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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:
2016-11-08 to 2017-01-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to the OECD 301F guideline. All the validity criteria were successful.
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 (incl. certificate)
Remarks:
14 October 2013
Specific details on test material used for the study:
None
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, adapted
Remarks:
Pre-conditioning consisted in aerating sludge (in mineral medium) for 5-7 days before the test and at the test temperature
Details on inoculum:
- Source of inoculum/activated sludge: The inoculum was a fresh sample of activated sludge and was collected from the aeration tank of a sewage treatment plant receiving predominantly domestic sewage.
- Sample site: Water treatment plant "STEP de Lille", 33290 Blanquefort (France).
- Laboratory culture: A filtration through a fine sieve (about 1 mm) was performed in order to remove coarse particles. After removal of any coarse particles, the sludge was washed by decantation in a mineral medium until sludge was considered free from excess substrate or inhibitor.
- Inoculum preconditioning: Pre-conditioning consisted in aerating sludge (in mineral medium) for 5-7 days before the test and at the test temperature.
Duration of test (contact time):
60 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST ITEM PREPARATION:
- the test item was poorly soluble in water and in order to improve the dissolution the test item was dissolved in silicone oil AR 20 up to final concentration of 1 % v/v. Around 25 mg of test item was stirred with 2.5 mL silicone oil AR 20 by magnetically stirring until completely dissolved. Then, this solution was directly supplemented or completed into 250 mL with mineral medium and sludge directly in each test vessels.
The test medium was the mineral medium, a weakly saline aqueous medium.
The test item was tested at 100 mg/L in mineral medium containing 1 % v/v of silicone oil AR 20.

TEST CONDITIONS
- Composition of medium: Mineral medium according to OECD 301F guideline
- Test temperature: The test was performed in darkness between 22.0 to 22.5 °C (constant temperature +/- 1°C).
- Mineral medium - pH: 7.6
- Inoculum pH: 6.9
- Dissolved oxygen: 9.2 mg/L
- Suspended solids concentration: 3.65 g/L
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: WTW Oxitop
- Number of culture flasks/concentration: 3 flasks (test item, silicone oil 1% and inoculum)
- Test performed in closed vessels: Yes
- A measured volume of inoculated medium containing a known concentration of the test item (100 mg/L) as the nominal sole source of Carbon, was stirred in a closed vessel at a constant temperature up to 28 days. The experiment was prolonged up to 60 days.
- Details of trap for CO2: Microbial respiration consumes O2 and releases CO2 which were absorbed by soda lime pellets present in the headspace of flasks. This produced a decrease in pressure in the flask proportional to the amount of oxygen taken up by the microbial population during biodegradation, which was expressed as a percentage of ThOD (Theoretical Oxygen Demand).
- Allowance was made for the endogenous activity of the inoculum by running parallel blanks with inoculum but without the test item. Furthermore, in order to check the procedure, a reference item control (sodium benzoate + inoculum) and a toxicity control (sodium benzoate + inoculum + test item+ silicone oil AR 20) were run in parallel. Moreover, a control with silicone oil AR 20 1% was performed to check that silicon was non toxic to bacteria and non biodegradable.
The pH of the contents was measured at the start and the end of the experiment.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes; 3 flasks (inoculum only)
- Inoculum silicone control: 3 flasks (inoculum silicone oil AR 20 1% v/v)
- Reference item: 2 flasks (Sodium benzoate and inoculum)
- Toxicity control: Yes; 2 flasks (test item, silicone oil AR 20 1% v/v, sodium benzoate and inoculum)
Reference substance:
benzoic acid, sodium salt
Preliminary study:
An elementary analysis of the test item (non GLP data) has been performed and gave the following results:
- % carbon = 81.16 and 81.32 for the measurement 1 and the measurement 2 respectively
- % hydrogen = 10.74 and 10.76 for the measurement 1 and the measurement 2 respectively
- % nitrogen = Absence for the measurement 1 and 2
- % oxygen = 7.61 and 7.57 for the measurement 1 and the measurement 2 respectively
Key result
Parameter:
% degradation (O2 consumption)
Value:
47.76
St. dev.:
4.66
Sampling time:
28 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
61.88
St. dev.:
4.66
Sampling time:
60 d
Details on results:
Biodegradation of the test item:
The test item “CARROT SEED OIL” contains no nitrogen according to the elementary analysis, the evaluation of biodegradation has to be based in the following expressed as ThODNH4.
The degradation for the 10-day window beginning was reached on day 3, but at the end of the 10-day window (reaching at least 60%), on day 13, the mean degradation was 33.92% following the ThODNH4, therefore the 10-day window failed.
The degradation rate of the test item “CARROT SEED OIL” reached 47.76%, following the ThODNH4, after 28 days of incubation.
According to the study monitor, and as it had been anticipated in the study plan, the testing period was extended to 60 days. After 60 days, the degradation rate of the test item “CISTUS ORGANIC OIL” reached 61.88%, following the ThODNH4.
According to the OECD guideline 301F, the test item is not considered as readily biodegradable, nethertheless, on day 60, the degradation rate of the test item “CISTUS ORGANIC OIL” reached 61.88%, following the ThODNH4. From the 52nd day, the degradation rate of the test item reached 60.45% to 61.88% at the end of the test: 60 days.

Biodegradation of the Toxicity Control:
A biodegradation of 38.18% and 36.44% after 14 days of incubation was noted in the Toxicity control (containing both of the test item and the reference item) based on ThODNH4.
At the end on the test, 60 days of incubation, the results were respectively 66.58% and 64.11%.
Therefore, the test item was not inhibitory at the tested concentration on the aerobic activated sludge micro-organism because degradation was superior to 25% within 14 days (validity criterion).
Results with reference substance:
The reference item “Sodium benzoate” was degraded up to 82.91% and 78.72%( mean value: 81.82%) after 14 days and 77.89% in the both replicates after 60 days of incubation at the end of the test. Therefore, it confirms the suitability of the used aerobic sludge inoculum.

Table 5.2.1/2: pH values

Treatment

Flask number

pH
at the start

pH
at the end of the test

(60 days)

Inoculum Control

3

7.6

7.4

9

7.6

7.4

1

7.6

7.4

Reference Item

5

7.6

7.9

8

7.6

7.9

Test Item

17

7.6

7.5

18

7.6

7.5

20

7.6

7.5

Toxicity Control

22

7.6

8.0

16

7.6

8.0

Inoculum silicon Control

2

7.5

7.4

24

7.5

7.4

13

7.5

7.4

 

Table 5.2.1/3: Percentage (%) Biodegradation during 60 days for Toxicity Control and Test item (“PYCNOGENOL”) with ThODNH4=1.524 mgO2/mg Test item

Time (Days)

Tox Control

Test item

Flask 22

Flask 16

Flask 17

Flask 18

Flask 20

Average

0

0,00

0,00

0,00

0,00

0,00

0,00

1

6,03

5,66

0,00

0,00

0,00

0,00

2

13,45

15,94

0,47

0,00

0,00

0,16

3

15,58

17,34

7,09

4,74

2,86

4,89

4

18,78

19,83

11,83

10,42

7,09

9,78

5

21,97

22,67

17,04

15,63

11,83

14,83

6

25,39

25,39

21,30

19,42

15,63

18,78

7

28,40

28,40

24,63

23,19

19,42

22,41

8

30,92

30,41

29,84

27,45

22,72

26,67

9

33,51

32,50

33,37

31,01

25,34

29,91

10

35,67

34,41

36,26

32,90

27,69

32,28

11

36,93

36,67

38,61

35,25

30,07

34,64

12

38,33

38,08

40,49

37,13

31,49

36,37

13

39,49

39,24

42,37

38,68

32,96

38,00

14

41,25

40,49

44,39

40,02

34,64

39,68

15

42,40

41,15

45,26

41,57

35,85

40,89

16

43,47

42,47

47,03

42,66

36,95

42,21

17

44,55

43,30

48,14

44,10

37,72

43,32

18

46,56

44,55

49,82

45,78

39,06

44,89

19

47,71

45,19

51,68

46,98

39,58

46,08

20

48,79

45,77

53,46

48,42

40,69

47,52

21

50,05

46,28

54,81

49,76

41,70

48,76

22

51,63

46,85

56,25

50,87

42,47

49,87

23

52,46

47,43

56,69

52,65

43,58

50,97

24

53,72

48,19

57,69

54,33

44,59

52,21

25

55,05

48,76

58,13

55,78

45,36

53,09

26

55,88

49,59

58,57

56,22

46,47

53,75

27

57,21

50,42

59,34

56,99

47,24

54,53

28

58,54

51,00

59,78

57,76

47,68

55,07

29

59,55

52,01

60,45

58,10

49,36

55,97

30

59,95

52,41

59,98

58,30

49,56

55,95

31

60,55

53,01

60,45

58,77

50,71

56,64

32

61,38

53,59

60,89

58,87

50,81

56,85

33

62,14

54,60

61,22

59,88

51,81

57,64

34

62,54

55,50

61,42

59,74

52,35

57,84

35

63,29

56,25

61,42

60,42

52,69

58,18

36

63,54

56,76

62,10

60,42

53,36

58,62

37

63,95

57,66

61,63

60,28

53,90

58,60

38

64,20

58,17

62,30

60,95

54,57

59,27

39

64,52

58,74

62,40

61,39

55,01

59,60

40

64,78

59,25

62,40

61,39

55,34

59,71

41

65,10

59,57

62,84

61,83

55,11

59,92

42

65,35

60,33

62,84

62,16

56,12

60,37

43

65,18

60,65

62,60

61,93

56,22

60,25

44

65,61

61,33

62,84

62,84

57,12

60,93

45

65,43

61,91

62,94

62,94

57,22

61,03

46

65,76

62,49

63,37

63,37

57,66

61,47

47

65,76

62,99

63,37

63,37

58,00

61,58

48

65,76

63,49

63,71

63,71

58,00

61,81

49

65,58

63,82

63,14

63,47

58,10

61,57

50

65,58

64,07

63,47

63,47

58,10

61,68

51

65,58

64,57

63,47

63,81

58,77

62,02

52

65,58

64,83

63,81

64,48

58,77

62,35

53

64,90

64,90

63,58

64,25

58,87

62,23

54

64,90

65,40

63,58

64,58

59,54

62,57

55

64,90

65,40

63,58

65,26

59,88

62,90

56

64,90

66,16

64,25

65,26

59,88

63,13

57

64,73

65,98

64,01

65,02

59,98

63,00

58

64,46

65,97

64,00

65,00

59,96

62,99

59

64,73

66,23

64,35

65,36

60,65

63,45

60

64,29

66,05

63,76

65,44

60,74

63,31

Table 5.2.1/4: Validity criteria conformity

Criteria

Validity
for OECD 301F

Validity for the test

Oxygen uptake of the inoculum control
in 28 days and up to 60 days

< 60 mg/L

Day 28

Day 60

Yes

Yes

Silicon Control
12.7 & 15.5 mg/L

Silicon Control
22.5 to 24.0 mg/L

Control
19.7 to 24.0 mg/L

Control
26.8 to 36.6 mg/L

Variation between the test item replicates at the end of the test

< 20%

Yes
(9.8%)

Yes
(7.5%)

Biodegradability of the reference item
within 14 days

> 60%

Yes(78.72% to 82.91%)

Biodegradability of the toxicity control
within 14 days

> 25%

Yes(36.44% to 38.18%)

pH of the inoculum control at 60 days

6 to 8.5

Yes(7.4 to 8.0)

All the validity criteria were successful.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The degradation rate of the test item “carrot seed oil” reached 47.76%, following the ThODNH4, after 28 days of incubation. Under conditions of OECD Guideline 301F, the test item “CARROT SEED OIL” is not considered as readily biodegradable. Nevertheless, the degradation rate of the test item was above 60% (61.88%) at Day 60.
Executive summary:

The aim of this study was to determine the effects of the test item “CARROT SEED OIL”, according to the OECD 301F guideline, ready biodegradability within an experimental period of 28 days by respirometric method. According to the study monitor’s decision, the experiment was prolonged up to 60 days.

The amount of oxygen taken up by the microbial population during the biodegradation of the test item (corrected by blank inoculum, run in parallel) was determined from the change in pressure in apparatus and is expressed as percentage of theorical Oxygen Demand (ThOD).

 

Results are presented as follows:

The degradation rate of the test item “CARROT SEED OIL” reached 47.76%, following the ThODNH4, after 28 days of incubation. Therefore, according to the OECD guideline 301F, the test item is not considered as readily biodegradable. Nevertheless, the test was prolonged up to 60 days. After 60 days of incubation the pass level of 60% was reached with a degradation rate of the test item equal to 61.88%, following the ThODNH4.

Validity criteria conformity:

Criteria

Validity
for OECD 301F

Validity for the test

Oxygen uptake of the inoculum control
in 28 days and up to 60 days

< 60 mg/L

Day 28

Day 60

Yes

Yes

Silicon Control
12.7 & 15.5 mg/L

Silicon Control
22.5 to 24.0 mg/L

Control
19.7 to 24.0 mg/L

Control
26.8 to 36.6 mg/L

Variation between the test item replicates at the end of the test

< 20%

Yes
(9.8 %)

Yes
(7.5 %)

Biodegradability of the reference item
within 14 days

> 60%

Yes(78.72 % to 82.91 %)

Biodegradability of the toxicity control
within 14 days

> 25%

Yes(36.44 % to 38.18 %)

pH of the inoculum control at 60 days

6 to 8.5

Yes (7.4 to 8.0)

 

All the validity criteria were successful.

Description of key information

According to the OECD guideline 301F, the test item is not considered readily biodegradable. Nevertheless, at day 60, the degradation rate of the test item “CARROT SEED OIL” was above 60%, following the ThODNH4.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

A reliable experimental study was available to determine the ready biodegradability of the test item “CARROT SEED OIL”, according to the OECD 301F guideline and in compliance with GLP.

The amount of oxygen taken up by the microbial population during the biodegradation of the test item (corrected by blank inoculum, run in parallel) was determined from the change in pressure in apparatus and is expressed as percentage of theorical Oxygen Demand (ThOD).

 

The degradation rate of the test item “CARROT SEED OIL” reached 47.76%, following the ThODNH4, after 28 days of incubation and 61.88 %, at Day 60.

 

All the validity criteria of the study were successful. This study was considered as acceptable for that endpoint.