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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 October 2012 to 30 November 2012
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
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: activated sludge used for this study was obtained from the Wareham Waste Water Treatment Plant, Wareham, Massachusetts, which receives primarily domestic waste
- Preparation of inoculum for exposure: the sludge was passed through a 2 mm sieve and centrifuged at 1000 rpm for 10 minutes. The supernatant was discarded, the sludge was washed with 1000 mL mineral medium and the contents were centrifuged at least once again and the supernatant was discarded. A 15 mg solids/mL inoculum solution was prepared (3.0 g dry weight sludge brought to 200 mL with mineral media) and aerated until used. The test substance flasks, the blank flasks, the procedural control flask and the toxicity control flask all received 6.0 mL of the inoculum to produce an activated sludge concentration of 30 mg/L.

In addition, a 50-g aliquot of fresh soil, a 25-g aliquot of Weweantic River sediment and a 25-g of Taunton River sediment were collected near Smithers Viscient. The soil and sediment were suspended in 1.0 L of Weweantic River water. The suspension was filtered through glass wool prior to use. A 3.0 mL aliquot of the soil/sediment filtrate was added to each test vessel containing 3-L of mineral medium and 6.0 mL of activated sludge.
Duration of test (contact time):
28 d
Initial conc.:
9.5 other: mg C/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium:
Substance Final concentration in test solution
KH2PO4 8.50 g//L
K2HPO4 21.75 g/L
Na2HPO4 33.40 g/L
NH4Cl 0.50 g/L
MgSO4.7H2O 22.50 g/L
CaCl2 27.50 g/L
FeCl3.6H2O 0.25 g/L


- Test temperature: 20.4 - 22.2 °C
- pH: 7.37 - 7.48
- pH adjusted: no
- Aeration of dilution water: yes
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 4 L glass bottles
- Number of culture flasks/concentration: two for the test material, two inoculum blanks, one sodium benzoate control and one toxicity control
- Method used to create aerobic conditions: all test systems were aerated continuously under positive pressure using CO2-free air
- Measuring equipment: KOH carbon dioxide trap. The amount of evolved CO2 in each trap was determined using a Shimadzu TOC V-CPH Carbon Analyzer.
- Test performed in closed vessels: yes. All vessels were sealed with rubber stoppers
- Details of trap for CO2 and volatile organics if used: The outlet port of each system was connected to two CO2 efluent gas traps, the first consisting of 200 mL of 0.2-N potassium hydroxide (KOH) and the second trap containing 100 mL of 0.2 N KOH

SAMPLING
- Sampling frequency: on test days 1, 3, 7, 9, 12, 14, 19, 23 and 28, a 7-mL sample was removed from the first KOH carbon dioxide trap on each test system and analysed for CO2 evolution

ANALYSIS AND CALCULATIONS
The Shimadzu TOC-V-CPH Carbon Analyzer was used to analyse the samples removed from the KOH traps. Total inorganic carbon (TIC) values were compared to a standard curve for at least five concentrations of sodium bicarbonate in order to determine the quantity of CO2 evolved.
The amount of carbon dioxide evolved from each test system was adjusted by subtracting the mean CO2 production value from the blank controls. The percent biodegradability for each test system was calculated using the following equation and is expressed as cumulative percent biodegradation (or percent of theoretical CO2 production).

% Ultimate biodegradability = (mg CO2 produced / mg TOC added x 3.67) x 100

where 3.67 is the molecular weight conversion factor for carbon to carbon dioxide

CONTROL AND BLANK SYSTEM
- Blank controls: containing inoculum
- Procedural control: containing inoculum and reference substance
- Toxicity control: containing inoculum, reference and test substances
Reference substance:
benzoic acid, sodium salt
Test performance:
The cumulative net CO2 evolved from the sodium benzoate procedural control was 67.48% of theoretical by day 7, thus exceeding the “pass” criteria of the test (reaching 60% or greater CO2 evolution within a 10-day window of reaching 10% biodegradation). This rapid biodegradation of sodium benzoate confirmed the presence of an active microbial population and system integrity.
Parameter:
% degradation (CO2 evolution)
Value:
101.63
St. dev.:
3.41
Sampling time:
28 d
Details on results:
The mean cumulative CO2 from the test material, procedural control, toxicity control and blank control test vessels was 53.28, 44.49, 77.56 and 17.88 mg/L, respectively, at day 28. The cumulative net percent CO2 production, or percent ultimate biodegradation, for the test material, procedural control, and toxicity control was calculated to be 101.63, 72.56 and 87.97 %, respectively. Greater than 60 % generation of CO2 occurred in the flasks containing test material within 10 day window of reaching 10 % biodegradation, therefore meeting the criteria for ‘ready biodegradability’. The cumulative net CO2 evolved from the toxicity control was 30.71 % on day 3 and therefore the test material was not considered toxic to the inoculums.
Results with reference substance:
More than 60 % degradation was achieved within the 10 day window as expected for a biodegradable substance, confirming the activated sludge contained viable organisms. 67.48 % degredation was achieved by day 7, with 72.56 % degradation by day 28.

Table 2: Cumulative CO2(mg/L) evolved from the test vessels

Vessel

Substance

Day

1

3

7

9

12

14

19

23

28

1

Test material

3.83

11.08

34.2

36.43

39.63

37.51

39.61

45.8

52.44

2

Test material

3.65

10.91

33.32

36.65

39.05

38.45

39.46

45.71

54.12

Mean

3.74

10.99

33.76

36.54

39.34

37.98

39.54

45.76

53.28

Std. Dev.

0.12

0.12

0.62

0.15

0.41

0.67

0.1

0.06

1.19

5

Procedural Control

8.11

21.1

30.02

30.34

32.21

30.78

33.28

38.53

44.49

3

BLANK

3.4

5.05

8.26

9.12

10.82

10.38

11.8

14.38

17.46

4

BLANK

2.95

4.34

7.24

8.36

9.92

9.03

12.37

14.45

18.3

Mean

3.17

4.69

7.75

8.74

10.37

9.71

12.08

14.42

17.88

Std. Dev.

0.32

0.5

0.72

0.54

0.64

0.95

0.41

0.05

0.59

6

Toxicity Control

8.41

25.53

52.18

59.77

60

57.65

62.2

65

77.56

Table 3: Cumulative net percent CO2 evolved (ultimate biodegradation)

Cumulative CO2 Evolved (mg/L)

Vessel

Substance

Day

Day

Day

Day

Day

Day

Day

Day

Day

Day

0

1

9

12

14

19

23

28 

1

Test Material

0

1.88

18.33

75.92

79.49

83.99

79.79

79.02

90.08

99.22

2

Test Material

0

1.38

17.85

73.41

80.09

82.31

82.51

78.59

89.83

104.04

4

Mean

0

1.63

18.09

74.66

79.79

83.15

81.15

78.8

89.95

101.63

Std. Dev.

-

0.35

0.34

1.78

0.43

1.19

1.92

0.3

0.18

3.41

5

Procedural Control

0

14.98

49.72

67.48

65.43

66.17

63.86

64.22

73.06

72.56

 6

Toxicity Control

0

7.72

30.71

65.5

75.22

73.16

70.67

73.88

74.56

87.97

          

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the conditions of the study the test material was determined to be ‘readily biodegradable’ (reaching 60 % or greater CO2 evolution within a 10-day window of exceeding 10 % biodegradation).
Executive summary:

The ready biodegradability of the test material was determined in a GLP study which was conducted in accordance with the standardised guideline OCED 301 B in a CO2 evolution test.

The mean cumulative net percent CO2 evolved (percent biodegradation) from the aqueous test medium fortified with test material at 10 mg C/L was 101.63 % on day 28. The toxicity control on day 3 was 28.41 %, which indicates that the test material was not toxic to the inoculum in the test medium.

The cumulative net percent CO2 evolved from the sodium benzoate procedural control was 67.48 % of theoretical by day 7, thus meeting the “pass” criteria of the test (reaching 60 % or greater CO2 evolution within a 10-day window of reaching 10 % biodegradation). This rapid biodegradation of sodium benzoate confirmed the presence of an active microbial population and system integrity.

Under the conditions of the study the test material was determined to be ‘readily biodegradable’ (reaching 60 % or greater CO2 evolution within a 10-day window of exceeding 10 % biodegradation).

Description of key information

Study conducted to recognised testing guideline with GLP certification.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

The ready biodegradability of the test material was determined in a GLP study which was conducted in accordance with the standardised guideline OCED 301 B in a CO2 evolution test.

 

The mean cumulative net percent CO2 evolved (percent biodegradation) from the aqueous test medium fortified with test material at 10 mg C/L was 101.63 % on day 28. The toxicity control on day 3 was 28.41 %, which indicates that the test material was not toxic to the inoculum in the test medium.

 

The cumulative net percent CO2 evolved from the sodium benzoate procedural control was 67.48 % of theoretical by day 7, thus meeting the “pass” criteria of the test (reaching 60 % or greater CO2 evolution within a 10-day window of reaching 10 % biodegradation). This rapid biodegradation of sodium benzoate confirmed the presence of an active microbial population and system integrity.

Under the conditions of the study the test material was determined to be ‘readily biodegradable’ (reaching 60 % or greater CO2 evolution within a 10-day window of exceeding 10 % biodegradation).

The study was performed in line with GLP and an accepted standardised guideline with a high standard of reporting. The study was assigned a reliability score of 1 and considered suitable for assessment as an accurate reflection of the substance.

The available data are considered to be complete and the conclusion, readily biodegradable, was taken forward for risk assessment.

No further degradation studies are required.

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