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

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:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study was performed according to OECD 301D study guidelines

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge, collected from STP, was used as inoculum for the CBT test. This was pre-conditioned at the test temperature. 1 mL supernatant was diluted to 100 mL with mineral medium and from this solution 0.05 mL was added to 125 mL test bottles. This gave the bacterial count as 104 to 106 CFU/L.
- Method of cultivation: Aeration of incoming domestic sludge with micro-organisms by means of diffuser
- Storage conditions: 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.
- Pretreatment: Sludge was pre-conditioned by decanting the supernatant of the activated sludge and later washed with sludge by mineral media followed by aerating for 1 day at the 20º C test temperature.
- Concentration of sludge: [11/15/2021 3:38 PM] Subodh Gaikwad (Guest)
Biomass Concentration: A concentration of 32 mL/400 mL of activated sludge solution was added to every flask. This concentration gave suspended solid of 1.0 g/L.
- Initial cell/biomass concentration:
- Water filtered: yes/no
- Type and size of filter used, if any:

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: Compositon of media is presented in table below
- Additional substrate:
- Solubilising agent (type and concentration if used): Not applicable
- Test temperature: 20 ± 1°C
- pH: Control 7.0-7.4
- pH adjusted: no
- Aeration of dilution water: no
- Suspended solids concentration:
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: BOD bottles
- Number of culture flasks/concentration: 2 bottles per concentration
- Method used to create aerobic conditions:
- Measuring equipment: BOD probe inducator which measures DO
- Test performed in closed vessels due to significant volatility of test substance: not applicable
- Test performed in open system: closed with bod bottle stoppers

SAMPLING
- Sampling frequency: 0th, 7th, 14th, 21st, 28th days.
- Sample storage before analysis: DO measured immediatly
- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank: Containing inoculum and mineral media
- Toxicity control: Containing test chemical, reference chemical (sodium benzoate),N-ATU, inoculum and mineral media.
- Other:

Reference substance:

benzoic acid, sodium salt

Test performance:

The solution of the test substance in the mineral medium, at 4 mg/L, is inoculated with a relatively small number of micro-organisms from a mixed population and kept in completely full, closed bottles in the dark at a constant temperature. Degradation is followed by analysis of dissolved oxygen over a 28-d period. The amount of oxygen taken up by the microbial population during biodegradation of the test substance, corrected for uptake by the blank inoculum run in parallel, is expressed as a percentage of ThOD. A reference compound is run in parallel to check the operation of the procedures. Normally, the test lasts for 28 d. Test however may be ended before 28 d, i.e. as soon as the biodegradation curve has reached a plateau for at least three determinations. Test may also be prolonged beyond 28 days when the curve shows that biodegradation has started but that the plateau has not been reached by day 28, but in such cases the chemical would not be classified as readily biodegradable.

Parameter:

% degradation (O2 consumption)

Value:

61.86

Sampling time:

28 d

Remarks on result:

other: readily biodegradable

Details on results:

D.O values (oxygen consumed mg O2/L) were recorded in presented in tables by using D.O meter. The amount of oxygen taken up by the microbial population during biodegradation of the test chemical was corrected for uptake by the inoculum blank control and BOD was calculated accordingly and represented in Table section. The % degradation values of 0th, 7th, 14th, 21st, and 28th day were calculated and represented in Table 4. ThOD, BOD28 and % degradation of both reference and the test chemical, calculated using the formula given in OECD guideline 301 D Closed Bottle Test, are summarized in Table sections.

Results with reference substance:

Reference chemical degraded upto 97 % with in 28 days of exposure

D.O VALUES (mg/L)

No. of Days	Blank Control	Test Chemical	Procedure Control(Reference Chemical)	Toxicity Control
0	7.3	7.9	7.4	7.4
7	7.2	5.2	5.0	5.2
14	7.0	1.4	2.0	1.2
21	6.8	0.8	1.0	0.9
28	7.0	0.4	0.6	0.6

BOD VALUES (mgO₂/mg)

No. of Days	Test Chemical	Procedure Control(Reference Chemical)	Toxicity Control
0	0	0	0
7	0.65	0.58	0.53
14	1.55	1.28	1.48
21	1.65	1.48	1.50
28	1.80	1.63	1.63

 

% BIODEGRADATION

No. of Days	Test Chemical	Procedure Control(Reference Chemical)	Toxicity Control
0	0	0	0
7	22.34	34.43	18.04
14	53.26	76.35	50.69
21	56.70	88.32	51.55
28	61.86	97.3	55.84

BOD28, ThOD AND % BIODEGRADATION VALUES

Test System	BOD28	ThOD (mgO2/mg)	% Biodegradation
Test Chemical	1.80	2.2	69.55
Procedure Control(Reference Chemical)	1.63	1.67	91.62
Toxicity Control	1.63	2.91	69.55

 

pH VALUES

No. of Days	Blank Control	Test Chemical	Procedure Control(Reference Chemical)	Toxicity Control
0	7.4	7.4	7.4	7.5
7	7.3	7.3	7.3	7.3
14	7.3	7.2	7.1	7.0
21	7.1	7.2	7.0	7.0
28	7.0	7.1	6.9	6.9

 

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

After exposure period of days test chemical is considered to be readily biodegradable in water and the % degradaibilty is 61.86 %

Executive summary:

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.

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.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

Various experimental studies has been investigated for the test chemical for reviewing the biodegradation endpoint which have been summarized 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.