide solution, even when freshly prepared, contains carbonates. The amount of CO2 evolved by the control chambers did not exceed the 40 mg/L value considered the acceptable limit for CO2 evolution tests.
Results with reference substance:
The viability of the inoculum and validity of the test were supported by the results of the reference substance, sodium benzoate, from which 99.0% of theoretical CO2 was evolved. An average percent biodegradation of greater than 60% was achieved within 8 days, thereby fulfilling the criteria for a valid test by reaching the pass level by day 14.

Table 5.2.1/1: Dissolved Organic Carbon Concentration and pH of Test Solutions at Test Termination

 

Test Chamber

DOC (mg C/L)

pH

Control Rep. A

<1.0

6.02

Control Rep. B

<1.0

5.53

Sodium Benzoate Rep. A (10 mg C/L)

<1.0

5.59

Sodium Benzoate Rep. B (10 mg C/L)

<1.0

5.48

ST 23 C 97 Rep. A (10 mg C/L)

<1.0

5.04

ST 23 C 97 Rep. B (10 mg C/L)

<1.0

5.46

ST 23 C 97 Killed Control (10 mg C/L)

1.0

5.75

ST 23 C 97 Toxicity Control (20 mg C/L)

<1.0

5.42

 

Table 5.2.1/2: Cumulative Percent of Theoretical Carbon Dioxide Evolved

 

Day

Control Rep. A

Control Rep. B

Benzoate Rep. A

Benzoate Rep. B

ST 23 C 97 Rep. A

ST 23 C 97 Rep. B

ST 23 C 97 Killed Control

ST 23 C 97 Toxicity Control

3

NA

NA

67.4

56.8

7.3

1.8

-0.5

21.8

6

NA

NA

75.7

73.9

50.6

45.0

-2.2

51.5

10

NA

NA

82.4

83.6

63.4

60.9

-4.2

69.3

13

NA

NA

84.7

86.9

70.8

69.8

-4.9

76.2

17

NA

NA

96.4

95.5

76.2

73.8

-6.1

82.6

20

NA

NA

97.3

96.9

78.2

76.0

-6.5

83.8

24

NA

NA

98.1

98.0

79.6

78.3

-6.3

84.5

27

NA

NA

98.3

98.5

81.0

79.5

-7.0

85.5

29

NA

NA

98.6

99.4

82.2

85.6

-7.2

85.8

Mean Cumulative Percent TCO2

99.0

83.9

NA

NA

Standard Deviation

0.6

2.4

NA

NA

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the test conditions, 83.9% biodegradation of the test substance was observed after 28 days, within the 10 days window. Therefore, the test substance is considered readily biodegradable.
Executive summary:

In a ready biodegradation study performed according to OECD Guideline 301 B and GLP, the test substance was tested at a concentration of 10 mg C/L and the inoculum was activated sludge. The degradation of the test material was assessed by the determination of the CO2 evolution.

The average cumulative percent of theoretical carbon dioxide produced by the test substance at 10 mg C/L was 83.9% and the 10d window was fulfilled (the pass value (60% degradation) was reached within 10 days of achieving 10% degradation). The killed control did not evolve a measurable amount of CO2. The toxicity control evolved approximately 85.8 % TCO2 indicating that the test substance was not toxic to the inoculum. In conclusion, the test substance is considered readily biodegradable under the test conditions.

Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-01-12 to 1999-02-09
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
This study was performed according to OECD Guideline 302 A with GLP statement. This study is considered reliable with restrictions as no reference substance was tested.
Qualifier:
according to
Guideline:
OECD Guideline 302 A (Inherent Biodegradability: Modified SCAS Test)
Deviations:
yes
Remarks:
reference substance not reported
Qualifier:
according to
Guideline:
other: TSCA Title 40 of the Code of Federal Regulations Part 796.3340
Deviations:
not specified
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Remarks:
Statement signed by the Study Director on 1999-03-24.
Specific details on test material used for the study:
- Theoretical carbon content: 81%
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge was collected from the Prospect Bay Wastewater Treatment Facility on January 07, 1999.
- The sludge was sieved through a 2 mm screen and then aerated The solids concentration of the sludge was measured and adjusted to approximately 2500 mg/L. Approximately 23.5 h after the start of aeration the air flow was stopped and the solids were allowed to settle for approximately 30 minutes. After the settling period the supernatant from above the settled solids was drained and the volume was replaced with 990 mL of tap water and 10 mL of synthetic sewage for each liter of effluent removed. Aeration of the sludge was continued after the addition of the influent. This cycle was repeated at intervals of approximately 23.5 h for five days to allow the sludge to become acclimated to laboratory conditions.
Five days after collection the sludge was settled for approximately 30 minutes and the effluent above the settled solids removed. The effluent was clear and had a measured DOC concentration of 6.5 mg C/L.
Five days after collection, the sludge was settled and the effluent drained. The total suspended solids (TSS) concentration of the settled was measured. Based on the average measured TSS concentration, the sludge was distributed among the test vessels so that the TSS concentration was approximately 2500 mg/L in a total test volume of 1.5 L.
Duration of test (contact time):
28 d
Initial conc.:
20 other: mg Carbon/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Synthetic sewage was used as a source of nutrients that are required for bacterial metabolism.
- Test temperature: 19-22 °C

EXPERIMENTAL DESIGN:
The test contained one control and one treatment group each containing two replicate SCAS test vessels. The control group was used to measure the residual DOC concentration in the activated sludge supernatant that was not exposed to the test substance. The two test vessels within the treatment group were dosed daily at 20 mg C/L. Synthetic sewage was added to all vessels to provide the necessary nutrients required for bacterial metabolism. A DOC analysis was performed on a sample of the effluent from each of the SCAS test vessels three times per week over a 4-week test period. The DOC values were used to determine the percent carbon removed from the test system.

TEST PROCEDURE:
At the experimental start, apply 0.423 L of settled sludge solids, 0.0247 g of test substance, 10 mL of synthetic sewage and adequate tap water to achieve a total volume of 1.5 L were combined in each of the treatment group vessels. Approximately 0.423 L of settled sludge solids, 10 mL of synthetic sewage and adequate tap water to achieve a total volume of 1.5 L were combined in each of the control group vessels. The resulting TSS concentration in the test and control vessels was approximately 2500 mg/L in a total volume of 1.5 L. The sludge within the vessels was aerated overnight (approximately 23 h) at a rate adequate to hold solids in suspension. At the end of the aeration period, the air flow was stopped and the sludge was allowed to settle for approximately 30 minutes. One liter of effluent above the settled solids was drained from a port located at the 500 mL mark. 0.0247 g of test substance, 10 mL of synthetic sewage and adequate tap water to achieve a total volume of 1.5 L were added to each of the vessels in the treatment group and 10 mL of synthetic sewage and adequate tap water to achieve a total volume of 1.5 L was added to each of the vessels in the control group. The sludge then was aerated for approximately 23.5 h at an air flow rate adequate to hold solids in suspension. This cycle was repeated for 28 days.

EFFLUENT ANALYSIS:
A sample of the effluent was collected from each of the treatment and control group test vessels at the end of the aeration period on days 1, 3, 6, 8, 10, 13, 15, 17, 20, 22, 24, and 27. The samples were analysed for DOC using a Shimadzu Model 5000 carbon analyzer.
Reference substance:
not specified
Preliminary study:
None
Test performance:
None
Key result
Parameter:
% degradation (DOC removal)
Remarks:
average percent removal
Value:
99.8
Sampling time:
28 d
Details on results:
The mean measured DOC concentration in the effluent from the control and treatment sludges during this phase of the study ranged from 5.3 to 7.5 mg C/L and 5.25 to 8.3 mg C/L, respectively. Carbon removal over the test period ranged from 96.0 to 101.8% with an average removal of 99.8%.
Results with reference substance:
None

Table 5.2.1/1: Cumulative Percent of Theoretical Carbon Dioxide Evolved

 

Day of test

Control Rep. 1

Control Rep. 2

Mean control DOC

ST 23 C 97 Rep. 1

ST 23 C 97 Rep. 2

Mean ST 23 C 97 DOC

Percent removal

1

6.3

6.2

6.3

6.5

6.3

6.4

99.5

3

7.2

6.9

7.1

6.7

6.7

6.7

101.8

6

6.9

6.8

6.9

6.8

6.7

6.8

100.5

8

6.7

6.5

6.6

6.6

6.4

6.5

100.5

10

6.2

6.1

6.2

6.0

6.5

6.3

99.5

13

6.3

6.0

6.2

6.0

6.3

6.2

100.0

15

5.6

5.6

5.6

5.8

5.8

5.8

99.0

17

5.8

5.4

5.6

5.5

5.5

5.5

100.5

20

7.6

7.4

7.5

7.9

8.7

8.3

96.0

22

5.5

5.4

5.5

5.3

5.6

5.5

100.0

24

5.4

5.2

5.3

5.4

5.3

5.4

99.5

27

5.3

5.4

5.4

5.2

5.3

5.3

100.5

Validity criteria fulfilled:
not specified
Interpretation of results:
other: Ultimately biodegradable
Conclusions:
Removal of the test substance at 20 mg C/L in activated sludge ranged from 96.0 to 101.8% with an average removal of 99.8% over a 28-day test period. A result of 20% or greater removal in the SCAS test is regarded as evidence of inherent biodegradability, whereas a result greater than 70% is evidence of ultimate biodegradability. Hence, this test substance should be classified as ultimate biodegradable.
Executive summary:

In inherent biodegradability study performed according to OECD Guideline 302 A and GLP, the test substance was tested at concentrations of 20 mg C/L and the inoculum was activated sludge. Percent carbon removal was calculated from effluent DOC analysis over a 28-day test period.

The mean measured DOC concentration in the effluent from the control and treatment sludges during this phase of the study ranged from 5.3 to 7.5 mg C/L and 5.25 to 8.3 mg C/L, respectively. Removal of the test substance at 20 mg C/L in activated sludge ranged from 96.0 to 101.8% with an average removal of 99.8% over a 28-day test period. A result of 20% or greater removal in the SCAS test is regarded as evidence of inherent biodegradability, whereas a result greater than 70% is evidence of ultimate biodegradability.

Hence, this test substance should be classified as ultimate biodegradable.

Description of key information

OECD Guideline 301B, GLP, key study, validity 1:

85.8% biodegradation after 28 days within the 10-day window.

Readily biodegradable.

OECD Guideline 302A, GLP, key study, validity 2:

99.8% biodegradation after 28 days.

Ultimately biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

To assess the biodegradation potential of the registered substance, two key studies are availabe.

The first key biodegradation study (Wildlife International, 1999a) was performed according to OECD Guideline 301 B and GLP to assess the ready biodegradability of the registered substance. In this study, 10 mg C/L of test substance was exposed with activated sludge during 28 days and the degradation of the test substance was assessed by the determination of the CO2 evolution.

The average cumulative percent of theoretical carbon dioxide produced by the test substance was 83.9%, within the 10d window. The toxicity control evolved approximately 85.8 % TCO2 indicating that the test substance was not toxic to the inoculum at the tested concentration. In conclusion, the test substance is considered readily biodegradable under the test conditions.

The second key biodegradation study (Wildlife International, 1999b) was performed according to OECD Guideline 302 A and GLP to assess the inherent biodegradability of the registered substance. In this study, 20 mg C/L of test substance was exposed with activated sludge and the percent carbon removal was calculated from effluent DOC analysis over a 28-day test period.

The mean measured DOC concentration in the effluent from the control and treatment sludges during this phase of the study ranged from 5.3 to 7.5 mg C/L and 5.25 to 8.3 mg C/L, respectively. Removal of the test substance at 20 mg C/L in activated sludge ranged from 96.0 to 101.8% with an average removal of 99.8% over a 28-day test period. A result of 20% or greater removal in the SCAS test is regarded as evidence of inherent biodegradability, whereas a result greater than 70% is evidence of ultimate biodegradability. Hence, the test substance should be classified as ultimate biodegradable.

In conclusion, the registered substance is considered as readily and ultimately biodegradable.