Ethyl 4-methyl-2-oxocyclohexanecarboxylate

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 Jun 2017 - 24 Aug 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 310 using the headspace test and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006, corrected 2014

Deviations:

yes

Remarks:

Different number of replicates in Procedure Control, see "Details on study design"

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: The freshly obtained sludge was used immediately. The concentration of suspended solids was determined to be 3.2 g/L in the concentrated sludge. Before use, the sludge was allowed to settle (40 minutes) and the liquid was decanted for use as inoculum at the amount of 10 mL/L of mineral medium.

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

inorg. C analysis

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral medium according to OECD 310
- Test temperature: 19.7 - 24.1 °C
- pH at start of test: 7.5-7.9
- pH adjusted: no
- Continuous darkness: yes
- Shaking: on an orbital shaker, with a shaking rate sufficient to keep the vessel well mixed and in suspension.

TEST SYSTEM
- Culturing apparatus: well-sealed glass serum vessels.
- Total number of culture flasks/concentration: Inoculum blank: 17, Procedure control: 16, Test Item: 17, Toxicity control: 14, Abiotic control: 8.
- Deviation from OECD Guideline 310: at day 14, 5 replicate vessels of Procedure control were analysed instead of 3. This was considered not to have impacted overall integrity of the study or the interpretation of the study results and conclusions.
- Method used to create aerobic conditions: headspace of air
- Measuring equipment: TOC total organic carbon analyser
- Test performed in closed vessels: yes

SAMPLING
- Sampling frequency: at day 1, 7, 14, 21, 28 for Inoculum blank and Test item; at day 1, 7, 14, 28 for Procedure control and Toxicity control; at day 1 and 28 for Abiotic control.
- Sampling method: Sacrificing replicate vessels.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Only inoculated medium.
- Abiotic sterile control: no inoculated medium, sterilized with 1 mL of a solution containing 10 g/L of HgCl2. Contained Test item.
- Toxicity control: Inoculated medium, reference item, and test item
- Procedure control: Inoculated medium and reference item

Reference substance:

other: Octan-1-ol

Key result

Parameter:

% degradation (inorg. C analysis)

Value:

46

St. dev.:

22.2

Sampling time:

28 d

Details on results:

For biodegradation percentage at all sampling points (i.e., day 1, 7, 14, 21, 28), see Table 1 in 'Any other information on results incl. tables'.
The standard deviation was not included in the report but has been calculated from the data in Table 5 of the report, see "Any other information of results incl. tables".

Table 1

Biodegradation in Percentage of theTheoretical maximum Inorganic Carbon Production (ThIC)

Nominal day	Microbial degradation in percentage of the ThIC			Degradation
	Reference Item	Test Item	Toxicity Control	Abiotic Control
1	0	0	0	0
7	83	2	38	-
14	105	4	39	-
21	-	30	-	-
28	96	46	60	0

- = Not measured

Table3          
Individual Concentrations of Inorganic Carbon (IC) in mg/L of the Inoculum Blank

Nominal day	Date of sampling / IC measure- ment	Individual measurements of inorganic carbon (IC)
		Vessel A	Vessel B	Vessel C	Vessel D	Vessel E	Mean	Std.Dev	CV%
1	28/Jul/17	0.9939	1.014	1.050	-	-	1.019	0.028	3
7	3/Aug/17	1.146	1.183	1.185	-	-	1.171	0.022	2
14	10/Aug/17	1.232	1.299	1.237	-	-	1.256	0.037	3
21	17/Aug/17	1.334	1.157	1.107	-	-	1.199	0.119	10
28	24/Aug/17	1.932	2.293	3.789	2.012	2.075	2.420	0.777	32

- = Not applicable

 

Table4          
Individual Concentrations of Inorganic Carbon (IC) in mg/L of the Procedure Control (Reference Item)

Nominal day	Date of sampling / IC measure- ment	Individual measurements of inorganic carbon (IC)
		Vessel A	Vessel B	Vessel C	Vessel D	Vessel E	Mean	Std.Dev	CV%
1	28/Jul/17	0.9449	1.051	1.018	-	-	1.005	0.054	5
7	3/Aug/17	15.52	18.57	18.92	-	-	17.67	1.870	11
14	10/Aug/17	19.01	20.80	21.45	24.66	24.85	22.15	2.538	11
28	24/Aug/17	18.14	23.53	27.67	23.23	15.90	21.69	4.681	22

- = Not applicable

 

Table5          
Individual Concentrations of Inorganic Carbon (IC) in mg/L of the Test Item

Nominal day	Date of sampling / IC measure- ment	Individual measurements of inorganic carbon (IC)
		Vessel A	Vessel B	Vessel C	Vessel D	Vessel E	Mean	Std.Dev	CV%
1	28/Jul/17	1.065	1.057	1.060	-	-	1.061	0.004	0
7	3/Aug/17	1.671	1.386	1.501	-	-	1.519	0.143	9
14	10/Aug/17	2.989	1.717	1.540	-	-	2.082	0.790	38
21	17/Aug/17	11.10	7.786	2.447	-	-	7.111	4.366	61
28	24/Aug/17	15.68	5.975	16.63	10.38	9.674	11.67	4.437	38

- = Not applicable

 

Table6          
Individual Concentrations of Inorganic Carbon (IC) in mg/L of the Toxicity Control

Nominal day	Date of sampling / IC measure- ment	Individual measurements of inorganic carbon (IC)
		Vessel A	Vessel B	Vessel C	Vessel D	Vessel E	Mean	Std.Dev	CV%
1	28/Jul/17	1.042	0.9931	1.063	-	-	1.033	0.036	3
7	3/Aug/17	15.56	15.69	18.14	-	-	16.46	1.453	9
14	10/Aug/17	15.45	15.99	19.49	-	-	16.98	2.193	13
28	24/Aug/17	25.01	26.30	26.55	27.34	26.77	26.39	0.864	3

- = Not applicable

Table7          
Individual Concentrations of Inorganic Carbon (IC) in mg/L of the Abiotic Control

Nominal day	Date of sampling / IC measure- ment	Individual measurements of inorganic carbon (IC)
		Vessel A	Vessel B	Vessel C	Vessel D	Vessel E	Mean	Std.Dev	CV%
1	28/Jul/17	0.6925	0.7274	0.6698	-	-	0.6966	0.029	4
28	24/Aug/17	1.497	2.469	1.710	1.850	1.737	1.853	0.367	20

- = Not applicable

 

Table8          
Average Concentrations of Inorganic Carbon (IC) in Each Group

Nominal day	Average IC concentration (mg/L)
	Inoculum blank	Procedure control	Test item	Toxicity control	Abiotic control
1	1.019	1.005	1.061	1.033	0.6966
7	1.171	17.67	1.519	16.46	-
14	1.256	22.15	2.082	16.98	-
21	1.199	-	7.111	-	-
28	2.420	21.69	11.67	26.39	1.853

- = Not applicable

 

Validity criteria fulfilled:

yes

Remarks:

For details on validity criteria see 'Overall remarks' section

Interpretation of results:

not readily biodegradable

Conclusions:

FRET 13-0545 was biodegraded (46%) during the test period. However, the criterion for ready biodegradability was not met. Thus, under the conditions of this test FRET 13-0545 was not readily biodegradable.

Executive summary:

The ready biodegradability of FRET 13-0545 was tested according to OECD Guideline 310 (CO₂ in Sealed Vessels (Headspace Test)). Average biodegradation of FRET 13-0545 was 46% during the test period. However, since at least 60% biodegradation was not reached within 10 days immediately following the attainment of 10% biodegradation (10-day window), the criterion for ready biodegradability was not met. Thus, under the conditions of this test FRET 13-0545 was not readily biodegradable. This study is considered to be reliable without restriction.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sept 19, 2016 - Mar 23, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Lot PDJ 378-68
Purity 93.2%

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Inoculum Preparation
Activated sludge was obtained from a sewage plant treating predominantly domestic sewage (Nanjing Chengdong Sewage Treatment Plant) (Batch No.: AS201611081).
The sludge was washed with basal mineral salt medium (BSM) by settling the sludge in a graduated cylinder, decanting the supernatant liquid, replacing with fresh BSM, aerating, and settling. The above washing process was repeated 3 times, then the sludge was centrifuged one time (2500 r/min, 4°C, 10 minutes) and the supernatant liquid decanted. Thereafter, a small amount of the washed sludge was weighed and then dried at 100°C until the weight of the dried sludge did not change significantly anymore. Then the dry weight percentage of suspended solids (p) of the activated sludge was calculated to be 11.5%. Finally 17.39 g wet sludge after centrifugation was mixed with 0.5 L BSM (Batch No.: BSM201611141) to obtain an activated sludge with a mixed liquor suspended solids level of 4 g/L. Cell density was determined to be 6.3×108 CFU/L using microscope.28

Duration of test (contact time):

28 d

Initial conc.:

ca. 33.2 - 34.8 mg/L

Based on:

test mat.

Details on study design:

4.1 Preparation of the Test Medium
Reference control stock solution of aniline (1003 mg/L): Dissolved 1.0028 g of aniline in 1 L BSM.
The test substance was added into test bottles directly.
4.2 Test Conditions
Duration: 28 days
Vessels: Glass BOD bottles of 510 mL
Water: Deionised water
Measuring apparatus: Automatic respirometer (BOD meter)
Test medium: Basal Mineral Salt Medium
Controls: Inoculated sample without test substance
pH: 6.86 ~ 7.65
Temperature: (22±1) °C
4.3 Test System
Vessels 1 & 2 (Test suspension): containing test substance (34.8 mg/L and 33.2 mg/L, respectively) and inoculum (mixed liquor suspended solids [MLSS]: 30 mg/L);
Vessels 3 & 4 (Inoculum control): containing only inoculum (MLSS: 30 mg/L);
Vessel 5 (Procedure control): containing reference substance (40 mg/L) and inoculum (MLSS: 30 mg/L);
Vessel 6 (Inhibition control): Inhibition control containing test substance (33.6 mg/L), reference substance (40 mg/L) and inoculum (MLSS: 30 mg/L).
4.4 Performance of the Test
Glass BOD bottles (volume of 510 mL) were used as test vessels.
Vessels 1 and 2 (Test suspension): 0.0087 g and 0.0083 g of test substance and 1.875 mL of the prepared activated sludge (MLSS = 4 g/L) were added. Then BSM was added to obtain the final volume of 250 mL to give concentration of test substance 34.8 mg/L and 33.2 mg/L, and inoculum (MLSS) 30 mg/L.
Vessels 3 and 4 (Inoculum control): 1.875 mL of the prepared activated sludge was added. Then BSM was added to obtain final volume of 250 mL to give a concentration of inoculum (MLSS) 30 mg/L.
Vessel 5 (Procedure control): 10 mL of the reference control parent solution of aniline (1003 mg/L) and 1.875 mL of the prepared activated sludge were added. Then BSM was added to obtain final volume of 250 mL to give concentration of reference substance 40 mg/L and inoculum (MLSS) 30 mg/L.
Vessel 6 (Inhibition control): 0.0084 g of test substance, 10 mL reference control stock solution, and 1.875 mL of the prepared activated sludge were added one by one. Then BSM was added to obtain final volume of 250 mL to give concentration of reference substance 40 mg/L, test substance of 33.6 mg/L and inoculum (MLSS) of 30 mg/L.
The following table shows the details of the test substance added:
No. Flask 1 Flask 2 Flask 6
Sample weight (g) 0.0087 0.0083 0.0084
Concentration(mg/L) 34.8 33.2 33.6
ThOD(mg/L) 75.5 72.0 72.9
All vessels contained a Teflon-coated magnetic stir bar for consistency. The equipment was assembled, the stirrer was started, and the equipment was checked for air-tightness. The vessels were kept at 22 ± 1ºC in darkness. Temperature, stirrer and operate of BOD equipment and the color of the contents in test vessels were checked and oxygen uptake was measured and recorded every day for each test vessel.
At the start and end of incubation (0th and 28th day), the pH of the contents in each vessel was measured.

Reference substance:

aniline

Test performance:

Duriug lhe test, the temperature was kept at (22± l) °C. The pH value of the content in the test container was maintained between 6.86 and 7.65 (see Table 1). The total oxygen uptake in the inoculum blank was 26.1 mg 0 2/L at the end of the test, not exceeding 60 mg 0 2/L.
Biodegradation of the reference substance, aniline, reached 85.3% in 14 days, exceeding the validation criteria of> 60%. The difference of replicate values during the test, was both less than 20%. Biodegradation of inhibition control was 88.4% at 14 days, exceeding the validation criteria of > 25%, and the oxygen consumption by the test substance in group of Test suspension is greater than 60% of inoculum blank during the test period, indicating that there was no inhibition
effect to the inoculum. Thus, the test was considered to be valid.

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. 82.1

Sampling time:

28 d

Details on results:

The results show that under the experimental conditions, biodegradation of the test substance (FRET 13-0545) was 82.1 % at the end of test, and 66.3% at 10-d window. The test substance was considered as readily biodegradable under the experimental conditions.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The results show that under the experimental conditions, following OECD TG 301F, biodegradation of the test substance (FRET 13-0545) was 82.1 % at the end of test, and 66.3% at 10-d window. The test substance was considered as readily biodegradable under the experimental conditions.

Executive summary:

The results show that under the experimental conditions, following OECD TG 301F, biodegradation of the test substance (FRET 13-0545) was 82.1 % at the end of test, and 66.3% at 10-d window. The test substance was considered as readily biodegradable under the experimental conditions.

Description of key information

The test item was biodegraded (46%) during the test period according to OECD TG 310. However, the criterion for ready biodegradability was not met. Thus, under the conditions of this test FRET 13-0545 was not readily biodegradable.

Biodegradation of the test substance (FRET 13-0545) was 82.1 % at the end of test (d28), and 66.3% at 10-d window, under the experimental conditions, following OECD TG 301F. The test substance was considered as ready biodegradable under the test condition.

Taken together the substance is considered as ready biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable