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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:
From 2018-01-17 to 2018-04-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study is performed according to the OECD 301F guideline.
Justification for type of information:
Details on study plan is provided in this study record.
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
None
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge, microorganisms from a domestic waste water treatment plant will be supplied by a municipal sewage treatment plant (e.g. Rossdorf, Germany).
- Conditioning:The aerobic activated sludge used for this study was deposited for 15 min, washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in test water and centrifuged again. This procedure was done three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water and aerated overnight. This suspension was used for the experiment.
The washed activated sludge will be pre-conditioned in test water for a maximum of 2 days.
Duration of test (contact time):
>= 28 - <= 60 d
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Surrounding Type: Climatic chamber
- Temperature: 22 °C ± 1°C
- Light Conditions: Darkness
- pH-Value: 6.5 to 6.6 (measured at the start of the test and adjusted to 7.4 to 7.6 with Sodium Hydroxide) 7.4 to 7.8 (measured at day 63)
- Recording: Test conditions (temperature) will be recorded continuously with suitable instruments, documented in the raw data and reported in the final report. (Short-term deviations (< 2 hours) from the recommended temperature range will normally not result in major disturbances of the test performance and will not be recorded).

TEST WATER
- Reconstitued pure water: Analytical grade salts were added to pure water to prepare the following stock solutions:
a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2 H2O, 0.5 g NH4Cl filled up with pure water to 1000 mL volume; the pH should be 7.4 ± 0.2.
b) 22.5 g MgSO4 x 7 H2O filled up with pure water to 1000 mL volume
c) 36.4 g CaCl2 x 2 H2O filled up with pure water to 1000 mL volume
d) 0.25 g FeCl3 x 6 H2O filled up with pure water to 1000 mL volume
In order to avoid precipitation of iron hydroxide in the stock solution d), one drop of concentrated HCl per litre was added before storage.
50 mL of stock solution a) and 5 mL of the stock solutions b) to d) were combined and filled up to a final volume of 5000 mL with pure water.

COURSE OF THE TEST
- Preparation of Test Flasks: The amounts of test item and reference item were directly weighed into the test flasks.
For a better distribution of the test item, silicone oil AR 20 at a concentration of about 1% of the final flask volume was be added to all flasks in order to improve the bioavailability of the mixture constituents.
- The closed test flasks were incubated in a climatised chamber under continuous stirring. The consumption of oxygen was determined by measuring the change of pressure in the flasks. Evolved carbon dioxide was absorbed in an aqueous solution (45%) of potassium hydroxide.
- Test Duration: 60 days (pH was measured at day 63)

TEST PARAMETERS
- Measurements of Oxygen: The change of pressure in the test flasks was measured by means of a manometric method (BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany) each day.
- Temperature: The temperature was recorded by means of the automated software AMR Wincontrol©.
- pH-Value: pH-values were measured in procedure control, a separately prepared test flask with test item (to prevent loss of test item in the test flasks) and a separately prepared test flask without test item (control) at test start and in all flasks at day 63, except in the abiotic and toxicity control, using a pH-electrode WTW pH 340i.

TEST SYSTEM
- The closed test flasks were incubated in a climatised chamber under continuous stirring. The consumption of oxygen was determined by measuring the change of pressure in the flasks. Evolved carbon dioxide was absorbed in an aqueous solution (45%) of potassium hydroxide.
- Number of culture flasks/concentration: 2
The test flasks were closed gas-tight by a measuring head. An appropriate CO2-absorber (KOH as an aqueous solution) was used for trapping the produced carbon dioxide. The amount of O2 consumed by the activated sludge was calculated from the decrease of pressure in the reaction vessel.

SAMPLING
O2 consumed daily by the activated sludge was calculated from the decrease of pressure in the reaction vessel.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 (activated sludge + silicone oil + test water)
- Abiotic sterile control: 1
- Toxicity control: 1
- Other: Procedure control : (Ref. Item + activated sludge + silicone oil + test water)
Reference substance:
benzoic acid, sodium salt
Remarks:
Sodium Benzoate
Preliminary study:
No data.
Test performance:
No data.
Parameter:
% degradation (O2 consumption)
Remarks:
ThODNH4/ThODNO3
Value:
10
Sampling time:
1 d
Parameter:
% degradation (O2 consumption)
Remarks:
ThODNO3
Value:
60
Sampling time:
11 d
Parameter:
% degradation (O2 consumption)
Remarks:
ThODNH4
Value:
61
Sampling time:
11 d
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
ThODNO3
Value:
70
Sampling time:
28 d
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
ThODNH4
Value:
72
Sampling time:
28 d
Details on results:
The mean biodegradation of 10% of Fenugreek Absolute was reached at day 1 (ThODNH4, ThODNO3). At the end of the 10-day window at day 11, the mean degradation of Fenugreek Absolute was 61% (ThODNH4) and 60% (ThODNO3) and therefore the 10 day window criterion was passed. The mean biodegradation after 28 days was 72% (ThODNH4) and 70% (ThODNO3), and at test end after 60 days 82% (ThODNH4) and 80% (ThODNO3).
Therefore, Fenugreek Absolute is considered to be readily biodegradable based on ThODNH4 and ThODNO3.
Results with reference substance:
The reference item sodium benzoate was sufficiently degraded to 85% after 14 days, to 89% after 28 days and to 103 % after 60 days of incubation.

pH-Values at the End of the Test

Flask No.

Treatment

pH-value

1

Fenugreek Absolute

7.4

2

Fenugreek Absolute

7.6

3

Inoculum control

7.5

4

Inoculum control

7.5

5

Reference item (procedure control)

7.8

Cumulative Biochemical Oxygen Demand (mg O2/L) in Test Flasks during the Test Period of 60 Days

Time

Flask No.

(days)

1

2

3

4

5

6

7

 

1

30

30

0

0

60

0

55

 

2

55

55

5

0

85

0

95

 

3

80

75

5

5

105

0

125

 

4

95

100

5

5

120

10

155

 

5

110

115

5

5

130

20

200

 

6

120

130

10

5

135

25

210

 

7

130

140

10

10

140

30

225

 

8

135

150

10

10

145

30

230

 

9

145

160

10

10

150

35

240

 

10

150

170

15

10

150

35

250

 

11

155

175

15

10

150

35

265

 

12

160

175

15

10

155

35

275

 

13

165

175

15

10

155

40

280

 

14

170

175

20

10

160

45

280

 

15

175

175

20

10

160

45

280

 

16

175

180

20

15

160

45

280

 

17

180

180

25

15

165

45

280

 

18

180

180

25

15

165

45

280

 

19

185

180

25

20

165

45

280

 

20

185

185

25

20

170

50

285

 

21

190

185

25

20

170

50

285

 

22

190

190

25

20

170

50

285

 

23

195

190

25

20

170

50

285

 

24

195

195

25

20

170

50

285

 

25

195

195

25

20

170

50

285

 

26

195

195

25

20

175

55

285

 

27

200

200

25

20

175

55

285

 

28

205

200

25

20

175

55

285

 

29

210

205

25

20

180

55

285

 

30

210

205

25

20

180

55

285

 

31

210

205

25

20

180

55

285

 

32

210

205

25

20

180

55

285

 

33

210

205

25

20

180

55

285

 

34

215

205

25

20

180

55

285

 

35

215

210

25

20

185

55

285

36

215

210

25

20

185

55

285

37

220

210

25

20

190

60

285

38

220

215

25

20

190

60

285

39

220

215

25

20

190

60

285

40

220

215

25

20

190

60

285

41

220

215

25

20

190

60

285

42

220

215

25

20

190

60

285

43

220

215

25

20

190

60

285

44

220

215

25

20

195

65

285

45

220

220

30

20

195

65

285

46

220

220

30

20

195

65

285

47

220

220

30

20

195

65

285

48

225

225

30

20

195

65

285

49

230

230

30

20

195

65

285

50

230

230

30

20

195

65

285

51

230

230

30

20

195

65

285

52

230

230

30

20

195

65

285

53

230

230

30

20

195

65

285

54

230

230

30

20

195

65

285

55

230

230

30

20

195

65

285

56

230

230

30

20

195

65

285

57

230

230

30

20

195

65

285

58

230

230

30

20

195

65

285

59

230

230

30

20

200

65

285

60

230

230

30

20

200

65

285

Flasks 1 and 2: Fenugreek Absolute

Flasks 3 and 4: inoculum control

Flask5: reference(procedure control)

Flask 6: abiotic control

Flask 7: toxicity control

Percentage Biodegradation of Test Item, of Sodium Benzoate and of the Toxicity Control based on ThODNH4:

Time

Percentage Biodegradation1

(Days)

Fenugreek Absolute1

Sodium Benzoate2

Toxicity
control1, 2

 

Flask 1 [%]

Flask 2 [%]

Flask 5 [%]

Flask 7 [%]

1

12

12

35

13

2

21

21

48

22

3

30

28

59

29

4

36

39

67

36

5

41

45

73

47

6

44

50

75

48

7

47

53

76

51

8

49

57

79

52

9

53

61

82

55

10

54

64

81

57

11

56

66

81

60

12

58

66

83

63

13

60

66

83

64

14

61

65

85

63

15

63

65

85

63

16

62

66

83

63

17

63

65

85

62

18

63

65

85

62

19

64

64

83

61

20

64

66

86

63

21

66

66

86

63

22

66

68

86

63

23

68

68

86

63

24

68

70

86

63

25

68

70

86

63

26

68

70

89

63

27

70

72

89

63

28

72

72

89

63

29

74

74

92

63

30

74

74

92

63

31

74

74

92

63

32

74

74

92

63

33

74

74

92

63

34

76

74

92

63

35

76

76

95

63

36

76

76

95

63

37

78

76

98

63

38

78

78

98

63

39

78

78

98

63

40

78

78

98

63

41

78

78

98

63

42

78

78

98

63

43

78

78

98

63

44

78

78

101

63

45

77

79

100

62

46

77

79

100

62

47

77

79

100

62

48

79

81

100

62

49

81

83

100

62

50

81

83

100

62

51

81

83

100

62

52

81

83

100

62

53

81

83

100

62

54

81

83

100

62

55

81

83

100

62

56

81

83

100

62

57

81

83

100

62

58

81

83

100

62

59

81

83

103

62

60

81

83

103

62

1ThODNH4of Fenugreek Absolute: 2.444 mg O2/mg test item
2ThODNH4of sodium benzoate: 1.666 mg O2/mg reference item

Percentage Biodegradation of Test Item, of Sodium Benzoate and of the Toxicity Control based on ThODNO3:

Time

Percentage Biodegradation1

(Days)

Fenugreek Absolute1

Sodium Benzoate2

Toxicity
control1, 2

 

Flask 1 [%]

Flask 2 [%]

Flask 5 [%]

Flask 7 [%]

1

12

12

35

13

2

20

21

48

22

3

29

28

59

28

4

35

38

67

35

5

40

44

73

46

6

43

49

75

48

7

46

52

76

51

8

48

56

79

52

9

52

59

82

54

10

53

62

81

56

11

55

64

81

59

12

57

64

83

62

13

59

64

83

63

14

60

63

85

62

15

62

63

85

62

16

61

64

83

62

17

62

63

85

61

18

62

63

85

61

19

63

62

83

61

20

63

64

86

62

21

65

64

86

62

22

65

66

86

62

23

66

66

86

62

24

66

68

86

62

25

66

68

86

62

26

66

68

89

62

27

68

70

89

62

28

70

70

89

62

29

72

72

92

62

30

72

72

92

62

31

72

72

92

62

32

72

72

92

62

33

72

72

92

62

34

74

72

92

62

35

74

74

95

62

36

74

74

95

62

37

76

74

98

62

38

76

76

98

62

39

76

76

98

62

40

76

76

98

62

41

76

76

98

62

42

76

76

98

62

43

76

76

98

62

44

76

76

101

62

45

75

77

100

61

46

75

77

100

61

47

75

77

100

61

48

77

79

100

61

49

79

81

100

61

50

79

81

100

61

51

79

81

100

61

52

79

81

100

61

53

79

81

100

61

54

79

81

100

61

55

79

81

100

61

56

79

81

100

61

57

79

81

100

61

58

79

81

100

61

59

79

81

103

61

60

79

81

103

61

1ThODNO3of Fenugreek Absolute: 2.502 mg O2/mg test item
2ThODNH4of sodium benzoate: 1.666 mg O2/mg reference item

Biodegradation in the Toxicity Control

In the toxicity control containing both, the test item and the reference item sodium benzoate, 63% (ThODNH4) biodegradation was noted within 14 and 28 days (62% based on ThODNO3) and 62% after 60 days (ThODNH4) as well as 61% (ThODNO3).

Abiotic Control

The oxygen demand in the abiotic control started at day 44 and reached a maximum of 65 mg/L during the test duration of 60 days.

Validity criteria

Inoculum Control:

The oxygen demand of the inoculum control (medium and inoculum) was 22.5 mg O2/L and thus not greater than 60 mg O2/L within 28 days as required by the test guideline.

pH-Value:

The pH-value of the test item flasks at day 63 was 7.4 to 7.6 and therefore within the range of pH 6.0 to 8.5 as required by the test guideline.

Reference Item:

The percentage degradation of the reference item should reach the level for ready biodegradability (>60%) within 14 days as required by the test guideline.

The reference item sodium benzoate was degraded to more than 60% after 4 days of incubation.

Test Item:

The difference of duplicate values for the degradation of the test item at the end of the test, at day 28 and at the end of the 10-day window was less than 20%. The difference of duplicate values at day 11 was 10%, at day 28 0% and at day 60 2%.

The validity criterion was fulfilled.

Toxicity Control:

If in a toxicity test, containing both the test item and a reference item less than 25% biodegradation (based on total ThOD) occurred within 14 days, the test item can be assumed to be inhibitory. The biodegradation was 63% (ThODNH4) and 62% (ThODNO3) at day 14; the test item was not inhibitory.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The degradation rate of Fenugreek Absolute reached 60% within the 10-day window and after 28 and 60 days.
Therefore, Fenugreek Absolute is considered to be readily biodegradable based on ThODNH4 and ThODNO3.
Executive summary:

The test item Fenugreek Absolute was investigated for its ready biodegradability in a manometric respirometry test, according to the OECD 301F guideline and under GLP compliance. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

Nitrogen is part of the molecular structure of the test item; therefore nitrification (ThODNO3) was considered for the evaluation of the results. The criterion for ready biodegradability under the conditions of a manometric respirometry test is the degradation of the test item of at least 60%, reached within a 10-day window; the 10-day window starts when the degradation of the test item reaches at least 10% degradation.

 

The mean biodegradation of 10% of Fenugreek Absolute was reached at day 1 (ThODNH4, ThODNO3). At the end of the 10-day window at day 11, the mean degradation of Fenugreek Absolute was 61% (ThODNH4) and 60% (ThODNO3) and therefore the 10 day window criterion was passed. The mean biodegradation after 28 days was 72% (ThODNH4) and 70% (ThODNO3), and at test end after 60 days 82% (ThODNH4) and 80% (ThODNO3).

Therefore, Fenugreek Absolute is considered to be readily biodegradable based on ThODNH4and ThODNO3.

In the toxicity control containing both, the test item and the reference item sodium benzoate, 63% (ThODNH4) biodegradation was noted within 14 and 28 days (62% based on ThODNO3) and 62% after 60 days (ThODNH4) as well as 61% (ThODNO3).

According to the test guidelines, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.

Description of key information

The registered substance is regarded as readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

The test item Fenugreek Absolute was investigated for its ready biodegradability in a manometric respirometry test, according to the OECD 301F guideline and under GLP compliance. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

Nitrogen is part of the molecular structure of the test item; therefore nitrification (ThODNO3) was considered for the evaluation of the results. The criterion for ready biodegradability under the conditions of a manometric respirometry test is the degradation of the test item of at least 60%, reached within a 10-day window; the 10-day window starts when the degradation of the test item reaches at least 10% degradation.

 

The mean biodegradation of 10% of Fenugreek Absolute was reached at day 1 (ThODNH4, ThODNO3). At the end of the 10-day window at day 11, the mean degradation of Fenugreek Absolute was 61% (ThODNH4) and 60% (ThODNO3) and therefore the 10 day window criterion was passed. The mean biodegradation after 28 days was 72% (ThODNH4) and 70% (ThODNO3), and at test end after 60 days 82% (ThODNH4) and 80% (ThODNO3).

Therefore, Fenugreek Absolute is considered to be readily biodegradable based on ThODNH4and ThODNO3.

In the toxicity control containing both, the test item and the reference item sodium benzoate, 63% (ThODNH4) biodegradation was noted within 14 and 28 days (62% based on ThODNO3) and 62% after 60 days (ThODNH4) as well as 61% (ThODNO3).

According to the test guidelines, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.