Reaction mass of (R*,R*)-7-methoxy-3,7-dimethyl-2-octanol and (R*,S*)-7-methoxy-3,7-dimethyl-2-octanol

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:

key study

Study period:

2017-03-29 to 2017-08-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline No. 301 D. All validity criteria were fulfilled

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

10 January 2017

Oxygen conditions:

aerobic

Inoculum or test system:

other: river water

Details on inoculum:

- Origin: River water was sampled from “La Mourachonne”, situated downstream of a plant treating domestic wastewater (Pegomas, France).
- Pre-conditioning: The river water was aerated for about 7 days before use as inoculum at the test temperature to reduce endogenous respiration.

Duration of test (contact time):

55 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Test water: Mineral medium, Reconstituted water, as prescribed by the OECD Guideline No. 301D
- Composition of medium: The mineral medium was prepared by adding one mL of each of the following stock solutions (a to d) to each litre of ultrapure water: a) 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.40 mg Na2HPO4•2H2O, 0.50 mg NH4Cl. The pH of this solution was approximately 7.4 ± 0.1; b) 36.40 mg CaCl2•2H2O, c) 22.50 mg MgSO4•7H2O and d) 0.25 mg FeCl3•6H2O.). This reconstituted water was aerated for at least 20 min and thereafter set aside at the test temperature for about 20 h before use.
- Test temperature: Temperatures recorded continuously and situated between 20.68 and 21.32°C throughout the test (average value: 21.07°C), and complied with the requirements as laid down in the study plan (22°C ± 2°C, constant within 1°C).
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: All-glass sealed with ground glass stoppers BOD (Biological Oxygen Demand) bottles of approximately 300 mL capacity. Each test vessel was uniquely identified with study code, replicate number, date of the start of the biodegradability test (=J0) and type of series.
- Inoculum: A concentration of inoculum of approximately 3.10^4 cells.L-1 was used.
- Number of culture flasks/concentration: 10 bottles containing test item, inoculum, mineral medium and silica gel (test suspension)

SAMPLING
- Sampling frequency: Determinations of the concentration of dissolved oxygen using an oxygen electrode in duplicate vessels were made at day 0, 7, 14, 21,
29, 35, 42, 49 and 55 for the “test suspension” and “inoculum blank” series, and only at the start of the test and after 7 and 14 days of incubation for the “procedure control” and “toxicity control” series.
- Temperature: Measured continuously in the growth chamber.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 18 bottles containing inoculum and mineral medium
- Test suspension: 18 bottles containing test item, inoculum and mineral medium
- Procedure control: 6 bottles containing reference substance, inoculum and mineral medium
- Toxicity control: 6 bottles containing test item, reference substance, inoculum and mineral medium

Reference substance:

benzoic acid, sodium salt

Remarks:

2 mg/L

Preliminary study:

None

Key result

Parameter:

% degradation (O2 consumption)

Value:

10

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

45

Sampling time:

55 d

Details on results:

Substances are considered to be readily biodegradable in the Closed Bottle test if oxygen consumption is equal to or greater than 60% of the ThOD value in the ten-day window within the 28-day period of the test (the 10-day window begins when the degree of biodegradation has reached 10% of the ThOD).
Test item 7-METHOXY-3,7-DIMETHYLOCTAN-2-OL MULTICONSTITUENT was biodegraded by 10% at day 28 in the Closed Bottle test. The test period was therefore extended to 55 days in order to demonstrate a potential inherent biodegradability of the test item. The biodegradation percentage at day 55 was only 45%. It should be noted that the test item had no inhibitory effect on the activity of the microbial inoculum, as demonstrated by the test results of the toxicity control, in agreement with OECD 301 (oxygen consumption in the toxicity control must be greater than 25% of the ThOD value within 14 days).

Calculated ThOD of the test item: 2.72 mg O2/mg

Results with reference substance:

Calculated ThOD of the reference substance: 1.67 mg O2/mg. The biodegradation percentage of the reference substance, sodium benzoate, was 94 % at day 14.

Table 5.2.1/1: Percentage biodegradation of the test item, the reference substance and of the toxicity control throughout the test

		Time (days)
		0	7	14	21	28	35	42	49	55
Test Suspension (2 mg test item.L^-1)	Replicate 1	0.00	-0.46	0.74	6.71	18.66	6.80	17.00	24.94	62.32
	Replicate 2	0.00	-0.83	4.78	-4.14	2.11	59.74	15.90	32.35	28.13
	Mean	0	-1	3	1	10	33	16	30	45
Procedure Control (2 mg reference substance.L^-1)	Replicate 1	0.00	87.87	106.89	-	-	-	-	-	-
	Replicate 2	0.00	81.89	80.54	-	-	-	-	-	-
	Mean	0	85	94	-	-	-	-	-	-
Toxicity Control * (2 mg reference substance.L^-1 + 2 mg test item.L^-1)	Mean	0	80	72	-	-	-	-	-	-

N.A.: not applicable

* % biodegradation_{tox. Control}=  (BOD tox. cont / ThODref. subst.) * 100

with BOD tox. cont = (mTox(o) - mTox(n)) - (ma(o) - ma(n)) / mg test ref susbst.L^-1in vessel

and where:

 n = number of days after the start of the test

mTox(0) = mean toxicity control value at day 0

 mTox (n) = mean toxicity control value at day n

 ma(0) = mean test suspension value at day 0

 ma(n) = mean test suspension value at day n.

Table 5.2.1/2: Dissolved oxygen concentrations (mg.L^-1) throughout the test

	Time (days)
	0	7	14	21	28	35	42	49	55
Inoculum Blank	9.09	8.68	8.45	8.52	7.90	7.81	7.28	7.52	7.50
	9.00	8.74	8.44	8.56	7.98	7.70	7.66	7.45	7.23
Mean	9.05	8.71	8.45	8.54	7.94	7.76	7.47	7.49	7.37
Test Suspension (2 mg test item.L^-1)	9.11	8.80	8.47	8.24	6.99	7.45	6.61	6.03	4.04
	9.05	8.76	8.19	8.77	7.83	4.51	6.61	5.73	5.84
Mean	9.08	8.78	8.33	8.51	7.41	5.98	6.61	5.88	4.94
Procedure Control(2 mg reference substance.L^-1)	9.37	6.10	5.20	-	-	-	-	-	-
	9.23	6.16	5.94	-	-	-	-	-	-
Mean	9.30	6.13	5.57	-	-	-	-	-	-
Toxicity Control (2 mg reference substance.L^-1 + 2 mg test item.L^-1)	9.33	6.22	6.47	-	-	-	-	-	-
	9.25	6.39	5.79	-	-	-	-	-	-
Mean	9.29	6.31	6.13	-	-	-	-	-	-

Validity criteria of the test:   

Inoculum blank: Oxygen depletion in the inoculum blank did not exceed 1.5 mg dissolved oxygen/L after 28 days.

Residual [O₂]: Oxygen concentrations were > 0.5 mg/L in all bottles during the test period.

Replicate values: The differences of the replicate (test suspension) values at day 28 were less than 20 %.

Reference substance: The biodegradation percentage of the reference substance, sodium benzoate, was 94% at day 14.

Thus all validity criteria of the test have been fulfilled in the present study.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Test item 7-METHOXY-3,7-DIMETHYLOCTAN-2-OL MULTICONSTITUENT was biodegraded by 10% at day 28 in the Closed Bottle test and 45% at day 55.
Therefore, 7-METHOXY-3,7-DIMETHYLOCTAN-2-OL MULTICONSTITUENT cannot be considered as readily biodegradable under the experimental conditions.

Executive summary:

A ready biodegradability study was performed to assess the biotic degradation of test item 7-METHOXY-3,7-DIMETHYLOCTAN-2-OL MULTICONSTITUENT according to the OECD Guideline 301 D and in compliance with GLP.

Eighteen BOD bottles were filled with mineral medium inoculated with river water, and without (inoculum blank) or with test item added at a nominal concentration of 2 mg.L^-1(test suspension). In the meantime, test vessels containing reference substance sodium benzoate (2 mg.L¹) were tested in order to check the procedure. A toxicity control, containing both the test item and the reference substance, was also performed in order to check the absence of test item effect on the microbial inoculum.The concentrations of dissolved oxygen in duplicate bottles from each group incubated at 22°C ± 2°C in darkness were measured at the start of the test and thereafter once a week until the end of the test.

The validity of the test was demonstrated by an endogenous respiration < 1.5 mgO₂.L^-1 and by a biodegradation of the reference substance of 94% of its ThOD after 14 days of incubation. Moreover, the differences of the replicate values at day 28 were less than 20% and oxygen concentrations were > 0.5 mg/L ^{in all bottles during the test period}. Furthermore,the toxicity control showed that the test item had no inhibitory effect on the activity of the microbial inoculum.

In this study, test item 7-METHOXY-3,7-DIMETHYLOCTAN-2-OL MULTICONSTITUENT was biodegraded by 10% at day 28. The test period was therefore extended to 55 days in order to demonstrate a potential inherent biodegradability of the test item. The biodegradation percentage at day 55 was 45%. Therefore, 7-METHOXY-3,7-DIMETHYLOCTAN-2-OL MULTICONSTITUENT cannot be considered as readily biodegradable under the experimental conditions

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 27th, 2010 to January 11th, 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed according to OECD Guideline No. 301 F. All validity criteria were fulfilled

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other 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:

yes

Remarks:

An optional toxicity control as suggested by OECD guideline 301 F was not performed. The validity of the study is not adversely affected.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

23 June 2008

Oxygen conditions:

not specified

Inoculum or test system:

activated sludge, domestic, 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 min, discarding the supernatant and resuspending in mineral medium) and kept aerobic until being used on the same day.

Duration of test (contact time):

70 d

Initial conc.:

30 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST PRINCIPLE
A measured volume of inoculated mineral medium, containing a known concentration of test substance 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 BOD (corrected for uptake by blank inoculum, run in parallel) is expressed as a percentage of ThOD.

TEST CONDITIONS
- Test 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: (A) 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.40 mg Na2HPO4•2H2O, 0.50 mg NH4Cl. Solution A dissolved in water and made up to 1 litre. (B) 36.40 mg CaCl2•2H2O. Solution B dissolved in water and made up to 1 L. (C) 22.50 mg MgSO4•7H2O. Solution C dissolved in water and made up to 1 L. (D) 0.25 mg FeCl3•6H2O dissolved in water and made up to 1 L.
- Mineral medium preparation: 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 5L with deionised water. The pH is measured and if necessary adjusted to 7.4 +/- 0.2 with phosphoric acid or potassium hydroxide.
- Test temperature: 21.0 - 21.5 °C
- pH: 7.6 -8.16
- Dry weight of suspended solids: 6.41 g/L, diluted to 1.53 g/L. To obtain a concentration of 30.0 mg/L (dry weight) in 103 ml total volume (Flasks 23 and 24), 2.00 mL of sludge was added (inoculum). To obtain a concentration of 30.0 mg/L (dry weight) in 255 mL total volume (Flasks 1, 2, 3 and 4), 5.00 mL of sludge was added (inoculum)

APPARATUS
The respirometer used during this study is an Oxitop Control System, made by Wissenschaftlich-Technische Werkstatten (WTW), Weilheim, Germany.

PREPARATION OF THE FLASKS
Test substance samples (7.65 mg, corresponding to 30.0 mg/L in 255 mL test medium) are weighed in small aluminium boats and added directly to the test flasks of the Oxitop, whereas reference is added as 1.00 mL of a 10.2 g/L solution in mineral medium, to give a total volume of 103 mL.
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 has 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 instryument starts the data acquisition (time zero of the experiment).

TEST AND REFERENCE SUBSTANCES
Nominal concentrations:
- test substance: 30.0 mg/L
- reference substance: 100 mg/L

Reference substance:

benzoic acid, sodium salt

Remarks:

purity min: 99.0%

Preliminary study:

None

Test performance:

Everyday, the oxygen consumption of each flask is recorded and correct temperature and stirring are checked.
At the end of the test period (28 days normally), the pH of each flask is measured again.

Key result

Parameter:

% degradation (O2 consumption)

Value:

49

Sampling time:

28 d

Details on results:

Osyrol undergoes 49% biodegradation after 28 days (77% after 56 days and 76% after 70 days) in the test conditions.
The 10-day window criterion is not fulfilled (14% biodegradation at day 6 and 41% at day 16).
Osyrol did not inhibit the intrinsic respiration of the inoculum at the test concentration and was therefore considered to be non-toxic to the incoculum at the test concentration.
Thus, osyrol should be regarded as not readily biodegradable according to this test. However, based on these results Osyrol should be regarded as inherently and ultimately biodegradable.

ThOD of the test item: 2.72 mg O2/mg.

Results with reference substance:

Calculated ThOD of the reference substance: 1.67 mg O2/mg. The biodegradation percentage of the reference substance, sodium benzoate, was 87 % at day 14.

Table 5.2.1/1: O2 uptake and % biodegradation of test substance

	Days:		5	6	16	21	28	56	70
O2 uptake of sludge (inoculum blank)	1	B1	18.8	20.2	29.6	32.3	36.3	49.8	55.2
	2	B2	17.5	18.8	29.6	35	41.7	61.9	72.7
	mean	B	18.2	19.5	29.6	33.7	39	55.9	64
O2 uptake of Test subst. + sludge	3	C1	24.2	31	64.6	72.6	80.7	117.9	124.9
	4	C2	24.2	31	61.9	67.3	76.7	120	127
O2 uptake of Test Substance		C1-B	6	11.5	35	39	41.7	62.1	61
		C2-B	6.1	11.5	32.3	33.7	37.7	64.2	63.1
% biodegradation of test substance		D1	7	14	43	48	51	76	75
		D2	7	14	40	41	46	79	77
	mean	D	7	14	41	45	49	77	76

Calculations:

B1, B2, C1, C2, A1, A2, E1: 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)

Table 5.2.1/2: O2 uptake and % biodegradation of reference substance

	Days:		5	7	14	21	28
O2 uptake of sludge (inoculum blank)	1	B1	18.8	21.5	28.3	32.3	36.3
	2	B2	17.5	20.2	26.9	35	41.7
	mean	B	18.2	20.9	27.6	33.7	39
O2 uptake of Ref. subst. + sludge	23	A1	141.2	152.3	172.4	188.5	193.5
	24	A2	146.2	152.3	172.4	188.5	193.5
O2 uptake of Ref. Substance		A1 - B	123	131.4	144.8	154.9	154.5
		A2 - B	128.1	131.4	144.8	154.9	154.5
% biodegradation of ref. substance		D1	74	79	87	93	93
		D2	77	79	87	93	93
	mean	D	75	79	87	93	93

Calculations:

B1, B2, C1, C2, A1, A2 : 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 test substance concentration (mg/L)

Validity criteria of the test:      

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.

Degradation of sodium benzoate exceeded 40% after day 7 and 65% after day 14: 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.

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

49% of biodegradation were obtained with the test item after 28 days (77% after 56 days and 76% after 70 days) in the test conditions.
The 10-day window criterion was not fulfilled (14% biodegradation at day 6 and 41% at day 16).
The test item did not inhibit the intrinsic respiration of the inoculum and was therefore considered to be non-toxic to the incoculum at the tested concentration.
Thus, the test item should be considered as not readily biodegradable according to this test. However, based on these results it should be considered as inherently and ultimately biodegradable.

Executive summary:

The Ready Biodegradability of the test item was determined by the Manometric Respirometry Test, following the OECD 301 F Guideline and performed with GLP compliance.

A measured volume of inoculated mineral medium, containing a known concentration of the test substance as the nominal sole source of organic carbon, was 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 was determined by measuring the pressure drop in the respirometer flask. The BOD (corrected for uptake by blank inoculum, run in parallel) was expressed as a percentage of ThOD.

Degradation of sodium benzoate (reference substance) exceeded 40% after day 7 and 65% after day 14: the activity of the inoculum was thus verified (validity criterion). The repeatability validity criterion (not more than 20% difference between replicates) was fulfilled. Therefore, the test is considered valid.

49% of biodegradation were measured after 28 days (77% after 56 days and 76% after 70 days) in the test conditions. The 10-day window criterion was not fulfilled (14% biodegradation at day 6 and 41% at day 16).

The test item did not inhibit the intrinsic respiration of the inoculum and was therefore considered to be non-toxic to the incoculum at the tested concentration.

Therefore, the test item should be considered as not readily biodegradable according to this test. However, based on these results it should be considered as inherently and ultimately biodegradable.

Description of key information

The Ready Biodegradability of the substance was first determined by the Manometric Respirometry Test, following Guideline OECD 301 F and performed according to GLP. The tested substance underwent 49% biodegradation after 28 days (77% after 56 days and 76% after 70 days) in the test conditions. The 10-day window criterion was not fulfilled (14% biodegradation at day 6 and 41% at day 16). The tested substance did not inhibit the intrinsic respiration of the inoculum at the test concentration and was therefore considered to be non-toxic to the incoculum at the test concentration. Thus, the test item should be considered as not readily biodegradable according to this test. However, based on these results it should be considered as inherently and ultimately biodegradable.

A second study was conducted according to Guideline OECD 301D. Test item 7-METHOXY-3,7-DIMETHYLOCTAN-2-OL MULTICONSTITUENT was biodegraded by 10% at day 28 in the Closed Bottle test and by 45% at day 55, confirming that the substance cannot be considered as readily biodegradable. However, the results at 55 days are not reliable as the difference between both replicates for the test substance degradation is higher than 20% (35%).

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable