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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:
27th May 1993 to 25th June 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Secondary effluent collected from the secondary effluent composite sampler at the Columbia Wastewater Treatment Facility (Columbia, Missouri).
An aliquot of the secondary effluent supernatant was used to perform a standard plate count and to enumerate the microbial population. Heterotrophic plate counts were determined on day 0 using a pour plate method. Plate count agar was used as plating medium.
Heterotrophic plate counts were performed on the secondary effluent supernatant. A dilution scheme that consisted of 1E-04, 1E-05, 1E-6 and 1E-07 dilutions of the secondary effluent supernatant was used. The plates were prepared and incubated in an environmental chamber at 20 -22 °C. The plate counts indicated that the inoculum contained 5.9 E+05 colony forming units (CFU)/mL.
- Storage conditions: Upon arrival, the scondary effluent was aerated and then allowed to settle for 1 hour. The supernatant was decanted and kept aerated at room temperature until use. The remaining fraction containing the precipitate was discarded.
- Water filtered: yes
- Type and size of filter used, if any: 0.2 µm hollow-fibre filter
Duration of test (contact time):
29 d
Initial conc.:
10 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium:
1 mL/L of FeCl3.6H20 (0.250 g/L)
10 mL/L of MgSO4.7H2O (22.5 g/L)
1 mL/L of CaCl2 (27.5 g/L)
10 mL/L of KH2PO4 (8.5 g/L), K2HPO4 (21.75 g/L), Na2HPO4 (26.639 g/L), NH4Cl (0.5 g/L)
2400 mL of test medium was added to each vessel. 30 mL of inoculum were added.
- Test temperature: 20-22 °C
- Aeration of dilution water: Incoming air was passed through a column containing Ascarite to remove CO2. Air was introduced into each bottle via a glass tube. Flow rates were measured with flow meters.
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 4 L carboys
- Number of culture flasks/concentration: 3 with the test material
- Method used to create aerobic conditions: Air was introduced into each bottle via a glass tube
- Details of trap for CO2: The outlet from each bottle was connected to three CO2 absorber gas washing bottles in series each filled with 100 mL of 0.2 N KOH.

SAMPLING
- Sampling frequency: Days 1, 3, 5, 7, 10, 14, 18, 22, 27 and 29 for CO2. On days 0 and 29, samples were taken from the carboys containing the reference substance and the inoculum blank to determine TOC.
- Sampling method: For each sample day, duplicate aliquots of the KOH solution from the gas-washing bottle nearest the carboy were placed in glass autosampler vials without headspace. The vials were capped with a Teflon-lined cap, sealed with parafilm and stored until analysis. The remaining KOH solution in the gas-washing bottle was discarded and replaced with a fresh bottle. The fresh gas-bottle was placed at the end of the trap series. TOC samples were taken by removing duplicate 7 Ml aliquots from the carboy liquid and filtering using a 0.45 µm nylon syringe filter.
A total organic carbon analyser with automatic sample injector were used for analysing CO2 and TOC (method: combustion (680 °C) and nondispersive infrared detection). CO2 evolution was quantified by measuring the inorganic cabon concentration in the KOH solution traps with the carbon analyser. CO2 analysis was performed by injecting the KOH solution using the inorganic carbon mode. Triplicate injections of each sample were analysed. The average carbon content for each sample was then calculated.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: Yes
- Toxicity control: Yes

STATISTICAL METHODS:
Inorganic carbon concentrations were calculated by the carbon analysed as mg C/L, based on carbon standards. The mg CO2 values were calculated from the mg C/L using the following:
Calculated mg C/L from TOC * 0.1 L volume of gas-washing bottle *(1 mmol CO2/mmol C) * (44 mg CO2/mmol CO2) = mg CO2
For carboys receiving test or reference substance, the net mg CO2 was determined by subtracting the average mg CO2 of the inoculum blank for a given day from the mg CO2 calculated per carboy per day. The net cumulative mg CO2 produced during the study was calculated. Percent theoretical CO2 (%THCO2) was also calculated and determined using the following:
%THCO2 = (net cumulative mg CO2 produced/ThCO2) * 100 %
Where:
ThCO2 = (dosed mg C/L) * (3L/carboy) x (1 mmol C/12.01 mg C) * (1 mmol CO2/mmol C) * (44.01 mg CO2/mmol CO2)
The dosed mg C was determined from the theoretical dosing rate for the test substance additions and the TOC analyses results for the reference substance additions.
Reference substance:
benzoic acid, sodium salt
Remarks:
20 mg C/L
Key result
Parameter:
% degradation (CO2 evolution)
Value:
27.99
Sampling time:
29 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
10.74
Sampling time:
10 d
Details on results:
Over the 29 day test period, the inoculum blank system evolved 15.95 mg of CO2. After correction for the background CO2 levels in the blank KOH. This value was within the acceptable range recommending in the guideline.
The carboys containing the test material and active inoculum exhibited a mean percent ThCO2 of 27.99 ± 2.48 % (SD). The sterile test carboy exhibited no significant CO2 evolution (< 1 % ThCO2).
The CO2 production from the inoculum blank system was subtracted from systems containing test and/or references substance. The reference control reached 60 % ThCO2 by day 3 of the test. This indicated that the inoculum was viable and active.

The termination TOC value for the toxicity control flask was 5.985 mg C/L. The source of this value is unclear. Although the test substance was not soluble in water, it is likely that the TOC in the test medium of this flask was due to the test substance remaining at termination. The high percent ThCO2 value and the fact that most of it was evolved in the first week of the test indicate that the reference substance was consumed rather rapidly and completely. The only other remaining source of carbon was the test substance. The test substance may have emulsified in the test medium to an extent that it was able to pass through the filter use don the TOC sample. It is also possible that the test substance was transformed into a more soluble compound by the action of the inoculum. This would also be supported by the slow and partial degradation observed. It is assumed that the test substance is not toxicity to the inoculum at the concentration tested because the reference substance in the toxicity control carboy did degrade in the presence of the test substance. The four carboys dosed only with the test substance were not tested for TOC because the test substance was insoluble in water.
Results with reference substance:
In the reference control, the evolution of 100.6 % ThCO2 was observed. The toxicity control exhibited 75.98 % ThCO2 evolution. It was estimated that 67 % was attributable to the reference substance and 9 % to the test material.
The day 29 TOC analysis indicated the inoculum blank had no significant carbon remaining in the medium. The reference carboy showed 0.261 mg C/L TOC in the test medium at day 29. The reference value indicates that all of the substance was consumed during the 29 day test.

Table 2: Percent Theoretical CO2 Evolved

Carboy

Day

1

3

5

7

10

14

18

22

27

29 - Trap 1

29 - Trap 2

29 - Trap 3

IB (cumulative mg CO2 evolved)

2.91

4.93

5.64

5.85

8.28

8.57

9.86

11.12

14.86

15.88

15.88

15.95

%ThCO2

R

5.53

61.33

82.76

87.78

92.69

95.61

97.65

99.00

100.10

100.50

100.60

100.60

T-1

0.00

1.52

2.58

5.91

12.55

16.96

20.24

22.68

28.55

29.86

30.40

30.69

T-2

0.00

0.00

0.61

1.12

7.78

11.87

16.39

19.84

22.16

23.44

24.14

24.70

T-3

0.00

1.52

2.65

5.19

11.88

18.77

23.23

24.92

26.31

27.25

28.16

28.57

STC

0.00

0.60

0.60

0.60

0.60

0.60

0.60

0.65

0.65

0.65

0.84

0.84

ToxC

1.91

37.43

48.03

53.04

59.89

64.44

68.87

71.90

74.67

75.79

75.88

75.88

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the conditions of the test, the 60 % threshold value was not attained by the test material during the test period. The test material was therefore classified as not readily biodegradable. An average of 27.99 % of the ThCO2 was evolved by the flasks containing the test material by the end of the test. The results indicate that the substance may biodegrade under different environmental conditions.
Executive summary:

The biodegradation potential of the test substance was investigated in a ready biodegradability study performed in accordance with OECD 301B. Under the conditions of the test, the 60 % threshold value was not attained by the test material during the test period. The test material was therefore classified as not readily biodegradable. An average of 27.99 % of the ThCO2 was evolved by the flasks containing the test material by the end of the test. The results indicate that the substance may biodegrade under different environmental conditions.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14th April 2003 to 30th September 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
other: ASTM D 5864-95: Standard test method for determining aerobic aquatic biodegradation of lubricants of their components.
Version / remarks:
CO2 evolution test
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
Reference substance: Canola oil
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, 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 (Denton, Maryland) on the 5th May 2003.
- Preparation of inoculum for exposure: The sludge was allowed to settle for approximately 30 minutes. The sludge was sieved through a 2 mm screen and then homogenised in a blender at a medium speed for 2 minutes. The sludge was allowed to settle for 30 minutes. 1L aliquot of supernatant was removed and supplemented with 25 mg vitamin free casamino acids and 26 mg yeast extract. A standard plate count was performed on the inoculum prior to adaptation. Plates were incubated at approximately 21 °C for approximately 48 hours.
The inoculum used in the study was composed of five different adaptation cultures. The cultures were maintained concurrently and were prepared as follows. For each treatment group, approximately 100 mL of supplemented inoculum was combined with approximately 900 mL of test medium within each 2 L Erlenmeyer flask. The solutions were continuously aerated with CO2 free air. The test substances were incrementally added at concentrations equivalent to 4, 8 and 8 mg C/L on days 0, 7 and 11, respectively. On day 14, an equal volume of each culture was combined o make composite inoculum. The composite inoculum was screened with glass wool and then homogenised in a blender at medium speed for approximately 2 minutes. A standard plate count was performed on the inoculum. Plates were incubated for 48 hours.
- Initial cell/biomass concentration: The standard plate count during the 14-day adaptation period was 8.7E+05 CFU/mL. The result of the standard plate count performed on the composite inoculum after the adaptation period was 5.7E+03 CFU/mL.
- Water filtered: yes
Duration of test (contact time):
29 d
Initial conc.:
10 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
CO2 evolution
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 of ferr4ic chloride solution (0.025 %)
3 mL of magnesium sulfate solution (2.25 %)
30 mL of phosphate buffer (pH 7.2)
30 mL of composite inoculum
- Test temperature: The test temperature range was recorded between 20 and 22 °C, within the range specified in the protocol.
- pH: The pH of the test chamber was determined to be 5.8 during the study.
-- Aeration of dilution water: All test chambers were aerated with CO2 free air for 24 hours at a rate of 50-100 mL/minute.

TEST SYSTEM
- Culturing apparatus: 4 L amber glass vessels
- Number of culture flasks/concentration: Triplicate
- Measuring equipment: Carbon analyser measuring the K2CO3 in the traps
- Details of trap for CO2: CO2 traps containing KOH

SAMPLING
- Sampling frequency: The traps were removed for analysis on days 1, 7, 10, 13, 15, 20, 22 and 27.
- Sampling method: The CO2 trap nearest the test chamber was removed and analysed for inorganic carbon. The two remaining traps were placed on position closer to the test chamber and new trap was placed at the end of the series. On the 28th day of the test, the contents of each test chamber was removed and the pH determined. The contents of each chamber were then acidified by adding 1 mL of concentrated HCl to drive off inorganic carbonate. The chambers were aerated overnight and the trapping solutions closes to the test chambers analysed for inorganic carbon.


CONTROL AND BLANK SYSTEM
- Inoculum blank: A single flask served as the inoculum blank control
STATISTICAL METHODS:
The results of the inorganic carbon analyses of the CO2 traps were converted to mg CO2 produced using the following equation:
mgCO2 = result(mg C/L) X vol. of KOH (L) X 3.67 mg CO2/mg C
The cumulative mg of CO2 for the test and reference substances were corrected for the amount of CO2 eveolved by the control, using the following equation:
Cumulative mg CO2 evolved = Σ mg CO2 test – mean Σ mg CO2 control
The percentage of theoretical carbon dioxide (%TCO2) evolved was calculated as follows:
%TCO2 = ((mg CO2 produced)/(mg of carbon in test) (3.67 mg CO2/mg Carbon)) X 100
Reference substance:
other: canola oil
Test performance:
The control chambers evolved a minimal amount of CO2 over the test period. This was corrected for the amount of CO2 in the traps to account for carbonates expected to be present in the potassium hydroxide solutions. The amount of CO2 evolved by the control chambers did not exceed 50 mg/3 L value considered to be the acceptable limit of CO2 evolution tests.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
29.8
St. dev.:
5.7
Sampling time:
28 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
10.2
St. dev.:
3.7
Sampling time:
10 d
Results with reference substance:
The vaibility of the inoculum and validity of the test were supported by the reference substance. 82.3 % degradation was observed with the reference substance at the end of the test period.

Table 1: Inorganic Carbon Present in the CO2 Traps (mg C/L)

Day

Control - A

Control - B

Control - C

Reference Canola - A

Reference Canola - B

Reference Canola - C

Test Material - A

Test Material -

Test material - C

Blank

1

5.8

5.7

4.6*

5.3

7.0

6.3

5.5

5.1

5.4

5.3

7

8.2

13.0

8.9

60.9

116.5

98.8

22.1

31.2

37.6

4.8*

10

8.6

11.1

8.4

69.0

70.0

76.7

16.3

19.6

22.9

5.6

13

9.1

10.0

7.2

65.0

55.8

55.7

8.7

6.7

26.4

16.6

15

9.5

9.4

7.5

37.0

26.8

24.2

24.2

11.7

18.3

15.8

20

9.3

12.0

7.7

46.3

29.2

25.8

51.9

40.9

31.3

6.7

22

8.2

10.8

8.3

11.7

7.4

11.0

8.2

6.9

13.3

13.2

27

10.9

14.3

7.8

14.4

20.8

13.2

19.1

25.8

20.2

8.1

29

11.7

11.5

10.2

10.8

14.1

10.8

9.5

12.3

16.5

6.7

* Results are less than lowest calibration standard.

 

Table 2: Cumulative amount of CO2 Evolved (mg)

Day

Control - A

Control - B

Control - C

Reference Canola - A

Reference Canola - B

Reference Canola - C

Test Material - A

Test Material -

Test material - C

Control - A²

Control - B²

Control - C²

Blank

1

2.1

2.1

1.7

0.0

0.6

0.3

0.0

-0.1

0.0

0.2

0.1

-0.3

1.9

7

5.1

6.9

5.0

18.6

39.7

32.9

4.5

7.7

10.1

1.4

3.2

1.2

3.7

10

8.3

10.9

8.0

40.5

61.9

57.6

7.0

11.4

15.1

2.5

5.2

2.3

5.8

13

11.6

14.6

10.7

61.2

79.2

74.9

7.0

10.7

21.6

-0.2

2.8

-1.2

11.9

15

15.1

18.1

13.4

71.5

85.8

80.5

12.6

11.7

25.1

-2.5

0.4

-4.2

17.7

20

18.5

22.5

16.3

85.0

93.0

86.4

28.1

23.2

33.0

-1.6

2.3

-3.9

20.1

22

21.5

26.4

19.3

85.9

92.3

87.1

27.8

22.4

34.5

-3.4

1.5

-5.7

25.0

27

25.5

31.7

22.2

87.2

95.9

87.9

30.8

27.8

37.9

-2.4

3.7

-5.8

27.9

29

29.8

35.9

25.9

87.0

97.0

87.8

30.2

28.2

39.9

-0.6

5.5

-4.5

30.4

* Corrected for the average amount of CO2 evolved by control to correct for the amount of CO2 attributed to the inoculum and the KOH
² Corrected for the amount of CO2 in the blank to determine the amount of CO2 evolved by the inoculum.

 

Table 3:

Day

Control - A

Control - B

Control - C

Reference Canola - A

Reference Canola - B

Reference Canola - C

Test Material - A

Test Material -

Test material - C

1

NA

NA

NA

0.0

0.5

0.3

0.0

-0.1

0.0

7

NA

NA

NA

16.9

36.0

29.9

4.1

7.0

9.2

10

NA

NA

NA

36.8

56.2

52.3

6.4

10.4

13.7

13

NA

NA

NA

55.6

71.9

68.0

6.4

9.7

19.6

15

NA

NA

NA

65.0

77.9

73.1

11.5

10.7

22.8

20

NA

NA

NA

77.2

84.4

78.5

25.6

21.1

30.0

22

NA

NA

NA

78.0

83.9

79.1

25.3

20.3

31.4

27

NA

NA

NA

79.2

87.1

79.9

28.0

25.3

34.4

29

NA

NA

NA

79.1

88.1

79.8

27.4

25.7

36.2

Average

82.3

29.8

Standard Deviation

5.0

5.7

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the conditions of the test, the test material acheived 29.8 % degradation on day 28 of the test. The test substance is therefore considered to be not readily biodegradable under the conditions of the test, however this doesn't necessarily mean that the substance will not biodegrade under relevant environmental conditions.
Executive summary:

The biodegradation of the test material was evaluated in a study conducted in accordance ASTM D 5864-95: Standard test method for determining aerobic aquatic biodegradation of lubricants of their components. Under the conditions of the test, the test material acheived 29.8 % degradation on day 28 of the test. The test substance is therefore considered to be not readily biodegradable under the conditions of the test, however this doesn't necessarily mean that the substance will not biodegrade under relevant environmental conditions.

Description of key information

The biodegradation potential of the test material was evaluated in two carbon dioxide evolution studies (OECD 301B). The test material acheived 29.8 and 27.99 % biodegradation in the two studies, respectively. It was therefore concluded that the test material does not meet the criteria for readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information