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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 25/09/2014 to 24/10/2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted according OECD guideline and in compliance with GPL
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
The temperature during the test was at 19 °C for 1.5 days (2014-09-27 to 2014-09-28). This deviation was considered to have no impact on the quality and integrity of the study.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 1720
- Expiration date of the lot/batch: 08/10/2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light, in the tightly closed original container under nitrogen

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was heated to 40 °C until homogeneity was reached before further use. The test item was treated with ultrasound for 10 minutes at room temperature.

OTHER SPECIFICS:
CAS No. 1119235-20-9
IUPAC Chemical Name Heat-treated distillate of avocado oil, Persea gratissima, Lauraceae
Type of substance UVCB substance
Chemical characterisation Fatty acids, glycerides, unsaponifiables
Main components Total sterol content : 1.4% / Total furanic compounds content : 26.7% / Total triols 1,2,4 content : 6.7% / Unsaponifiable content : 34.8%
Appearance Brown to orange coloured semi-liquid product which can be partially crystallized
Molecular weight Mean value 385 g/mol
Water solubility Insoluble
TOC* 63.5%
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
Municipal sewage treatment plant, 31137 Hildesheim, Germany. Activated sludge from the sewage plant at Hildesheim is well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste.

- Pretreatment:
The activated sludge was washed twice with chlorine free tap water. After the second washing the settled sludge was resuspended in mineral salts medium and was maintained in an aerobic condition by aeration for 20 hours. Thereafter the sludge was homogenized with a blender. After sedimentation the supernatant was decanted and maintained in an aerobic condition by aeration with CO2 free air until test start. Ten mL/L of this mixture were used to initiate inoculation.

- Initial cell/biomass concentration: Approx. 107 - 108 CFU/L
Duration of test (contact time):
28 d
Initial conc.:
15 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral salts medium acc. to OECD 301 B / CO2 Evolution Test
- Solubilising agent (type and concentration if used): Based on the results of pre-investigations performed prior to this test and based on the recommendations of relevant guidance from ECHA and ECETOC/UBA/EA for poorly water soluble substances (please refer to the point ‘Literature’), humic acid was used in additional incubation vessels in order to enhance the bioavailability of CAE.
- Test temperature: Nominal 22 ± 2 °C, actually measured 19 – 24 °C
- pH adjusted: no
- Aeration of dilution water: 30 - 100 mL/min
- Continuous darkness: yes. Low light conditions (brown glass bottles: test media excluded from light)

The following incubation vessels were prepared:

- two for the test item concentration (P1, P2)
- one for the functional control (R1)
- two for the inoculum control (C1, C2)
- one for the toxicity control (T1)
- two for the test item concentration with humic acid (P1HA, P2HA)
- two for the humic acid control (C1HA, C2HA)

TEST SYSTEM
- Method used to create aerobic conditions: The vessels were aerated for 24 h with CO2 free air.
- Test performed in open system: yes
- Details of trap for CO2 and volatile organics if used: After 24 h the CO2 adsorption vessels were connected to the air outlets of the incubation vessels via a series of 3 gas wash bottles, each containing 100 mL of a 0.0125 mol/L Ba(OH)2 solution.

Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (CO2 evolution)
Value:
100
Sampling time:
28 d
Remarks on result:
other: test item replicates with and without humic acid
Key result
Parameter:
% degradation (CO2 evolution)
Value:
60
Sampling time:
8 d
Remarks on result:
other: test item replicates without humic acid
Key result
Parameter:
% degradation (CO2 evolution)
Value:
60
Sampling time:
9 d
Remarks on result:
other: test item replicates with humic acid
Details on results:
The adaptation phase of the functional control changed after 2 days into the degradation phase (degradation >10%). The course of the degradation was rapid and the functional control reached the pass level of 60% within 6 days and a biodegradation of 100% after 28 days. The validity criterion degradation > 60% after 14 days was fulfilled.

In the toxicity control containing both test and reference item a biodegradation of 62% was determined within 7 days and it came to 96% after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

For the test item replicates without humic acid, the mean 10% level (beginning of biodegradation) was reached on day 2. The 60% pass level was reached by the mean of replicates on day 8 and the 10-day-window was fulfilled. The mean biodegradation after 28 days was 100%. For the test item replicates with humic acid, the mean 10% level (beginning of biodegradation) was reached on day 3. The 60% pass level was reached by the mean of replicates within 9 days and the 10-day-window was fulfilled. The mean biodegradation after 28 days was 100%.
Results with reference substance:
The adaptation phase of the functional control changed after 2 days into the degradation phase (degradation > 10%). The course of the degradation was rapid and the functional control reached the pass level of 60% within 6 days and a biodegradation of 100% after 28 days. The validity criterion degradation > 60% after 14 days was fulfilled.

Table 1:   CO2-Production and Biodegradation after 28 Days

CO2-Production

Functional

Test Item

Test Item

Toxicity Control

Control

with humic acid

test item +
reference item

reference item

1

2

1

2

Net        [mg/3 L]

128.7

106.2

126.3

101.2

120.6

222.6

               [mg/L]

42.9

35.4

42.1

33.7

40.2

74.2

Theor.   [mg/3 L]

127.8

104.9

104.9

232.7

               [mg/L]

42.6

35.0

35.0

77.6

Degradation [%]

100  

100

100

96

100  

96  

Table2:   CO2-Production and Biodegradation in the Inoculum Control, the Control and the Toxicity Control

Study Day

Date

Inoculum
Control

Inoculum
Control with humic acid

Functional Control

Toxicity Control

 

 

[mg CO2/3 L]

[mg CO2/3 L]

[mg CO2/3 L]

Degr.

[mg CO2/3 L]

Degr.

 

 

mv

mv

Gross

Net Sum

[%]

Gross

Net Sum

[%]

1

2014-09-26

4.1

3.2

5.8

1.7

1

4.7

0.6

0

4

2014-09-29

7.1

7.1

56.3

50.9

40

56.5

50.0

21

6

2014-10-01

4.9

5.3

44.3

90.3

71

56.5

101.6

44

7

2014-10-02

2.9

2.9

19.9

107.3

84

45.3

144.0

62

11

2014-10-06

8.0

7.9

17.4

116.7

91

41.3

177.3

76

14

2014-10-09

6.5

6.6

10.1

120.3

94

25.9

196.7

85

18

2014-10-13

6.7

8.2

11.0

124.6

97

22.6

212.6

91

21

2014-10-16

7.0

6.6

10.1

127.7

100

12.1

217.7

94

25

2014-10-20

7.3

6.9

8.1

128.5

100

9.9

220.3

95

28

2014-10-23

6.7

6.0

6.6

128.5

100

7.6

221.2

95

29*

2014-10-24

9.6

10.3

9.8

128.7

100

10.6

222.6

96

Degr. = degradation         mv = mean value *) results of last two gas wash bottles

Table3:      CO2-Production and Biodegradation in the Inoculum Control and Test Item Replicates (without humic acid)

Study Day Day

Date

Inoculum
Control

Test Item

Replicate 1

Replicate 2

 

 

[mg CO2/3 L]

[mg CO2/3 L]

Degr.

[mg CO2/3 L]

Degr.

 

 

mv

Gross

Net Sum

[%]

Gross

Net Sum

[%]

1

2014-09-26

4.1

3.4

0.0

0

3.9

0.0

0

4

2014-09-29

7.1

38.3

31.2

30

42.4

35.3

34

6

2014-10-01

4.9

26.1

52.4

50

24.0

54.4

52

7

2014-10-02

2.9

12.2

61.7

59

9.6

61.1

58

11

2014-10-06

8.0

28.2

81.9

78

35.2

88.3

84

14

2014-10-09

6.5

16.8

92.2

88

20.8

102.6

98

18

2014-10-13

6.7

14.1

99.6

95

16.7

112.6

100

21

2014-10-16

7.0

11.4

104.0

99

12.5

118.1

100

25

2014-10-20

7.3

7.7

104.4

100

9.4

120.2

100

28

2014-10-23

6.7

6.8

104.5

100

8.1

121.6

100

29*

2014-10-24

9.6

11.3

106.2

100

14.3

126.3

100

                           Degr. = degradation               mv = mean value           *) results of last two gas wash bottles

 Table 4:   CO2-Production and Biodegradation in the Inoculum Control and Test Item Replicates (with humic acid)

Study Day

Date

Inoculum
Control with humic acid

Test Item
with humic acid

Replicate 1

Replicate 2

 

 

[mg CO2/3 L]

[mg CO2/3 L]

Degr.

[mg CO2/3 L]

Degr.

 

 

mv

Gross

Net Sum

[%]

Gross

Net Sum

[%]

1

2014-09-26

3.2

3.4

0.2

0

4.1

0.9

1

4

2014-09-29

7.1

30.9

24.0

23

30.7

24.5

23

6

2014-10-01

5.3

16.7

35.4

34

26.8

46.0

44

7

2014-10-02

2.9

12.7

45.2

43

13.3

56.4

54

11

2014-10-06

7.9

32.9

70.2

67

32.7

81.2

77

14

2014-10-09

6.6

18.9

82.5

79

21.7

96.3

92

18

2014-10-13

8.2

17.2

91.5

87

18.7

106.8

100

21

2014-10-16

6.6

10.5

95.4

91

12.9

113.1

100

25

2014-10-20

6.9

9.4

97.9

93

10.5

116.7

100

28

2014-10-23

6.0

7.2

99.1

94

8.7

119.4

100

29*

2014-10-24

10.3

12.4

101.2

96

11.5

120.6

100

                           Degr. = degradation               mv = mean value           *) results of last two gas wash bottles

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the test conditions the test item CAE is classified as readily biodegradable in the 10-day-window and within 28 days.
Executive summary:

The ready biodegradability of the test item CAE (Batch No.1720) was determined with a non-adapted activated sludge over a test period of 28 days in the Modified Sturm Test. The study was conducted from 2014-09-25 to 2014-10-24, according to OECD 301 B at Dr.U.Noack-Laboratorien.

The Total Organic Content (TOC) of CAE was determined at an external test facility to be 63.5% acc. to ISO 10694:1995. The test item was tested at a concentration of 15 mg/L with 2 replicates, corresponding to a carbon content (TOC) of 9.53 mg C/L in the test vessels. Based on the results of pre-investigations performed prior to this test and based on the recommendations of relevant guidance from ECHA and ECETOC/UBA/EA for poorly water soluble substances, humic acid was used in additional incubation vessels (2 replicates) in order to enhance the bioavailability of CAE.The test vessels were incubated at low light conditions and at a temperature of 19 – 24 °C.

The biodegradation of the test item was followed by titrimetric analysis of the quantity of CO2 produced by the respiration of bacteria. The degradation was stopped on day 28 by acidification of the test solutions. The last titration was made on day 29, after residual CO2 had been purged from the test solutions over a period of 24 hours. The percentage CO2 production was calculated in relation to the theoretical CO2 production (ThCO2) of the test item. The biodegradation was calculated for each titration time.

 

To check the activity of the test system, sodium benzoate was used as functional control. The percentage degradation of the functional control reached the pass level of 60% within 6 days and a biodegradation of 100% after 28 days.

 

In the toxicity control containing both test and reference item a biodegradation of 60% was determined within 7 days and it came to 96% after 28 days. Hence the biodegradation of the reference item was not inhibited by the test itemin the toxicity control.

 

For the test item replicates without humic acid, the mean 10% level (beginning of biodegradation) was reached on day 2. The 60% pass level was reached by the mean of replicates on day 8 and the 10-day-window was fulfilled. The mean biodegradation after 28 days was 100%. For the test item replicates with humic acid, the mean 10% level (beginning of biodegradation) was reached on day 3. The 60% pass level was reached by the mean of replicates within 9 days and the 10-day-window was fulfilled. The mean biodegradation after 28 days was 100%.

 

Under the test conditions the test item is classified as readily biodegradable in the 10-day-window and within 28 days.

 

Description of key information

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information