2-ethyl-N-(3-methoxyphenyl)-N-methylbutanamide

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

Study period:

The study was conducted between 08 February 2018 and 08 March 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability 1 is assigned because the study conducted according to OECD TG 301F in compliance with GLP, without deviations that influence the quality of the results.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

On Day’s 21 and 23 of the test the temperature in the waterbath was recorded as being 18.9 and 19.4 °C respectively. This was a deviation from the Study Plan which states the test will be conducted at a temperature of between 20 to 24 °C with a maximum deviation of ± 1 ºC. This deviation was considered to have not affected the integrity or validity of the study given that all validation criterion were met.

Deviations:

yes

Remarks:

On Days 21 and 23 the waterbath temperature was recorded as 18.9 and 19.4 °C respectively. This was a deviation from the Study Plan which states the test will be conducted at a temperature of between 20 to 24 °C with a maximum deviation of ± 1 ºC.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FRET 15-0735
Physical state/Appearance: clear colorless liquid
Storage Conditions: Room temperature in the dark

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic (adaptation not specified)

Details on inoculum:

Test System and Supporting Information
A mixed population of sewage treatment micro-organisms was obtained on 5 February 2018 from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, 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 21 ± 1 ºC prior to use.

Mineral Medium
The mineral medium used in this study was that recommended in the OECD Guidelines.
The deionized reverse osmosis water used for the preparation of the mineral medium and the mineral medium used for the test contained less than 1 mg/L Total Organic Carbon (TOC).

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Test Item Preparation
The test item was dissolved directly in mineral medium.
An amount of test item (400 mg) was dissolved in mineral medium with the aid of ultrasonication for approximately 30 minutes and the volume adjusted to 2 liters to give a 200 mg/L stock solution. An aliquot (250 mL) of this stock solution was diluted with mineral medium (245 mL) and inoculum (5 mL) to give the final test concentration of 100 mg/L. The volumetric flasks containing the stock solution and the test concentration were inverted several times to ensure homogeneity.
A test concentration of 100 mg/L was selected for use in the study following the recommendations of the Test Guidelines.

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 the reference item directly in mineral medium with the aid of ultrasonication for approximately 10 minutes. An aliquot (50 mL) of this stock solution was diluted with mineral medium (445 mL) and the inoculum (5 mL), to give the test concentration of 100 mg/L. The volumetric flask containing the stock solution was inverted several times to ensure homogeneity.

Toxicity Control
A toxicity control, containing the test item and aniline, was prepared in order to assess any toxic effect of the test item on the sewage treatment micro-organisms used in the test.
An aliquot (250 mL) of the 200 mg/L test item stock solution and an aliquot (50 mL) of the 1000 mg/L aniline stock solution was diluted with mineral medium (195 mL) and inoculum (5 mL) to give the test concentration of 100 mg test item/L and 100 mg aniline/L.

Preparation of Test System
The following test preparations were prepared and inoculated in 500 mL bottles:
a) Three replicate bottles containing inoculated mineral medium to act as the inoculum control.
b) Two replicate bottles containing inoculated mineral medium and the reference item, aniline, at a concentration of 100 mg/L.
c) Three replicate bottles in inoculated mineral medium and the test item at a concentration of 100 mg/L.
d) Two replicate bottles containing in inoculated mineral medium, at a concentration of 100 mg/L and the test item at a concentration of 100 mg/L to act as toxicity control 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 and the pH of all vessels measured using a Hach HQ40d 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.
In order to confirm that the aniline and test item stock solutions were prepared correctly, a diluted, 100 mg/L stock solution (in reverse osmosis water) was also sampled for TOC analysis.
All remaining 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. 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 on the hard disk drive of a non-dedicated computer.
The test was conducted in diffuse light at temperatures of between 19 and 21 ºC.
On Day 28, two inoculum control, one procedure control, two test item and one toxicity control vessel were sampled for pH analysis.
The remaining vessels which were not sampled were discarded and are 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:

aniline

Remarks:

Test concentration: 100 mg/L

Test performance:

The mean BOD of the inoculated mineral medium (control) was 15.01 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 7.6 and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between extremes of replicate BOD values at the end of the test was less than 20% and therefore satisfied the validation criterion given in the OECD Test Guidelines.

Parameter:

% degradation (O2 consumption)

Value:

2

Sampling time:

28 d

Details on results:

The test item attained 2% biodegradation after 28 days, calculated from the oxygen consumption values, and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.
The toxicity control attained 31% biodegradation after 14 days and 35% biodegradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.

Parameter:

ThOD

Value:

265 other: mg O2/L

Results with reference substance:

Aniline (procedure control) attained 68% biodegradation after 14 days and 74% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Theoretical Oxygen Demand Values

Calculation of Theoretical Oxygen Demand (ThOD) for the test and reference items.

Test Item: FRET 15-0735           C₁₄H₂₁NO₂    mol wt = 235.33

ThOD (NO₃) = (16(28 +10.5 +2.5 -2))/235.33 = 2.65 mg O₂ / mg

Therefore for a test concentration of 100 mg/L, the ThOD will be 265 mg O₂/L.

 

Reference Item (Procedure Control) : Aniline C₆H₅NH₂      mol wt = 93.13

ThOD (NO₃) = (16(12 +3.5 +2.5))/93.13 = 3.09 mg O₂ / mg

Therefore, for a test concentration of 100 mg/L, the ThOD will be 309 mg O₂/L.

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	0	0	0	0	0
4	1	0	0	0	0
5	1	1	1	1	0
6	6	1	1	1	2
7	21	1	1	1	11
8	41	1	1	1	22
9	59	1	1	1	26
10	63	1	1	1	29
11	65	1	1	1	30
12	66	1	1	1	30
13	67	1	1	1	31
14	68	1	1	1	31
15	69	1	1	1	32
16	70	1	1	1	32
17	70	1	1	1	32
18	71	1	1	1	33
19	72	1	1	1	33
20	72	1	1	1	33
21	72	1	1	1	34
22	73	1	1	1	34
23	73	1	1	1	34
24	73	1	1	1	34
25	74	1	1	1	35
26	74	1	1	1	35
27	74	1	1	1	35
28	74	1	2	2	35

R= Replicate

Validity criteria fulfilled:

yes

Remarks:

The toxicity control attained 31% biodegradation after 14 days and 35% biodegradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.

Interpretation of results:

not readily biodegradable

Remarks:

The test item attained 2% biodegradation after 28 days, calculated from the oxygen consumption values, and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Conclusions:

The substance showed 2% biodegradation in an OECD TG 301F test and is considered to be not readily biodegradable.

Executive summary:

The ready biodegradability of FRET 15-0735 was investigated in a study conducted in accordance with OECD TG 301F and GLP. The concentration tested was 100 mg/L test substance. The test item attained 2% biodegradation after 28 days and therefore cannot be considered as readily biodegradable. Aniline (procedure control) attained 68% biodegradation after 14 days and 74% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Description of key information

The ready biodegradability of FRET 15-0735 was investigated in a study conducted in accordance with OECD TG 301F and GLP. The concentration tested was 100 mg/L test substance. The test item attained 2% biodegradation after 28 days and therefore cannot be considered as readily biodegradable. Aniline (procedure control) attained 68% biodegradation after 14 days and 74% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions. 

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater