Reaction mass of (3E)-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one and 4-(dodecylsulfanyl)-4-(2,6,6-trimethylcyclohex-1-en-1-yl)butan-2-one and 4-(dodecylsulfanyl)-4-(2,6,6-trimethylcyclohex-2-en-1-yl)butan-2-one

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:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. Extended biodegradation exposure to 60-days was conducted under non-GLP in accordance with established literature.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Extension to 60-days in a parallel screening using the same conditions of the main test.

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: February 2011; signature: September 2011

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A mixed population of activated sewage sludge micro-organisms (Test System) was obtained from the secondary treatment stage of the sewage treatment plant at Villette (Geneva, Switzerland)), which treats predominantly domestic sewage.
- Storage conditions: See pretreatment field.
- Storage length: < 1 week
- Preparation of inoculum for exposure:
- Pretreatment: The sample of activated sewage sludge was maintained on continuous aeration upon receipt. A sample of the activated sewage sludge was washed three times by settlement (centrifuge) and suspension in culture medium. To remove any excessive amounts of Dissolved Organic Carbon (DOC) that may have been present, the solution was stirred and maintained on with pure oxygen at room temperature. Determination of dry weight is made to inoculate final solution with 30mg/L dry weight activated sludge.
- Concentration of sludge: The sludge was diluted in the BOD bottles to 30 mg DW/L.

Duration of test (contact time):

28 d

Initial conc.:

>= 99 - <= 106.65 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: See table 1. 10 mL of Solution A [KH2PO4: 8.50 g/L; K2HPO4: 21.75g/L; Na2HPO4,2H2O: 33.40 g/L; NH4Cl: 0.50 g/L]; 1 mL of Solution B [CaCl2,2H2O: 36.40 g/L]; 1 mL of Solution C [MgSO4,7H2O: 22.50 g/L]; 1 mL of Solution D [FeCl3,6H2O: 0.25 g/L] and purified water diluted to 1 L.
- Solubilising agent (type and concentration if used): None.
- Test temperature: 22 ±1 °C
- pH: See table 1.
- pH adjusted: No.
- Aeration of dilution water: Not reported
- Continuous darkness: No. The test was conducted in diffuse light.

TEST SYSTEM
- Culturing apparatus: Flasks with continuous stirring.
- Number of culture flasks/concentration: In triplicate (test item); In duplicate (Inoculum blank); single flasks (Abiotic Sterile Control and toxicity control)
- Method used to create aerobic conditions: Sealed flasks wth sensor head/CO2 trap.
- Measuring equipment: The respirometer used during this study is an Oxitop Control System (WTW oxitopC). Evolved carbon dioxide is absorbed the CO2 trap.

SAMPLING
- Sampling frequency: Daily.
- Sampling method: The respirometer used during this study is an Oxitop Control System.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes. See table 1.
- Abiotic sterile control: Yes.
- Toxicity control: Yes.
- Other: Positive reference control (Sodium Benzoate).

Reference substance:

benzoic acid, sodium salt

Remarks:

105 mg/L

Test performance:

The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled for the flasks containing test substance. Therefore, the test is considered valid.

Parameter:

% degradation (O2 consumption)

Value:

39

Sampling time:

28 d

Remarks on result:

other: mean biodegradation (in triplicate): test 1: 47%; test 2: 39% and test 3: 33%

Parameter:

% degradation (O2 consumption)

Value:

70

Sampling time:

60 d

Remarks on result:

other: mean biodegradation (in triplicate): test 1: 77%; test 2: 74% and test 3: 59%; not under GLP conditions.

Details on results:

The test material (test 1) attained 47% degradation after 28 days.
The test material (test 2) attained 39% degradation after 28 days.
The test material (test 3) attained 33% degradation after 28 days.

Results with reference substance:

Degradation of sodium benzoate exceeded 40% after 2 days and 65% after 5 days: the activity of the inoculum was thus verified (validity criterion).

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable, not fulfilling specific criteria

Conclusions:

The test substance mean biodegradation in triplicate was 39 % at day 28. In an extended exposure to 60 days the test substance mean biodegradation in triplicate was 70% 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 301F guideline under GLP. The test substance, at a concentration of 106.65 mg/L in test 1, at a concentration of 105.70 mg/L in test 2 and at a concentration of 99.00 mg/L in test 3 was exposed to activated sewage sludge micro-organisms obtained from the secondary treatment stage of the sewage treatment which treats predominantly domestic sewage (Villette: Geneva, Switzerland) with culture medium in sealed culture vessels in diffused light at 22°C ± 1°C for 28 days. The sludge was diluted in the BOD bottles to 30 mg DW/L. The degradation of the test substance was assessed by the measurement of daily oxygen consumption on days 0 and 28 using an Oxitop control system. Control solutions with inoculum and the reference substance, sodium benzoate, together with a toxicity control were used for validation purposes. The oxygen uptake was < 32 mg O2/L in all of the test systems and the pH value at the end of the test period 28 days did not exceed 7.20. The test system therefore met the validation criteria of the guideline. The toxicity test attained 54 % degradation after 28 days thereby confirming that the test substance was not inhibitory to the sewage treatment micro-organisms used in the study. Sodium Benzoate attained 88% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The mean biodegradation for triplicate test flasks at 28 days for the test substance was 39%. Under the conditions of the study, test substance could not be considered as readily biodegradable. A parallel series of tests (not under GLP) was conducted and using the same conditions as the main study with a prolonged exposure to 60-days. The test substance (test 1, 2 and 3) attained respectively 77%, 74% and 59% at day 60, demonstrating that the substance was not persistent and indicative of the substance inherent, ultimate biodegradability.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

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 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: GB/T 2180 I - 2008: Chemicals - Ready Biodegradability: Manometric Respirometry Test. Standards Press of China (2008).

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge from Datansha Sewage Treatment Plant of Guangzhou, China. The inoculum was selected as it was recommended by "The Guidelines for the Testing of Chemicals". The batch number of the inoculum is included in the full study report.
- Storage conditions: See pretreatment field.
- Storage length: < 1 week; 3 days from sampling to test initiation.
- Preparation of inoculum for exposure:
- Pretreatment: The sludge was removed any coarse particles and impurities on the surface, then was washed with the test medium (1100 g, 10 min, repeated for 6 times), the supernatant liquid phase was decanted and the suspended solids re-suspended in the test medium. The dry weight of the suspended solid was determined as 5.1 g/L. Before the test, 47 mL of the above inoculum was taken into a triangular flask and suspended in 53 mL of the test medium to obtain a concentration equivalent to 2.4 g suspended solids per litre, keeping the sludge aerobic until used. The inoculum was added into the test suspensions and the procedure control to give a final concentration of 27.6 mg suspended solids per litre and those of the inoculum blanks and the toxicity control to give a final concentration of 27.8 mg suspended solids per litre.
- Concentration of sludge: The sludge was diluted in flasks to 27.6 mg SS/L. within the test systems; 27.8 mg SS/L in the negative and toxicity controls.

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: See table 1. Solution A [KH2PO4: 8.50 g; K2HPO4.3H2O: 28.50 g; Na2HPO4.12H2O: 67.15 g; NH4Cl: 0.50 g - in 1 L water]; Solution B [CaCl2: 13.75 g in 0.5 L water]; Solution C [MgSO4.7H2O: 11.25 g in 0.5 L water]; Solution D [FeCl3,6H2O: 0.125 g in 0.5 L water]. 50 mL of solution A was mixed with 4 L deionised water; then 5 ml of solutions B, C and D and made up to 5 L with deionised water.
- Solubilising agent (type and concentration if used): None.
- Test temperature: 22 ±1 °C
- pH: See table 1.
- pH adjusted: If necessary adjusted the pH values of bottles I, 2, 3, 4, 5 and 6 to 7.4 ± 0.2 at the start of the test
- Aeration of dilution water: Not reported
- Continuous darkness: Yes.

TEST SYSTEM
- Culturing apparatus: Flasks with continuous stirring.
- Number of culture flasks/concentration: In duplicate (Inoculum blank); single flasks (procedure control and toxicity control)
- Method used to create aerobic conditions: Sealed flasks wth sensor head/CO2 trap.
- Measuring equipment: The respirometer used during this study is an Oxitop Control System.

SAMPLING
- Sampling frequency: Daily.
- Sampling method: Respirometry measuring the oxygen uptake of the test medium.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes. See table 1.
- Abiotic sterile control: No.
- Toxicity control: Yes.
- Other: Positive reference control ('Procedure control' ; Sodium Benzoate).

Reference substance:

benzoic acid, sodium salt

Remarks:

32.9 mg/L

Test performance:

(1) The oxygen uptake of the inoculum blanks were 18.2 mg/L and 18.0 mg/L, the mean was 18.1 mg/L, less than 60 mg/L in 28d.
(2) At the end of the test, the pH values of each test vessel were 7.43-7.64, within the range of 6.0-8.5.
(3) The difference of extremes of replicate values of the removal of the test substance at the plateau, at end of the test or at the end of the 10-d window was less than 20%.
(4) The percentage degradation of the procedure control and the toxicity control were 93.4% and 60.4%, which had reached the pass levels of 60% and 25% of ThOD on 14d.

Parameter:

% degradation (O2 consumption)

Value:

57.6

Sampling time:

28 d

Results with reference substance:

Degradation of sodium benzoate exceeded 93.4 % after 14 days: the activity of the inoculum was thus verified (validity criterion).

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test substance mean biodegradation in duplicate was 57.6 % at day 28.

Executive summary:

The ready biodegradability test was carried out according to OECD TG 301F guideline under GLP. The test substance, at a concentration of 20 mg/L was exposed to activated sewage sludge micro-organisms obtained from the Datansha Sewage Treatment Plant of Guangzhou, China, with culture medium in sealed culture vessels in the dark at 22°C ± 1°C for 28 days. The sludge was diluted in flasks to 27.6 mg SS/L. within the test systems; 27.8 mg SS/L in the negative and toxicity controls. The degradation of the test substance was assessed by the measurement of daily oxygen consumption on days 0 and 28 using an Oxitop control system. Control solutions with inoculum and the reference substance, sodium benzoate, together with a toxicity control were used for validation purposes. The oxygen uptake of the inoculum blanks were 18.2 mg/L and 18.0 mg/L, the mean was 18.1 mg/L, less than 60 mg/L in 28d. At the end of the test, the pH values of each test vessel were within the range of 6.0-8.5. The test system therefore met the validation criteria of the guideline. The toxicity test attained 60 % degradation after 28 days thereby confirming that the test substance was not inhibitory to the sewage treatment micro-organisms used in the study. Sodium Benzoate attained 95 % degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The mean biodegradation for duplicate test flasks at 28 days for the test substance was 57.6 %. Under the conditions of the study, test substance could not be considered as readily biodegradable.

Description of key information

1. Biodegradation: mean biodegradation 39% (28-days) not readily biodegradable, mean degradation 70%, substance within 60-day extended time period; substance is inherently biodegradable, OECD TG 301F, 2013

2. Biodegradation: mean biodegradation 57.6% (28-days) not readily biodegradable, OECD TG 301F, 2014

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, not fulfilling specific criteria

Additional information

OECD TG 301F, 2013 - The ready biodegradability test was carried out according to OECD TG 301F guideline under GLP. The test substance, at a concentration of 106.65 mg/L in test 1, at a concentration of 105.70 mg/L in test 2 and at a concentration of 99.00 mg/L in test 3 was exposed to activated sewage sludge micro-organisms obtained from the secondary treatment stage of the sewage treatment which treats predominantly domestic sewage (Villette: Geneva, Switzerland) with culture medium in sealed culture vessels in diffused light at 22°C ± 1°C for 28 days. The sludge was diluted in the BOD bottles to 30 mg DW/L. The degradation of the test substance was assessed by the measurement of daily oxygen consumption on days 0 and 28 using an Oxitop control system. Control solutions with inoculum and the reference substance, sodium benzoate, together with a toxicity control were used for validation purposes. The oxygen uptake was < 32 mg O2/L in all of the test systems and the pH value at the end of the test period 28 days did not exceed 7.20. The test system therefore met the validation criteria of the guideline. The toxicity test attained 54 % degradation after 28 days thereby confirming that the test substance was not inhibitory to the sewage treatment micro-organisms used in the study. Sodium Benzoate attained 88% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The mean biodegradation for triplicate test flasks at 28 days for the test substance was 39%. Under the conditions of the study, test substance could not be considered as readily biodegradable. A parallel series of tests (not under GLP) was conducted and using the same conditions as the main study with a prolonged exposure to 60-days. The test substance (test 1, 2 and 3) attained respectively 77%, 74% and 59% at day 60, demonstrating that the substance was not persistent and indicative of the substance inherent, ultimate biodegradability.

 

OECD TG 301F, 2014 - The ready biodegradability test was carried out according to OECD TG 301F guideline under GLP. The test substance, at a concentration of 20 mg/L was exposed to activated sewage sludge micro-organisms obtained from the Datansha Sewage Treatment Plant of Guangzhou, China, with culture medium in sealed culture vessels in the dark at 22°C ± 1°C for 28 days. The sludge was diluted in flasks to 27.6 mg SS/L. within the test systems; 27.8 mg SS/L in the negative and toxicity controls. The degradation of the test substance was assessed by the measurement of daily oxygen consumption on days 0 and 28 using an Oxitop control system. Control solutions with inoculum and the reference substance, sodium benzoate, together with a toxicity control were used for validation purposes. The oxygen uptake of the inoculum blanks were 18.2 mg/L and 18.0 mg/L, the mean was 18.1 mg/L, less than 60 mg/L in 28d. At the end of the test, the pH values of each test vessel were within the range of 6.0-8.5. The test system therefore met the validation criteria of the guideline. The toxicity test attained 60 % degradation after 28 days thereby confirming that the test substance was not inhibitory to the sewage treatment micro-organisms used in the study. Sodium Benzoate attained 95 % degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The mean biodegradation for duplicate test flasks at 28 days for the test substance was 57.6 %. Under the conditions of the study, test substance could not be considered as readily biodegradable.

 

The weight of evidence indicates that: the substance is not-readily biodegradable under the conditions of the OECD TG 301F test. However, there is sufficient evidence to indicate that the substance is inherently biodegradable (not fulfilling specific criteria), since the substance attained 70% degradation under the prolonged OECD TG 301F (2013) study. This can be attributed to evidence of inherent, ultimate biodegradation which provides data for extrapolation to degradation rate constants within environmental compartments.

 

References:

1. ECHA Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.7b: Endpoint Specific Guidance, R.7.9.4.1 and R7.9.4.4, version 4.0, June 2017)

2. Introduction to the section of the OECD Guidelines for Testing of Chemicals on Degradation and Accumulation, Section 3 (OECD, September 2006)

3. ECHA Guidance on Application on the CLP Criteria, section 4.1.3.2.3.2 (version 5.0, June 2017)