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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:
weight of evidence
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
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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:
- Fresh activated sludge from a biological waste water treatment plant treating predominantly domestic sewage (Bois-de-Bay, Satigny, Switzerland) was used.
- 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:
60
Sampling time:
42 d
Remarks on result:
other: Taken from % biodegradation timeplot (Appendix 2)
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:
weight of evidence
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
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:
- Fresh activated sludge from a biological waste water treatment plant treating predominantly domestic sewage (City of Geneva , Aire) was used.
- 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.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 April 2019 to 25 March 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD 301F guideline study to GLP. In addition substance specific analysis was performed to quantifying residual concentration of the parent test item and of two expected primary metabolites, COOH-metabolite and CONH2-metabolite.
Justification for type of information:
Following an ECHA decision CCH-D-2114394631-45-01/F on EC:263-214-5 (3,7-dimethylnona-2,6-dienenitrile), it was requested to conduct additional toxicological studies, which included Identification of degradation products. A prolonged 301F study was performed with additional chemical specific analysis to support the PBT assessment of the registered substance i.e. that the registered substance undergoes complete degradation and that any potential metabolites are transient, not persistent and not bioaccumulative. As such the registrant considers there is no need to do further work to identify degradation products.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
In addition to the standard 301F study performed to GLP. This study includes non GLP sequential analyses for quantifying residual concentration of the test item and of two expected primary metabolites, COOH-metabolite and CONH2-metabolite
Deviations:
yes
Remarks:
Optional toxicity control not performed, measuring heads different to those foreseen in study plan, analysis for nitrite & nitrate at test end not performed - validity of the study is not adversely/negatively affected (see "Overall Remarks"for details)
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Names of test material (as cited in study report): Lemonile
- Aspect: Colourless to pale yellow liquid
- Analytical purity: 99.7 % (sum of the components)
- Lot No: PE00223799
- Expiration date of the lot: November 04, 2020
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Fresh activated sludge from a biological waste water treatment plant treating predominantly domestic sewage (Bois-de-Bay, Satigny, Switzerland) was used.
- 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):
60 d
Initial conc.:
30 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
APPARATUS
- The respirometer used during this study is an Oxitop Control System, made by Wissenschaftllch-Technische Werkstatten (WTW), Weilheim, Germany.

WATER
- The water used during this study is ultrapure water, containing less than 5 ppb total organic carbon, produced by using a Millipore Direct-Q 3 UV purification system.

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
- Prepared by mixing 50 ml of solution A and 2000 ml deionised water, adding 5 ml of each of the solutions B, C and D and making up to 5 litres with deionised water. The pH is measured and if necessary adjusted to 7.4 ± 0.2 with phosphoric acid or potassium hydroxide.

DETERMINATION OF DRY WEIGHT OF SUSPENDED SOLIDS
- The dry weight of suspended solids is determined by taking two 50 ml samples of the homogenised sludge, evaporating water on a steam bath, drying in an oven at 105 - 110°C for two hours and weighing the residue.

FLASK PREPARATION
- Test substance samples (7.65 mg, corresponding to 30 mg/L in 255 mL) were weighed in small aluminium boats and added to the contents of the test flasks.
- For reference substance samples 12.75 mg (corresponding to 50.0 mg/I in 255 ml of test medium) were weighed in small aluminium boats and added directly to the test flasks.
- Flasks were filled with 250 mL of mineral medium.
- 5.00 ml of suspended sludge diluted to a concentration of 1.53 g/L dry matter was added.
- As the substance is a neutral test item, the pH of each flask at the start of the test was not measured, in order not to remove any floating undissolved test substance from the test. The pH was assumed to be the same as the mineral medium. Neutral test substances, even sodium benzoate, have been shown not to affect the pH of the medium by more than 0.1 pH unit.
- Two sodium hydroxide pellets were placed in the quivers on top of the bottle and the flasks were closed tightly with the measuring heads.
- The flasks were allowed to equilibrate to the test temperature.
- The measurement was started by programming the measuring unit of the Oxitop test flasks, and the test flasks were placed in the temperature controlled cupboard of the Oxitop system. After temperature equilibration, the controller of the instrument starts the data acquisition (time zero of the experiment).

For non GLP sequential analyses during the biodegradation testing procedure, additional study flasks, with the test substance, were prepared and placed under the same conditions with no respirometry Oxitop system, but sealed by a cap. These additional flasks were placed in a thermostatic cabinet under the same temperature conditions.

TEST CONDITIONS
- Everyday the oxygen consumption of each flask was recorded and correct temperature and stirring checked.
- The temperature throughout the test was 21.72°C- 22.14°C
- At the end of the test period, the pH of each flask was measured

NOMINAL CONCENTRATIONS
- Test material: 30 mg/L
- Reference substance: 50 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:
- Degradation of sodium benzoate exceeded 40% after 7 days and 65% after 14 days: the activity of the inoculum was thus verified (validity criterion).
- The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled. Therefore, the test is considered valid.
Parameter:
% degradation (O2 consumption)
Value:
26
Sampling time:
28 d
Remarks on result:
other: Ultimate biodegradation
Remarks:
Taken from Table 2
Parameter:
% degradation (O2 consumption)
Value:
60
Sampling time:
45 d
Remarks on result:
other: Ultimate biodegradation
Remarks:
Taken from % biodeg timeplot (Appendix 2)
Parameter:
% degradation (O2 consumption)
Value:
73
Sampling time:
60 d
Remarks on result:
other: Ultimate biodegradation
Remarks:
Taken from Table 2
Parameter:
% degradation (test mat. analysis)
Value:
31.8
Sampling time:
22 d
Remarks on result:
other: Primary biodegradation of parent substance
Parameter:
% degradation (test mat. analysis)
Value:
27.5
Sampling time:
28 d
Remarks on result:
other: Primary biodegradation of parent substance
Parameter:
% degradation (test mat. analysis)
Value:
100
Sampling time:
60 d
Remarks on result:
other: Primary biodegradation of parent substance
Details on results:
From the main study:
- Oxygen uptakes, as read on the Oxitop controller, are corrected to account for the small differences between actual and nominal concentrations of test and reference substances. Oxygen uptake curves can be found in Appendix 1 of the attached full study report. Calculated % biodegradation curves and detailed results are reported in Appendixes 2 to 4. The oxygen uptake tables for the test item and reference substance are reproduced below in "any other information on results incl tables" - see table 2 and 3.
- Lemonile undergoes 26% biodegradation after 28 days (73% after 60 days) in the test conditions. At the test concentration, Lemonile has reduced the intrinsic respiration of the inoculum by more than 20% on the days 1 to 10 of the test. Toxicity towards the inoculum can therefore not be excluded.
- Lemonile should be regarded as not readily biodegradable according to this test. However, based on these results Lemonile should be regarded as inherently biodegradable.

From analytical investigations during biodegradation testing procedure (Appendix 5 of full study report):
- Evolution of concentrations for the test item, Lemonile, and for the two expected primary metabolites, COCH-Metabolite and CONH2Metabolite, during biodegradation testing procedure was performed using a non-GLP analytical part of the study.
- As observed on Table 29 and Figure 10 from Appendix 5 of the full study report (and in "attached background informaion"), concentration of the test item,Lemonile, decreased from TO to being below the Limit of Detection at Day 60. The measured concentrations of Lemonile (average of two replicates) were 27.39 mg/L (day 0), 28.10 mg/L (day 1), 25.83 mg/L (day 4), 25.01 mg/L (day 7), 27.31mg/L (day 14), 18.67 mg/L (day 21), 19.86 mg/L (day 28) and < LOD (day 60). This indicates that the biodegradation of Lemonile starts after day 14. The measured concentrations at day 21 and 28 are respectively 68.2 and 72.5% of the initial measured concentration, which corresponds to 31.8 and 27.5% primary biodegradation. At the end of the test, Lemonile is below the limit of detection, indicating complete (100%) primary biodegradation.
- No significant evolution in the COOH-Metabolite was observed from TO to Day 60, with the specific analytical signal remaining very low throughout the test period (determined concentration <=0.1 mg/L). In contrast, although the measured concentrations for the CONH2-Metabolite were also very low throughout the test, there was clear concentration trend with an observed peak for this specific analyte at Days 14 and 21 (0.71 mg/Land 0.61 mg/L ); it's signal was below the Limit of Detection at both TO and Day 60.
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 (validity criterion).

Table 1:Actual concentrations

Flask No.

Concentration of test substance

(mg/L)

Concentration of reference substance

(mg/L)

pH initial

pH final

51 (A1)

0

50.0

7.6

7.8

51 (A2)

0

49.9

7.6

7.8

49 (B1)

0

0

7.6

7.6

50 (B2)

0

0

7.6

7.6

53 (C1)

30.0

0

7.6

7.5

54 (C2)

30.2

0

7.6

7.5

 

 

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

-

Days

5

7

14

21

28

60

BOD of sludge

1

B1

17.5

21.5

28.3

32.3

36.3

45.8

2

B2

17.5

20.2

26.9

32.3

36.3

40.4

Mean

B

17.5

20.9

27.6

32.2

36.3

43.1

BOD test substance

1

C1

10.8

13.5

25.6

40.4

64.6

113.9

2

C2

10.8

14.8

28.3

40.4

57.8

113.6

1 corr.

C1-B

-6.7

-7.3

-2.0

8.1

28.3

70.8

2 corr

C2-B

-6.7

-6.0

0.7

8.1

21.5

70.5

% biodegradation of test substance

1

D1

-7

-8

-2

8

29

73

2

D2

-7

-6

1

8

22

73

Mean

D

-7

-7

-1

8

26

73

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

 60

O2uptake of sludge (inoculum blank)

49

B1

17.5

21.5

28.3

32.3

36.3

 45.8

50

B2

17.5

20.2

26.9

32.3

36.3

 40.4

Mean

B

17.5

20.9

27.6

32.3

26.3

 43.1

O2uptake of Reference Substance + sludge

51

A1

78.1

86.1

98.2

104.9

108.9

122.9

52

A2

79.5

86.3

101.1

108.1

114.1

129.1

O2uptake of Reference Substance

-

A1-B

60.6

65.3

70.6

72.6

72.6

79.8

-

A2-B

62.0

65.5

73.5

75.8

77.8

 86.0

% biodegradation of reference substance

-

D1

73

78

85

87

87

  96

-

D2

74

79

88

91

93

  103

Mean

D

74

79

87

89

90

  100

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)

 

Table 29 from Appendix 5. Results of determined concentrations for analytical targets in study flasks with sequential analyses during biodegradation testing procedure (dilution factor for vial preparation taken into account see section 1.3.4. in Appendix 5).

Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable
Conclusions:
Lemonile undergoes 26% biodegradation after 28 days (73% after 60 days) in the test conditions. At the test concentration, Lemonile has reduced the intrinsic respiration of the inoculum by more than 20% on the days 1 to 10 of the test. Toxicity towards the inoculum can therefore not be excluded.

Lemonile should be regarded as not readily biodegradable according to this test. However, based on these results Lemonile should be regarded as inherently biodegradable.

In a non-GLP part of the study, the concentrations of Lemonile and two expected primary metabolites were monitored as a function of time. The concentration of the parent test item, Lemonile, decreased from 27.4 mg/I (test start) to 19.9 mg/I at day 28 to become not detectable at day 60. Thus showing that Lemonile undergoes complete primary degradation.

Very low levels of the two metabolites were detected. The maximum concentration of the CONH2-metabolite was 0. 7mg/L at days 14 and 21 and was not detected at the end of the test (day 60) while the COCH-Metabolite was below the limit of quantification throughout the test. These results coupled with the high BOD at the end of the test indicate that any metabolites formed following primary degradation are transient and not persistent.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
Not applicable
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification
Remarks:
The value is not an experimental result, however the QSAR model is well documented with regard to validation parameters according to OECD principles. Moreover, the substance is fully characterised and falls within the parameter and metabolic domain. There are 8.3% structural fragments not present in the training set but this is not considered to significantly affect the predictions.
Justification for type of information:
Following an ECHA decision CCH-D-2114394631-45-01/F on EC:263-214-5 (3,7-dimethylnona-2,6-dienenitrile), it was requested to conduct additional toxicological studies, which included Identification of degradation products. A QSAR was used to support the PBT assessment of the registered substance i.e. that the registered substance undergoes complete degradation and that any potential metabolites are transient, not persistent and not bioaccumulative). As such the registrant considers there is no need to do further work to identify degradation products.

1. SOFTWARE
available in OASIS Catalogic v.5.14.1.5

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
C(#N)C=C(CCC=C(CC)C)C (3,7-dimethylnona-2,6-dienenitrile, main component of registered substance, 4 stereisomers)
N#CCC(C)=CCC=C(CC)C (3,7-dimethyl-3,6-nonadienenitrile, impurity component of regsitered substance, 2 E/Z isomers)

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached QMRF

5. APPLICABILITY DOMAIN
See attached QPRF

6. ADEQUACY OF THE RESULT
See attached QPRF
Principles of method if other than guideline:
QSAR, CATALOGIC Kinetic 301F v.14.17. More details are given in QMRF/QPRF attached to the dossier.
GLP compliance:
no
Specific details on test material used for the study:
No additional information
Oxygen conditions:
aerobic
Inoculum or test system:
other: Aerobic microorganisms
Details on inoculum:
not applicable
Duration of test (contact time):
28 d
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
not applicable
Preliminary study:
not applicable
Test performance:
not applicable
Parameter:
% degradation (O2 consumption)
Value:
83
Sampling time:
28 d
Remarks on result:
other: Prediction for 3,7-dimethylnona-2,6-dienenitrile, includes Primary Half Life = < 1 day, Ultimate Half Life = 8.63 day, Start day of 10 days window =2, BOD at 10 days window =0.6041, Classification=Ready. Structural domain = 91.67% correct ACFs
Remarks:
CATALOGIC ALERT: Nitrile suspected BOD inhibition effect
Parameter:
% degradation (O2 consumption)
Value:
82
Sampling time:
28 d
Remarks on result:
other: Prediction for 3,7-dimethylnona-3,6-dienenitrile, includes primary Half Life = < 1d, Ultimate Half Life=11.13d, Start day of 10 days window=3, BOD at 10 days window =0.5629, Classification=Not Ready. Structural domain = 91.7% correct ACFs
Details on results:
- The model provides results of % BOD and quantities of parent and biodegradation products.
- It predicts that 3,7-dimethylnona-2,6-dienenitrile will achieve 83% BOD and that 0% of the parent will remain at the end of a 28 day ready test. A similar result is obtained for 3,7-dimethylnona-3,6-dienenitrile, the isomeric impurity in Lemonile (82% BOD, 0% parent remaining).
- CATALOGIC warns of a suspected BOD inhibition effect due to the presence of the nitrile function. Models within CATALOGIC do not allow for this in predicting when biodegradation starts and thus the Kinetic 301F v14.7 model predicts that biodegradation starts on day 2 (main component) / day 3 (isomeric impurity). Experimental studies (see other endpoint study records) show that biodegradation, in fact, does not start until day 17-28 and that toxicity to the inoculum is an issue.
- In terms of predicted metabolites present in quantities of >0.1% (the PBT threshold for relevant degradation products), CATALOGIC predicts only one metabolite from the parent substance, 3,7-dimethylnona-2,6-dienenitrile, and two from the parent impurity substance, 3,7-dimethylnona-3,6-dienenitrile. These predicted metabolites are butan-2-ol (0.026 mol/mol of parent, 3,7-dimethylnona-2,6-dienenitrile and 0.027 mol/mol of parent, 3,7-dimethylnona-3,6-dienenitrile) and 6-methyloct-5-en-2-ol (0.026 mol/mol of parent, 3,7-dimethylnona-3,6-dienenitrile).
- The estimated log Kow values for the two predicted metabolites are 0.768 and 3.064, compared with 3.957 for the parent components.
- The CATALOGIC Kinetic 301F v14.7 model predicts that the butan-2-ol metabolite is readily biodegradable and the 6-methyloct-5-en-2-ol metabolite as readily biodegadable but failing the 10 day window. Both metabolites are in the parameter domain, metabolic domain and 100% in the structural domain
- The two parent substances are in the parameter domain and metabolic domain but are not completely in the structural domain. Both targets have 91.67% correct atom centre fragments (ACFs) and 8.33% unknown fragments compared to the training set. As neither target substance has incorrect fragments (i.e. fragments present in training chemicals that are incorrectly predocted), the quality of the prediction is not considered to be significantly affected. The only unknown fragment is where the center of the fragment is the methyl group in the beta-position to the nitrile. Based on an expert assessment of biotransformations described in the literature for aliphatic nitriles, the presence of the methyl group in the beta-position is not expected to affect the biotransformation pathway of nitrile hydrolysis. Therefore, the simulated metabolism is considered correct and it is premise for correct model prediction.
- Three close structural analogues were identified from a search of the CATALOGIC 301F model training set and OECD Toolbox. The observed and predicted BOD results for structural analogues are similar, supporting the reliability of the prediction for the target molecules.
2-tridecenenitrile, CAS 22629-49-8, BOD obs.= 87% (28d, 301F, Givaudan Study), BOD pred.= 83%.
3,7-dimethyloct-6-enenitrile, CAS 51566-62-2, BOD obs.= 69% (28d, 301F, Givaudan Study), BOD pred.= 82%.
3,7-dimethylocta-2,6-dienenitrile,CAS 5146-66-7, BOD obs,.= 80% (28d, 301F, Givaudan Study), BOD pred.= 82%

For additional information, see the attached QPRFs, CATALOGIC SHORT REPORT, and ANALYSIS OF UNKNOWN FRAGMENTS
Results with reference substance:
not applicable

Four potential biotransformation pathways are predicted for 3,7 -dimethylnona-2,6 -dienenitrile and three potential pathways for 3,7-dimethyl-3,6-nonadienenitrile (See Appendix 3 of respectives QPRFs and Predicted Metabolic Maps file). Similar probabilities are given for the alternative inital steps, which are:

3,7 -dimethylnona-2,6 -dienenitrile, main component (4 stereoisomers) of Lemonile

nitrile hydrolysis to the corresponding amide (Prob=0.999)

nitrile hydrolysis to the corresponding acid (Prob=0.999)

epoxide formation and hydrolysis (Prob=1.000)

methyl group hydroxylation (Prob=0.900)

3,7 -dimethyl-3,6 -nonadienenitrile, impurity component (E/Z isomers) of Lemonile

nitrile hydrolysis to the corresponding amide (Prob=0.999)

nitrile hydrolysis to the corresponding acid (Prob=0.999)

methyl group hydroxylation (Prob=0.900)

The EAWAG-BBD Pathway Prediction System was also used to predict plausible pathways for microbial degradation. The same same likelihood of Nitrile -> Carboxylate(including via the amide); aliphatic Methyl [H2] -> primary Alcohol; and secondary Aliphatic -> secondary Alcohol was predicted (i.e. neutal likelihood - metabolic maps attached).

Validity criteria fulfilled:
not applicable
Interpretation of results:
readily biodegradable
Conclusions:
3,7 -Dimethylnona-2,6 -dienenitrile is predicted to be readily biodegradable (BOD 83% after 28d) and 3,7 -dimethylnona-3,6 -dienenitrile (isomeric impurity, BOD 82% after 28d) readily but failing the 10d window. The predicted quantity of parent remaining at the end of a test is 0%. Predicted metabolites present in quantities of >0.1% are butan-2-ol and 6-methyloct-5-en-2-ol . The log Kow obtained from CATALOGIC shows that all predicted metabolites (log Kow 0.768 and 3.064) are more polar than the parent and considered as non B. The CATALOGIC Kinetic 301F v14.7 model predicts both metabolites are readily biodegradable (i.e. not P). 
Executive summary:

A QSAR was used to support the PBT assessment of the registered substance.The CATALOGIC Kinetic 301F v.13.16, plug-in from OASIS CATALOGIC v.5.13.1. has been used to predict the biodegradation properties. The model provides results of % BOD and quantities of parent and biodegradation products. 3,7 -Dimethylnona-2,6 -dienenitrile is predicted to be readily biodegradable (BOD 83% after 28d) and 3,7-dimethylnona-3,6 -dienenitrile (isomeric impurity, BOD 82% after 28d) readily but failing the 10d window. The target compounds fulfil the general properties requirements (in terms of log Kow, molecular weight and water solubility) and are in the metabolic domain of the model. The targets are not completely in the structural domain (8.3% unknown fragments). The only unknown fragment is where the center of the fragment is the methyl group in the 3-position (i.e. beta to the nitrile). From an expert assessment of biotransformations described in the literature for aliphatic nitriles, the presence of this methyl group is not expected to affect the biotransformation pathway of nitrile hydrolysis. Therefore, the simulated metabolism is considered correct and it is premise for correct model prediction. The latter is supported by experimental data for three close structural analogues.

 

CATALOGIC has also been used to explore plausible pathways for microbial degradation, likely primary biodegradation products and whether any metabolites are potentially persistent. The predicted quantity of parent remaining at the end of a test is 0%. In terms of predicted metabolites present in quantities of >0.1% (the PBT threshold for relevant degradation products), only one metabolite is predicted from 3,7-dimethylnona-2,6-dienenitrile and two from the impurity, 3,7-dimethylnona-3,6-dienenitrile. These predicted metabolites are butan-2-ol and 6 -methyloct-5-en-2-ol . The metabolites identified have estimated log Kow of 0.7676 and 3.064 respectively, and are considered as non B / vB. The CATALOGIC Kinetic 301F v14.7 model also predicts both metabolites are readily biodegradable (i.e. not P). The not P and not B status for metabolite butan-2-ol is confirmed by data on the ECHA website for CAS 78-92-2. 

Description of key information

Three reliable ready biodegradability tests according to OECD 301F are available for the registered substance. At the end of the standard test duration of 28 days, the level of biodegradation (measured as % ThOD) was 10, 32 and 26%. Thus, the registered substance cannot be considered as readily biodegradable.

Since degradation had started and a plateau had not been reached, the tests were extended for the purpose of assessing inherent, ultimate biodegradability. The biodegradation continued reaching levels of 60% (day 42), 70% (day 57) and 73% (day 60) at the end of each respective test. Based on these results, the registered substance is considered to be inherently and ultimately biodegradable. Furthermore, given that a result of more than 60% ultimate biodegradability (ThOD) was obtained in enhanced ready biodegradability tests, indicates that the registered substance and its underlying constituents are not P/vP for the purposes of the PBT/vPvB assessment. This is supported by chemical specific analysis in one of the tests (Study No 19-E098), which showed that no parent substance remained at the end of the test.

Two potential primary metabolites, the corresponding acid and corresponding amide, were also quantitatively analysed by LC-MS in Study No 19-E098. The concentration of the acid metabolite was below the limit of quantification throughout the test. The measured concentrations for the amide metabolite were also very low throughout the test. However, there was a concentration trend for this specific analyte with maximum values at Days 14 and 21. It's signal was below the Limit of Detection at both the start (day 0) and end (Day 60) of the test. These results coupled with the high BOD at the end of the test (73%, day 60) indicate that any metabolites formed following primary degradation are transient and not persistent. This is supported by CATALOGIC predictions.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, fulfilling specific criteria

Additional information

Three reliable ready biodegradability tests according to OECD 301F are available for the registered substance (Givaudan Roure 1995, Study No. 95-E04; Givaudan Suisse 2010, Study No. 09-E174; Dutriez 2020, Study No. 19-E098). At the end of the standard test duration of 28 days, the level of biodegradation (measured as BOD) was 10, 32 and 26% respectively. Thus, the registered substance cannot be considered readily biodegradable.   

 

All three studies were extended beyond the 28 days for the purpose of assessing inherent, ultimate biodegradability. Specific chemical analysis of the parent substance and two target primary metabolites (the corresponding amide and acid) was included in Study No. 19-E098 to address an ECHA decision (CCH-D-2114394631-45-01/F) requesting the “Identification of degradation products (Annex IX, 9.2.3.) using an appropriate test method with the registered substance”. The identification of the degradation products is a standard information requirement according to column 1, Section 9.2.3. of Annex IX of the REACH Regulation for a substance that is not readily biodegradable and ECHA considered that this information was needed in relation to the PBT/vPvB assessment of the registered substance. CATALOGIC and the EAWAG-BBD Pathway Prediction System have also been used to predict plausible pathways for microbial degradation, likely primary biodegradation products and whether any metabolites are potentially persistent.The results of the three extended ready biodegradability tests (60%-73% BOD), indicate the high likelihood of ultimate biodegradation. Specific chemical analysis confirmed that no parent substance remained at the end of a prolonged 301F test indicating that the registered substance undergoes complete primary degradation. Further no significant evolution of the corresponding acid metabolite was observed, while the corresponding amide metabolite increased slightly to a maximum at days 14/21 and then declined to be non-detectable by the end of the test. These results coupled with the high BOD at the end of the test (73%, day 60) indicate any metabolites formed following primary degradation are transient and not persistent. This is supported by CATALOGIC predictions. Given that the parent substance itself is not bioaccumulative (measured log Kow 3.1 to 3.2, estimated BCF values of 37 to 105) and that biodegradation typically leads to more polar metabolites, any transient metabolites are likely to have a lower bioaccumulation potential than the parent substance. Therefore there is a low likelihood of degradation products with PBT properties (i.e. any metabolites formed following primary degradation are transient, not persistent and not bioaccumulative). As such the registrant considers there is no need to do further work to identify degradation products. A more detailed justification is outlined in the following paragraphs.  

 

An extended ready biodegradability test is a recognised “enhanced biodegradation screening test”.  These enhancements are designed to improve the environmental relevance of biodegradability assessment for persistence assessment without the requirement for simulation testing. The ECHA guidance (version 3.0, June 2017, section R.11.4.1.1) outlines the conditions that should be met when positive results from an enhanced screening test may be used to conclude that a substance is not P/vP. These are: 1) the enhancements should only be about an extended test duration or an increased test vessel size, 2) the test should be performed with non-pre-adapted/non pre-exposed inocula, 3) the test duration should never be extended beyond 60 days, and 4) the test criteria set for ready biodegradability tests should be applied, i.e. 60% ultimate biodegradability (ThOD, CO2 evolution) or 70% ultimate biodegradability (DOC removal), without the 10-day window.  

 

The above conditions were met in all extended OECD 301F studies performed on the registered substance. The pass criteria of 60% ThOD was achieved well before day 60 (i.e. on day 42 for Studies 95-E04 and 09-E714 and day 45 for study 19-E098), and the inoculum was fresh activated sludge from biological waste water treatment plants (WWTPs) treating predominantly domestic sewage that do not receive releases from sites using the substance (City of Geneva , Aire in Study 95-E04;  Bois-de-Bay, Satigny, Switzerland in Studies 09-E174 and 19 -E098). Furthermore, the three studies were performed at different times with different inoculum batches. Therefore the results provide a good weight-of-evidence of the biodegradation properties of the registered substance.

 

Study No 95-E04 was stopped on day 42 when the level of biodegradation had reached the 60% pass criteria to conclude that a substance is not P/vP. However, the other two studies were continued for a further 15 and 18 days. The final levels of degradation achieved were 70% (day 57, Study 09-E714) and 73% (day 60, Study 19-E098) respectively. The high levels of biodegradation obtained in the extended tests indicate that i) any degradation products formed following primary degradation of the parent substance are further metabolised and ii) both the parent substance and any transient breakdown products or metabolites are not persistent. Furthermore, microbial attack generally leads to more polar metabolites. Given that the parent substance itself is not bioaccumulative (measured log Kow 3.1 to 3.2, estimated BCF values of 37 to 105), there is a low likelihood of bioaccumulative degradation products.

 

The registered substance, tradename Lemonile, is a multi-constituent substance (four isomers). The registrant acknowledges that standard biodegradation screening tests are not ideally suited to mixtures because they measure ultimate biodegradation as a function of either the CO2 evolved or O2 consumed and as such do not provide information on the biodegradability of individual constituents. However, in the case of substances that are composed of structurally similar constituents that are expected to have similar degradation potential, standard screening biodegradation tests are still suitable. The OECD guideline (2006) specifically describes such tests being suitable for a substance which consists of “constituents with different chain-lengths, degree and/or site of branching or stereoisomers”. The ECHA guidance (version 3.0, June 2017, section R.11.4.2.2.3) states that “If the selected test item consists of sufficiently similar structures and is shown to meet the stringent ultimate ready biodegradation test criterion (>60% in 28 days), it can be concluded that the underlying constituents comprising the complex substances are not expected to be persistent”. It follows, that the same argument may be applied to positive results from an enhanced biodegradation screening test. The four isomers present in the registered substance (3,7-dimethylnona-2,6-dienenitrile) vary only in the stereochemistry of the two double bonds present i.e.  2Z/6Z, 2Z/6E, 2E/6Z and 2E/6E. The substance also contains an isomeric impurity, 3,7-dimethyl-3,6-nonadienenitrile (mixture of two E/Z isomers). The only difference between this impurity and the main constituent isomers is the position of the double bond. Neither the difference in stereochemistry nor the position of the double bond is expected to significantly affect the biodegradation properties of these isomers. As such the registered substances and its underlying constituents are considered not P/vP based on the positive results of three enhanced biodegradation screening tests.

 

The constituents of the registered substance are organic nitriles. The biodegradation of organic nitriles requires the presence of microbes that are endowed with nitrilase, nitrile hydratase and amidase systems which transform nitriles to acids and amides (Bhalla et al, 2012). There are two different enzymatic pathways: 1) Nitrile hydratase catalyzes the hydration of nitriles to corresponding amides and amidase further to acid and ammonia and 2) nitrilase catalyzes the direct hydrolysis of nitriles to corresponding acid and ammonia. The widespread natural occurrence of these bacterial enzymes (soil, plant material, fungi) is well documented (Kobayashi et al., 1995; Gong et al., 2012) and it can be assumed that these enzymes will be broadly present in sewage treatment plant microbial populations. Therefore, potential primary steps in the biodegradation of Lemonile are conversion of the nitrile functionality to the amide and/or carboxylic acid. CATALOGIC and the EAWAG-BBD Pathway Prediction System support this hypothesis. 

 

Reference samples of 3,7-dimethylnona-2,6-dienoic acid (COOH-metabolite) and 3,7-dimethylnona-2,6-dienamide (CONH2-metabolite) were synthesised by Givaudan’s research centre. These reference substances were synthesised from the parent substance, Lemonile, and therefore the isomeric ratio of the corresponding constituents are expected to remain similar. In a non-GLP part of study 19-E098, the concentrations of Lemonile and the two expected primary metabolites were quantitatively analysed by LC-MS at the start of the test and on days 1, 4, 7, 14, 21, 28 and 60. The test item and the two expected primary degradation compounds gave a chromatographic profile consisting of several peaks, thereof two main peaks, for which the area changed accordingly with known concentration. The concentration calculations were based on the sum of these peaks. The measured concentrations of Lemonile (average of two replicates) were 27.39 mg/L (day 0), 28.10 mg/L (day 1), 25.83 mg/L (day 4), 25.01 mg/L (day 7), 27.31mg/L (day 14), 18.67 mg/L (day 21), 19.86 mg/L (day 28) and < LOD (day 60). This indicates that the biodegradation of Lemonile starts after day 14. The measured concentrations at day 21 and 28 are respectively 68.2 and 72.5% of the initial measured concentration, which corresponds to 31.8 and 27.5% primary biodegradation. At the end of the test, Lemonile is below the limit of detection, indicating complete primary biodegradation. Very low levels of the two metabolites were detected. The maximum concentration of the CONH2-metabolite was 0. 7mg/L at days 14 and 21 and was not detected at the end of the test (day 60) while the COOH-Metabolite was below the limit of quantification throughout the test. The substance specific analysis confirms that the parent test item undergoes complete primary biodegradation and is therefore not persistent. The target metabolite analysis coupled with the high BOD at the end of the test (73% day 60) indicates that any metabolites formed following primary degradation are transient and not persistent. This is supported by CATALOGIC predictions.

 

Probable biodegradation pathways have been simulated using the CATALOGIC Kinetic 301F v14.17 model. Two chemical structures were assessed, 3,7-dimethylnona-2,6-dienenitrile (the isomeric main constituents) and 3,7-dimethylnona-3,6-dienenitrile (the isomeric impurity). The target compounds fulfil the general properties requirements (in terms of log Kow, molecular weight and water solubility) and are in the metabolic domain of the model. The targets are not completely in the structural domain (8.3% unknown fragments). The only unknown fragment is where the center of the fragment is the methyl group in the 3-position (i.e. beta to the nitrile). From an expert assessment of biotransformations described in the literature for aliphatic nitriles, the presence of this methyl group is not expected to affect the biotransformation pathway of nitrile hydrolysis. Therefore, the simulated metabolism is considered correct and it is premise for correct model prediction. The latter is supported by experimental data for three close structural analogues. See QPRF for details.

The model provides results of % BOD and quantities of parent and biodegradation products. It predicts that 3,7-dimethylnona-2,6-dienenitrile will achieve 83% BOD and that 0% of the parent will remain at the end of a 28 day ready test. A similar result is obtained for 3,7-dimethylnona-3,6-dienenitrile, the isomeric impurity in Lemonile. These results support complete primary biodegradation of the registered substance. CATALOGIC warns of a suspected BOD inhibition effect due to the presence of the nitrile function. However, models within CATALOGIC cannot allow for this in predicting when biodegradation starts and thus the Kinetic 301F v14.7 model predicts that biodegradation starts on day 2 (main component) / 3 (isomeric impurity). Experimental studies show that biodegradation, in fact, does not start until day 17-28 and that toxicity to the inoculum is an issue (see following paragraphs for details). Further they show that following the onset of biodegradation, the pass criterion of 60% is achieved within approximately 14-25 days (i.e. on day 42-45). Therefore, if one ignores the start day, the biodegradation profile predicted by CATALOGIC is aligned to that observed experimentally. In terms of predicted metabolites present in quantities of >0.1% (the PBT threshold for relevant degradation products) at the end of a 301F test, CATALOGIC predicts only one metabolite from the parent substance, 3,7-dimethylnona-2,6-dienenitrile, and two from the parent impurity substance, 3,7-dimethylnona-3,6-dienenitrile. These predicted metabolites are butan-2-ol (0.026 mol/mol of parent, 3,7-dimethylnona-2,6-dienenitrile and 0.027 mol/mol of parent, 3,7-dimethylnona-3,6-dienenitrile) and 6-methyloct-5-en-2-ol (0.026 mol/mol of parent, 3,7-dimethylnona-3,6-dienenitrile). Given that the impurity component is present in the registered substance at < 10%, then the total predicted quantities for the two metabolites are approximately 2.61% (0.9*2.6% + 0.1*2.7%) butan-2-ol and 0.26% 6-methyloct-5-en-2-ol. The estimated log Kow values for the two predicted metabolites are 0.768and 3.064, compared with 3.957 for the parent components (i.e. not B). The CATALOGIC Kinetic 301F v14.7 model also predicts both metabolites are readily biodegradable (i.e. not P). The not P and not B status for metabolite butan-2-ol is confirmed by data on the ECHA website for CAS 78-92-2. 

 

One rationale for prolongation of the test duration in an enhanced biodegradation screening test is to give the microorganisms more time for accessing and degrading the substance. This is particularly pertinent for poorly water soluble substances, where poor bioavailability of the substance can limit the degradation rate. The water solubility of the registered substance is 42 mg/L. The test substance concentrations employed in the aforementioned biodegradation screening tests were 100 mg/L (Study No 95-E04), 30 mg/L (Study No 19-E098) and 20 mg/L (Study No 09-E174). The time taken for biodegradation to officially start (defined as 10%) was respectively 28, 23 and 17 days. The trend in the onset of biodegradation for the three tests indicates that solubility may have played a part in limiting the degradation rate, particularly for the test performed at 100mg/L, which was above the water solubility of the test item.

 

Another explanation for the delayed start in biodegradation is toxicity to the inoculum. All three tests showed inhibition to the inoculum at the start of the test, with negative biodegradation levels observed during the first 10 days (Study No 95-E04), 14 days (Study No 19-E098) and 4 days (Study No 09-E174). In Study No 95-E04 the toxicity was assessed, as suggested in the OECD 301F method, by comparing the respiration of a flask containing the test chemical and a readily biodegradable reference substance with the respiration of a flask containing only the readily biodegradable reference substance. Since the flask containing test substance + reference item did not have a lower respiration than the flask containing only the reference item, it was concluded that the test chemical was not inhibitory. However, a toxicity test which is based on the effect of a test substance on the degradation of a readily biodegradable reference item may be less sensitive than assessing the intrinsic respiration of the inoculum to the test substance alone. Two key issues in using the former method to evaluate the toxicity of a substance to be assessed for biodegradability are described by Reynolds et al (1987). The first is that for easily degradable substrates the initial number of active microorganisms in the inoculum will not be critical. Even if a large proportion of the inoculum is killed, the remaining organisms will proliferate and degrade the substance, still giving a high degradation rate with no indication of the toxic effect of the test substance added. The second is that the effect of the test substance on the degradation of a reference substance may not reflect the effects of the test substance on those microorganism species responsible for degrading it. Therefore, the registrant considers that the influence of toxicity in delaying the start of degradation cannot be ruled out for this substance.  In addition, under realistic environmental exposure conditions where the substance will be present at lower non-toxic concentrations, it is hypothesised that a lag-phase will not be observed, resulting in more efficient and rapid biodegradation kinetics.  The Environmental Risk Assessment in the Chemical Safety Report accompanying the submission, derives a maximum worse-case scenario Predicted Environmental Concentration entering a Sewage Treatment Plant (PECSTP) of 0.023mg/L (local PEC for fragrance compounding scenarios) which is approx. 870 to 4,350 times less than that employed in the biodegradation screening studies (20-100mg/L).

 

A further rationale for the delayed start and initial slow rate of biodegradation as evidence by the % biodegradation time plots (based on O2 uptake of the test substance), is that the first step(s) in the biodegradation pathway may be rate limiting and that once the products of primary degradation are formed they are more easily metabolised. The chemical specific analytical results from the 19-E098 study support this hypothesis in that no significant evolution of the COOH-Metabolite analytical target was observed, while the CONH2-metabolite analytical target increased slightly to a maximum at days 14/21 and was not detected by the end of the test. The acid and amide are two expected primary metabolites. However, other potential sites for initial microbial attack are possible, such as hydroxylation of methyl/CH2 groups or epoxidation of double bonds. For instance, the CATALOGIC Kinetic 301F v14.17 predicts similar probabilities for the amide, the acid, an epoxide or a hydroxylated metabolite being primary biodegradation products; while the EAWAG-BBD Pathway Prediction System assigns the same likelihood of Nitrile -> Carboxylate(including via the amide); aliphatic Methyl [H2] -> primary Alcohol; and secondary Aliphatic -> secondary Alcohol (metabolic maps attached). Whatever the exact identity of the metabolites from primary degradation, the more rapid degradation observed in the second phase of the degradation process (as indicated by the study time plots) indicates that the metabolites formed are degraded further and, based on the high levels of ultimate biodegradation achieved, can be assumed to be minimal and transient.

 

Biodegradation screening tests are performed under very stringent conditions compared to relevant environmental conditions. Thus the results from such tests are considered conservative and worst case.  Given that the registration substance achieved ≥ 60% biodegradation in three independent enhanced biodegradation screening tests it is consider that the substance and its underlying constituents are not P/vP and that any metabolites formed are transient (i.e. not potentially persistent). Furthermore, microbial attack generally leads to more polar metabolites and given that the parent substance itself is not bioaccumulative (measured log Kow 3.1 to 3.2, estimated BCF values of 37 to 105), any transient metabolites are likely to have a lower bioaccumulation potential than the parent substance. Thus, the registrant considers that there is no need to identify degradation products in order to assess their potential PBT/vPvB properties.

 

References:

 

Bhalla T.C., Sharma N., Bhatia R.K. (2012) Microbial Degradation of Cyanides and Nitriles. In: Satyanarayana T., Johri B., Anil Prakash (eds) Microorganisms in Environmental Management. Springer, Dordrecht

 

Gong J. N., Lu Z-M.,Li H., Shi J-S., Zhou Z-M., Xu Z-H. 2012.  Nitrilases in nitrile biocatalysis: Recent progress and forthcoming research.  Microb. Cell Fact., 11, 142

 

Kobayashi M., Suzuki T, Fujita T., Masuda M.,Shimizu S. (1995).  Occurrence of enzymes involved in biosynthesis of Indole-3-Acetic Acid from Indole-3 -Acetonitrile in plant-associated bacteria,AgrobacteriumandRhizobium.  Proc. Natl. Acad. Sci. USA, Vol. 92, 714-718.

 

OECD (2006). OECD Guidelines for the Testing of Chemicals, Revised Introduction To The OECD Guidelines For Testing Of Chemicals, Section 3 Part I: Principles And Strategies Related To The Testing Of Degradation Of Organic Chemicals

 

Reynolds L., Blok J., de Morsier A., Gerike P., Wellens H., Bontinck, W.J. (1987).Evaluation of the toxicity of substances to be assessed for biodegradability. Chemosphere, 16, 2259.