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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 2018-01-08 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. Validity criteria 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, non-adapted
Details on inoculum:
- Species/Origin: Aerobic activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the sewage treatment plant 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.
Duration of test (contact time):
60 d
Initial conc.:
3.282 other: mg oxygen per mg test item
Based on:
test mat.
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 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
- Culturing apparatus: 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)
Remarks:
ThODNH4
Value:
42.5
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Remarks:
ThODNH4
Value:
58
Sampling time:
60 d
Details on results:
TEST ITEM: The mean biodegradation of 10% of PATCHOULI EO FRACTION ALPHA GUAIENE 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 ALPHA GUAIENE RICH was 31% (ThODNH4). The mean biodegradation after 28 days was 43% (ThODNH4) and at test end after 60 days 58% (ThODNH4).
TOXICITY CONTROL: In the toxicity control containing both, the test item and the reference item sodium benzoate, 34% (ThODNH4) biodegradation was noted within 14 days, 45% (ThODNH4) biodegradation after 28 days and 52% after 60 days of incubation.
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, 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 ALPHA GUAIENE RICH

7.2

2

PATCHOULI EO FRACTION ALPHA GUAIENE RICH

7.4

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

5

5

5

105

0

105

 

4

10

10

5

5

120

0

120

 

5

20

15

5

5

130

0

130

 

6

35

35

10

5

135

0

140

 

7

45

60

10

10

140

0

145

 

8

55

75

10

10

145

0

150

 

9

60

80

10

10

150

0

155

 

10

65

85

15

10

150

0

160

 

11

70

90

15

10

150

0

170

 

12

75

95

15

10

155

0

175

 

13

85

105

15

10

155

0

180

 

14

95

115

20

10

160

0

185

 

15

100

125

20

10

160

0

190

 

16

105

135

20

15

160

0

190

 

17

110

140

25

15

160

0

195

 

18

115

145

25

15

165

0

200

 

19

120

150

25

20

165

0

205

 

20

125

155

25

20

170

0

210

 

21

130

160

25

20

170

0

215

 

22

135

160

25

20

170

0

220

 

23

140

165

25

20

170

0

225

 

24

145

165

25

20

170

0

230

 

25

145

165

25

20

170

0

240

 

26

150

170

25

20

175

0

240

 

27

155

170

25

20

175

0

245

 

28

160

170

25

20

175

0

250

 

29

165

175

25

20

180

0

255

 

30

165

175

25

20

180

0

255

 

31

170

180

25

20

180

0

260

 

32

175

180

25

20

180

0

265

33

175

180

25

20

180

0

265

34

175

180

25

20

180

0

270

35

180

185

25

20

185

0

275

36

180

185

25

20

185

0

275

37

185

190

25

20

190

0

280

38

185

190

25

20

190

0

280

39

190

190

25

20

190

0

285

40

190

190

25

20

190

0

285

41

190

190

25

20

190

0

285

42

195

190

25

20

190

0

285

43

195

195

25

20

190

0

285

44

195

195

25

20

195

0

285

45

195

195

30

20

195

0

285

46

195

200

30

20

195

0

285

47

200

200

30

20

195

0

285

48

200

200

30

20

195

0

285

49

200

200

30

20

195

0

285

50

200

200

30

20

195

0

285

51

200

200

30

20

195

0

285

52

200

205

30

20

195

0

285

53

205

205

30

20

195

0

285

54

205

205

30

20

195

0

285

55

205

210

30

20

195

0

285

56

210

210

30

20

195

0

285

57

210

210

30

20

195

0

285

58

210

215

30

20

195

0

285

59

215

220

30

20

200

0

285

60

215

220

30

20

200

0

285

Flasks 1 and 2: PATCHOULI EO FRACTION ALPHA GUAIENE 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 ALPHA GUAIENE RICH1

Sodium Benzoate2

Toxicity control1, 2

 

Flask 1 [%]

Flask 2 [%]

Flask 5 [%]

Flask 7 [%]

1

0

0

35

12

2

1

1

48

16

3

0

0

59

20

4

1

1

67

23

5

4

3

73

25

6

8

8

75

26

7

10

15

76

27

8

13

19

79

28

9

15

21

82

29

10

16

21

81

29

11

17

23

81

31

12

19

24

83

32

13

22

27

83

33

14

24

30

85

34

15

25

32

85

35

16

26

35

83

34

17

27

35

82

35

18

28

37

85

36

19

29

38

83

36

20

31

39

86

37

21

32

41

86

38

22

34

41

86

39

23

35

42

86

40

24

37

42

86

41

25

37

42

86

43

26

38

44

89

43

27

40

44

89

44

28

41

44

89

45

29

43

45

92

46

30

43

45

92

46

31

44

46

92

47

32

46

46

92

48

33

46

46

92

48

34

46

46

92

49

35

47

48

95

50

36

47

48

95

50

37

49

49

98

51

38

49

49

98

51

39

50

49

98

52

40

50

49

98

52

41

50

49

98

52

42

52

49

98

52

43

52

51

98

52

44

52

51

101

52

45

51

50

100

52

46

51

52

100

52

47

52

52

100

52

48

52

52

100

52

49

52

52

100

52

50

52

52

100

52

51

52

52

100

52

52

52

53

100

52

53

54

53

100

52

54

54

53

100

52

55

54

55

100

52

56

55

55

100

52

57

55

55

100

52

58

55

56

100

52

59

57

58

103

52

60

57

58

103

52

1ThODNH4of PATCHOULI EO FRACTION ALPHA GUAIENE RICH: 3.282 mg O2/mg test item
2
ThODNH4of 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, 34% (ThODNH4) biodegradation was noted within 14 days, 45% (ThODNH4) biodegradation after 28 days and 52% after 60 days of incubation.

Abiotic Control

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 7.4 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 8%, 3% and 1%, 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 34% 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 42.5% after 28 days of incubation, the test item is considered not readily biodegradable.
Executive summary:

The test item Patchouli EO Fraction Alpha Guaiene 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 ALPHA GUAIENE 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 ALPHA GUAIENE RICH was 31% (ThODNH4). The mean biodegradation after 28 days was 43% (ThODNH4) and at test end after 60 days 58% (ThODNH4). Therefore, the test item is considered not readily biodegradable according to test guideline.

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.

In the toxicity control containing both, the test item and the reference item sodium benzoate, 34% biodegradation was noted within 14 days and 45% 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.

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 Alpha Guaiene 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 ALPHA GUAIENE 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 ALPHA GUAIENE RICH was 31% (ThODNH4). The mean biodegradation after 28 days was 43% (ThODNH4) and at test end after 60 days 58% (ThODNH4).Therefore, the test item is considered not readily biodegradable according to test guideline.

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.

In the toxicity control containing both, the test item and the reference item sodium benzoate, 34% biodegradation was noted within 14 days and 45% 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.