Reaction mass of 4-methyl-2-phenyl-3,6-dihydro-2H-pyran and 4-methyl-6-phenyl-3,6-dihydro-2H-pyran and 4-methylene-2-phenyltetrahydro-2H-pyran

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:

08-05-2013 to 05-06-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

read-across: supporting information

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: July 2012 ; signature: November 2012

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
- Storage conditions: ca. 21°C
- Storage length: Used on day of collection
- Preparation of inoculum for exposure: Aerobic activated sludge: washed two times by settlement and resuspension inmineral medium to remove any excessive amounts of dissolved organic carbon (DOC)
- Concentration of sludge: 3.0 g/L solids concentration prior to use.
- Initial cell/biomass concentration: Not reported.
- Water filtered: yes
- Type and size of filter used, if any: preweighed GF/A filter paper ; rinsed three times with 20 mL deionized reverse osmosis water prior to drying in an oven prior to weighing

Duration of test (contact time):

28 d

Initial conc.:

24.5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

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 ; 1 mL of Solution b ; 1 mL of Solution c ; 1 mL of Solution d. Reverse osmosis purified and deionized water.
Test item concentration: 24.5 mg/L, equivalent to 20 mg carbon/L ; Procedure control: Reference substance sodium benzoate, concentration 17.1 mg/L, equivalent to 10 mg carbon/L ; Toxicity control: concentration, 24.5 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 30 mg carbon/L
- Solubilising agent (type and concentration if used): None
- Test temperature: 21 °C, maintained in temperature controlled room
- pH: Measured prior to and completion of test. See table 1
- pH adjusted: Yes. 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
- Aeration of dilution water: Not reported
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: Preparations were prepared and inoculated in 5 liter test culture vessels each containing 3 liters of solution
- Number of culture flasks/concentration: In duplicate, except for Abiotic Control and Toxictity Control
- Method used to create aerobic conditions: The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 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 analyzer 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 (2ml) 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 TC: Samples (30ml) 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 0 and 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:

46

Sampling time:

28 d

Table 2: Percentage Biodegradation Values

Day	Mean % Degradation Sodium Benzoate Procedure Control	% Degradation Test Substance			Mean % Degradation Test Substance plus Sodium Benzoate Toxicity Control
		R1	R2	Mean (R1 and R2)
0	0	0	0	0	0
2	54	7	6	7	17
6	67	7	13	10	21
8	79	5	19	12	23
10	78	8	20	14	25
14	84	15	26	20	31
21	75	12	36	24	29
28	76	9	55	32	35
29*	79	38	55	46	38

* Day 29 values corrected to include any carry-over of CO2 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)
Test Item 20 mg C/L R1	20.81**	0.75	4
Test Item 20 mg C/L R2	20.95**	0.83	4

R1 – R2 = Replicates 1 and 2

* Corrected for 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

Validity criteria fulfilled:

yes

Interpretation of results:

other: not ready biodegradable

Conclusions:

The test item mean biodegradation in duplicate was 46 % at day 28.

Executive summary:

The ready biodegradability test was carried out according to OECD 301B Ready Biodegradability: CO2 Evolution (Modified Sturm Test) guidelines under GLP. The test substance, at a concentration of 20 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 substance 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 32.64 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 31% degradation after 14 days and 38% degradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test. Sodium benzoate attained 84% degradation after 14 days and 79% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The test substance attained 46% degradation after 28 days and therefore cannot be considered to be readily biodegradable.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

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

Adequacy of study:

key study

Study period:

08-05-2013 to 05-06-2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included in attachment to IUCLID section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across is based on the hypothesis that the source and target substances have common structural features in the same relative positions. The source and target have similar physico-chemical, toxicological properties and because of common metabolism they share common or have similar breakdown products and therefore potential mechanisms of action. Further information is included in attachment to IUCLID section 13.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source and target chemicals have comparable chemical similarity. Further information is included in attachment to IUCLID section 13

3. ANALOGUE APPROACH JUSTIFICATION
The source substance is a chemically similar substance with common metabolism and common or similar degradants of the target substance. Further information is included in attachment to IUCLID section 13

4. DATA MATRIX
Further information is included in attachment to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

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: July 2012 ; signature: November 2012

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
- Storage conditions: ca. 21°C
- Storage length: Used on day of collection
- Preparation of inoculum for exposure: Aerobic activated sludge: washed two times by settlement and resuspension inmineral medium to remove any excessive amounts of dissolved organic carbon (DOC)
- Concentration of sludge: 3.0 g/L solids concentration prior to use.
- Initial cell/biomass concentration: Not reported.
- Water filtered: yes
- Type and size of filter used, if any: preweighed GF/A filter paper ; rinsed three times with 20 mL deionized reverse osmosis water prior to drying in an oven prior to weighing

Duration of test (contact time):

28 d

Initial conc.:

24.5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

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 ; 1 mL of Solution b ; 1 mL of Solution c ; 1 mL of Solution d. Reverse osmosis purified and deionized water.
Test item concentration: 24.5 mg/L, equivalent to 20 mg carbon/L ; Procedure control: Reference substance sodium benzoate, concentration 17.1 mg/L, equivalent to 10 mg carbon/L ; Toxicity control: concentration, 24.5 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 30 mg carbon/L
- Solubilising agent (type and concentration if used): None
- Test temperature: 21 °C, maintained in temperature controlled room
- pH: Measured prior to and completion of test. See table 1
- pH adjusted: Yes. 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
- Aeration of dilution water: Not reported
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: Preparations were prepared and inoculated in 5 liter test culture vessels each containing 3 liters of solution
- Number of culture flasks/concentration: In duplicate, except for Abiotic Control and Toxictity Control
- Method used to create aerobic conditions: The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 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 analyzer 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 (2ml) 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 TC: Samples (30ml) 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 0 and 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:

46

Sampling time:

28 d

Table 2: Percentage Biodegradation Values

Day	Mean % Degradation Sodium Benzoate Procedure Control	% Degradation Test Substance			Mean % Degradation Test Substance plus Sodium Benzoate Toxicity Control
		R1	R2	Mean (R1 and R2)
0	0	0	0	0	0
2	54	7	6	7	17
6	67	7	13	10	21
8	79	5	19	12	23
10	78	8	20	14	25
14	84	15	26	20	31
21	75	12	36	24	29
28	76	9	55	32	35
29*	79	38	55	46	38

* Day 29 values corrected to include any carry-over of CO2 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)
Test Item 20 mg C/L R1	20.81**	0.75	4
Test Item 20 mg C/L R2	20.95**	0.83	4

R1 – R2 = Replicates 1 and 2

* Corrected for 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

Validity criteria fulfilled:

yes

Interpretation of results:

other: not ready biodegradable

Conclusions:

The target substance biodegradation in duplicate is expected to be 46 % at day 28.

Executive summary:

The ready biodegradability test was carried out on a source substance according to OECD 301B Ready Biodegradability: CO2 Evolution (Modified Sturm Test) guidelines under GLP. The test substance, at a concentration of 20 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 substance 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 32.64 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 31% degradation after 14 days and 38% degradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test. Sodium benzoate attained 84% degradation after 14 days and 79% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The test substance attained 46% degradation after 28 days and therefore cannot be considered to be readily biodegradable.

Description of key information

Weight of evidence: not readily biodegradable, 2019

1. Biodegradation (Read-Across: 4-methyl-2-phenyltetrahydro-2H-pyran): 46% (28-days; 10-d window not met) not readily biodegradable, OECD TG 301B, 2013

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

Key study : OECD 301B, 2013 : Read-Across - SOURCE (4-methyl-2-phenyltetrahydro-2H-pyran) : The ready biodegradability test was carried out according to OECD 301B Ready Biodegradability: CO2 Evolution (Modified Sturm Test) guidelines under GLP. The test substance, at a concentration of 20 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 substance 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 32.64 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 31% degradation after 14 days and 38% degradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test. Sodium benzoate attained 84% degradation after 14 days and 79% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The test substance attained 46% degradation after 28 days and therefore cannot be considered to be readily biodegradable.