Reaction mass of 4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and 4-(2,6,6-trimethylcyclohex-1-ene-1-yl)-but-3-ene-2-one

Endpoint summary

Description of key information

Biodegradation in water

Biodegradation study was conducted for 28-days following the OECD guideline 301 D for determining the ready biodegradability of the test chemical. The study was performed at a temperature of 20°C under aerobic conditions. Aerobic conditions was provided by means of mineral media which is aerated for 20 hours prior to start of the experiment. The test system included control, test chemical and reference substance. The activated sludge was collected from SMS Municipal sewage treatment plant (130 MLD STP) in a thoroughly cleansed container. The sampling site for collection of the activated sludge was selected ensuring that no known history of its contamination with the test item within the previous four years considering the history of possible agricultural, industrial or domestic inputs. The sampling depth was 1-2 feet from the aeration tank. The temperature of the activated sludge was measured (38º C) at the site of collection. Oxygen concentration of the activated sludge sample was 2.9 mg/L. The sample was transported to the test facility within 3 hours from collection and kept it aerobic during transport. The concentration of test and reference substance (Sodium Benzoate) chosen for both the study was 4 mg/L. OECD mineral medium was used for the study. ThOD (Theoretical oxygen demand) of test and reference chemical was determined by calculation. % degradation was calculated using the values of BOD and ThOD for test chemical and reference substance. The % degradation of procedure control (reference substance) was also calculated using BOD & ThOD and was determined to be 97.3 %. Degradation of Sodium Benzoate exceeds 34.43 % on 7 days & 76.35 % on 14th day. The activity of the inoculum is thus verified and the test can be considered as valid. The BOD28 value of test chemical was observed to be 1.63 mgO2/mg. ThOD was calculated as 2.91 mgO2/mg. Accordingly, the % degradation of the test chemical after 35 days of incubation at 20 ± 1°C according to Closed Bottle test was determined to be 47.36 %. Based on the results, the test chemical under the test conditions, was considered to be readily biodegradable in nature.

Biodegradation in water and sediment

Primary biodegradation in surface water from a Danish stream was determined by compound specified analysis. The study was performed following the modified OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test) under aerobic conditions. Natural water (stream of good ecological quality and without known point sources close to sampling point) sampled from Gudenåen (DK) 16th of June 2020. WGS84: 56°6'23.2"N 9°43'17.4"E at a dept of 5 -20 cm below surface was used as a surface water. The water was quite clear with a slight light brown hint. Total suspended solids was 2.0 mg/L, total dissolved solids was 210 mg/L. No pre-treatment was done of the natural water. Water kept at 20 ± 1.5°C during transport to lab (4 hours), then stored at 12°C until test setup within 30 hours of sampling. The temperature and pH at the time of collection of the sample was 20.0°C and 6.5 pH. Biomass i.e., plate count at 24 and 72 hr was 36 CFU/ml and 1281 CFU/ml, respectively. Water was not filtered. Dissolved non-volatile organic carbon (NVOC) was measured instead of DOC/TOC and was 4.4 mg/L. Study was performed using 20 mL amber headspace closed vials. A large no. of biotic test systems (15 -18 replicates) consisting of 14.5 ml surface water and 0.5 ml stock solution was prepared test vials. Stock solution was prepared in water by equilibrium partioning from a pre-loaded silicone rod (passive dosing). This method for preparing stock solutions avoids the need for solvents for spiking. A similar number of abiotic control test systems were prepared using 14.5 ml MilliQ water and 0.5 ml stock solution. Blanks were prepared with 15 ml MilliQ water. Aerobic conditions created by using aerobic natural water (initial oxygen content 9.0 mg/L at 20 °C)  and incubating using a headspace of 5 mL with a low test substance concentration of 0.2 mg/l. Test was conducted at a temperature of 12°C and pH of 6.5 under continuous darkness with conductivity of 334 µS/cm while rolling. pH of the test medium (natural water) was not adjusted. No aeration was provided during the study. Suspended solid concentrations uded in the study was 2.0 mg/l. At 5 -6 time-points, 3 biotic and 3 abiotic test systems were sacrificed for analysis (day 2, 6, 9, 14 and 21). Tests systems were put directly on the autosampler of the GCMS without storage. Blank test systems were prepared and incubated with biotic and abiotic test systems and analyzed at each time-point (signal well below the 3% dilution of initial test concentration). Analysis was carried out using direct immersion solid phase microextraction coupled to GC-MS. Carryover was < 1.0% and since test systems were analyzed alternating biotic and abiotic test systems this constitutes the detection limit in this test. Biodegradation was then evaluated by the ratio between peak areas in the biotic and abiotic test systems. Triplicate ratios of biotic/abiotic test system peak areas were calculated for each time point and used in Graph-pad Prism 8.4.3 to fit the first order degradation model with lag phase. The (pseudo-)first order (= half-life) (DT50) of the test chemical was evaluated to be 6.8 days at 12°C. Primary percentage degradation of the test chemical was determined to be >99% at 21 days. Thus, on the basis of results, test chemical was considered to be not persistent in surface water.

Primary biodegradation in surface water from a Danish stream was determined by compound specified analysis. The study was performed following the modified OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test) under aerobic conditions. Natural water (stream of good ecological quality and without known point sources close to sampling point) sampled from Gudenåen (DK) 17th of august 2020 and 15 of june 2020, WGS84: 56°6'23.2"N 9°43'17.4"E at a dept of 5 -20 cm below surface was used as a surface water. The water was quite clear with a slight light brown hint. Total suspended solids, total dissolved solids was 4.3 mg/L and 2.2 mg/l, 150 mg/L and 210 mg/l in month of august and june respectively. No pre-treatment was done of the natural water. Water kept at 20 ± 1.5°C during transport to lab (4 hours), then stored at 12°C until test setup within 30 hours of sampling. The temperature and pH at the time of collection of the sample was 24.0°C, 7.4 (aug) and 6.5(june). Biomass i.e., plate count at 24 and 72 hr was 270 CFU/ml and 2100 CFU/ml in august and 36 CFU/ml and 1300 CFU/ml in june, respectively. Water was not filtered. Dissolved non-volatile organic carbon (NVOC) was measured instead of DOC/TOC and was 5.7 mg/L (aug) and 4.4 (june). Study was performed using 20 mL amber headspace closed vials. A large no. of biotic test systems (15 -18 replicates) consisting of 13.5 ml surface water and 0.5 ml stock solution and 1 ml spiking mixture. Stock solution was prepared in water by equilibrium partioning from a pre-loaded silicone rod (passive dosing). A similar number of abiotic control test systems were prepared using 14.5 ml MilliQ water and 0.5 ml stock solution. Blanks were prepared with 15 ml MilliQ water. Aerobic conditions created by using aerobic natural water (initial oxygen content 7.4 mg/L at 20 °C)  and incubating using a headspace of 5 mL with a low test substance concentration of 0.033 mg/l. Test was conducted at a temperature of 12°C and pH of 7.4 under continuous darkness with conductivity of 334 µS/cm while rolling. pH of the test medium (natural water) was not adjusted. No aeration was provided during the study. Suspended solid concentrations used in the study was 4.3 mg/l. At 5 -6 time-points, 3 biotic and 3 abiotic test systems were sacrificed for analysis (day 1, 5, 8, 13, 20,  24 and 28). Tests systems were put directly on the autosampler of the GCMS without storage. Blank test systems were prepared and incubated with biotic and abiotic test systems and analyzed at each time-point (signal well below the 3% dilution of initial test concentration). Analysis was carried out using direct immersion solid phase microextraction coupled to GC-MS. Carryover was < 1.0% and since test systems were analyzed alternating biotic and abiotic test systems this constitutes the detection limit in this test. Biodegradation was then evaluated by the ratio between peak areas in the biotic and abiotic test systems. Triplicate ratios of biotic/abiotic test system peak areas were calculated for each time point and used in Graph-pad Prism 8.4.3 to fit the first order degradation model with lag phase. The (pseudo-)first order (= half-life) (DT50) of the test chemical was evaluated to be ~55.79 and 34.72 days at 12°C in month of august and june respectively. Primary percentage degradation of the test chemical was determined to be<12% amd <60%  in august and june month within 28 days. Hence, test chemical was considered to be not persistent in surface water.

In accordance with column 2 of Annex IX of the REACH regulation, testing for this end point is scientifically not necessary and does not need to be conducted since the test chemical is readily biodegradable in water.

 

Biodegradation in soil

In accordance with column 2 of Annex IX of the REACH regulation, testing for this end point is scientifically not necessary and does not need to be conducted since the test chemical is readily biodegradable in water.

Additional information

Biodegradation in water

Various experimental studieshas been investigated for the test chemicalfor reviewing the biodegradation endpointwhich have beensummarized as below;

 

Biodegradation study was conducted for 28-days following the OECD guideline 301 D for determining the ready biodegradability of the test chemical. The study was performed at a temperature of 20°C under aerobic conditions. Aerobic conditions was provided by means of mineral media which is aerated for 20 hours prior to start of the experiment. The test system included control, test chemical and reference substance. The activated sludge was collected from SMS Municipal sewage treatment plant (130 MLD STP) in a thoroughly cleansed container. The sampling site for collection of the activated sludge was selected ensuring that no known history of its contamination with the test item within the previous four years considering the history of possible agricultural, industrial or domestic inputs. The sampling depth was 1-2 feet from the aeration tank. The temperature of the activated sludge was measured (38º C) at the site of collection. Oxygen concentration of the activated sludge sample was 2.9 mg/L. The sample was transported to the test facility within 3 hours from collection and kept it aerobic during transport. The concentration of test and reference substance (Sodium Benzoate) chosen for both the study was 4 mg/L. OECD mineral medium was used for the study. ThOD (Theoretical oxygen demand) of test and reference chemical was determined by calculation. % degradation was calculated using the values of BOD and ThOD for test chemical and reference substance. The % degradation of procedure control (reference substance) was also calculated using BOD & ThOD and was determined to be 97.3 %. Degradation of Sodium Benzoate exceeds 34.43 % on 7 days & 76.35 % on 14th day. The activity of the inoculum is thus verified and the test can be considered as valid. The BOD28 value of test chemical was observed to be 1.63 mgO2/mg. ThOD was calculated as 2.91 mgO2/mg. Accordingly, the % degradation of the test chemical after 35 days of incubation at 20 ± 1°C according to Closed Bottle test was determined to be 47.36 %. Based on the results, the test chemical under the test conditions, was considered to be readily biodegradable in nature.

The Ready Biodegradability of test chemical was determined by the Manometric Respirometry Test according to the OECD Guidelines for Testing of Chemicals, Method No. 301 F. The test chemical undergoes 71% biodegradation after 28 days in the test conditions. The 10 -day window criterion is also fulfilled (16% biodegradation on day 4 and 65% on day 14). The curves obtained with the reference substance alone and with test chemical + reference substance show no toxic effect to the micro-organisms at the test concentration (100 mg/l). Thus, the test chemical should be regarded as readily biodegradable according to this test.

 

The another Manometric Respirometry Test was conducted for test chemical to assess its percent degradation as per the OECD Guidelines for Testing of Chemicals, Method No. 301 F. The Test chemical undergoes 60% biodegradation after 28 days (65% after 36 days) in the test conditions. Biodegradation exceeds 10% on day 8 and reaches only 52% at the end of the 10-day window (days 8 to 18).The curves obtained with the reference substance alone and with Test chemical + reference substance show no toxic effect of Test chemical to the micro-organisms at the test concentration (100 mg/I).Thus, Test chemical should be regarded as readily biodegradable according to this test.

 

The above results were supported by Biodegradation study conducted according to OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test) for 28 days for evaluating the percentage biodegradability of test substance. The percentage degradation of the test substance was determined to be 75.4% by using CO2 evolution parameter. Thus, based on percentage degradation of test chemical, the chemical was considered to be readily biodegradable in nature.

By considering results of all the studies mentioned above it can be concluded that the test chemical can be expected to be readily biodegradable in nature.

 

Biodegradation in water and sediment

Primary biodegradation in surface water from a Danish stream was determined by compound specified analysis. The study was performed following the modified OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test) under aerobic conditions. Natural water (stream of good ecological quality and without known point sources close to sampling point) sampled from Gudenåen (DK) 16th of June 2020. WGS84: 56°6'23.2"N 9°43'17.4"E at a dept of 5 -20 cm below surface was used as a surface water. The water was quite clear with a slight light brown hint. Total suspended solids was 2.0 mg/L, total dissolved solids was 210 mg/L. No pre-treatment was done of the natural water. Water kept at 20 ± 1.5°C during transport to lab (4 hours), then stored at 12°C until test setup within 30 hours of sampling. The temperature and pH at the time of collection of the sample was 20.0°C and 6.5 pH. Biomass i.e., plate count at 24 and 72 hr was 36 CFU/ml and 1281 CFU/ml, respectively. Water was not filtered. Dissolved non-volatile organic carbon (NVOC) was measured instead of DOC/TOC and was 4.4 mg/L. Study was performed using 20 mL amber headspace closed vials. A large no. of biotic test systems (15 -18 replicates) consisting of 14.5 ml surface water and 0.5 ml stock solution was prepared test vials. Stock solution was prepared in water by equilibrium partioning from a pre-loaded silicone rod (passive dosing). This method for preparing stock solutions avoids the need for solvents for spiking. A similar number of abiotic control test systems were prepared using 14.5 ml MilliQ water and 0.5 ml stock solution. Blanks were prepared with 15 ml MilliQ water. Aerobic conditions created by using aerobic natural water (initial oxygen content 9.0 mg/L at 20 °C)  and incubating using a headspace of 5 mL with a low test substance concentration of 0.2 mg/l. Test was conducted at a temperature of 12°C and pH of 6.5 under continuous darkness with conductivity of 334 µS/cm while rolling. pH of the test medium (natural water) was not adjusted. No aeration was provided during the study. Suspended solid concentrations uded in the study was 2.0 mg/l. At 5 -6 time-points, 3 biotic and 3 abiotic test systems were sacrificed for analysis (day 2, 6, 9, 14 and 21). Tests systems were put directly on the autosampler of the GCMS without storage. Blank test systems were prepared and incubated with biotic and abiotic test systems and analyzed at each time-point (signal well below the 3% dilution of initial test concentration). Analysis was carried out using direct immersion solid phase microextraction coupled to GC-MS. Carryover was < 1.0% and since test systems were analyzed alternating biotic and abiotic test systems this constitutes the detection limit in this test. Biodegradation was then evaluated by the ratio between peak areas in the biotic and abiotic test systems. Triplicate ratios of biotic/abiotic test system peak areas were calculated for each time point and used in Graph-pad Prism 8.4.3 to fit the first order degradation model with lag phase. The (pseudo-)first order (= half-life) (DT50) of the test chemical was evaluated to be 6.8 days at 12°C. Primary percentage degradation of the test chemical was determined to be >99% at 21 days. Thus, on the basis of results, test chemical was considered to be not persistent in surface water.

Primary biodegradation in surface water from a Danish stream was determined by compound specified analysis. The study was performed following the modified OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test) under aerobic conditions. Natural water (stream of good ecological quality and without known point sources close to sampling point) sampled from Gudenåen (DK) 17th of august 2020 and 15 of june 2020, WGS84: 56°6'23.2"N 9°43'17.4"E at a dept of 5 -20 cm below surface was used as a surface water. The water was quite clear with a slight light brown hint. Total suspended solids, total dissolved solids was 4.3 mg/L and 2.2 mg/l, 150 mg/L and 210 mg/l in month of august and june respectively. No pre-treatment was done of the natural water. Water kept at 20 ± 1.5°C during transport to lab (4 hours), then stored at 12°C until test setup within 30 hours of sampling. The temperature and pH at the time of collection of the sample was 24.0°C, 7.4 (aug) and 6.5(june). Biomass i.e., plate count at 24 and 72 hr was 270 CFU/ml and 2100 CFU/ml in august and 36 CFU/ml and 1300 CFU/ml in june, respectively. Water was not filtered. Dissolved non-volatile organic carbon (NVOC) was measured instead of DOC/TOC and was 5.7 mg/L (aug) and 4.4 (june). Study was performed using 20 mL amber headspace closed vials. A large no. of biotic test systems (15 -18 replicates) consisting of 13.5 ml surface water and 0.5 ml stock solution and 1 ml spiking mixture. Stock solution was prepared in water by equilibrium partioning from a pre-loaded silicone rod (passive dosing). A similar number of abiotic control test systems were prepared using 14.5 ml MilliQ water and 0.5 ml stock solution. Blanks were prepared with 15 ml MilliQ water. Aerobic conditions created by using aerobic natural water (initial oxygen content 7.4 mg/L at 20 °C)  and incubating using a headspace of 5 mL with a low test substance concentration of 0.033 mg/l. Test was conducted at a temperature of 12°C and pH of 7.4 under continuous darkness with conductivity of 334 µS/cm while rolling. pH of the test medium (natural water) was not adjusted. No aeration was provided during the study. Suspended solid concentrations used in the study was 4.3 mg/l. At 5 -6 time-points, 3 biotic and 3 abiotic test systems were sacrificed for analysis (day 1, 5, 8, 13, 20,  24 and 28). Tests systems were put directly on the autosampler of the GCMS without storage. Blank test systems were prepared and incubated with biotic and abiotic test systems and analyzed at each time-point (signal well below the 3% dilution of initial test concentration). Analysis was carried out using direct immersion solid phase microextraction coupled to GC-MS. Carryover was < 1.0% and since test systems were analyzed alternating biotic and abiotic test systems this constitutes the detection limit in this test. Biodegradation was then evaluated by the ratio between peak areas in the biotic and abiotic test systems. Triplicate ratios of biotic/abiotic test system peak areas were calculated for each time point and used in Graph-pad Prism 8.4.3 to fit the first order degradation model with lag phase. The (pseudo-)first order (= half-life) (DT50) of the test chemical was evaluated to be ~55.79 and 34.72 days at 12°C in month of august and june respectively. Primary percentage degradation of the test chemical was determined to be<12% amd <60%  in august and june month within 28 days. Hence, test chemical was considered to be not persistent in surface water.

In accordance with column 2 of Annex IX of the REACH regulation, testing for this end point is scientifically not necessary and does not need to be conducted since the test chemical is readily biodegradable in water.

 

Biodegradation in soil

In accordance with column 2 of Annex IX of the REACH regulation, testing for this end pointis scientifically not necessary and does not need to be conducted since the test chemicalis readily biodegradable in water.