2-(3,5-dimethylhex-3-en-2-yloxy)-2-methylpropyl cyclopropanecarboxylate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 29 April 2008 to 16 July 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 17 and 18 March 2008 / Date of signature: 23 June 2008

Specific details on test material used for the study:

- Theoretical Oxygen Demand (ThOD): 2.56 mg O2/mg test substance

Batch No. : LDP0800001
Purity : 97.0%
Date of Expiry : 01 May, 2010

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Fresh activated sludge from a biological waste water treatment plant treating predominantly domestic sewage (City of Geneva, Peney-Dessous) was used.
The sludge is 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) and kept aerobic until being used on the same day.
To obtain a concentration of 30 mg/L (dry weight) in a 250 mL flask, 1.69 mL of slude is needed (inoculum).

Duration of test (contact time):

62 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Water: The water used during this study is deionised water containing less than 10 mg/L dissolved organic carbon.
- Composition of medium:
The following stock solutions were prepared:
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 litre.
Solution B: CaCl2: 27.5 g; dissolved in water and made up to 1 litre.
Solution C: MgSO4.7H2O: 22.5 g; dissolved in water and made up to 1 litre.
Solution D: FeCl3.6H2O: 0.25 g; HCl Conc.: one drop; dissolved in water and made up to 1 litre.
The mineral medium was 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.
- Test temperature: 22°C
- pH: See table 5.2.1 in "Any other information on materials and methods incl. tables".
- Suspended solids concentration: dry weight of suspended solids = 4.444 g/L. 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.

TEST SYSTEM
- Culturing apparatus: 250mL-flask
- Number of culture flasks/concentration: 2
- Measuring equipment: The respirometers used during the study are SAPROMAT D 12, made by J.M. VOITH GmbH, Heidenheim, Germany (Serial Nos.: SAPROMAT 1: 9316, controller 1: 1921, SAPROMAT 2: 9511, controller 2: 2067).
- Test performed in closed vessels: yes
- Preparation of the flasks: Test substance samples (25 mg, corresponding to 100 mg/L in a 250mL-flask) are weighed in small aluminium boats and added directly to the test flasks of the SAPROMAT, whereas reference substance samples (sodium benzoate) are added as 1.0 mL of a 25 mg/mL solution in mineral medium. All flasks are filled with 250 mL of mineral medium. Samples of test or reference substance, or both, are added. Then, a volume of suspended sludge corresponding to 7.5 mg dry weight is added. Except when the test substance had an acid or alkaline character, the pH of each flask is not measured but assumed to be the same as the mineral medium, in order not to remove any floating undissolved test substance from the test medium by dipping a glass electrode in it. Neutral test substances (even sodium benzoate which is slightly alcaline) were shown not to affect the pH of the medium by more than 0.1 pH unit. About 2 g of soda lime are placed in an attachment of the stopper and the flasks are closed and placed in the water bath of the SAPROMAT. After temperature and pressure equilibration, the oxygen-meters of the instrument are set to zero (time zero of the experiment).

SAMPLING
- Sampling frequency: everyday the oxygen consumption of each flask is recorded and correct temperature and stirring are checked. At the end of the test period, the pH of each flask is measured again.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes. A pair of flasks of the volumetric respirometer are filled with mineral medium + test substance (100 mg/L) + reference substance (100 mg/L) + inoculum and their respirations are recorded as for the other flasks.
- Other: See table 5.2.1 in "Any other information on materials and methods incl. tables".

Reference substance:

benzoic acid, sodium salt

Remarks:

Nominal concentration used in the test: 100 mg/L

Parameter:

% degradation (O2 consumption)

Value:

50

Sampling time:

28 d

Remarks on result:

other: Average of two incubates (53% BOD, 47% BOD)

Key result

Parameter:

% degradation (O2 consumption)

Value:

72

Sampling time:

62 d

Remarks on result:

other: Average of two incubates (72% BOD, 72% BOD)

Details on results:

Oxygen uptakes, as read on the SAPROMAT meters, are corrected to account for the small differences between actual and nominal concentrations of test and reference substances.
The test substance undergoes 50% and 72% biodegradation after 28 and 62 days, respectively, in the test conditions.
The curves obtained with the reference substance alone and with the test substance + reference substance show no toxic effect of the test substance to the microorganisms at the test concentration of 100 mg/L.
See tables 5.2.1/2 and 5.2.1/3 in "Any other information on results incl. tables".

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 5.2.1/2: Biodegradability of the test substance

Days			5	7	15	21	28	62
O2 uptake of sludge (inoculum blank)	2/3 2/4 Mean	B1 B2 B	24.0 17.0 20.5	28.0 21.0 24.5	36.0 28.0 32.0	39.0 30.0 34.5	43.0 34.0 38.5	59.0 48.0 53.5
O2 uptake of test substance + sludge	2/7 2/8	C1 C2	30.0 27.0	41.9 39.0	93.8 93.8	131.6 122.7	173.5 157.6	237.3 238.4
O2 uptake of test substance		C1-B C2-B	9.5 6.5	17.4 14.5	61.8 61.8	97.1 88.2	135.0 119.1	183.8 184.9
% biodegradation of test substance	Mean	D1 D2 D	4 3 3	7 6 6	24 24 24	38 34 36	53 47 50	72 72 72
% degradation of test substance in presence of reference substance (co-metabolism)		Dco1 Dco2 Dco	10 9 9	12 11 12	17 15 16	19 17 18	25 23 24	44 50 41

Calculations:

B1, B2, C1, C2, A1, A2, E1, E2: experimental O2 uptake values

B = (B1 + B2) / 2

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

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

D = (D1 + D2) / 2

Dco1 = 100 * (E1 - A1) / ThOD * [S]

Dco2 = 100 * (E2 - A2) / ThOD * [S]

Dco = (Dco1 + Dco2) / 2

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

Test substance: Formula C16H28O3; Molecular weight 268.4 g/mol; ThOD 2.56 mg O2/mg

Table 5.2.1/3: Biodegradability of the reference substance and the toxicity control

Days			5	7	14	21	28	62
O2 uptake of sludge (inoculum blank)	2/3 2/4 Mean	B1 B2 B	24.0 17.0 20.5	28.0 21.0 24.5	36.0 27.0 31.5	39.0 30.0 34.5	43.0 34.0 38.5	59.0 48.0 53.5
O2 uptake of reference substance + sludge	1/5 1/6	A1 A2	132.0 138.0	148.0 154.0	176.0 181.0	191.0 196.0	200.0 203.0	229.0 230.0
O2 uptake of reference substance		A1-B A2-B	111.5 117.5	123.5 129.5	144.5 149.5	156.5 161.5	161.5 164.5	175.5 176.5
O2 uptake of reference substance + test substance + sludge	2/5 2/6 Mean	E1 E2 E	157.0 161.0 159.0	180.0 183.0 181.5	218.0 220.0 219.0	239.0 240.0 239.5	263.0 262.0 262.5	340.9 358.0 349.5
% biodegradation of reference substance	Mean	D1 D2 D	67 71 69	74 78 76	87 90 88	94 97 95	97 99 98	105 106 106

Calculations:

B1, B2, C1, C2, A1, A2, E1, E2: experimental O2 uptake values

B = (B1 + B2) / 2

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

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

D = (D1 + D2) / 2

E = (E1 + E2) / 2

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

Sodium benzoate: Formula C7H5O2Na1; Molecular weight 144.1 g/mol; ThOD 1.67 mg O2/mg

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

The test substance undergoes 50% and 72% biodegradation after 28 and 62 days, respectively, in the test conditions. Thus, the test substance should be regarded as inherently biodegradable based on the fact that biodegradation exceeded the pass level of 60% after day 28 . In addition, no toxic effect of the test substance to the microorganisms at the test concentration of 100 mg/L was observed.

Executive summary:

The ready biodegradability of the test substance was determined by the Manometric Respirometry Test according to the OECD Guideline 301F and EU Method C.4-D with GLP compliance.

In this study, a measured volume of inoculated mineral medium, containing a known concentration of the test substance (100 mg/L) as the nominal sole source of organic carbon, is stirred in a closed flask at a constant temperature (+/- 1°C) for up to 62 days. The consumption of oxygen is determined by measuring the quantity of oxygen (produced electrolytically) required to maintain constant the gas volume in the respirometer flask. Evolved carbon dioxide is absorbed in soda lime pellets. The amount of oxygen taken up by the microbial population during biodegradation of the test substance (corrected for uptake by blank inoculum, run in parallel) is expressed as a percentage of ThOD (Theoretical Oxygen Demand, calculated from the elemental composition, assuming that carbon is oxidized to carbon dioxide and hydrogen to water).

According to the results of this study, the test substance undergoes 50% and 72% biodegradation after 28 and 62 days, respectively, in the test conditions.

The curves obtained with the reference substance alone and with the test substance + reference substance show no toxic effect of the test substance to the microorganisms at the test concentration of 100 mg/L.

Thus, the test substance should be regarded as not readily biodegradable according to this test method. However, based on the fact that biodegradation exceeded the pass level of 60% after day 28 the test substance should be regarded as inherently biodegradable.
Guidance given in the OECD Guidelines for Testing of Chemicals, Section 3, Part 1: Principles and strategies related to the testing of degradation of organic chemicals (adopted 23 March 2006), paragraphs 21 and 36 indicates that fulfilling the pass level criterion after 28 days indicates that the test substance is inherently biodegradable.

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 29 April 2008 to 16 July 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))

Deviations:

yes

Remarks:

Fresh activated sludge was used instead of sludge cultivated as described in the guideline and no quantitative analysis of the test substance was performed.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 17 and 18 March 2008 / Date of signature: 23 June 2008

Specific details on test material used for the study:

- Theoretical Oxygen Demand (ThOD): 2.56 mg O2/mg test substance

Batch No. : LDP0800001
Purity : 97.0%
Date of Expiry : 01 May, 2010

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Fresh activated sludge from a biological waste water treatment plant treating predominantly domestic sewage (City of Geneva, Peney-Dessous) was used.
The sludge is 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) and kept aerobic until being used on the same day.
To obtain a concentration of 100 mg/L (dry weight) in a 250 mL flask, 5.63 mL of slude is needed (inoculum).

Duration of test (contact time):

62 d

Initial conc.:

30 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Water: The water used during this study is deionised water containing less than 10 mg/L dissolved organic carbon.
- Composition of medium:
The following stock solutions were prepared:
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 litre.
Solution B: CaCl2: 27.5 g; dissolved in water and made up to 1 litre.
Solution C: MgSO4.7H2O: 22.5 g; dissolved in water and made up to 1 litre.
Solution D: FeCl3.6H2O: 0.25 g; HCl Conc.: one drop; dissolved in water and made up to 1 litre.
The mineral medium was 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.
- Test temperature: 22°C
- pH: See table 5.2.1 in "Any other information on materials and methods incl. tables".
- Suspended solids concentration: dry weight of suspended solids = 4.444 g/L. 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.

TEST SYSTEM
- Culturing apparatus: 250mL-flask
- Number of culture flasks/concentration: 2
- Measuring equipment: The respirometers used during the study are SAPROMAT D 12, made by J.M. VOITH GmbH, Heidenheim, Germany (Serial Nos.: SAPROMAT 1: 9316, controller 1: 1921, SAPROMAT 2: 9511, controller 2: 2067).
- Test performed in closed vessels: yes
- Preparation of the flasks: Test substance samples (7.5 mg, corresponding to 30 mg/L in a 250mL-flask) are weighed in small aluminium boats and added directly to the test flasks of the SAPROMAT, whereas reference substance samples (sodium benzoate) are added as 1.0 mL of a 25 mg/mL solution in mineral medium. All flasks are filled with 250 mL of mineral medium. Samples of test or reference substance, or both, are added. Then, a volume of suspended sludge corresponding to 25 mg dry weight is added. Except when the test substance had an acid or alkaline character, the pH of each flask is not measured but assumed to be the same as the mineral medium, in order not to remove any floating undissolved test substance from the test medium by dipping a glass electrode in it. Neutral test substances (even sodium benzoate which is slightly alcaline) were shown not to affect the pH of the medium by more than 0.1 pH unit. About 2 g of soda lime are placed in an attachment of the stopper and the flasks are closed and placed in the water bath of the SAPROMAT. After temperature and pressure equilibration, the oxygen-meters of the instrument are set to zero (time zero of the experiment).

SAMPLING
- Sampling frequency: everyday the oxygen consumption of each flask is recorded and correct temperature and stirring are checked. At the end of the test period, the pH of each flask is measured again.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Other: See table 5.2.1 in "Any other information on materials and methods incl. tables".

Reference substance:

benzoic acid, sodium salt

Remarks:

Nominal test concentration: 100 mg/L (during the OECD Guideline 301F study: Givaudan, 2008a, K)

Key result

Parameter:

% degradation (O2 consumption)

Value:

22

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Value:

19

Sampling time:

62 d

Details on results:

Oxygen uptakes, as read on the SAPROMAT meters, are corrected to account for the small differences between actual and nominal concentrations of test and reference substances.
The test substance undergoes 22% and 19% biodegradation after 28 and 62 days, respectively, in the test conditions.
See tables 5.2.1/2 and 5.2.1/3 in "Any other information on results incl. tables".

Results with reference substance:

According to the OECD Guideline 301F study (Givaudan, 2008a, K): 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 5.2.1/2: Biodegradability of the test substance

Days			5	7	14	21	28	62
O2 uptake of sludge (inoculum blank)	1/9 1/10 Mean	B1 (100) B2 (100) B (100)	79.0 82.0 80.5	95.0 94.0 94.5	124.0 113.0 118.5	134.0 124.0 129.0	140.0 131.0 135.5	179.0 160.0 169.0
O2 uptake of test substance + sludge	1/11 1/12	C1 C2	86.0 85.0	104.1 103.0	130.1 130.0	143.1 144.0	151.1 153.0	184.5 183.0
O2 uptake of test substance		C1-B (100) C2-B (100)	5.5 4.5	9.6 8.5	11.6 11.5	14.1 15.0	15.6 17.5	15.5 14.0
% biodegradation of test substance	Mean	D1 D2 D	7 6 7	12 11 12	15 15 15	18 20 19	20 23 22	20 18 19

Calculations:

B1 (100), B2 (100), C1, C2: experimental O2 uptake values

B (100) = [B1 (100) + B2 (100)] / 2

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

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

D = (D1 + D2) / 2

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

Test substance: Formula C16H28O3; Molecular weight 268.4 g/mol; ThOD 2.56 mg O2/mg

Validity criteria fulfilled:

not specified

Interpretation of results:

not inherently biodegradable

Conclusions:

The test substance undergoes 22 and 19% biodegradation after 28 and 62 days, respectively, in the test conditions. Thus, the test substance should be regarded as not inherently biodegradable according to this test method.

Executive summary:

The inherent biodegradability of the test substance was determined by the Manometric Respirometry Test. The method used is basically the one described under OECD Guideline 302C with GLP compliance. However, fresh activated sludge was used instead of sludge cultivated as described in the guideline and no quantitative analysis of the test substance was performed.

In this study, a measured volume of inoculated mineral medium, containing a known concentration of the test substance (30 mg/L) as the nominal sole source of organic carbon, is stirred in a closed flask at a constant temperature (+/- 1°C) for up to 62 days. The consumption of oxygen is determined by measuring the quantity of oxygen (produced electrolytically) required to maintain constant the gas volume in the respirometer flask. Evolved carbon dioxide is absorbed in soda lime pellets. The amount of oxygen taken up by the microbial population during biodegradation of the test substance (corrected for uptake by blank inoculum, run in parallel) is expressed as a percentage of ThOD (Theoretical Oxygen Demand, calculated from the elemental composition, assuming that carbon is oxidized to carbon dioxide and hydrogen to water).

According to the results of this study, the test substance undergoes 22% and 19% biodegradation after 28 and 62 days, respectively, in the test conditions.

Thus, the test substance should be regarded as not inherently biodegradable according to this study.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 20 January 2011 to 18 March 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 26 and 27 October 2010 / Date of signature: 08 February 2011

Specific details on test material used for the study:

- Theoretical Oxygen Demand (ThOD): 2.56 mg O2/mg

Batch No. : LDP0800001
Purity : 97.7%
Date of Expiry : 16 December, 2012

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Fresh activated sludge from a biological waste water treatment plant treating predominantly domestic sewage (Bois-de-Bay, Satigny, Switzerland) was used.
The sludge is 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) and kept aerobic until being used on the same day.
To obtain a concentration of 30 mg/L (dry weight) in 103 mL total volume (Flasks 1 and 2), 2.00 mL of sludge was added (inoculum).
To obtain a concentration of 30 mg/L (dry weight) in 255 mL total volume (Flasks 3 to 28), 5.00 mL of sludge was added (inoculum).

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

test mat.

Remarks:

Tes substance concentration of 20 mg/L equivalent to a ThOD of 51.2 mg O2/L

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Water: The water used during this study is deionised water containing less than 10 mg/L dissolved organic carbon.
- Composition of medium:
The following stock solutions were prepared:
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 litre.
Solution B: CaCl2: 27.5 g; dissolved in water and made up to 1 litre.
Solution C: MgSO4.7H2O: 22.5 g; dissolved in water and made up to 1 litre.
Solution D: FeCl3.6H2O: 0.25 g; HCl Conc.: one drop; dissolved in water and made up to 1 litre.
The mineral medium was 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.
- Test temperature: 22.3 - 23.0°C
- pH: See table 5.2.1 in "Any other information on materials and methods incl. tables".
- Suspended solids concentration: dry weight of suspended solids = 4.37 g/L, diluted to 1.53 g/L. 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.

TEST SYSTEM
- Culturing apparatus: 250mL-flask
- Measuring equipment: The respirometer used during the study is an Oxitop Control System, made by Wissenschaftlich-Technische Werkstätten (WTW), Weilheim, Germany.
- Test performed in closed vessels: yes
- Preparation of the flasks: Test substance samples (5.10 mg, corresponding to 20 mg/L in 255 mL of test medium) are weighed in small aluminium boats and added directly to the test flasks of the Oxitop, whereas reference substance samples (sodium benzoate) are added as 1.0 mL of a 10.2 g/L solution in mineral medium, to give a total volume of 103 mL. All flasks are filled with 250 mL of mineral medium (flasks containing reference substance: 100 mL). Samples of test or reference substance are added. Then, suspended sludge diluted to a concentration of 1.53 g/L dry matter is added. Except when the test substance had an acid or alkaline character, the pH of each flask is not measured but assumed to be the same as the mineral medium, in order not to remove any floating undissolved test substance from the test medium by dipping a glass electrode in it. Neutral test substances, even sodium benzoate, were shown not to affect the pH of the medium by more than 0.1 pH unit. Two sodium hydroxide pellets are placed in the quivers on top of the bottle, and the flasks are closed tightly with the measuring heads. The flasks are allowed to equilibrate to the test temperature. The measurement is started by programming the measuring unit of the Oxitop test flasks, and the test flasks are 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).

SAMPLING
Respirometry - Sampling frequency: everyday the oxygen consumption of each flask is recorded and correct temperature and stirring are checked. At the end of the test period, the pH of each flask is measured again;
Substance-specific Analyses : Time 0, 1 hour, 3, 19, 24, 43, 48, 72, 144, 168 and 672 hours (see Table 5.2.1).

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: no
See table 5.2.1 in "Any other information on materials and methods incl. tables".

Reference substance:

benzoic acid, sodium salt

Remarks:

nominal concentration used in the test: 100 mg/L

Parameter:

% degradation (test mat. analysis)

Value:

90.7

Sampling time:

24 h

Remarks on result:

other: 9.3% of Parent remaining after 24 hours compared with Time 0 nominal

Parameter:

% degradation (test mat. analysis)

Value:

59

Sampling time:

1 h

Remarks on result:

other: 41% of Parent remaining after 1 hour compared with Time 0 nominal

Key result

Parameter:

% degradation (O2 consumption)

Value:

14

Sampling time:

28 d

Details on results:

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.
The test substance undergoes 14% biodegradation based on BOD after 28 days in the test conditions.

Substance specific Analytics - Accompanying quantitative analysis for the test substance eivdences rapid primary biodegradation of the test substance. 2-methyl-2-(1,2,4-trimethylpent-2 -enyloxy)propan-1 -ol was identified as primary metabolite by comparison to authentic material. Quantitive analysis for this substance confirms further degradation of this primary metabolite.
From the results of the substance specific analyses, the DT50 and DT90 for the test substance are the order of 1 hour and 24 hours, respectively. Detectable quantities of the primary metabolite appear after 1 hour of incubation and attain a maximum equivalent to 30% of the applied starting material after 48 hours, before demonstrating a gradual decline to less than one-third of this value within the following 4 day period. The expected DT50 for this primary metabolite is less than 96 hours.
At the end of the test (day 28), neither the test substance nor the primary metabolite were observed in the test solutions (See tables in "Any other information on results incl. tables".

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 5.2.1/2: Biodegradability of the test substance

Days			5	7	8	14	18	21	28
O2 uptake of sludge (inoculum blank)	3 4 Mean	B1 B2 B	17.5 18.8 18.2	20.2 21.5 20.9	20.2 21.5 20.9	25.6 28.3 27.0	29.6 31.0 30.3	31.0 32.3 31.7	35.0 35.0 35.0
O2 uptake of test substance + sludge	23 24	C1 C2	22.9 20.2	25.6 24.2	28.3 26.9	35.0 32.3	39.0 37.7	41.7 40.4	43.1 41.7
O2 uptake of test substance		C1-B C2-B	4.8 2.1	4.8 3.4	7.5 6.1	8.1 5.3	8.7 7.4	10.1 8.8	8.1 6.7
% biodegradation of test substance	Mean	D1 D2 D	9 4 7	9 4 7	15 12 13	16 10 13	17 14 16	20 17 18	16 13 14

Calculations:

B1, B2, C1, C2, A1, A2: experimental O2 uptake values

B = (B1 + B2) / 2

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

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

D = (D1 + D2) / 2

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

Test substance: Formula C16H28O3; Molecular weight 268.4 g/mol; ThOD 2.56 mg O2/mg

Table 5.2.1/3: Biodegradability of the reference substance

Days			5	7	14	21	28
O2 uptake of sludge (inoculum blank)	3 4 Mean	B1 B2 B	17.5 18.8 18.2	20.2 21.5 20.9	25.6 28.3 27.0	31.0 32.3 31.7	35.0 35.0 35.0
O2 uptake of reference substance + sludge	1 2	A1 A2	136.1 131.1	146.2 141.2	167.4 162.3	188.5 188.5	193.5 198.6
O2 uptake of reference substance		A1-B A2-B	118.0 112.9	125.4 120.3	140.4 135.4	156.9 156.9	158.5 163.6
% biodegradation of reference substance	Mean	D1 D2 D	71 68 69	75 72 74	84 81 83	94 94 94	95 98 97

Calculations:

B1, B2, C1, C2, A1, A2, E1, E2: experimental O2 uptake values

B = (B1 + B2) / 2

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

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

D = (D1 + D2) / 2

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

Sodium benzoate: Formula C7H5O2Na1; Molecular weight 144.1 g/mol; ThOD 1.67 mg O2/mg

Table 5.2.1/4: Concentration of the test substance (% of initial concentration) and the primary metabolite (as % of theory) as a function of time

Time (h)	Test flask	Test substance		Primary metabolite
		Relative concentration vs. Nominal concentration time t=0	Average relative concentration vs. Nominal concentration time t=0	Relative concentration vs. Nominal equivalent parent time t=0	Average relative concentration vs. Nominal equivalent parent time t=0
0	25 / 26	82.2 / 93.7	88.0	0.0 / 0.0	0.0 (<LOD)
1	5 / 6	48.5 / 33.5	41.0	1.1 / 0.9	1.0
3	7 / 8	39.4 / 14.8	27.1	3.0 / 1.1	2.0
19	9 / 10	6.5 / 8.2	7.4	6.9 / 8.2	7.5
24	11 / 12	37.1* / 9.3	9.3	12.1 / 13.4	12.7
43	13 / 14	3.3 / 2.7	3.0	23.4 / 14.1	18.7
48	15 / 16	2.5 / 6.6	4.6	25.1 / 34.1	29.6
72	17 / 18	0.9 / 40.7*	0.9	24.7 / 15.0	19.9
144	19 / 20	0.6 / 0.6	0.6	6.4 / 12.1	9.3
168	21 / 22	0.4 / 0.4	0.4	5.4 / 7.0	6.2
672	23 / 24	0.0 / 0.0	0.0 (<LOD)	0.0 / 0.0	0.0 (<LOD)

* considered outlier

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test substance undergoes 14% biodegradation after 28 days in the test conditions. Thus, the test substance should be regarded as not readily biodegradable according to this test method.
Accompanying quantitative analysis for the test substance evidences rapid primary biodegradation of the test substance. 2-methyl-2-(1,2,4-trimethylpent-2 -enyloxy)propan-1 -ol was identified as primary metabolite by comparison to authentic material. Quantitive analysis for this substance confirms further degradation of this primary metabolite. At the end of the test (day 28), neither the test substance nor the primary metabolite were observed in the test solutions.

Based on substance specific analyses the DT50 and DT90 of the Parent material was observed to be, respectively, the order of 1 hour and 24 hours.

Executive summary:

The ready biodegradability of the test substance was determined by the Manometric Respirometry Test according to the OECD Guideline 301F and EU Method C.4-D with GLP compliance.

In this study, a measured volume of inoculated mineral medium, containing a known concentration of the test substance (here 20 mg/L) as the nominal sole source of organic carbon, is stirred in a closed flask at a constant temperature (+/- 1°C) for up to 28 days. Evolved carbon dioxide is absorbed in sodium hydroxide pellets. The consumption of oxygen is determined by measuring the pressure drop in the respirometer flask. The Biological Oxygen Demand (BOD), amount of oxygen taken up by the microbial population during biodegradation of the test substance (corrected for uptake by blank inoculum, run in parallel) is expressed as a percentage of ThOD (Theoretical Oxygen Demand, calculated from the elemental composition, assuming that carbon is oxidized to carbon dioxide and hydrogen to water).

According to the results of this study, the test substance undergoes 14% biodegradation after 28 days in the test conditions, thus, the test substance should be regarded as not readily biodegradable according to this test method.

Accompanying quantitative analysis for the test substance evidences rapid primary biodegradation of the test substance. 2-methyl-2-(1,2,4-trimethylpent-2 -enyloxy)propan-1 -ol was identified as primary metabolite by comparison to authentic material. Quantitive analysis for this substance confirms further degradation of this primary metabolite. From the results of the substance specific analyses, the DT50 and DT90 for the test substance are the order of 1 hour and 24 hours, respectively. Detectable quantities of the primary metabolite appear after 1 hour of incubation and attain a maximum equivalent to 30% of the applied starting material after 48 hours, before demonstrating a gradual decline to less than one-third of this value within the following 4 day period. The expected DT50 for this primary metabolite is less than 96 hours.

At the end of the test (day 28), neither the test substance nor the primary metabolite were observed in the test solutions (<LOD, equivalent to <0.015% of parent starting concentration).

Reference 4

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 20 June 2008 to 08 October 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: SEPA HJ/T 153-2004, "The guidelines for the testing of chemicals". Beijing: China Environmental Sciences Publishing House, 2004.

Deviations:

not specified

GLP compliance:

yes

Specific details on test material used for the study:

- Theoretical Oxygen Demand (ThOD): 2.56 mg O2/mg
- Storage conditions: Refrigerator 4°C

Batch No. : LDP0800001
Purity : 97%
Date of Expiry : 01 May, 2010

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, non-adapted

Details on inoculum:

Samples of secondary effluent were obtained from a sewage plant treating predominantly domestic sewage. Aerobic conditions were maintained in the effluent by aeration from a laboratory supply of oil-free compressed air.
On the day of the test, the test inoculum was prepared by filtering the sample of sewage effluent through a coarse filter, (discarding the first 200 mL) and collecting the remaining filtrates. This inoculum was maintained under aerobic conditions in the test area until use.

Duration of test (contact time):

28 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

PREPARATION OF TEST MEDIUM
The dilution medium was prepared by adding one mL of each of the following stock solutions, prepared in pre-aerated pure water to each litre of pure water.
Solution A: KH2PO4: 8.5 g; K2HPO4: 21.75 g; Na2HPO4.2H2O: 33.4 g; NH4Cl: 0.5 g. The pH of this solution should be 7.4.
Solution B: CaCl2: 27.5 g.
Solution C: MgSO4.7H2O: 22.5 g.
Solution D: FeCl3.6H2O: 0.25 g.

PREPARATION OF SOLUTIONS OF THE TEST SUBSTANCE
Because the test substance is very insoluble in the dilution medium used in the test, a concentrated stock solution at 1000 mg/L was prepared with acetone. A concentrated solution of the reference substance (aniline) was prepared in ultra-pure water and added to the mineral salts medium. Appropriate volumes of this solution were added to test solutions to give a test concentration of 2 mg/L. In line with normal practice in this type of study, an assessment of the stability of the test substance formulation and achieved concentration were not conducted.

TEST METHODS
The stored solutions of the test substance will be added to the vessels designed "test" and "inhibition" to give the required final test concentration (2 mg/L). After volatilize the solvent with N2, the BOD bottles would be filled 1/3 volume with the dilution medium that had been aerated for 20 minutes and allowed to stand at the test temperature for at least 20h. Following the additions of test and reference substance, each vessel will be inoculated with secondary effluent at a level of one mL/L. The pH will be adjusted to 7.4 with NaOH or HCl, as appropriate and pure water added to give the required final volume. Finally, all BOD bottles would be filled with the dilution medium.
The test substance were added to the vessels designated "test" and "inhibition" to give the required final test concentration (2 mg/L).
The reference substance (aniline) were added to the "inhibition" and "reference" vessels to give a final test concentration (2 mg/L).
Then, transfer each prepared solution to the required number of pre-calibrated BOD bottles (four "inhibition", eighteen "control", "test" or "reference").
Common-controls were shared between this and a concurrent study of identifical test design.
The concentration of dissolved oxygen in duplicate vessels from each "test", "control" and "reference" group were determined after addition of the prepared solutions to the BOD bottles (0d) and the remaining bottles were incubated at 20 +/- 2°C in darkness. Subsequent determinations of the concentration of dissolved oxygen in duplicate vessels taken from each group were made on 5, 7, 11, 14, 18, 21, 25 and 28 days/
The pH and temperature of the contents of each bottle were measured after the concentration of dissolved oxygen had been determined.
Determinations of the oxygen level in the "inhibition" group were made at the start and after five days.

Reference substance:

aniline

Remarks:

Nominal concentration used in the test: 2 mg/L

Test performance:

The test is valid because the level of biodegradation of the reference substance achieves more than 60% within 14 days from the start of the test and the level of dissolved oxygen does not fall below 0.5 mg O2/L. Oxygen consumption in inoculated blanks must not exceed 1.5 mg O2/L after 28 days of incubation.

Key result

Parameter:

% degradation (O2 consumption)

Value:

67.3

Sampling time:

28 d

Remarks on result:

other: Average value (mean of replicate 1 = 66.5% and replicate 2 = 68.1%) but not fulfilling the 10-day and 14-day window criterion.

Details on results:

During the test, the temperature was kept at 20°C.
See tables in "Any other information on results incl. tables".

Results with reference substance:

The percentage degradation of aniline has reached the pass level by day 14: the activity of the inoculum was thus verified (validity criterion).

Table 5.2.1/1: Concentrations of Dissolved Oxygen

Sample	DO after n days (mg/L)
	0d	5d	7d	11d	14d	18d	21d	25d	28d
Control1	8.17	7.95	7.68	7.73	8.01	7.91	7.70	7.31	6.81
Control2	8.16	7.91	7.61	7.58	7.82	7.82	7.21	6.69	6.78
Test	8.17	7.59	6.94	6.33	6.21	5.83	5.23	4.18	3.38
	8.15	7.54	6.87	6.21	6.02	5.52	4.93	4.07	3.28
Reference	8.18	7.21	5.92	5.30	5.03	4.81	4.32	3.68	3.21
	8.17	7.16	5.33	5.13	5.01	4.81	4.28	3.46	3.11
Inhibition	8.17	6.81
	8.16	6.67

Table 5.2.1/2: Biodegradability of the test and reference substances

Sample	Biodegradation after n days (%)
	5d	7d	11d	14d	18d	21d	25d	28d
Test	6.71	13.8	25.9	33.3	39.7	43.4	55.0	66.5
	7.30	14.8	27.8	36.6	45.3	48.8	56.7	68.1
Reference	15.3	36.3	49.4	60.4	64.0	65.6	69.5	75.0
	16.1	48.3	52.7	60.6	63.8	66.3	73.9	76.9

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

The biodegradation of the test substance reached 66.5 - 68.1% (mean of 67.3%), readily biodegradable, but without the 10-day and 14-day window criterion.

Executive summary:

The ready biodegradability of the test substance was determined according to OECD Guideline 301D, SEPA HJ/T 153 -2004, EU Method C.4 -E and US EPA OPPTS Method 835.3110 with GLP statement, in a 28-day dissolved oxygen depletion using secondary effluent from a domestic waste water treatment plant.

During the test, the temperature was kept at 20°C. The test is valid because the level of biodegradation of the reference substance achieves more than 60% within 14 days from the start of the test and the level of dissolved oxygen does not fall below 0.5 mg O2/L. Oxygen consumption in inoculated blanks did not exceed 1.5 mg O2/L after 28 days of incubation. Finally, the results showed that biodegradation of the test substance reached 66.5 - 68.1% (mean of 67.3%) but without the 10 -day and 14-day window criterion.

Description of key information

The biodegradability of the registered substance has been tested in four GLP compliant studies and following OECD guidelines.

Three valid studies tested readily biodegradability: extended 301F “Manometric Respirometry test” (Givaudan, 2008a), advanced 301F "Manometric Respirometry test" with substance specific analyses (Givaudan, 2011) and 301D “Closed bottle test” (PEAPC, 2008). In addition, one valid study tested inherent biodegradability: 302C (Givaudan, 2008b).

During the 301D “Closed bottle test” key study, the level of biodegradation of the substance reached 66.5 to 68.1% by the end of the test (28 days), however this level was not reached within the required 14-day window.  In conclusion, the registered substance is considered as being readily biodegradable but failing the 14-day window criterion.

The extended 301F (Givaudan, 2008a) the 302C (Givaudan, 2008b) and the advanced 301F (Givaudan, 2011) studies have been assessed as supporting studies. The 301F study allows to conclude that the registered substance is inherently biodegradable, and thus is not persistent in the environment as 50% of biodegradation was observed after 28 days and more than 60% of biodegradation was observed in the extended test period (72% biodegradation after 62 days).

In the advanced OECD 301F study (Givaudan, 2011) accompanying quantitative analysis for the registered substance showed evidence of rapid primary biodegradation of the substance. A primary metabolite was identified (2-methyl-2-(1,2,4-trimethylpent-2-enyloxy)propan-1-ol) and a quantitive analysis for this substance confirms further degradation of this primary metabolite. From the results of the substance specific analyses, DT50 and DT90 for the registered substance are the order of 1 hour and 24 hours, respectively. Detectable quantities of the primary metabolite appear after 1 hour of incubation and attain a maximum equivalent to 30% of the applied starting material after 48 hours, before demonstrating a gradual decline to less than one-third of this value within the following 4 day period. The expected DT50 for this primary metabolite is less than 96 hours. At the end of the test (day 28), neither the registered substance nor the primary metabolite were observed in the test solutions.

The 302C study demonstrated only 22% and 19% biodegradation after 28 and 62 days, respectively.

The substance is not persistent in the environment.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable but failing 10-day window

Type of water:

freshwater