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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 November 2009 to 09 February 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
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
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Names of test material (as cited in study report): Lemonile; 3,7-dimethyl-2,6-nonadienenitrile
- Substance type: Colourless liquid
- Physical state: Liquid
- Analytical purity: 99.3 % (sum of components)
- Lot No: VE00066885
- Expiration date of the lot: 2012-02-06
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Sludge was collected in the morning, washed three times in the mineral medium by centrifuging at 1000 g for 10 minutes, discarding the supernatant and resuspending in mineral medium.
- The sludge was kept under aerobic conditions and used the same day.
Duration of test (contact time):
57 d
Initial conc.:
20 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
APPARATUS
- Respirometer: Oxitop Control System

WATER
- Deionised water containing less than 10 mg/L dissolved organic carbon was used during the study.

STOCK SOLUTIONS
- Solution A: KH2PO4 8.5 g; K2HPO4 21.75 g, Na2HPO4.2H2O 33.4 g; NH4Cl 0.5 g (dissolved in water and made up to 1 L).
- Solution B: CaCl2 27.5 g (dissolved in water and made up to 1 L).
- Solution C: MgSO4.7H2O 22.5 g (dissolved in water and made up to 1 L).
- Solution D: FeCl3.6H2O 0.25 g; concentrated HCl one drop (dissolved in water and made up to 1 L).

MINERAL MEDIUM
- Solution A (50 mL) and deionised water (2000 mL) were mixed and Solution B (5 mL), Solution C (5 mL) and Solution D (5 mL) added before making up to 5 L with deionised water.

DETERMINATION OF DRY WEIGHT OF SUSPENDED SOLIDS
- Two 50 mL samples of the homogenised sludge were taken and water was evaporated on a steam bath, dried in an oven at 105-110 °C for two hours, and weighing the residue.

FLASK PREPARATION
- Test substance samples (5.1 mg, corresponding to 20 mg/L in 255 mL) were weighed in small aluminium boats and added to the contents of the test flasks.
- The reference substance was added as 1.00 mL of a 10.0 g/L solution in mineral medium to give a total volume of 103 mL.
- Flasks were filled with 250 mL of mineral medium (flasks containing the reference substance: 100 mL).
- Suspended sludge diluted to a concentration of 1.53 g/L dry matter was added.
- pH of each flask was measured and, if necessary, adjusted to 7.4 ± 0.2 with phosphoric acid or potassium hydroxide.
- Two sodium hydroxide pellets were placed in the quivers on top of the bottle.
- The flasks were then closed tightly with the measuring heads and cooled to about 18-20 °C.
- After temperature equilibration, the controller of the instrument starts the data acquisition (time zero of the experiment).

TEST CONDITIONS
- Correct temperature (22 °C) and stirring were checked daily.
- Oxygen consumption of each flask was recorded daily.
- pH was measured for a second time in each flask at the end of the study.

NOMINAL CONCENTRATIONS
- Test material: 20 mg/L
- Reference substance: 100 mg/L

TOXICITY CONTROL
- An optional toxicity control was not performed. The validity of the study was not adversely affected.
Reference substance:
benzoic acid, sodium salt
Test performance:
- The activity of the inoculum was verified.
- The repeatability validity criterion (not more than 20% difference between replicates) was fulfilled.
Parameter:
% degradation (O2 consumption)
Value:
32
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
70
Sampling time:
54 d
Details on results:
Lemonile undergoes 32% degradation after 28 days (70% after 54 days) under the test conditions. The 10-day window criterion is not fulfilled (11% biodegradation on day 17 and 29% on day 27).
Results with reference substance:
Degradation of sodium benzoate exceeded 40% after 7 days and 65% after 14 days. The activity of the inoculum was thus verified.

Table 1:Actual concentrations

Flask No.

Concentration of test substance

(mg/L)

Concentration of reference substance

(mg/L)

pH initial

pH final

17

20.0

0

7.6

7.32

18

20.0

0

7.6

7.33

1

0

0

7.6

7.35

2

0

0

7.6

7.33

17

0

99.0

7.6

7.99

18

0

99.0

7.6

7.99

 

 

Table 2:O2uptake for Lemonile (mg O2/L, adjusted to nominal concentrations)

-

Days

7

14

16

17

27

28

57

O2uptake of sludge (inoculum blank)

1

B1

14.8

21.5

21.5

21.5

28.3

28.3

37.7

2

B2

13.5

20.2

21.5

21.5

31.0

31.0

44.4

Mean

B

14.2

20.9

21.5

21.5

29.7

29.7

41.1

O2uptake of Test Substance + sludge

13

C1

16.2

24.2

26.9

28.3

55.2

56.5

88.8

14

C2

17.5

25.6

26.9

28.3

41.7

44.4

83.5

O2uptake of Test Substance

-

C1-B

2.1

3.4

5.4

6.8

25.6

26.9

47.8

-

C2-B

3.4

4.8

5.4

6.8

12.1

14.8

42.5

% biodegradation of test substance

-

D1

3

5

8

11

40

42

74

-

D2

5

7

8

11

19

23

66

Mean

D

4

6

8

11

29

32

70

Calculations:

B1, B2, C1, C2: experimental O2uptake values

B = (B1+B2)/2

D1 = 100*(C1-B)/ThOD*[S]

D2 = 100*(C2-B)/ThOD*[S]

D = (D1+D2)/2

ThOD: 3.23 mg O2/mg

[S]: Initial test substance concentration (mg/L)

 

 

Table 3:O2uptake for Sodium Benzoate (mg O2/L, adjusted to nominal concentrations)

Days

5

7

14

21

28

O2uptake of sludge (inoculum blank)

1

B1

14.8

14.8

21.5

25.6

28.3

2

B2

13.5

13.5

20.2

26.9

31.0

Mean

B

14.2

14.2

20.9

26.3

29.7

O2uptake of Reference Substance + sludge

17

A1

131.2

146.3

162.4

172.4

183.5

18

A2

141.3

152.4

172.5

188.6

198.7

O2uptake of Reference Substance

-

A1-B

117.0

132.2

141.6

146.2

153.9

-

A2-B

127.1

138.2

151.7

162.4

169.0

% biodegradation of reference substance

-

D1

70

79

85

88

92

-

D2

76

83

91

97

101

Mean

D

73

81

88

93

97

Calculations:

B1, B2, A1, A2: experimental O2uptake values

B = (B1+B2)/2

D1 = 100*(A1-B)/ThOD*[S]

D2 = 100*(A2-B)/ThOD*[S]

D = (D1+D2)/2

ThOD: 1.67 mg O2/mg

[S]: Initial reference substance concentration (mg/L)

 

Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable
Conclusions:
Lemonile is considered as not readily biodegradable but inherently biodegradable according to this test.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
05 January 1995 to 17 February 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Names of test material (as cited in study report): Lemonile; 3,7-dimethyl-2(3),6-nonadienonitrile
- Substance type: Yellow liquid
- Physical state: Liquid
- Analytical purity: 98.8 % (GC, sum of components)
- Lot No: 001165
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Sludge was collected in the morning, washed three times in the mineral medium by centrifuging at 1000g for 10 minutes, discarding the supernatant and resuspending in mineral medium.
- The sludge was kept under aerobic conditions and used the same day.
Duration of test (contact time):
42 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
APPARATUS
- Respirometer: SAPROMAT D2

WATER
- Deionised water containing less than 10 mg/L dissolved organic carbon was used during the study.

STOCK SOLUTIONS
- Solution A: KH2PO4 8.5 g; K2HPO4 21.75 g, Na2HPO4.2H2O 33.4 g; NH4Cl 0.5 g (dissolved in water and made up to 1 L).
- Solution B: CaCl2 27.5 g (dissolved in water and made up to 1 L).
- Solution C: MgSO4.7H2O 22.5 g (dissolved in water and made up to 1 L).
- Solution D: FeCl3.6H2O 0.25 g; concentrated HCl one drop (dissolved in water and made up to 1 L).

MINERAL MEDIUM
- Solution A (50 mL) and deionised water (2000 mL) were mixed and Solution B (5 mL), Solution C (5 mL) and Solution D (5 mL) added before making up to 5 L with deionised water.

DETERMINATION OF DRY WEIGHT OF SUSPENDED SOLIDS
- Two 50 mL samples of the homogenised sludge were taken and water was evaporated on a steam bath, dried in an oven at 105-110 °C for two hours, and weighing the residue.
- Dry weight of suspended solids: 2.583 g/L

TOXICITY OF THE TEST CHEMICAL
- The toxicity of the test chemical to the inoculum was checked.
- A pair of flasks of the volumetric respirometer were filled with mineral medium, test chemical (100 mg/L), aniline (100 mg/L) and inoculum.
- Respiration was recorded.
- If respiration is lower than that of the flasks containing mineral medium, aniline (100 mg/L) and inoculum then the test chemical is assumed to be inhibitory to the inoculum used.

FLASK PREPARATION
- A volume of suspended sludge corresponding to 7.5 mg dry weight was placed in a 250 mL flask and made up with mineral medium.
- Test substance and reference samples were weighed in small aluminium boats and added to the contents of the test flasks.
- pH of each flask was measured and, if necessary, adjusted to 7.4 ± 0.2 with phosphoric acid or potassium hydroxide.
- About 2 g of soda lime was placed in an attachment of the stopper.
- The flasks were then closed and placed in the water bath of the SAPROMAT.
- After temperature and pressure equilibration, the oxygen meters of the instrument were set to zero (time zero of the experiment).

TEST CONDITIONS
- Correct temperature (22 °C) and stirring were checked daily.
- Oxygen consumption of each flask was recorded daily.
- pH was measured for a second time in each flask at the end of the study.

NOMINAL CONCENTRATIONS
- Test material: 100 mg/L
- Reference substance: 100 mg/L
Reference substance:
aniline
Test performance:
Since there was an increase of the pH in the flasks containing Lemonile, the nitrile nitrogen was supposed to become an ammonium ion (alkaline) and not a nitrile or nitrate ion (which are both acidic). The theoretical oxygen demand (ThOD) was calculated accordingly.
Parameter:
% degradation (O2 consumption)
Value:
10
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
60
Sampling time:
42 d
Details on results:
Under the conditions of the test, Lemonile undergoes 60% biodegradation after 42 days but only 10% after 28 days. Biodegradation starts after a long (28 day) lag phase and reaches 53% at the end of the 10 day window (days 28 to 38). Thus, Lemonile should be regarded as not readily biodegradable according to this test. However, the results show that biodegradation occurs after adaptation of the micro-organisms.
At the concentration used in the test (100 mg/L), Lemonile was not inhibitory to the micro-organisms.
Results with reference substance:
Degradation of aniline exceeded 40% after 7 days and 65% after 14 days. The activity of the inoculum was thus verified.

Table 1:Actual concentrations

Flask No.

Concentration of test substance

(mg/L)

Concentration of reference substance

(mg/L)

pH initial

pH final

2/9

99.6

0

7.35

7.51

2/10

101.8

0

7.36

7.51

1/9

100.3

100.7

7.58

8.09

1/10

99.9

100.1

7.46

8.12

1/1

0

0

7.52

7.45

2/1

0

0

7.23

7.42

1/2

0

104.2

7.48

7.68

2/2

0

101.3

7.37

7.75

 

 

Table 2:Biological oxygen demand for Lemonile (BOD, mg O2/L, adjusted to nominal concentrations)

-

Days

7

14

21

28

38

42

BOD sludge

1

B1

16.0

18.0

18.0

18.0

18.0

18.0

2

B2

23.0

29.0

31.0

34.0

36.0

37.0

Mean

B

19.5

23.5

24.5

26.0

27.0

27.5

BOD test substance

1

C1

17.0

29.0

42.1

57.1

167.6

196.7

2

C2

15.1

23.0

33.8

50.5

185.1

198.8

1 corr.

C1-B

-2.5

5.5

17.6

31.1

140.6

169.2

2 corr

C2-B

-4.4

-0.5

9.3

24.5

158.1

171.3

% biodegradation of test substance

1

D1

-1

2

6

11

49

60

2

D2

-2

0

3

9

56

60

Mean

D

-1

1

5

10

53

60

Calculations:

B1, B2, C1, C2: experimental O2uptake values

B = (B1+B2)/2

D1 = 100*(C1-B)/ThOD*[S]

D2 = 100*(C2-B)/ThOD*[S]

D = (D1+D2)/2

ThOD: 2.84 mg O2/mg

[S]: Initial test substance concentration (mg/L)

 

 

Table 3:Biological oxygen demand for aniline (BOD, mg O2/L, adjusted to nominal concentrations)

-

Days

3

5

10

14

21

28

BOD sludge

1

B1

16.0

16.0

17.0

18.0

18.0

18.0

2

B2

18.0

21.0

26.0

29.0

31.0

34.0

Mean

B

17.0

18.5

21.5

23.5

24.5

26.0

BOD reference substance

1

A1

13.2

141.9

173.7

174.7

176.7

178.6

2

A2

18.0

156.2

185.8

190.8

198.7

202.7

1 corr.

A1-B

-3.8

123.4

152.2

151.2

152.2

152.6

2 corr

A2-B

1.0

137.7

164.3

167.3

174.2

176.7

% biodegradation of reference substance

1

D1

-2

51

63

63

63

63

2

D2

0

57

68

69

72

73

Mean

D

-1

54

66

66

68

68

Calculations:

B1, B2, A1, A2: experimental O2uptake values

B = (B1+B2)/2

D1 = 100*(A1-B)/ThOD*[S]

D2 = 100*(A2-B)/ThOD*[S]

D = (D1+D2)/2

ThOD: 2.41 mg O2/mg

[S]: Initial reference substance concentration (mg/L)

 

Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable
Conclusions:
Lemonile is regarded as not readily biodegradable, however, it is considered to be ultimately and inherently biodegradable.

Description of key information

Lemonile is considered to be inherently biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, fulfilling specific criteria

Additional information

A reliable ready biodegradability study was performed in accordance with GLP and OECD Guideline 301F (Givaudan Suisse SA, 09-E174). Lemonile underwent 32% biodegradation after 28 days (70% after 57 days) under the test conditions and the 10-day window criterion was not fulfilled (11% biodegradation on day 17 and 29% on day 27). Lemonile is therefore considered to be not readily biodegradable, but is considered to be inherently biodegradable and not persistent.

This result is supported by a second ready biodegradation study (Givaudan Roure SA, 95-E04) in which Lemonile underwent 60% biodegradation after 42 days but only 10% after 28 days. Again suggesting that Lemonile is inherently biodegradable and not persistent.

These studies demonstrate that Lemonile undergoes biodegradation but it is not considered to be rapidly biodegraded.