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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 2017-03-23 to 2017-06-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
All validity criteria was successful
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)
Remarks:
2017-03-13
Specific details on test material used for the study:
Not applicable
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Test Species: Aerobic activated sludge (microorganisms from a domestic waste¬water treatment plant) was supplied by the sewage treatment plant of Rossdorf, Germany.

- Conditioning: The aerobic activated sludge used for this study was 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 (see 6.5) and aerated overnight. This suspension was used for the experiment.
Duration of test (contact time):
60 d
Initial conc.:
102.7 mg/L
Based on:
other: Test Item Loading Rate (initial concentration in medium C0)
Initial conc.:
280.5 mg/L
Based on:
ThOD
Remarks:
ThOD(NH4)
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Aliquots of washed sludge suspension were mixed with reconstitued 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-value was 7.4.
b) 11.25 g MgSO4 x 7 H2O filled up with pure water to 500 mL volume
c) 18.2 g CaCl2 x 2 H2O filled up with pure water to 500 mL volume
d) 0.125 g FeCl3 x 6 H2O filled up with pure water to 500 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.
- Solubilising agent: Silicone oil AR20 was used as an emulsifying agent. (1%)
- Test temperature: 22°C ± 1°C
- pH: 7.3 to 7.9
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: The closed test flasks were incubated in a climatised chamber under continuous stirring.
- Number of culture flasks/concentration: 2
The test flasks were closed gas-tight by a measuring head. Potassium hydroxide solution (45%) 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.
- Measuring equipment: BSB/BOD-Sensor- System, Aqualytic Dortmund, Germany
- Test performed in closed vessels due to significant volatility of test substance:

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
Value:
56
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
70
Sampling time:
60 d
Details on results:
The mean biodegradation of 10% of Artemisia herba-alba oil (camphor type) was reached at day 3. At the end of the 10-day window at day 13, the mean degradation of Artemisia herba-alba oil (camphor type) was 41% and therefore the 10 day window criterion was not passed. The mean biodegradation at day 28 was 56%; therefore the test was prolonged till day 60. 60% biodegradation or more was reached at day 32, when the mean degradation was 61%. Afterwards an increase of degradation could be determined till day 52, when the plateau of biodegradation was reached; the mean degradation was 70% at day 52. Afterwards the degradation of Artemisia herba-alba oil (camphor type) remained almost constant and was 70% at test end after 60 days.
Results with reference substance:
The reference item sodium benzoate was sufficiently degraded to 77% after 14 days, to 77% after 28 days and to 78 % after 60 days of incubation.
The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.

Cumulative Biochemical Oxygen Demand (mg O2/L) in Test Flasks during the Test Period of 28 Days

Time

Flask No.

(days)

1

2

3

4

5

6

7

 

1

0

0

0

0

45

0

55

 

2

5

0

5

5

85

0

85

 

3

30

35

5

5

105

0

105

 

4

65

70

5

10

115

0

120

 

5

75

80

10

10

125

0

130

 

6

85

90

10

10

135

0

140

 

7

95

95

10

15

140

0

145

 

8

100

105

10

15

145

0

155

 

9

110

110

15

20

150

0

160

 

10

115

120

15

20

150

0

165

 

11

125

125

15

20

150

0

170

 

12

130

135

15

20

155

0

180

 

13

130

135

15

20

155

0

185

 

14

135

140

20

25

155

0

190

 

15

140

140

20

25

155

0

200

 

16

140

145

20

25

160

0

210

 

17

140

145

20

25

160

0

215

 

18

140

150

20

25

160

0

225

 

19

145

155

25

25

160

0

230

 

20

145

155

25

25

160

0

235

 

21

150

160

25

25

160

0

245

 

22

155

160

25

25

160

0

250

 

23

160

170

25

25

160

0

260

 

24

165

175

25

30

160

0

270

 

25

170

180

25

30

160

0

280

 

26

175

185

25

30

160

0

280

 

27

175

190

25

30

160

0

285

 

28

180

190

25

30

160

0

290

 

29

180

190

25

30

160

0

290

 

30

180

195

25

30

160

0

290

 

31

185

200

25

30

160

0

290

32

190

205

25

30

160

0

295

33

195

210

25

30

160

0

295

34

200

215

25

30

160

0

300

35

200

215

25

30

160

0

300

36

200

215

25

30

160

0

300

37

205

215

25

30

160

0

300

38

205

220

25

30

160

0

300

39

210

220

25

30

160

0

300

40

210

220

25

35

160

0

300

41

210

220

25

35

160

0

300

42

215

220

25

35

160

0

300

43

215

220

25

35

160

0

300

44

215

225

25

35

160

0

300

45

215

225

25

35

160

0

300

46

215

225

25

35

160

0

300

47

215

225

25

35

160

0

300

48

215

225

25

35

160

0

300

49

215

225

25

35

160

0

300

50

220

225

25

35

160

0

300

51

220

225

25

35

165

0

300

52

225

225

25

35

165

0

300

53

225

225

25

35

165

0

300

54

225

225

25

35

165

0

300

55

225

225

25

35

165

0

300

56

225

225

25

35

165

0

300

57

225

225

25

35

165

0

300

58

225

225

25

35

165

0

300

59

225

225

25

35

165

0

300

60

225

225

25

35

165

0

300

Flasks 1 and 2: Artemisia herba-alba oil (camphor type)

Flasks 3 and 4: inoculum control

Flask 5: 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)

Artemisia herba-alba oil (camphor type)1

Sodium Benzoate2

Toxicity
control1, 2

 

Flask 1 [%]

Flask 2 [%]

Flask 5 [%]

Flask 7 [%]

1

0

0

26

12

2

0

0

46

18

3

9

11

58

22

4

21

22

62

25

5

23

25

67

27

6

27

28

73

29

7

29

29

74

29

8

31

33

77

31

9

33

33

77

31

10

35

36

77

33

11

38

38

77

34

12

40

42

80

36

13

40

42

80

37

14

40

42

77

37

15

42

42

77

39

16

42

44

80

41

17

42

44

80

43

18

42

45

80

45

19

43

46

78

45

20

43

46

78

46

21

45

48

78

49

22

46

48

78

50

23

48

52

78

52

24

49

52

77

54

25

51

54

77

56

26

53

56

77

56

27

53

58

77

57

28

54

58

77

58

29

54

58

77

58

30

54

60

77

58

31

56

61

77

58

          

32

58

63

77

59

33

60

65

77

59

34

62

67

77

60

35

62

67

77

60

36

62

67

77

60

37

63

67

77

60

38

63

68

77

60

39

65

68

77

60

40

64

68

76

60

41

64

68

76

60

42

66

68

76

60

43

66

68

76

60

44

66

69

76

60

45

66

69

76

60

46

66

69

76

60

47

66

69

76

60

48

66

69

76

60

49

66

69

76

60

50

68

69

76

60

51

68

69

78

60

52

70

69

78

60

53

70

69

78

60

54

70

69

78

60

55

70

69

78

60

56

70

69

78

60

57

70

69

78

60

58

70

69

78

60

59

70

69

78

60

60

70

69

78

60

1ThODNH4of Artemisia herba-alba oil (camphor type): 2.732 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, 37% biodegradation was noted within 14 days and 58% biodegradation after 28 days of incubation. 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.

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.

Validity Criteria of the Study

- Inoculum Control: The oxygen demand of the inoculum control (medium and inoculum) was 27.5 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 the end of the test was 7.3 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 10-day window (day 13), at day 28, at the plateau (day 52) and at the end of the test and was less than 20%. The difference of duplicate values was not greater than 4% for any time point. The validity criterion was fulfilled.

- Toxicity Control: If in a toxicity test, containing both the test substance and a reference compound less than 25% biodegradation (based on total ThOD) occurred within 14 days, the test substance can be assumed to be inhibitory. The biodegradation was 37% at day 14; the test item was not inhibitory.

All validity criteria are fulfilled.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Artemisia herba-alba oil (camphor type) is considered to be not readily biodegradable after 28 days, but reached a degradation of 70% after 60 days.
Executive summary:

The test item Artemisia herba-alba oil (camphor type) was investigated for its ready biodegradability in a manometric respirometry test over a period of 28 days, according to the OECD 301F guideline, and further prolongated to 60 days. 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.

The mean biodegradation of 10% of Artemisia herba-alba oil (camphor type) was reached at day 3. At the end of the 10-day window at day 13, the mean degradation of Artemisia herba-alba oil (camphor type) was 41% and therefore the 10 day window criterion was not passed. The mean biodegradation at day 28 was 56%; therefore the test was prolonged till day 60. 60% biodegradation or more was reached at day 32, when the mean degradation was 61%. Afterwards an increase of degradation could be determined till day 52, when the plateau of biodegradation was reached; the mean degradation was 70% at day 52. Afterwards the degradation of Artemisia herba-alba oil (camphor type) remained almost constant and was 70% at test end after 60 days.

The degradation rate of Artemisia herba-alba oil (camphor type) did reach 60% after 32 days, but not within 28 days.

Therefore, Artemisia herba-alba oil (camphor type) is considered to be not readily biodegradable after 28 days, but reached a degradation of 70% after 60 days.

The reference item sodium benzoate was sufficiently degraded, thus confirming the suitability of the aerobic activated sludge inoculum used.

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 in the toxicity control.

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

pH values were in the range required in the guideline. Therefore, all validity criteria of the study were fulfilled.

Therefore, this study is considered valid for that endpoint.

Results synopsys:

Test item: Artemisia herba-alba oil (camphor type)

Biodegradation endpoint: Not readily biodegradable based on the results at 28 days, but reached a degradation of 70% after 60 days.

Description of key information

Artemisia herba-alba oil (camphor type) is considered to be not readily biodegradable after 28 days, but reached a degradation of 70% after 60 days.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

A reliable experimental study was available for that endpoint.

The test item Artemisia herba-alba oil (camphor type) was investigated for its ready biodegradability in a manometric respirometry test over a period of 28 days, according to the OECD 301F guideline, further extended to 60 days.

All validity criteria were fulfilled. Moreover, the reference item sodium benzoate was sufficiently degraded, thus confirming the suitability of the aerobic activated sludge inoculum used.

In addition, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days in the toxicity control.

Therefore, this study is considered valid for that endpoint.