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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:
30 June 2010 - 29 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Qualifier:
according to guideline
Guideline:
ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Pretreatment: The freshly obtained sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was 3.8 g/L in the concentrated sludge (information obtained from the municipal sewage treatment plant). Before use, the sludge was allowed to settle (48 minutes) and the liquid was decanted for use as inoculum at the amount of 10 mL/L of mineral medium.
- Concentration of sludge: 3.8 g/L
- Water filtered: no
Duration of test (contact time):
29 d
Initial conc.:
12 mg/L
Based on:
other: Total Organic Carbon (TOC)
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: 1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli-RO water
Stock solutions of mineral components
A) 8.50 g KH2PO4; 21.75 g K2HPO4; 67.20 g Na2HPO4.12H2O; 0.50 g NH4Cl; dissolved in Milli-Q water and made up to 1 litre, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli-Q water and made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli-Q water and made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli-Q water and made up to 1 litre.

- Test temperature: between 21.6 and 22.5 °C.
- pH:
At t=0 d: 7.5-7.6
At t=28 d: 7.7-8.0
- pH adjusted: no
- Aeration of dilution water: Not before the test, the test is aerated continously.
- The concentration of suspended solids was 3.8 g/L in the concentrated sludge (information obtained from the municipal sewage treatment plant). Before use, the sludge was allowed to settle (48 minutes) and the liquid was decanted for use as inoculum at the amount of 10 mL/L of mineral medium.
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 2 litre all-glass brown coloured bottles
- Number of culture flasks/concentration:
Test suspension: containing test substance and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Positive control: containing reference substance and inoculum (1 bottle).
Toxicity control: containing test substance, reference substance and inoculum (1 bottle).
- Method used to create aerobic conditions:
Synthetic air (a mixture of oxygen (ca. 20%) and nitrogen (ca. 80%)) was sparged through the solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
- Test performed in open system: yes
- Details of trap for CO2 and volatile organics if used:
CO2 was trapped in barium hydroxide solution. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampul). Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day, for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made at least 14 days.

SAMPLING
- Sampling frequency: Titration were made on day: 2, 5, 7, 9, 14, 19, 23, 28 and 29
- Sampling method: Titration of the whole volume of CO2-absorber

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes
Reference substance:
acetic acid, sodium salt
Parameter:
% degradation (CO2 evolution)
Value:
70
Sampling time:
29 d
Remarks on result:
other: Bottle A: HCl added on the 28th day (last CO2-measurement on the 29th day)
Parameter:
% degradation (CO2 evolution)
Value:
75
Sampling time:
29 d
Remarks on result:
other: Bottle B: HCl added on the 28th day (last CO2-measurement on the 29th day)
Details on results:
In the toxicity control more than 25% biodegradation occurred within 14 days (61%, based on ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity.
Results with reference substance:
The positive control substance was biodegraded by 82% within 14 days.

The relative biodegradation values calculated from the measurements performed during the test period revealed 70 and 75% biodegradation after 29 days of Vinasses but missed the 10-day window. According to “OECD Guidelines for the testing of chemicals – Revised introduction to the OECD guidelines for testing of chemicals, Section 3 – Part 1: Principles and strategies related to the testing of degradation of organic chemicals degradation” published by OECD (2006) the ready biodegradability data for mixtures of structurally similar chemicals should be evaluated carefully. For these substances the 10-day window should not be used for the interpretation of results. It is possible that a sequential biodegradation took place which might have led to a prolonged lag-phase and therefore a slower biodegradation period although the test substance was biodegraded in a large amount after the duration of the test. In this case the pass level can be applied after test termination. Thus, Vianasses is considered to be readily biodegradable.

In the toxicity control more than 25% biodegradation occurred within 14 days (61%, based on ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity. The reference substance was degraded to 82% after 14 days.

Table 1: CO2 production and percentage biodegradation of the test substance (bottle A)

Day

HCl (0.05 N) titrated (mL)

Produced CO2

(mL HCl)

Produced CO2

(mg)

Cumulative CO2

(mg)

Biodegradation1)

(%)

Blank (mean)

bottle A

2

45.84

38.35

7.49

8.2

8.2

9

5

46.50

28.51

17.99

19.8

28.0

32

7

44.51

36.23

8.28

9.1

37.1

42

9

46.43

41.29

5.14

5.6

42.8

49

14

45.76

39.83

5.93

6.5

49.3

56

19

46.56

40.71

5.85

6.4

55.7

64

23

44.90

43.54

1.36

1.5

57.2

65

28

45.20

43.26

1.94

2.1

59.4

68

29

44.81

44.72

0.09

0.1

59.5

68

29

47.17

46.95

0.22

0.2

59.7

68

29

48.36

47.15

1.21

1.3

61.0

70

1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2 of the test substance: 87.6 mg CO2/2 L

Table 2: CO2 production and percentage biodegradation of the test substance (bottle B)

Day

HCl (0.05 N) titrated (mL)

Produced CO2

(ml HCl)

Produced CO2

(mg)

Cumulative CO2

(mg)

Biodegradation1)

(%)

Blank (mean)

bottle B

2

45.84

40.55

5.29

5.8

5.8

7

5

46.50

27.63

18.87

20.8

26.6

30

7

44.51

35.14

9.37

10.3

36.9

42

9

46.43

41.63

4.79

5.3

42.1

48

14

45.76

40.35

5.41

6.0

48.1

55

19

46.56

39.20

7.36

8.1

56.2

64

23

44.90

42.55

2.35

2.6

58.8

67

28

45.20

41.33

3.87

4.3

63.0

72

29

44.81

44.14

0.67

0.7

63.8

73

29

47.17

46.54

0.63

0.7

64.5

74

29

48.36

46.90

1.46

1.6

66.1

75

1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test substance: 87.6 mg CO2/2 L

 

Table 3: CO2 production and percentage biodegradation of the positive control substance

Day

HCl (0.05 N) titrated (mL)

Produced CO2

(ml HCl)

Produced CO2

(mg)

Cumulative CO2

(mg)

Biodegradation1)

(%)

Blank (mean)

Positive control

2

45.84

41.69

4.15

4.6

4.6

5

5

46.50

16.08

30.42

33.5

38.0

44

7

44.51

28.86

15.65

17.2

55.2

64

9

46.43

37.69

8.74

9.6

64.8

76

14

45.76

40.51

5.25

5.8

70.6

82

1): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of sodium acetate: 85.7 mg CO2/2 L

 

Table 4: CO2 production and percentage biodegradation of the toxicity control

Day

HCl (0.05 N) titrated (mL)

Produced CO2

(ml HCl)

Produced CO2

(mg)

Cumulative CO2

(mg)

Biodegradation1)

(%)

Blank (mean)

toxicity control

2

45.84

28.60

17.24

19.0

19.0

11

5

46.50

10.05

36.45

40.1

59.0

34

7

44.51

21.55

22.96

25.3

84.3

49

9

46.43

34.44

11.99

13.2

97.5

56

14

45.76

37.74

8.02

8.8

106.3

61

1): Calculated as the ratio between CO2 produced (cumulative) and the sum of the ThCO2 of the test substance

and positive control: 173.2 mg CO2/2 L (ThCO2test substance: 87.6 mg CO2/2 L + ThCO2sodium acetate: 85.7 mg CO2/2 L)

 

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Biodegradation testing revealed 70 and 75% degradation after 29 days but missed the 10-day window. Because the substance is a UVCB the 10-day window criterion should not be applied according to “OECD Guidelines for the testing of chemicals – Revised introduction to the OECD guidelines for testing of chemicals, Section 3 – Part 1: Principles and strategies related to the testing of degradation of organic chemicals degradation” published by OECD (2006). The biodegradation rate at the end of the test should be used for the assessment. Thus, Vianasses is readily biodegradable.

Description of key information

Vinasses, residue of fermentation is readily biodegradable. Therefore, based on read-across by grouping of substances, the other members of the category are considered to be readily biodegradable as well.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

The biodegradation of Vinasses, residue of fermentation was tested according to the OECD guideline 301B “Ready Biodegradability: CO2 Evolution Test”. Activated sludge (domestic, non-adapted) was used as inoculum and the test substance was applied at a concentration of 12 mg/L total organic carbon. A biodegradation of 70 – 75% was observed after 29 days without fulfilling the 10-day window criterion. But since Vinasses, residue of fermentation is a UVCB the 10-day window should not be used for interpretation of biodegradability test results of UVCBs according to “OECD Guidelines for the testing of chemicals – Revised introduction to the OECD guidelines for testing of chemicals, Section 3 – Part 1: Principles and strategies related to the testing of degradation of organic chemicals degradation” published by OECD (2006). It is possible that a sequential biodegradation took place which might have led to a prolonged lag-phase and therefore to a slower biodegradation period although the test substance was biodegraded in a large amount during the test. In this case the pass level can be applied after test termination. Therefore, Vinasses, residue of fermentation is considered to be readily biodegradable.

OECD (2006) Guidelines for the testing of chemicals – Revised introduction to the OECD guidelines for testing of chemicals, Section 3 – Part 1: Principles and strategies related to the testing of degradation of organic chemicals degradation.