3-methyl-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-buten-2-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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experimental result using OECD guidelines.

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

Biodegradation study was conducted according to OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test) for evaluating the percentage biodegradability of test chemical.

GLP compliance:

no

Oxygen conditions:

not specified

Inoculum or test system:

other: Mixed inoculum

Details on inoculum:

Mixed Inoculum Preparation:Polyseed were used for this study. 1 polyseed capsule was added in 500 ml D.I water and then stirred for 1 hour for proper mixing and functioning of inoculum. This gave the bacterial count as 10E7 to 10E8 CFU/ml. At the regular interval microbial plating was also performed on agar to confirm the vitality and CFU count of microorganism.

Duration of test (contact time):

28 d

Initial conc.:

4 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: OECD mineral medium was used for the study
- Test temperature: 20°C
- Continuous darkness: Yes
- Other: The water used in this study is deionized water.

TEST SYSTEM
- Culturing apparatus: The apparatus used in this study is BOD bottles; with glass stoppers (125 ml), BOD incubator & oxygen electrode and meter.

CONTROL AND BLANK SYSTEM
- Inoculum blank: it contains only test inoculum
- Procedure control: contains reference compound and inoculum

Reference substance:

other: Sodium Benzoate

Key result

Parameter:

% degradation (O2 consumption)

Value:

42.51

Sampling time:

28 d

Remarks on result:

other: Other details not known

Details on results:

The oxygen consumed by the test systems was corrected for oxygen consumption occurring in the blank test systems.The BOD Values (mgO2/mg) and percent biodegradation results for each test system are reported in Tables 2 and 3, respectively. The BOD28 value of test chemical was observed to be 1.25 mgO2/mg. ThOD was determined by calculation as 2.94 mgO2/mg. % degradation was calculated using the values of BOD and ThOD for test item and was determined to be 42.51% at 20 ± 1°C. The % degradation of procedure control (reference item) was also calculated using BOD & ThOD and was determined to be 76.5%. Degradation of Sodium Benzoate exceeds 39.15% on 7 days & 70.48% on 14th day. The activity of the inoculum was thus verified and the test can be considered as valid.

Results with reference substance:

The % degradation of procedure control (reference item) was also calculated using BOD & ThOD and was determined to be 76.5%. Degradation of Sodium Benzoate exceeds 39.15% on 7 days & 70.48% on 14th day. The activity of the inoculum was thus verified and the test can be considered as valid.

TABLE 1: D.O Values (mg/L)

 

No. of Days	Inoculum Blank (Control)	Test Suspension	Procedure Control (Reference Item)
0	6.6	6.4	6.5
7	6.3	4.7	3.6
14	6	2	1.2
21	5.7	1.3	0.8
28	5.4	0.2	0.2

TABLE 2: BOD Values (mg O2/mg)

 

No. of Days	Test Suspension	Procedure Control (Reference Item)
0	0	0
7	0.35	0.65
14	0.95	1.17
21	1.15	1.2
28	1.25	1.27

 

TABLE 3: PERCENT BIODEGRDATION RESULTS

No. of Days	Test Suspension	Procedure Control (Reference Item)
0	0%	0%
7	11.9%	39.15%
14	32.31%	70.48%
21	39.11%	72.28%
28	42.51%	76.5%

 

Table 4: BOD28, THOD AND % BIODEGRADATION VALUES

 

Method details	BOD28(mgO2/mg)	ThOD (mgO2/mg)	%Biodegradation
Test Item	1.25	2.94	42.51
Reference Item	1.27	1.66	76.5

Validity criteria fulfilled:

yes

Interpretation of results:

other: ultimate inherently biodegradable

Conclusions:

The test chemical undergoes 42.51% biodegradation after 28 days in the test condition. Thus, the test item was considered to be ultimate inherently biodegradable in nature.

Executive summary:

28-days Closed Bottle test following the OECD guideline 301 D to determine the ready biodegradability of the test chemical. The study was performed at a temperature of 20°C. The test system included control, test item and reference item. Polyseed were used for this study. 1 polyseed capsule were added in 500 ml D.I water and then stirred for 1 hour for proper mixing and functioning of inoculum. This gave the bacterial count as 10E7 to 10E8 CFU/ml. At the regular interval microbial plating was also performed on agar to confirm the vitality and CFU count of microorganism. The concentration of test and reference item (Sodium Benzoate) chosen for both the study was 4 mg/L, while that of inoculum was 32 ml/l. OECD mineral medium was used for the study. ThOD (Theoretical oxygen demand) of test and reference item was determined by calculation. % degradation was calculated using the values of BOD and ThOD for test item and reference item. The % degradation of procedure control (reference item) was also calculated using BOD & ThOD and was determined to be 76.5%. Degradation of Sodium Benzoate exceeds 39.15% on 7 days & 70.48% on 14th day. The activity of the inoculum was thus verified and the test can be considered as valid.The BOD28 value of test chemical was observed to be 1.25 mgO2/mg. ThOD was calculated as 2.94 mgO2/mg. Accordingly, the % degradation of the test item after 28 days of incubation at 20 ± 1°C according to Closed Bottle test was determined to be 42.51%. Based on the results, the test item, under the test conditions, was considered to be ultimate inherently biodegradable in nature.

Description of key information

28-days Closed Bottle test following the OECD guideline 301 D to determine the ready biodegradability of the test chemical (Experimental study report, 2018). The study was performed at a temperature of 20°C. The test system included control, test item and reference item. Polyseed were used for this study. 1 polyseed capsule were added in 500 ml D.I water and then stirred for 1 hour for proper mixing and functioning of inoculum. This gave the bacterial count as 10E7 to 10E8 CFU/ml. At the regular interval microbial plating was also performed on agar to confirm the vitality and CFU count of microorganism. The concentration of test and reference item (Sodium Benzoate) chosen for both the study was 4 mg/L, while that of inoculum was 32 ml/l. OECD mineral medium was used for the study. ThOD (Theoretical oxygen demand) of test and reference item was determined by calculation. % degradation was calculated using the values of BOD and ThOD for test item and reference item. The % degradation of procedure control (reference item) was also calculated using BOD & ThOD and was determined to be 76.5%. Degradation of Sodium Benzoate exceeds 39.15% on 7 days & 70.48% on 14th day. The activity of the inoculum was thus verified and the test can be considered as valid.The BOD28 value of test chemical was observed to be 1.25 mgO2/mg. ThOD was calculated as 2.94 mgO2/mg. Accordingly, the % degradation of the test item after 28 days of incubation at 20 ± 1°C according to Closed Bottle test was determined to be 42.51%. Based on the results, the test item, under the test conditions, was considered to be ultimate inherently biodegradable in nature.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Additional information

Various experimental studies of the test chemical were reviewed for the biodegradation end point which are summarized as below:

 

In an experimental key study report (2018) for the test chemical,28-days Closed Bottle test following the OECD guideline 301 D to determine the ready biodegradability of the test chemical. The study was performed at a temperature of 20°C. The test system included control, test item and reference item. Polyseed were used for this study. 1 polyseed capsule were added in 500 ml D.I water and then stirred for 1 hour for proper mixing and functioning of inoculum. This gave the bacterial count as 10E7 to 10E8 CFU/ml. At the regular interval microbial plating was also performed on agar to confirm the vitality and CFU count of microorganism. The concentration of test and reference item (Sodium Benzoate) chosen for both the study was 4 mg/L, while that of inoculum was 32 ml/l. OECD mineral medium was used for the study. ThOD (Theoretical oxygen demand) of test and reference item was determined by calculation. % degradation was calculated using the values of BOD and ThOD for test item and reference item. The % degradation of procedure control (reference item) was also calculated using BOD & ThOD and was determined to be 76.5%. Degradation of Sodium Benzoate exceeds 39.15% on 7 days & 70.48% on 14th day. The activity of the inoculum was thus verified and the test can be considered as valid. The BOD28 value of test chemical was observed to be 1.25 mgO2/mg. ThOD was calculated as 2.94 mgO2/mg. Accordingly, the % degradation of the test item after 28 days of incubation at 20 ± 1°C according to Closed Bottle test was determined to be 42.51%. Based on the results, the test item, under the test conditions, was considered to be ultimate inherently biodegradable in nature.

 

Another biodegradation study was conducted for 56 days for evaluating the percentage biodegradability of test chemical (from secondary source, 2017). The study was performed according to OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test) at a temperature of 14-22°C. Initial test chemical conc. Used for the study was10 mg DOC/L. Activated sludge (Unacclimatized) was used as a test inoculum for the study. The test material (10 mg DOC/L) and secondary effluent from an unacclimatized activated sludge were shaken in a sealed vessel at 14-22 °C for 28 days. The headspace carbon dioxide level and inorganic carbon in the test medium was analysed. The percentage degradation of test chemical was determined to be 61.8% by CO2 evolution parameter in 28 days. Thus, based on percentage degradation, test chemical is considered to be readily biodegradable in nature.

 

In a supporting study from secondary source (2017), biodegradation experiment was conducted according to OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II)) for evaluating the percentage biodegradability of test chemical. Activated sludge mixture(1:1) from city sewage plant and chemical sewage plant was used as a test inoculum for the study. Concentration of inoculum i.e, sludge used was 100 mg/l and initial test substance conc. used in the study was 30 mg/l, respectively. Sodium benzoate (100 mg/l) was used as reference substance.The test substance (30 mg/L) or reference substances (100 mg/L) sodium benzoate was inoculated with a mixture of activated sludge (100 mg dry sludge/L) in a closed oxygen consumption measuring device for 56 days at 25 °C. Oxygen concentration was monitored continuously using a BOD meter. Percent biodegradation values were arithmetic mean of 2 or 3 independent measurements. The reference substance was 100% degraded after 28 days. Test chemical undergoes 63.4% degradation after 56 days. Time required for 10% degradation of test substance was 12 days. Thus, the test chemical was considered as inherently biodegradable in nature.

 

For the test chemical,biodegradation study was conducted for 28 days for evaluating the percentage biodegradability of test chemical (Susumu Ishizaki et. al., 2004). Activated sludge was used as a test inoculum for the study. Basic culture medium was used as a test medium for the study. The medium consisted of the following components (per liter of distilled water): 0.063g of K2HPO4, 0.026g of KH2PO4, 0.134g of Na2HPO4.7H2O, 0.0005g of NH4Cl, 0.068f of MgSO4, 0.083g of CaCl2, and 0.0008g of FeCl3.6H2O. The study was performed at a temperature of 25°C.To the resulting standard medium (900 ml), 108 mg of test chemical and 120 mg of standard activated sludge were added. The mixture was incubated at 25°C for 28 days undercontinuous shaking. The metabolites were extracted with ether, and the organic layer was washed with brine, dried over MgSO4 and concentrated in vacuo. The residue was chromatographed on silica gel, eluting with n-hexane-ethyl acetate (10:1) to give the product A. An analytical sample was further purified by GLC analysis. The IR absorption band at 1730/cm indicated the presence of carbonyl group. The 1H-NMR spectrum showed the presence of a geminal dimethyl at 0.76 & 0.91 ppm, a methyl on a double bond at 1.62 ppm, a methyl of methyl ketone at 2.81 ppm, and a proton on a double bond at 5.33 ppm. The 13C-NMR spectrum indicated the presence of 12 carbons. Thus, the product A was identified as (±)-1-(2,6,6-trimethyl-2-cyclohexen-1-yl)propan-2-one. Although percentage degradation of test chemical was not known, but major degradation product of test chemical was isolated and identified as(±)-1-(2,6,6-trimethyl-2-cyclohexen-1-yl)propan-2-one, indicating that the test chemical is biodegradable in nature.

 

Additional supporting biodegradation study (from peer reviewed journal Staci L. Simonich et. al., 2000) was conducted in activated sludge & trickling filter wastewater treatment for 3 days for evaluating the percentage biodegradability of test chemical. Wastewater was used as a test inoculum for the study. Initial test chemical conc. used for the study were 0.00367 mg/l and 0.00267 mg/l, respectively. The percentage degradation of test chemical was determined to be 99.5 and 94.6% degradation in activated sludge & trickling filter wastewater treatment over a period of 3 days. Thus, based on percentage degradation, test chemical is considered to be readily biodegradable in nature.

 

On the basis of above overall results for test chemical, it can be concluded that the test chemical can be expected to be ultimate inherently biodegradablein nature.