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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 2018-01-09 to 2018-04-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to the OECD 301F guideline and under GLP conditions. The validity criteria were fulfilled.
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes
Specific details on test material used for the study:
None
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Species/Origin: 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 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 was pre-conditioned in test water for a maximum of 2 days.
Duration of test (contact time):
60 d
Initial conc.:
3.021 other: mg oxygen per mg test item
Based on:
ThOD
Remarks:
NH4
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 6.7 (measured at the start of the test and adjusted to 7.4 with Sodium Hydroxide) 7.2 to 7.8 (measured at day 63)
- Reconstituted Water: The following stock solutions with analytical grade salts was prepared:
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 during storage, one drop of concentrated HCl per litre was added to stock solution d) before storage. 10 mL of stock solution a) and 1 mL of the stock solutions b) - d) were combined and filled up to a final volume of 1000 mL with pure water.
- Solubilising agent: Silicone oil AR20 was used as an emulsifying agent. (1%)

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
Preliminary study:
No data.
Test performance:
No data.
Parameter:
% degradation (O2 consumption)
Value:
54
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
68
Sampling time:
60 d
Details on results:
TEST ITEM: The mean biodegradation of 10% of PATCHOULI EO FRACTION PATCHOULOL RICH was reached at day 7 (ThODNH4). At the end of the 10-day window at day 17, the mean degradation of PATCHOULI EO FRACTION PATCHOULOL RICH was 38% (ThODNH4).The mean biodegradation after 28 days was 54% (ThODNH4) and 68% (ThODNH4) after 60 days.
TOXICITY CONTROL: In the toxicity control containing both, the test item and the reference item sodium benzoate, 37% (ThODNH4) biodegradation was noted within 14 days, 53% (ThODNH4) biodegradation after 28 days and 57% (ThODNH4) after 60 days of incubation.
According to the test guideline, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.
Results with reference substance:
The reference item sodium benzoate was sufficiently degraded to 85% after 14 days and to 89% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

pH-Values at the End of the Test

Flask No.

Treatment

pH-value

1

PATCHOULI EO FRACTION PATCHOULOL RICH

7.3

2

PATCHOULI EO FRACTION PATCHOULOL RICH

7.2

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

0

0

0

0

60

0

60

 

2

5

5

5

0

85

0

85

 

3

5

10

5

5

105

0

100

 

4

20

15

5

5

120

0

120

 

5

25

35

5

5

130

0

130

 

6

30

45

10

5

135

0

135

 

7

40

65

10

10

140

0

145

 

8

55

75

10

10

145

0

150

 

9

70

85

10

10

150

0

160

 

10

85

95

15

10

150

0

160

 

11

90

105

15

10

150

0

170

 

12

95

115

15

10

155

0

175

 

13

100

120

15

10

155

0

180

 

14

110

130

20

10

160

0

190

 

15

115

140

20

10

160

0

195

 

16

120

145

20

15

160

0

200

 

17

120

155

25

15

165

0

205

 

18

125

160

25

15

165

0

210

 

19

130

170

25

20

165

0

215

 

20

135

175

25

20

170

0

225

 

21

140

185

25

20

170

0

230

 

22

140

190

25

20

170

0

240

 

23

150

195

25

20

170

0

245

 

24

150

200

25

20

170

0

250

 

25

155

205

25

20

170

0

250

 

26

160

205

25

20

175

0

260

 

27

160

210

25

20

175

0

265

 

28

165

215

25

20

175

0

275

 

29

170

220

25

20

180

0

285

 

30

170

220

25

20

180

0

285

 

31

170

225

25

20

180

0

285

 

32

175

225

25

20

180

0

285

33

175

230

25

20

180

0

285

34

175

230

25

20

180

0

285

35

175

235

25

20

185

0

290

36

175

240

25

20

185

0

290

37

180

245

25

20

190

0

290

38

180

245

25

20

190

0

290

39

180

245

25

20

190

0

290

40

185

245

25

20

190

0

290

41

185

245

25

20

190

0

290

42

190

245

25

20

190

0

290

43

190

250

25

20

190

0

290

44

190

250

25

20

195

0

290

45

190

250

30

20

195

0

290

46

195

255

30

20

195

0

290

47

195

255

30

20

195

0

290

48

195

255

30

20

195

0

290

49

195

255

30

20

195

0

290

50

195

255

30

20

195

0

290

51

200

260

30

20

195

0

290

52

200

260

30

20

195

0

290

53

200

260

30

20

195

0

290

54

200

260

30

20

195

0

290

55

200

260

30

20

195

0

290

56

200

260

30

20

195

0

290

57

205

260

30

20

195

0

290

58

205

260

30

20

195

0

290

59

210

260

30

20

200

0

295

60

210

260

30

20

200

0

295

Flasks 1 and 2: PATCHOULI EO FRACTION PATCHOULOL RICH

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)

PATCHOULI EO FRACTION PATCHOULOL RICH1

Sodium Benzoate2

Toxicity control1, 2

 

Flask 1 [%]

Flask 2 [%]

Flask 5 [%]

Flask 7 [%]

1

0

0

35

13

2

1

1

48

17

3

0

2

59

20

4

5

3

67

24

5

7

10

73

26

6

7

12

75

27

7

10

18

76

28

8

15

21

79

29

9

20

24

82

32

10

24

26

81

31

11

25

30

81

33

12

27

33

83

34

13

29

34

83

35

14

31

37

85

37

15

33

40

85

38

16

34

41

83

38

17

33

43

85

39

18

34

45

85

40

19

35

47

83

41

20

37

49

86

43

21

38

52

86

44

22

38

53

86

46

23

42

55

86

47

24

42

57

86

48

25

43

58

86

48

26

45

58

89

50

27

45

60

89

51

28

47

61

89

53

29

48

63

92

55

30

48

63

92

55

31

48

65

92

55

32

50

65

92

55

33

50

66

92

55

34

50

66

92

55

35

50

68

95

56

36

50

69

95

56

37

52

71

98

56

38

52

71

98

56

39

52

71

98

56

40

53

71

98

56

41

53

71

98

56

42

55

71

98

56

43

55

73

98

56

44

55

73

101

56

45

54

72

100

56

46

56

73

100

56

47

56

73

100

56

48

56

73

100

56

49

56

73

100

56

50

56

73

100

56

51

57

75

100

56

52

57

75

100

56

53

57

75

100

56

54

57

75

100

56

55

57

75

100

56

56

57

75

100

56

57

59

75

100

56

58

59

75

100

56

59

60

75

103

57

60

60

75

103

57

           1ThODNH4of PATCHOULI EO FRACTION PATCHOULOL RICH: 3.021 mg O2/mg test item
           2ThODNH4of sodium benzoate: 1.666 mg O2/mg reference item

Biodegradation in the Toxicity Control

Percentage Biodegradation:

In the toxicity control containing both, the test item and the reference item sodium benzoate, 37% (ThODNH4) biodegradation was noted within 14 days, 53% (ThODNH4) biodegradation after 28 days and 57% (ThODNH4) after 60 days of incubation.

Conclusion:

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.

Abiotic Control

Oxygen Demand:

The oxygen demand in the abiotic control was 0 mg/L during the test duration. There was no need to correct the degradation of the test item and toxicity control.

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 7.2 to 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, at day 28 and at day 60 of the test was less than 20%. The difference of duplicate values at days 17, 28 and 60 differed by 10%, 14% and 5%, respectively.

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 ThODNH4) occurred within 14 days, the test item can be assumed to be inhibitory. The biodegradation was 37% at day 14; the test item was not inhibitory.

 

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
With a mean biodegradation rate (ThOD-NH4) of 54% after 28 days of incubation, the test item is considered not readily biodegradable.
Executive summary:

The test item Patchouli EO Fraction Patcoulol Rich 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.

The mean biodegradation of 10% of PATCHOULI EO FRACTION PATCHOULOL RICH was reached at day 7 (ThODNH4). At the end of the 10-day window at day 17, the mean degradation of PATCHOULI EO FRACTION PATCHOULOL RICH was 38% (ThODNH4).The mean biodegradation after 28 days was 54% (ThODNH4) and 68% (ThODNH4) after 60 days.

Therefore, the test item is considered not readily biodegradable according to test guideline.

The reference item sodium benzoate was sufficiently degraded to 67% after 14 days and to 89% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

In the toxicity control containing both, the test item and the reference item sodium benzoate, 37% biodegradation was noted within 14 days and 53% 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.

Description of key information

With a mean biodegradation rate (ThOD-NH4) < 60% after 28 days of incubation, the test item is considered not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

The test item Patchouli EO Fraction Patcoulol Rich 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.

The mean biodegradation of 10% of PATCHOULI EO FRACTION PATCHOULOL RICH was reached at day 7 (ThODNH4). At the end of the 10-day window at day 17, the mean degradation of PATCHOULI EO FRACTION PATCHOULOL RICH was 38% (ThODNH4).The mean biodegradation after 28 days was 54% (ThODNH4) and 68% (ThODNH4) after 60 days.

Therefore, the test item is considered not readily biodegradable according to test guideline.

The reference item sodium benzoate was sufficiently degraded to 67% after 14 days and to 89% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

In the toxicity control containing both, the test item and the reference item sodium benzoate, 37% biodegradation was noted within 14 days and 53% 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 degradationwas >25% within 14 days.