Reaction mass of (E)-1-(3,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one and (E)-1-(4,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted on 02 January 2019 and 30 January 2019.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)

Deviations:

yes

Remarks:

This deviation was considered to have had no effect on the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: PG-RAW-90-032
Physical State/Appearance: Clear colorless liquid
Storage Conditions: Approximately 4°C in the dark

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic (adaptation not specified)

Details on inoculum:

Test System and Supporting Information
A mixed population of sewage treatment micro organisms was obtained on 02 January 2019 from the secondary treatment stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Preparation of Inoculum
Upon receipt in the laboratory, the sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and the pH measured to be 7.5 using a Hach HQ40d Flexi handheld meter.
In order to reduce the inorganic carbon (IC) content of the inoculum, the filtrate was sparged with CO2 free air* for approximately 1-Hour whilst maintaining its pH at 6.5 using concentrated orthophosphoric acid. After sparging, the pH was restored to its original value of 7.5 using 7 M sodium hydroxide and the inoculum allowed to settle for approximately 1 hour prior to removal of an aliquot (2 liters) of the supernatant for use in the test. The supernatant was maintained on aeration using CO2 free air until use.

Mineral Medium
The mineral medium used in this study was that recommended in the OECD and ISO Guidelines.
The deionized reverse osmosis water used for the preparation of the mineral medium and the mineral medium used for the test contained less than 1 mg/L Total Organic Carbon (TOC).

Mineral 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 CaCl2.2H2O 36.40 g/L
Solution c MgSO4.7H2O 22.50 g/L
Solution d FeCl3.6H2O 0.25 g/L
(In order to avoid having to prepare solution (d) immediately before use, one drop of concentrated HCl per liter was added as a preservative).
To 1 liter (final volume) of purified water* was added the following volumes of solutions a to d.
10 mL of Solution a
1 mL of Solution b
1 mL of Solution c
1 mL of Solution d

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:

Experimental Design and Study Conduct
Preliminary Solubility Work
Information provided by the Sponsor indicated that the water solubility of the test item was unknown. Therefore preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation.
The following preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation
i) Ultrasonication and High Shear Mixing: An amount of test item (50 mg) was dispersed in 1 liter of deionized reverse osmosis purified water with the aid of shaking by hand for approximately 30 seconds prior to ultrasonication for 15 minutes. This formed a clear colorless water column with an oily film of test item on the surface. This was then subjected to high shear mixing (7500 rpm, 15 minutes) and formed a cloudy dispersion with oily specks floating at the surface.
This work confirmed that the test item was insoluble in water. Therefore the following additional solubility work was conducted to ascertain the best method to employ in the biodegradation test.
ii) Direct addition: An amount of test item (2.6 mg equivalent to 2.9 µL determined from preliminary work of 15 separate weighings) was added to 107 mL of mineral media. This formed a clear colorless media column with many oily globules of test item on the surface.
iii) Adsorption onto an Inert Support: An amount of test item (2.6 mg equivalent to
2.9 µL) was weighed on to 500 mg of silica gel. The silica gel/test item was added to 107 mL of mineral media This formed a cloudy dispersion with many oily globules of test item visible at the surface.
iv) Adsorption onto an Inert Support: An amount of test item (2.6 mg equivalent to
2.9 µL) was weighed onto a filter paper. The filter paper was added to 107 mL of mineral media. This formed a clear colorless media column with the filter paper settled on the bottom of the vessel and many oily globules of test item visible at the surface.
v) Preliminary Solution in a Volatile Solvent: The addition of a test item solvent stock to glass fiber filter paper was attempted.
An amount of test item (524 mg) was dissolved in acetone (10 mL), and formed a clear colorless solution. An aliquot (50 µL) of this solvent stock solution was dispensed to filter paper. The solvent was allowed to evaporate to dryness for approximately 15 minutes. The filter paper was then added to 107 mL of mineral medium. This formed a clear colorless media column with oily globules of test item visible floating at the surface.
vi) Preliminary Solution in a Non Volatile, Non Degradable Solvent: A nominal amount of test item (100 mg) was dispersed in silicone oil (10 mL) with the aid of shaking by hand for approximately 30 seconds. A clear colorless solution was formed. An aliquot (262 µL) of this solvent stock solution was added to 107 mL of mineral medium. This formed a clear colorless media column with an oily slick of test item/silicone oil visible floating at the surface and large oily globules of test item/silicone oil visible settled on the bottom of the vessel.
From the pre study solubility work and following the recommendations of the International Standards Organisation (ISO, 1995) and Handley et al (2002) it was concluded that the best testable dispersion was found to be obtained when using the solvent and filter paper method of preparation.

Test Item Preparation
Following preliminary solubility work and the recommendations of the International Standards Organisation (ISO 10634, 1995) and in the published literature (Handley et al, 2002) the test item was dissolved in an auxiliary solvent prior to adsorption onto filter paper**. Using this method the test item is evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms is increased thereby increasing the potential for biodegradation.
A nominal amount of test item (1049 mg) was dissolved in 10 mL of acetone to give a 1049 mg/10 mL solvent stock solution. An aliquot (25 µL) of this solvent stock solution was dispensed separately onto each filter paper** and the solvent allowed to evaporate to dryness for approximately 15 minutes prior to the addition to each test bottle. Inoculum (350 mL) was dispersed in mineral medium to a final volume 3.5 liters and an aliquot (107 mL) added to each of the 29 replicate test bottles to give a test concentration of 24.5 mg/L, equivalent to 20 mg carbon/L. Each test vessel was sealed using Teflon lined silicon septa and aluminum crimp caps.
The volumetric flask containing the solvent stock solution was inverted several times to ensure homogeneity of the solution.
A test concentration of 20 mg carbon/L was employed in the study following the recommendations of the test guidelines.
The inoculum control vessels were prepared in a similar manner using acetone without the addition of test item.

Reference Item Preparation
A reference item, sodium benzoate (C6H5COONa), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium. An aliquot (171.5 mL) of this stock solution was dispersed with inoculum (400 mL) and mineral medium to a final volume 4 liters, to give a test concentration of 42.9 mg/L, equivalent to 25 mg carbon/L. The pH of the reference item stock solution was measured to be 7.5 using a Hach HQ40d Flexi handheld meter. Aliquots (107 mL) of the 42.9 mg/L test concentration, equivalent to 25 mg carbon/L were dispensed to each of the 33 replicate test vessels and the vessels sealed using Teflon lined silicon septa and aluminum crimp caps.
A filter paper* was added to each procedure control vessel in order to maintain consistency between the test and procedure control vessels. Acetone (25 µL) was dispensed onto each filter paper and evaporated to dryness for approximately 15 minutes. The filter paper was added to each vessel.
The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

Toxicity Control
A toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro organisms used in the study.
An aliquot (25 µL) of the 1049 mg/10 mL test item solvent stock solution was dispensed separately onto each of the nine replicate filter papers* and the solvent allowed to evaporate to dryness for approximately 15 minutes prior to the addition to each test bottle. An aliquot (34.3 mL) of the reference item stock solution was dispersed with inoculum (100 mL) and mineral medium, final volume 1 liter, to give a reference item concentration of 34.3 mg/L, equivalent to 20 mg carbon/L. Aliquots (107 mL) of the 34.3 mg/L reference item concentration were dispensed to nine replicate test vessels and each test vessel sealed using Teflon lined silicon septa and aluminum crimp caps.
The final concentration in the toxicity control vessels was 24.5 mg test item/L plus 34.3 mg reference item/L, equivalent to 40 mg carbon/L.
All volumetric flasks were inverted several times to ensure homogeneity of the solution.

Preparation of Test System
The following test preparations incubated in 125 mL glass Wheaton bottles (160 mL capacity when full) each containing 107 mL of solution and they were inoculated with the prepared inoculum at a final concentration of 100 mL/L.
a) An inoculated control consisting of inoculated mineral medium, plus a filter paper*, 33 replicate vessels.
b) The procedure control containing the reference item (sodium benzoate) in inoculated mineral medium, plus a filter paper*, to give a final concentration of 25 mg carbon/L, 33 replicate vessels.
c) The test item on a filter paper* in inoculated mineral medium, to give a final concentration of 20 mg carbon/L, 29 replicate vessels.
d) The test item on a filter paper* plus the reference item in inoculated mineral medium, to give a final concentration of 40 mg carbon/L to act as a toxicity control, nine replicate vessels.
A filter paper with acetone evaporated to dryness was added to the inoculum control and procedure control vessels in order to maintain consistency between these vessels and the test item vessels.
The test media were dispensed to give a headspace to liquid ratio of 1:2. Sufficient vessels were prepared to allow a single IC determination per vessel with triplicate vessels for the inoculum control, procedure control, test item and toxicity control at each sampling occasion (five replicates for analysis on Day 28). Additional inoculum control and procedure control vessels were prepared to provide samples for Dissolved Organic Carbon (DOC) analyses on Days 0 and 28 (duplicate vessels per sampling occasion).
All vessels were sealed using Teflon lined silicon septa and aluminum crimp tops and incubated in darkness at 20 ±1 °C with constant shaking at approximately 125 rpm (INFORS Version 2 Multitron® Incubator).

Assessments
Inorganic Carbon Analysis
CO2 production in the vessels was determined by measuring the increase in the concentration of IC in the headspace.
Triplicate inoculum control, procedure control and test item vessels were sacrificed on Days 0, 2, 5, 7, 9, 14, 16 and 21 for IC analysis. On Day 28, five replicate vessels were sacrificed for IC analysis. Triplicate toxicity control vessels were sacrificed on Days 0, 7 and 14 for IC analysis.
An aliquot (1.0 mL) of concentrated orthophosphoric acid was injected through the septum of each vessel taken for analysis in order to lower the pH of the medium to below 3*. The vessels were then shaken at approximately 125 rpm (INFORS Version 2 Multitron® Incubator) for 1-Hour at 21 °C in darkness prior to samples being withdrawn from the headspace and analyzed for IC.
The principle of this method is that after acidification to a pH value of below 3 and equilibration at 20±1 °C, the equilibrium constant for the distribution of CO2 between the liquid and gaseous phases in the test vessels is 1.0 and hence only the CO2 concentration in the headspace needs to be determined.
The samples were analyzed using a Shimadzu TOC VCSH TOC analyzer. Samples (50 µL) taken from the headspace were injected into the IC channel of the TOC analyzer. IC analysis occurs by means of the conversion of the gaseous sample to CO2 by orthophosphoric acid using zero grade air as the carrier gas. Calibration was by reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in at least triplicate with three replicates being used in the calculation.

Dissolved Organic Carbon Analysis
On Days 0 and 28 samples (30 mL) were removed from the inoculum control and procedure control vessels prepared for DOC analysis and filtered through 0.45 µm Acrocap filters (approximately 5 mL discarded) prior to DOC analysis.
DOC analysis of the test item and toxicity control vessels was not possible due to the insoluble nature of the test item in water.
The samples were analyzed for DOC using a Shimadzu TOC VCPH TOC analyzer. Samples (50 µL) were injected into the Total Carbon (TC) and IC channels of the TOC analyzer. TC analysis is carried out at 680 °C using a platinum based catalyst and zero grade air as the carrier gas. IC analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using standard solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionized water. Each analysis was carried out in at least triplicate with three replicates being used in the calculation.

*Confirmed by preliminary work conducted where the pH of 107 mL of mineral medium containing varying concentrations of Na2CO3, acidified by the addition of 1 mL concentrated orthophosphoric acid, was determined to be less than 2.0. This work was shared with a similar study.

Reference substance:

benzoic acid, sodium salt

Test performance:

The mean TIC in the inoculum control vessels on Day 28 was 1.68 mg/L; equivalent to 8% of the organic carbon added initially as test item to the test vessels and therefore satisfied the validation criterion given in the Test Guideline.
The test item attained 4% biodegradation (95% confidence limits of 1.6 to 6.8 mg/L biodegradation) of the ThIC yield after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 310.
The toxicity control attained 32% biodegradation of the ThIC yield after 14 days, thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro organisms used in the test.
Variation in the biodegradation rates obtained on different sampling days was considered to be the result of normal biological variation between the respiration rates of replicate vessels. Due to the sacrificial nature of the study design, the biodegradation rates obtained on each sampling occasion were for individual replicate vessels and not the result of cumulative biodegradation values determined from a single vessel sampled on numerous occasions and as such variation in biodegradation rates on different sampling days is not uncommon.
DOC analyses conducted on samples taken from the reference item vessels on Days 0 and 28 showed that the replicate reference item vessels attained 100% biodegradation for each replicate vessel. The biodegradation rates for the reference item were higher than those determined by IC analyses. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to biodegradation and hence CO2 evolution occurring.

Parameter:

% degradation (CH4 evolution)

Value:

4

Sampling time:

28 d

Results with reference substance:

Sodium benzoate attained 77% biodegradation after 14 days and 66% biodegradation after 28 days of the ThIC yield thereby confirming the suitability of the inoculum and test conditions.

Total Inorganic Carbon Values on Each Analysis Occasion

Day	Total Inorganic Carbon (mg IC)
	Inoculum Control					Procedure Control					Test Item					Toxicity Control
	R1	R2	R3	R4	R5	R1	R2	R3	R4	R5	R1	R2	R3	R4	R5	R1	R2	R3	R4	R5
0	0.18	0.17	0.18	-	-	0.17	0.18	0.18	-	-	0.16	0.17	0.16	-	-	0.18	0.19	0.17	-	-
2	0.17	0.17	0.18	-	-	1.51	1.49	1.47	-	-	0.17	0.16	0.16	-	-	-	-	-	-	-
5	0.27	0.27	0.24	-	-	2.48	2.34	2.19	-	-	0.23	0.22	0.24	-	-	-	-	-	-	-
7	0.23	0.19	0.21	-	-	2.03	2.09	2.21	-	-	0.21	0.20	0.21	-	-	1.55	1.46	1.46	-	-
9	0.18	0.18	0.21	-	-	1.91	1.88	1.95	-	-	0.18	0.16	0.17	-	-	-	-	-	-	-
14	0.24	0.22	0.23	-	-	2.31	2.23	2.35	-	-	0.22	0.22	0.22	-	-	1.56	1.66	1.63	-	-
16	0.20	0.18	0.20	-	-	2.15	2.07	1.91	-	-	0.18	0.17	0.17	-	-	-	-	-	-	-
21	0.20	0.21	0.20	-	-	2.18	2.04	2.07	-	-	0.20	0.20	0.21	-	-	-	-	-	-	-
28	0.20	0.18	0.17	0.18	0.18	1.71	1.77	1.86	2.09	2.25	0.31	0.25	0.17	0.35	0.27	-	-	-	-	-

R   =     Replicate

 -   =     No measurement made

Percentage Biodegradation Values

Day	Biodegradation (%)
	Procedure Control	Test Item	Toxicity Control
0	0	0	0
2	49	0	-
5	78	0	-
7	71	0	30
9	64	0	-
14	77	0	32
16	69	0	-
21	71	0	-
28	66	4	-

 -      =   No Measurement made

Dissolved Organic Carbon (DOC) Values on Day 0 and Day 28

DOC Concentration
Test Vessel		Day 0			Day 28
		mg C/L	mg C/L Corrected for Mean Inoculum Control Value	Nominal Carbon Content (%)	mg C/L	mg C/L Corrected for Mean Inoculum Control Value	Biodegradation (%)
Inoculum Control	R1	1.12	-	-	<LOQ	-	-
	R2	1.06	-	-	<LOQ	-	-
Procedure Control	R1	24.71	23.62	94	<LOQ	<LOQ	100
	R2	23.38	23.29	93	<LOQ	<LOQ	100

R             =           Replicate

-              =           Not Applicable

LOQ      =             Limit of Quantitation(determined down to 1.0 mg Carbon/L).

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The substance PG-RAW-90-032 showed 4% biodegradation in an OECD TG 310 test and is considered to be not biodegradable.

Executive summary:

The ready biodegradability of PG-RAW-90-032 was investigated in a study conducted in accordance with OECD TG 310 (Headspace Test) and GLP. The concentration tested was 24.5 mg/L test substance. The test item attained 4% biodegradation (95% confidence limits of 1.6 to 6.8 mg/L biodegradation) of the ThIC yield after 28 days and therefore cannot be considered to be readily biodegradable.

Description of key information

The ready biodegradability of PG-RAW-90-032 was investigated in a study conducted in accordance with OECD TG 310 (Headspace Test) and GLP. The concentration tested was 24.5 mg/L test substance. The test item attained 4% biodegradation (95% confidence limits of 1.6 to 6.8 mg/L biodegradation) of the ThIC yield after 28 days and therefore cannot be considered to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater