Cyclohexanepropanol, 2,2,3,6-tetramethyl-.alpha.-propyl-

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:

17-07-2020 to 12-10-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

The Closed Bottle test was prolonged : REACH enhanced biodegradation screening test reference: ECHA (2017) European Chemicals Agency, Guidance on information requirements and chemical safety assessment Chapter R.7b: Endpoint specific guidance, ECHA-12-G-22-EN

Deviations:

no

Principles of method if other than guideline:

The study was extended using established inherent biodegradation determination literature methodology.

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: November 2019 ; signature: December 2019

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): 1. Secondary activated sludge was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge plant treating predominantly domestic wastewater.
- Storage conditions: See pretreatment field.
- Storage length: < 1 week
- Preparation of inoculum for exposure: See pre-treatment field.
- Pretreatment: The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 0.4 g Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted in the bottles to 2.0 mg/L.
- Concentration of sludge: The sludge was diluted in the BOD bottles to 2 mg DW/L

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:

TEST CONDITIONS
- Composition of medium: The nutrient medium of the Closed Bottle test contained per litre of deionized water; 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.3 mg Na2HPO4.2H2O, 22.5 mg MgSO4.7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3.6H2O. Ammonium chloride was omitted from the medium to prevent nitrification.
- Solubilising agent (type and concentration if used): None.
- Test temperature: Temperatures were within the prescribed temperature range of 22 to 24°C (Actual: 22.6 to 22.9°C).
- pH: The pH of the media (activated sludge) was 7.4 at the start of the test. The pH of the medium at day 28 was 7.2 (controls), 7.1 (controls plus silica gel) and 6.8 (test)
- pH adjusted: No.
- Aeration of dilution water: Not reported
- Suspended solids concentration: 2 mg/L dry weight
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.
- Number of culture flasks/concentration: 2 bottles containing only inoculum per time point (10 bottles total) ; 2 bottles containing test item with inoculum per time point (10 bottles total) ; 2 bottles containing test item and reference item and inoculum per time point (10 bottles total). The Closed Bottle test was prolonged. See SAMPLING for further information.
- Measuring equipment: See details on analytical methods. The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter. The pH was measured using a pH meter. The temperature was measured and recorded with a sensor connected to a data logger.
- Test performed in closed vessels due to significant volatility of test substance: No.
- Test performed in open system: No.

SAMPLING
- Sampling frequency: Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28. The Closed Bottle test was prolonged (REACH enhanced biodegradation screening tests, ECHA 2017), by measuring the course of the oxygen decrease at day 42, 60 and 84 using the bottles of day 28 and a specially designed funnel. This funnel fitted exactly in the BOD bottle. Subsequently, the oxygen electrode was inserted in the BOD bottle to measure the oxygen concentration. The medium dissipated by the electrode was collected in the funnel. After withdrawal of the oxygen electrode the medium collected flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo, Simple method to prolong the Closed Bottle test for the determination of the inherent biodegradability. Ecotoxicol. Environmen. Saf. 24 319-327. 1992).
- Sampling method: Analyses of the dissolved oxygen concentration
- Sterility check if applicable: No.
- Sample storage before analysis: Not applicable.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes.
- Abiotic sterile control: No.
- Toxicity control: Yes (where applicable)
- Other: Positive reference control (Sodium Acetate)

Reference substance:

acetic acid, sodium salt

Remarks:

6.7 mg/L

Test performance:

1. The endogenous respiration was 0.85 mg/L at day 28 (i.e. < 30 mgO2/L after 28 days)
2. The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled for the flasks containing test item on day 28 and/or reference item at end of 14-day window and on day 28.
3. The pH at day 84 (end of testing) was in the range of 6.0 to 8.5 (actual 7.1 to 7.2 for controls and 6.8 test item vessels)
4. Sodium Acetate attained 84% degradation at 14 days thereby confirming the suitability of the inoculum and test conditions.
5. Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test is optional in the OECD guideline and was not determined because possible toxicity of the test substances to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test substance was detected at day 7 and day 14 (Table I). Therefore, it is expected that the biodegradation in the first two weeks of the test was hampered due to the "high" initial test substance concentration.

Key result

Parameter:

% degradation (O2 consumption)

Value:

4

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

26

Sampling time:

42 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

60

Sampling time:

60 d

Parameter:

% degradation (O2 consumption)

Value:

68

Sampling time:

84 d

Results with reference substance:

Sodium Acetate attained 84% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions.

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable, not fulfilling specific criteria

Conclusions:

The test item mean biodegradation in duplicate was 4 % at day 28. In an extended exposure to 60 days the test item mean biodegradation in duplicate was 60% indicating that the substance is not persistent and demonstrates inherent, ultimate biodegradability.

Executive summary:

The ready biodegradability test was carried out according to OECD TG 301D guideline under GLP. The Closed Bottle test was prolonged : REACH enhanced biodegradation screening test reference: ECHA (2017) European Chemicals Agency, Guidance on information requirements and chemical safety assessment Chapter R.7b: Endpoint specific guidance, ECHA-12-G-22-EN. The test item at a concentration of 2 mg/L was exposed to activated sewage sludge micro-organisms obtained from the secondary treatment stage of the sewage treatment which treats predominantly domestic sewage in sealed BOD vessels and in the dark at 22°C ± 1°C for 84 days. The inoculum concentration was 2 mg/L DW in the BOD bottles. Degradation was assessed by the measurement of oxygen consumption via dissolved oxygen concentration measurement. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28. The Closed Bottle test was prolonged by measuring the course of the oxygen decrease at day 42, 60 and 84 using the bottles of day 28 and a specially designed funnel. This funnel fitted exactly in the BOD bottle. Subsequently, the oxygen electrode was inserted in the BOD bottle to measure the oxygen concentration. The medium dissipated by the electrode was collected in the funnel. After withdrawal of the oxygen electrode the medium collected flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo, 1992). Control solutions with inoculum and the reference substance: sodium acetate, together with a toxicity control (where applicable) were used for validation purposes. The endogenous respiration was 0.85 mg/L at day 28 (i.e. < 30 mgO2/L after 28 days). The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled for the flasks containing test item on day 28 and/or reference item at end of 14-day window and on day 28. The pH at day 84 (end of testing) was in the range of 6.0 to 8.5 (actual 7.1 to 7.2 for controls and 6.8 test item vessels). Sodium Acetate attained 84% degradation at 14 days thereby confirming the suitability of the inoculum and test conditions. The mean biodegradation for the test item at 28 days was 4%. In the prolonged Closed Bottle test (enhanced biodegradability test) the test item was biodegraded by 60% and 68% at day 60 and 84, respectively. The biodegradation percentage of ≥ 60% at day 60 demonstrates that the test item is ultimately (completely) biodegradable. Moreover, the biodegradation of 60% measured at day 60 allows classification of the test item as not persistent. Under the conditions of the study, test item is considered to be Inherently biodegradable.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10-04-2007 to 31-05-2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: August 2005 ; signature: November 2005

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Severn Trent Water Plc, Loughborough, UK, mixed population of activated sewage sludge micro-organisms from aeration stage which treats predominantly domestic sewage
- Laboratory culture: See below.
- Method of cultivation: See below.
- Storage conditions: Room temperature (at ca. 21°C).
- Storage length: Used on day of collection (< 1 week).
- Preparation of inoculum for exposure: Aerobic activated sludge: washed three times by settlement and resuspension in mineral medium to remove any excessive amounts of dissolved organic carbon (DOC)
- Concentration of sludge: 1.9 g/L solids concentration determined prior to use (and 30 mg SS/L final concentration in test vessels).
- Initial cell/biomass concentration: Not reported.
- Water filtered: yes (water used to prepare culture medium).
- Type and size of filter used, if any: Aerated reverse osmosis purified and deionised water. The 'dilution water' was kept in a temperature controlled room, at approximately 21 °C, under gentle aeration for 24 hours prior to use

Initial conc.:

12.5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Parameter followed for biodegradation estimation:

inorg. C analysis

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Composition of medium: Solution a: KH2PO4 8.50 g/L; K2HPO4 21.75 g/L; Na2HPO4.2H2O 33.40 g/L ; NH4Cl 0.50 g/L ; pH = 7.4 ; Solution b: CaCl2 27.50 g/L ; Solution c: MgSO4.7H2O 22.50 g/L ; Solution d: FeCl3.6H2O 0.25 g/L. To 1 litre (final volume) of purified water was added the following volumes of solutions: a – d. 10 mL of Solution a, with 1 mL of each of Solution b, Solution c and Solution d. Reverse osmosis purified and deionized water.
Test item concentration: 12.5 mg/L, equivalent to 10 mg C/L ;
Procedure control: Reference substance sodium benzoate, concentration 17.1 mg/L, equivalent to 10 mg C/L
Toxicity control: concentration, 12.5 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg C/L
- Solubilising agent (type and concentration if used): None
- Test temperature: 21 °C, maintained in temperature controlled room
- pH: ; pH of the test preparations was determined on Day 28 prior to acidification. See table 1
- pH adjusted: No. If necessary the pH was adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution, prior to the volume in all the vessels
being adjusted to 3 litres by the addition of mineral medium which had been purged overnight with CO2 free air. pH of the test preparations was also determined on day 28, prior to acidification with HCl, using a WTW pH/Oxi 3401 pH and dissolved oxygen meter
- Aeration of dilution water: Yes.
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: Preparations were prepared and inoculated in 5 litre test culture vessels each containing 3 litres of solution
- Number of culture flasks/concentration: In duplicate, except for Toxicity Control
- Method used to create aerobic conditions: The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 40 mL/min per vessel and stirred continuously by magnetic stirrer. The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime granules.
- Measuring equipment: Samples were analyzed for CO2 using a Tekmar-Dohrmann Apollo 9000 TOC analyser and a Shimadzu TOC-VCSH TOC analyser. Samples were analyzed for IC and TC using a Shimadzu TOC-VCPH TOC Analyzer. The pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification using a WTW pH/Oxi 340I pH and dissolved oxygen meter.
- Test performed in closed vessels due to significant volatility of test substance: No.
- Test performed in open system: Yes.
- Details of trap for CO2 and volatile organics if used: Not applicable.

SAMPLING
- Sampling frequency: CO2: samples (2 mL) were taken from the first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessels were all sampled on Days 0 and 29. ; IC and DOC: Samples (20 mL) were removed from the test item vessels on Day 0 prior to the addition of the test item in order to calculate the Inorganic Carbon content in the test media, but was not possible after dosing. ; pH of the test preparations was determined on Day 28 prior to acidification
- Sampling method: See measuring equipment above.
- Sterility check if applicable: Not reported.
- Sample storage before analysis: No.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes.
- Abiotic sterile control: No.
- Toxicity control: Yes.
- Other: Procedure control - using reference item (Sodium Benzoate only).

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (CO2 evolution)

Value:

6

Sampling time:

28 d

Parameter:

% degradation (CO2 evolution)

Remarks:

following end of day 28 acidification

Value:

10

Sampling time:

29 d

Remarks on result:

other: The final degradation value for day 28 following acidification was 10% degradation by CO2 evolution.

Details on results:

1. The IC content of the test item suspension in the mineral medium at the start of the test was below 5% of the TC content.
2. The total CO2 evolution in the control vessels on Day 28 was 24.29 mg/L and therefore < 40 mg/L medium.
3. The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled on day 28.
4. Sodium Benzoate attained > 60% at 14 days and 92% degradation at 28 days thereby confirming the suitability of the inoculum and test conditions.
5. Inhibition of the degradation of a well-degradable compound, e.g. sodium Benzoate by the test item in the Closed Bottle test was not found, the toxicity control attained in excess of > 25% degradation by day 14. After 28 days the toxicity control achieved 54% degradation.

The test item vessel attained 6% degradation by CO2 evolution on day 28.

The final value for day 28 following acidification was 10% degradation by CO2 evolution.

Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the OECD Test Guidelines. This acidification kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item.

Results with reference substance:

Sodium benzoate attained 87% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions.

Table 2: Percentage Biodegradation Values

Day	% Degradation Sodium Benzoate	% Degradation Test Item + Sodium Benzoate Toxicity Control	% Degradation Test Item
0	0	0	0
1	31	19	0
2	58	35	0
3	59	29	0
6	77	46	0
8	77	49	8
10	78	48	11
14	93	54	6
16	93	54	10
22	93	56	7
24	95	53	6
27	95	56	7
28	92	56	6
29*	96	59	10

* Day 29 values corrected to include any carry-over of C02 detected in Absorber 2

 

Table 3: Total and Inorganic Carbon Values in the Culture Vessels on Day 0

Test Vessel	Total Carbon* (mg/L)	Inorganic Carbon* (mg/L)	IC Content (% of TC)
Sodium Benzoate 10 mg C/L R1	8.52	-0.35	0
Sodium Benzoate 10 mg C/L R2	9.04	0.39	4
Test Item 10 mg C/L R1	8.75**	-0.50	0
Test Item 10 mg C/L R2	8.84**	-0.30	0
Test Item + Sodium Benzoate Toxicity Control 20 mg C/L	19.15**	0.17	1

Where:

R1 - R2 = Replicates 1 and 2

* = Corrected for control values. Negative values are due to measured concentrations being less than control values

** = Total carbon value given is the sum of the TC value obtained from analysis and the nominal TC contribution of the test item and sodium benzoate where applicable

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item mean biodegradation in duplicate was 6 % at day 28. The final value for day 28 following acidification was 10% degradation by CO2 evolution.

Executive summary:

The ready biodegradability test was carried out according to OECD 301B Ready Biodegradability: CO2 Evolution (Modified Sturm Test), EU Method C.4-C and USEPA OPPTS 835.3110 guidelines under GLP. The test item, at a concentration of 12.5 mg/L or 10 mg Carbon/L, was exposed to activated domestic sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at approximately 21 °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 substance, sodium benzoate, together with a toxicity control were used for validation purposes. The IC content of the test item suspension in the mineral medium at the start of the test was below 5% of the TC content and hence satisfied the validation criterion. The total CO2 evolution in the inoculum control vessels on Day 28 was 24.29 mg/L and the difference between the values for CO2 production at the end of the test for the replicate vessels was < 20% and therefore satisfied the validation criterion. The toxicity control attained 54% degradation after 14 days and 59% degradation after 28 days thereby confirming that the test item was no toxic to the sewage treatment micro-organisms used in the test. Sodium benzoate attained > 60% degradation after 14 days and 92% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The test item attained 6% degradation after 28 days. The final value for day 28 following acidification was 10% degradation by CO2 evolution. Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the OECD Test Guidelines. This acidification kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item. Under the conditions of this study, the test item is not readily biodegradable.

Description of key information

Weight of evidence: inherently biodegradable

1. Biodegradation: not readily biodegradable, mean biodegradation 10% (28 -days, following acidification), OECD TG 301B, CO2 Evolution test, 2007

2. Biodegradation: mean biodegradation 4% (28-days) not readily biodegradable, mean degradation 60%, substance within 60-day extended time period; substance is inherently biodegradable, OECD TG 301D, 2021

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, not fulfilling specific criteria

Type of water:

freshwater

Additional information

Key study : OECD TG 301B, 2007 : The ready biodegradability test was carried out according to OECD 301B Ready Biodegradability: CO2 Evolution (Modified Sturm Test), EU Method C.4-C and USEPA OPPTS 835.3110 guidelines under GLP. The test item, at a concentration of 12.5 mg/L or 10 mg Carbon/L, was exposed to activated domestic sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at approximately 21 °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 substance, sodium benzoate, together with a toxicity control were used for validation purposes. The IC content of the test item suspension in the mineral medium at the start of the test was below 5% of the TC content and hence satisfied the validation criterion. The total CO2 evolution in the inoculum control vessels on Day 28 was 24.29 mg/L and the difference between the values for CO2 production at the end of the test for the replicate vessels was < 20% and therefore satisfied the validation criterion. The toxicity control attained 54% degradation after 14 days and 59% degradation after 28 days thereby confirming that the test item was no toxic to the sewage treatment micro-organisms used in the test. Sodium benzoate attained > 60% degradation after 14 days and 92% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The test item attained 6% degradation after 28 days. The final value for day 28 following acidification was 10% degradation by CO2 evolution. Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the OECD Test Guidelines. This acidification kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item. Under the conditions of this study, the test item is not readily biodegradable.

 

Key study : OECD TG 301D - enhanced biodegradability study, 2021 : The ready biodegradability test was carried out according to OECD TG 301D guideline under GLP. The Closed Bottle test was prolonged : REACH enhanced biodegradation screening test reference: ECHA (2017) European Chemicals Agency, Guidance on information requirements and chemical safety assessment Chapter R.7b: Endpoint specific guidance, ECHA-12-G-22-EN. The test item at a concentration of 2 mg/L was exposed to activated sewage sludge micro-organisms obtained from the secondary treatment stage of the sewage treatment which treats predominantly domestic sewage in sealed BOD vessels and in the dark at 22°C ± 1°C for 84 days. The inoculum concentration was 2 mg/L DW in the BOD bottles. Degradation was assessed by the measurement of oxygen consumption via dissolved oxygen concentration measurement. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28. The Closed Bottle test was prolonged by measuring the course of the oxygen decrease at day 42, 60 and 84 using the bottles of day 28 and a specially designed funnel. This funnel fitted exactly in the BOD bottle. Subsequently, the oxygen electrode was inserted in the BOD bottle to measure the oxygen concentration. The medium dissipated by the electrode was collected in the funnel. After withdrawal of the oxygen electrode the medium collected flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo, 1992). Control solutions with inoculum and the reference substance: sodium acetate, together with a toxicity control (where applicable) were used for validation purposes. The endogenous respiration was 0.85 mg/L at day 28 (i.e. < 30 mgO2/L after 28 days). The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled for the flasks containing test item on day 28 and/or reference item at end of 14-day window and on day 28. The pH at day 84 (end of testing) was in the range of 6.0 to 8.5 (actual 7.1 to 7.2 for controls and 6.8 test item vessels). Sodium Acetate attained 84% degradation at 14 days thereby confirming the suitability of the inoculum and test conditions. The mean biodegradation for the test item at 28 days was 4%. In the prolonged Closed Bottle test (enhanced biodegradability test) the test item was biodegraded by 60% and 68% at day 60 and 84, respectively. The biodegradation percentage of ≥ 60% at day 60 demonstrates that the test item is ultimately (completely) biodegradable. Moreover, the biodegradation of 60% measured at day 60 allows classification of the test item as not persistent. Under the conditions of the study, test item is considered to be Inherently biodegradable.