Methyl [1α,2β(Z)]-(±)-3-oxo-2-(pent-2-enyl)cyclopentaneacetate

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 22nd March 2021
Experimental completion date 03rd June 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-A (Determination of the "Ready" Biodegradability - Dissolved Organic Carbon (DOC) Die-Away Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl [1α,2β(Z)]-(±)-3-oxo-2-(pent-2-enyl)cyclopentaneacetate

EC Number:

243-497-1

EC Name:

Methyl [1α,2β(Z)]-(±)-3-oxo-2-(pent-2-enyl)cyclopentaneacetate

Cas Number:

20073-13-6

Molecular formula:

C13H20O3

IUPAC Name:

methyl 2-[(1S,2S)-3-oxo-2-[(2Z)-pent-2-en-1-yl]cyclopentyl]acetate

Specific details on test material used for the study:

Identification: Methyljasmonate
CAS Number: 39924-52-2
Batch: 0900175
Purity: 99.03%
Physical State/Appearance: Clear colorless liquid
Expiry Date: 25 March 2023
Storage Conditions: Approximately 4 °C in the dark

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

A mixed population of sewage treatment micro-organisms was obtained on 05 May 2021 from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, UK, which treats predominantly domestic sewage.

Preparation of Inoculum
The sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and maintained on aeration in a temperature controlled room at approximately 20 °C prior to use.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Experimental Design and Study Conduct
Test Item Preparation
Following preliminary solubility work and the recommendations of the International Standards Organisation (ISO, 2018) and in the published literature (Handley et al, 2002) the test item was dissolved in an auxiliary solvent prior to adsorption onto filter paper*. High shear mixing was also applied to break up the filter paper containing the test item. 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 (1000 mg) was dissolved in 100 mL of acetone to give a 1000 mg/10 mL solvent stock solution. An aliquot (500 μL) of this solvent stock solution was dispensed onto a filter paper and the solvent allowed to evaporate to dryness for approximately 15 minutes. The filter paper was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes) prior to addition of inoculum. The volume was then adjusted to 500 mL to give a final concentration of 100 mg/L. The volumetric flask containing the solvent stock solution was inverted several times to ensure homogeneity of the solution.
The inoculum control vessels were prepared in a similar manner without the addition of test item.
A test concentration of 100 mg/L was selected for use in the study following the recommendations of the Test Guidelines.
As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Reference Item Preparation
A reference item, aniline (C6H5NH2), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving a nominal amount of freshly distilled reference item (500 mg) directly in mineral medium (500 mL) with the aid of ultrasonication for approximately 15 minutes. A filter paper* was added to each vessel in order to maintain consistency between the test and procedure control vessels. Acetone (500 μL) was dispensed onto each filter paper and evaporated to dryness for approximately 15 minutes. The filter paper was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes) prior to addition to each vessel. An aliquot (50 mL) of the 1000 mg/L aniline stock solution was added to each test vessel with the inoculum (5 mL) and the volume adjusted to 500 mL with mineral media, to give the test concentration of 100 mg/L. The volumetric flask containing the stock solution was inverted several times to ensure homogeneity.
The pH of the reference item stock solution was 7.5. The pH value was measured using a Hach HQ30d Flexi handheld meter.

Toxicity Control
A toxicity control, containing the test item and aniline, was prepared in order to assess any toxic effects of the test item on the sewage treatment micro-organisms used in the test.
An aliquot (500 μL) of the test item solvent stock solution was dispensed onto a filter paper, Whatman GF/A (70 mm diameter), and the solvent allowed to evaporate for approximately 15 minutes. The filter paper was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes) prior to addition to the test vessel with an aliquot (50 mL) of the 1000 mg/L aniline stock solution and inoculum (5 mL). The volume was adjusted to 500 mL with mineral medium to give a final concentration of 100 mg test item/L plus 100 mg aniline/L.

Preparation of Test System
The following test preparations were prepared and inoculated in 500 mL glass bottles:
a) Three replicate bottles containing inoculated mineral medium to act as the inoculum control plus a filter paper, Whatman GF/A (70 mm diameter)
b) Two replicate bottles containing inoculated mineral medium plus a filter paper, Whatman GF/A (70 mm diameter), and the reference item, aniline, at a concentration of 100 mg/L to act as the procedure control
c) Three replicate bottles containing the test item on filter paper, Whatman GF/A (70 mm diameter), and inoculated mineral medium and the test item at a concentration of 100 mg/L
d) Two replicate bottles containing the test item on filter paper, Whatman GF/A (70 mm diameter), at a concentration of 100 mg/L and the test item at a concentration of 100 mg/L reference item, aniline, at a concentration of 100 mg/L to act as toxicity control 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.
All vessels were inoculated with the prepared inoculum at a rate of 1% v/v.
On Day 0, the test and reference items were added to the mineral medium. The pH of all vessels was measured using a Hach HQ30d Flexi handheld meter, prior to the addition of the inoculum and the volume in all the vessels being adjusted to 500 mL by the addition of mineral medium.
All inoculum control, test item, procedure control and toxicity control vessels were placed in a CES Multi-Channel Aerobic Respirometer.
The system consists of a sample flask sealed by a sensor head/CO2 trap immersed in a temperature controlled water bath. The samples were stirred for the duration of the test with a magnetically coupled stirrer.
As biodegradation progresses, the micro-organisms convert oxygen to carbon dioxide which is absorbed into the ethanolamine solution (50% v/v) causing a net reduction in gas pressure within the sample flask (see Figure 1). The pressure reduction triggers the electrolytic process, generating oxygen and restoring the pressure in the sample flask. The magnitude of the electrolyzing current and the duration of the current is proportional to the amount of oxygen supplied to the micro-organisms. The data generated from the respirometer’s own battery backed memory was collected four times a day on the hard disk drive of a non-dedicated computer.
The test was conducted in diffuse light at a temperature of approximately 20 to 22 °C.
On Day 28, two inoculum controls, one procedure control, two test item and one toxicity
control vessel were sampled for pH analysis.
The remaining vessels which were not sampled were not reported. Additional replicate
vessels were prepared and incubated in order that in the event of a leak in the test system a
replicate vessel could be discarded without jeopardizing the integrity of the test.

Reference substance

Reference substance:

aniline

Results and discussion

% Degradation

Key result

Parameter:

% degradation (O2 consumption)

Value:

108

Sampling time:

28 d

Details on results:

Validation Criteria and Biodegradation
The mean BOD of the inoculated mineral medium (control) was 28.36 mg O2/L after 28 days and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The pH of the inoculated test item vessels on Day 28 ranged from 7.5 to 7.7 and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between extremes of replicate BOD values at the time the plateau was reached was less than 20% and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The test item attained 108% biodegradation after 28 days, calculated from the oxygen consumption values, and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained with 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.
The toxicity control attained 44% biodegradation after 14 days and 46% biodegradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.


Total Organic Carbon Confirmation
Total Organic Carbon of the diluted aniline stock solution confirmed that it had been prepared correctly.

BOD5 / COD results

Results with reference substance:

Aniline (procedure control) attained 72% biodegradation after 14 days with greater than 60% degradation being attained in a 10-day window. After 28 days 77% biodegradation was attained thereby confirming the suitability of the inoculum and test conditions

Any other information on results incl. tables

Biological Oxygen Demand Values

Day	BOD (mg O2/L)
	Inoculum Control		Procedure Control	Test Item		Toxicity Control
	R1	R2		R1	R2
0	0.00	0.00	0.00	0.00	0.00	0.00
1	1.00	1.30	1.66	1.42	1.50	2.42
2	1.34	2.04	2.80	1.92	2.04	3.08
3	2.24	2.08	11.08	2.54	3.08	9.96
4	3.50	3.38	34.62	4.16	4.78	36.36
5	4.70	4.58	82.00	5.74	6.54	93.88
6	5.74	5.58	166.56	7.24	8.38	142.32
7	6.74	6.78	182.76	8.82	11.50	169.10
8	8.04	8.16	195.76	10.82	13.82	187.06
9	8.62	8.88	208.20	12.54	15.46	200.46
10	9.58	9.82	218.08	15.70	17.46	213.24
11	10.66	10.92	224.28	22.74	19.58	226.00
12	11.82	12.12	229.16	36.94	21.86	236.86
13	12.86	13.28	233.32	61.64	24.12	248.02
14	14.04	14.46	236.98	90.88	26.36	252.36
15	14.92	15.46	239.70	116.04	28.58	255.28
16	16.20	16.74	242.94	139.06	31.28	258.14
17	17.58	18.20	246.10	159.40	34.40	260.94
18	18.12	18.70	248.16	176.98	37.40	262.64
19	18.90	19.74	250.28	196.04	44.90	264.64
20	20.12	20.82	252.56	211.92	72.30	266.60
21	21.08	21.82	254.56	217.66	144.70	268.30
22	22.00	22.58	256.18	220.92	195.54	269.98
23	22.94	23.40	258.10	223.78	228.96	271.60
24	24.50	24.78	260.40	227.50	254.82	273.68
25	25.66	25.94	262.52	231.28	276.22	275.30
26	26.66	26.70	264.02	234.48	295.10	276.48
27	27.62	27.74	265.68	237.12	310.16	277.98
28	28.28	28.44	267.18	239.12	322.82	279.22

 

R = Replicate

 

Percentage Biodegradation Values

Day	Biodegradation (%)
	Procedure Control	Test Item			Toxicity Control
		R1	R2	Mean
0	0	0	0	0	0
1	0	0	0	0	0
2	0	0	0	0	0
3	3	0	0	0	1
4	10	0	1	1	6
5	25	0	1	1	16
6	52	1	1	1	25
7	57	1	2	2	30
8	61	1	2	2	33
9	65	2	3	3	35
10	67	3	3	3	37
11	69	5	4	5	40
12	70	11	4	8	41
13	71	21	5	13	43
14	72	33	5	19	44
15	73	43	6	25	44
16	73	52	6	29	44
17	74	60	7	34	45
18	74	67	8	38	45
19	75	75	11	43	45
20	75	81	22	52	45
21	75	83	52	68	45
22	76	85	74	80	46
23	76	85	88	87	46
24	76	86	98	92	46
25	77	87	107	97	46
26	77	88	114	101	46
27	77	89	120	105	46
28	77	90	125	108	46

 

R = Replicate

pH Values of the Test Preparations on Days 0 and 28

Test Vessel	pH
	Day 0	Day 28
Inoculum Control R1	7.4	7.8
Inoculum Control R2	7.4	7.7
Procedure Control	7.4	8.5
Test Item R1	7.4	7.7
Test Item R2	7.4	7.5
Toxicity Control	7.4	8.4

 

 R   =    Replicate

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item attained 108% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained with 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Executive summary:

The study was performed to assess the ready biodegradability of the test item in an aerobic aqueous media. 

The test item at a concentration of 100 mg/L was exposed to sewage treatment micro‑organisms with mineral medium in sealed culture vessels in diffuse light at approximately 20 to 22 ºC for 28 days.  Following the recommendations of the International Standards Organisation (ISO 2018), the test item was dissolved in an auxiliary solvent prior to being adsorbed onto a filter paper and subsequent dispersal in test media.  Using this method the test item was evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms was increased thereby increasing the potential for biodegradation.

The biodegradation of the test item was assessed by the measurement of daily oxygen consumption values for 28 days.  Control solutions with inoculum and the reference item, aniline, and a toxicity control were used for validation purposes.

The test item attained 108% biodegradation after 28 days and satisfied the 10‑Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%.  The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Aniline (procedure control) attained 72% biodegradation after 14 days with greater than 60% degradation being attained in a 10-Day window.  After 28 days 77% biodegradation was attained.