3-{3-[(3,5,5-trimethylhexanoyl)oxy]-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propoxy}-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate

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:

2012-08-07 to 2012-09-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

GLP compliance:

yes (incl. QA statement)

Remarks:

19-21 July 2011

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Aeration stage of Severn Trent Water Plc sewage treatment plant, Loughborough, Leicestershire, UK on 06-08-2012
- Storage conditions: Washed twice by settlement and resuspension in mineral medium, maintained on continuous aeration at 21°C.
- Storage length: Used on day of collection
- Preparation of inoculum for exposure: As above
- Pretreatment: None
- Concentration of sludge: 30 mg suspended solids/L
- Initial cell/biomass concentration: Suspended solids concentration = 4.0 g/L prior to use
- Water filtered: no

Duration of test (contact time):

28 d

Initial conc.:

10 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: as per OECD Guideline
- Additional substrate: No
- Solubilising agent (type and concentration if used): dispersed in mineral medium
- Test temperature: 21- 23°C
- pH: 7.4 - 7.7
- pH adjusted: yes to pH 7.4 prior to inoculm inclusion if needed.
- Suspended solids concentration: 30 mg ss/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 5L test culture vessels
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: CO2 free air bubbled through solution at rate of 30 to 100 mL/min per vessel
- Measuring equipment: See analytical method
- Details of trap for CO2 and volatile organics if used: two 500 mL Dreschel bottles containing 350 mL of 0.05M NaOH, prepared using purified de-gassed water

SAMPLING
- Sampling frequency:
Ist absorber: Days 0, 2, 4, 6, 8, 10, 14, 21, 28 and 29
2nd absorber: Days 0 and 29
- Sample storage before analysis: No

CONTROL AND BLANK SYSTEM
- Inoculum blank: inoculated mineral medim (x2)
- Abiotic sterile control: No
- Toxicity control: test item plus reference item in incoculated medium to give concentration of 20 mg C/L (x1)


STATISTICAL METHODS:

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

2 d

Parameter:

% degradation (CO2 evolution)

Value:

28

Sampling time:

6 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

69

Sampling time:

14 d

Parameter:

% degradation (CO2 evolution)

Value:

106

Sampling time:

21 d

Parameter:

% degradation (CO2 evolution)

Value:

114

Sampling time:

28 d

Details on results:

See attached document for IC values and biodegradation curve.

Results with reference substance:

The toxicity control attained 68% degradation after 14 days and 114% degradation after 28 days thereby confirming the test item was not toxic to sewage treatment micro-organisms.

See attached document for IC values and biodegradation curve.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item attained 114% degradation after 28 days and satisfied the 10-day window validation criterion. The test item can therefore be considered to be readily biodegradable under the terms and conditions of the Guideline.

Executive summary:

Test Guidance

OECD Guideline 301B, EC Method C.4 -C and US EPA OPPTS 835.3110

Method

The test item at a concentration of 10 mg C/L was exposed to activated sewage sludge micro-organisms with a mineral medium in sealed culture vessels in the dark at 21 to 23°C for 28 days.

The degradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

Results

The test item attained 104% degradation after 28 days and satisfied the 10-day window validation criterion.

Conclusions

The test item can therefore be considered to be readily biodegradable under the terms and conditions of the Guideline.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum: Station Epuration Wavre, 2nd Stage, 23.02.1999
- Concentration of sludge: >1000000 CFU/mL (verification with Sanicheck) (CFU = Colony Forming Unit)

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: Clearly chemically defined medium
- Test temperature: 22.1-22.4 °C
- pH: 7.1-7.2
- Aeration of dilution water: CO2-free air, using a debit of 50 to 100 mL/min


TEST SYSTEM
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: CO2-free air
- Details of trap for CO2: The produced CO2 reacts with BA(OH)2 to give a precipitate of Barium carbonate. Then the produced CO2 could be
determinated in titrating the remaining BA(OH)2 with HCL and phenolphtaleine.

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (CO2 evolution)

Value:

63.1

Sampling time:

28 d

Results with reference substance:

The reference substance was degraded to 91.1% by day 28 at a concentration of 3g/l.

The test substance is readily biodegradable under conditions tested. The biodegradation was > 60% within 28 days, but the 10-day-window was not fulfilled. As the test substance is a mixture of constituents with different chain-lengths, sequential biodegradation of the individual structures is taking place. Due to a degradation of > 60% within 28 days Fatty acids, C5-9 esters with pentaerythritol (CAS-No.68424-30-6) can be regarded as readily biodegradable. The 10 -day window should not be applied to interpret the test results (Annex I, OECD Guideline for The Testing of Chemicals 'Revised introduction to the OECD guidelines for testing of chemicals, section 3', OECD, March 2006).

 

Table 1: Percentage CO2 total of reference and test substance

Day no°	Reference	Testsubstance
0	0.0	0.0
2	40.5	0.9
6	69.1	18.4
9	77.7	41.4
15	81.7	54.0
19	86.8	58.0
23	89.9	60.6
28	91.1	63.1

 

 

Interpretation of results:

readily biodegradable

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

23 January - 20 February 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Activated sludge from the aeration tank of a municipal waste water treatment plant, ARA Werdhölzli, 8048
Zürich, Switzerland (22 January 2007)
- Pretreatment: The sludge was washed twice with tap water.

Duration of test (contact time):

28 d

Initial conc.:

20.7 - 20.9 mg/L

Based on:

other: TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: Aerobic mineral salts medium prepared with deionised water
- Test temperature: 22 ± 0.5 °C
- pH adjusted: Yes, the pH-value was checked at the beginning and at the end of the test and adjusted to pH 7.4 (± 0.2) with NaOH or HCL.
- Suspended solids concentration: 30 mg/l
- Continuous darkness: Yes, temperature-controlled dark room

TEST SYSTEM
- Culturing apparatus: 2500 ml closed glass bottle containing a total volume of test solution of 2000 ml
- Number of culture flasks/concentration: 2/concentration
- Method used to create aerobic conditions: Aerated with synthetic CO2-free air for a maximum test period of 28 days.
- Measuring equipment: Shimadzu 5050 TOC-Analyzer using the NPOC-mode for DOC-determination
- Details of trap for CO2: The air leaving the individual vessels was passed through gas-absorption bottles filled with NaOH.

SAMPLING
- Sampling frequency: on day 0, 1, 3, 8, 10, 14, 17, 21, 24 and 28
- Sampling method: Samples were centrifuged (15 min at 4500g) and acidified to pH<2. Prior to analysis the samples were sparged with CO2-free
high purity air for 10 min to remove inorganic carbon.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (CO2 evolution)

Value:

72

Sampling time:

28 d

Results with reference substance:

The reference substance was degraded to 90.6% at day 14 and to 87.9% at day 28.

The test substance is readily biodegradable under conditions tested. The biodegradation was > 60% after 28 days and the

10 -day-window was fulfilled, because the biodegradation reached 61% at the end of the 10 -day-window.

Table 1: Percentage biodegradation (mean of two replicates) of test and reference substance

Day	Test substance (% degradation)	Reference substance (% degradation)
0	0.0	0.0
1	1.9	11.2
3	4.7	48.8
8	39.1	75.1
10	48.5	78.2
14	61.7	90.6
17	67.7	90.1
21	74.7	88.1
24	72.8	88.4
28	72.0	87.9

 

Interpretation of results:

readily biodegradable

Reference 4

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Activated sludge from the aeration basin of the domestic sewage treatment plant, 67435 Neustadt, Wstr.,
Lachen-Speyerdorf, 08 August 2008
- Preparation of inoculum for exposure: aerated continually, washed with and re-suspended in mineral medium

Duration of test (contact time):

29 d

Initial conc.:

32.3 mg/L

Based on:

test mat.

Initial conc.:

32.2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral medium as described in the OECD-Guideline
- Test temperature: 22 ± 1°C
- Suspended solids concentration: 25 mg/L

TEST SYSTEM
- Culturing apparatus: 2000 ml Schott-flasks
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: All flasks were aerated for > 24 hours with purified, CO2-free, moistened air.
- Measuring equipment: TOC multi N/C 2100S, Analytik Jena
- Details of trap for CO2 : The scrubbing of carbon dioxide was achieved by bubbling the purified air through a flask containing 1-m-NaOH. The
emitted CO2 was trapped in 0.25-m-NaOH.

SAMPLING
- Sampling frequency: day 0, 2, 4, 7, 9, 11, 14, 18, 23, 25 and 29

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2
- Abiotic sterile control: 1
- Toxicity control: 1

Reference substance:

aniline

Remarks:

26.1 mg/L

Parameter:

% degradation (CO2 evolution)

Value:

78.9

Sampling time:

29 d

Results with reference substance:

The reference substance was degraded to 84% at day 14 and to 72.1% at day 29.

The test substance is readily biodegradable under conditions tested. The biodegradation was 79% after 29 days and the 10 -day-window was fulfilled. The 10 -day-window began on day 1 and at its end 78% were reached.

In one reference flask was a leakage. Therefore the values for this replicate were not evaluated. As the test substance is readily biodegradable, the inoculum is healthy. The reference flask with the leakage can be disregarded.

The IC content of test item in medium was higher (35.2%) then demanded in the guideline. This due to the poorly water solubility of the test item. The nominal concentration of the measured solution was 1142 mg/L, corresponding to a theoretical organic carbon content of 811 ppm. The carbon content was measured with 11.69 ppm, with IC content being 5.1 ppm. The measured IC corresponds to 0.6% of nominal TC. Therefore the result is considered valid.

Table 1: Percentage biodegradation of test substance (average of 2 replicates) and reference substance,abiotic and toxicity control (respectively 1 replicate)

Day	Test Substance (% degradation)	Reference substance (% degradation)	Abiotic control (% degradation)	Toxicity control (% degradation)
2	20.7	27.8	-0.2	29.7
4	41.8	68.5	1.1	57.3
7	60.8	76.5	0.1	71.6
9	70.9	86.7	1.6	78.8
11	77.7	86.2	2.1	83.5
14	84.6	84.0	2.0	84.1
18	84.7	84.4	9.0	87.9
23	82.2	78.5	10.6	84.5
25	78.3	75.6	13.2	86.6
29	78.9	72.1	17.0	88.0

 

 

 

Interpretation of results:

readily biodegradable

Reference 5

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

To register EC453-460-3 under REACH program, a read-across approach for fulfillment of the endpoints has been adopted. EC453-460-3 is an aliphatic ester and fits the description of polyol esters category in HPV program (High Production Volume, US-EPA). The distinguishing feature of polyol esters category is that the fatty acids were linked to one or more of the multiple hydroxyl groups present in the polyol (alcohol portion of ester). The focus of this correspondence is to support read across to EC613-848-7 (target) which provided key studies to determine hazard profile of EC453-460-3 (target), published information on other structurally analogous polyol esters were served as supporting evidence.

Reason / purpose for cross-reference:

read-across source

Parameter:

% degradation (CO2 evolution)

Value:

78.9

Sampling time:

29 d

Results with reference substance:

The reference substance was degraded to 84% at day 14 and to 72.1% at day 29.

The test substance is readily biodegradable under conditions tested. The biodegradation was 79% after 29 days and the 10 -day-window was fulfilled. The 10 -day-window began on day 1 and at its end 78% were reached.

In one reference flask was a leakage. Therefore the values for this replicate were not evaluated. As the test substance is readily biodegradable, the inoculum is healthy. The reference flask with the leakage can be disregarded.

The IC content of test item in medium was higher (35.2%) then demanded in the guideline. This due to the poorly water solubility of the test item. The nominal concentration of the measured solution was 1142 mg/L, corresponding to a theoretical organic carbon content of 811 ppm. The carbon content was measured with 11.69 ppm, with IC content being 5.1 ppm. The measured IC corresponds to 0.6% of nominal TC. Therefore the result is considered valid.

Table 1: Percentage biodegradation of test substance (average of 2 replicates) and reference substance,abiotic and toxicity control (respectively 1 replicate)

Day	Test Substance (% degradation)	Reference substance (% degradation)	Abiotic control (% degradation)	Toxicity control (% degradation)
2	20.7	27.8	-0.2	29.7
4	41.8	68.5	1.1	57.3
7	60.8	76.5	0.1	71.6
9	70.9	86.7	1.6	78.8
11	77.7	86.2	2.1	83.5
14	84.6	84.0	2.0	84.1
18	84.7	84.4	9.0	87.9
23	82.2	78.5	10.6	84.5
25	78.3	75.6	13.2	86.6
29	78.9	72.1	17.0	88.0

 

 

 

Interpretation of results:

readily biodegradable

Reference 6

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

To register EC453-460-3 under REACH program, a read-across approach for fulfillment of the endpoints has been adopted. EC453-460-3 is an aliphatic ester and fits the description of polyol esters category in HPV program (High Production Volume, US-EPA). The distinguishing feature of polyol esters category is that the fatty acids were linked to one or more of the multiple hydroxyl groups present in the polyol (alcohol portion of ester). The focus of this correspondence is to support read across to EC613-848-7 (target) which provided key studies to determine hazard profile of EC453-460-3 (target), published information on other structurally analogous polyol esters were served as supporting evidence.

Reason / purpose for cross-reference:

read-across source

Parameter:

% degradation (CO2 evolution)

Value:

63.1

Sampling time:

28 d

Results with reference substance:

The reference substance was degraded to 91.1% by day 28 at a concentration of 3g/l.

The test substance is readily biodegradable under conditions tested. The biodegradation was > 60% within 28 days, but the 10-day-window was not fulfilled. As the test substance is a mixture of constituents with different chain-lengths, sequential biodegradation of the individual structures is taking place. Due to a degradation of > 60% within 28 days Fatty acids, C5-9 esters with pentaerythritol (CAS-No.68424-30-6) can be regarded as readily biodegradable. The 10 -day window should not be applied to interpret the test results (Annex I, OECD Guideline for The Testing of Chemicals 'Revised introduction to the OECD guidelines for testing of chemicals, section 3', OECD, March 2006).

 

Table 1: Percentage CO2 total of reference and test substance

Day no°	Reference	Testsubstance
0	0.0	0.0
2	40.5	0.9
6	69.1	18.4
9	77.7	41.4
15	81.7	54.0
19	86.8	58.0
23	89.9	60.6
28	91.1	63.1

 

 

Interpretation of results:

readily biodegradable

Reference 7

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

To register EC453-460-3 under REACH program, a read-across approach for fulfillment of the endpoints has been adopted. EC453-460-3 is an aliphatic ester and fits the description of polyol esters category in HPV program (High Production Volume, US-EPA). The distinguishing feature of polyol esters category is that the fatty acids were linked to one or more of the multiple hydroxyl groups present in the polyol (alcohol portion of ester). The focus of this correspondence is to support read across to EC613-848-7 (target) which provided key studies to determine hazard profile of EC453-460-3 (target), published information on other structurally analogous polyol esters were served as supporting evidence.

Reason / purpose for cross-reference:

read-across source

Duration of test (contact time):

d

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

2 d

Parameter:

% degradation (CO2 evolution)

Value:

28

Sampling time:

6 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

69

Sampling time:

14 d

Parameter:

% degradation (CO2 evolution)

Value:

106

Sampling time:

21 d

Parameter:

% degradation (CO2 evolution)

Value:

114

Sampling time:

28 d

Details on results:

See attached document for IC values and biodegradation curve.

Results with reference substance:

The toxicity control attained 68% degradation after 14 days and 114% degradation after 28 days thereby confirming the test item was not toxic to sewage treatment micro-organisms.

See attached document for IC values and biodegradation curve.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item attained 114% degradation after 28 days and satisfied the 10-day window validation criterion. The test item can therefore be considered to be readily biodegradable under the terms and conditions of the Guideline.

Executive summary:

Test Guidance

OECD Guideline 301B, EC Method C.4 -C and US EPA OPPTS 835.3110

Method

The test item at a concentration of 10 mg C/L was exposed to activated sewage sludge micro-organisms with a mineral medium in sealed culture vessels in the dark at 21 to 23°C for 28 days.

The degradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

Results

The test item attained 104% degradation after 28 days and satisfied the 10-day window validation criterion.

Conclusions

The test item can therefore be considered to be readily biodegradable under the terms and conditions of the Guideline.

Reference 8

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

To register EC453-460-3 under REACH program, a read-across approach for fulfillment of the endpoints has been adopted. EC453-460-3 is an aliphatic ester and fits the description of polyol esters category in HPV program (High Production Volume, US-EPA). The distinguishing feature of polyol esters category is that the fatty acids were linked to one or more of the multiple hydroxyl groups present in the polyol (alcohol portion of ester). The focus of this correspondence is to support read across to EC613-848-7 (target) which provided key studies to determine hazard profile of EC453-460-3 (target), published information on other structurally analogous polyol esters were served as supporting evidence.

Reason / purpose for cross-reference:

read-across source

Parameter:

% degradation (CO2 evolution)

Value:

72

Sampling time:

28 d

Results with reference substance:

The reference substance was degraded to 90.6% at day 14 and to 87.9% at day 28.

The test substance is readily biodegradable under conditions tested. The biodegradation was > 60% after 28 days and the

10 -day-window was fulfilled, because the biodegradation reached 61% at the end of the 10 -day-window.

Table 1: Percentage biodegradation (mean of two replicates) of test and reference substance

Day	Test substance (% degradation)	Reference substance (% degradation)
0	0.0	0.0
1	1.9	11.2
3	4.7	48.8
8	39.1	75.1
10	48.5	78.2
14	61.7	90.6
17	67.7	90.1
21	74.7	88.1
24	72.8	88.4
28	72.0	87.9

 

Interpretation of results:

readily biodegradable

Description of key information

The test item attained 114% degradation after 28 days and satisfied the 10-day window validation criterion. The test item can therefore be considered to be readily biodegradable under the terms and conditions of the Guideline.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable