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REACH

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

EC number: 840-202-3 | CAS number: 2101947-22-0

Life Cycle description
Uses advised against

Ecotoxicological information

Toxicity to microorganisms

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Administrative data

Link to relevant study record(s)

Reference

Endpoint:: activated sludge respiration inhibition testing
Type of information:: experimental study
Adequacy of study:: key study
Study period:: The study was conducted between 01 February 2018 and 27 March 2018.
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: other: Study conducted according to OECD 209 guidelines and in compliance with GLP without deviations that influence the result.
Qualifier:: according to guideline
Guideline:: OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:: no
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
Analytical monitoring:: no
Vehicle:: no
Details on test solutions:: Range-Finding Test:
The test concentrations to be used in the definitive test were determined by a preliminary range finding test.
In the range finding test, activated sewage sludge micro organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L. The test item was dispersed directly in water.
Nominal amounts of test item (5, 50 and 500 mg (500 mg in triplicate)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at room temperature, in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (three replicates at 1000 mg/L).
The pH of the test item dispersions was measured after stirring using Hach HQ40d Flexi handheld meter and if necessary, adjusted to between pH 7.0 and 8.0.
The control group was maintained under identical conditions but not exposed to the test item.

Definitive Test:
The test item was dispersed directly in water.
Nominal amounts of test item (50, 90, 160, 280 and 500 mg (three replicates of each)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at room temperature, in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 100, 180, 320, 560 and 1000 mg/L (three replicates of each).
The pH of the test item dispersions was measured after stirring using Hach HQ40d Flexi handheld meter and if necessary, adjusted to between pH 7.0 and 8.0.
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.
The control group was maintained under identical conditions but not exposed to the test item.
Test organisms (species):: activated sludge of a predominantly domestic sewage
Details on inoculum:: Inoculum
A mixed population of activated sewage sludge micro-organisms was obtained on 1 February 2018 for the range finding test and on 26 March 2018 for the definitive test from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.

Preparation
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC overnight prior to use in the test. On the day of collection the activated sewage sludge (9.5 liters) was fed synthetic sewage (475 mL). The pH of the sample on the day of the test was 7.4 measured using a Hach HQ40d Flexi handheld meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper* using a Buchner funnel which was then rinsed three times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 ºC for at least 1-Hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L prior to use.
* Rinsed 3 times with 20 mL deionized reverse osmosis water prior to drying in an oven

Test water:
The test water used for the range finding and definitive tests was deionized reverse osmosis water containing less than 1 mg/L Total Organic Carbon (TOC).

SYNTHETIC SEWAGE
A synthetic sewage of the following composition, was added to each test vessel to act as a respiratory substrate:
16 g Peptone
11 g Meat extract
3 g Urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
Each constituent was dissolved in a final volume of 1 liter of water with the aid of ultrasonication.
The pH of the synthetic sewage stock used to feed the activated sewage sludge and used in the range finding and definitive tests was between pH 7.2 and 7.3. The pH values were measured using a Hach HQ40d Flexi handheld meter.
Test type:: static
Water media type:: freshwater
Limit test:: no
Total exposure duration:: 3 h
Test temperature:: 20 °C
pH:: pH 7.0-8.0
Nominal and measured concentrations:: Test substance
Range-Finding Test: Nominal: 10, 100 and 1000 mg/l.
Definitive Test: 100, 180, 320, 560 and 1000 mg/l.

Reference substance
Range-Finding Test: 3.2, 10 and 32 mg/l.
Definitive Test: 3.2, 10 and 32 mg/l.
Details on test conditions:: At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the reference item. Two additional control vessels were then prepared prior to the test item vessels being prepared. Finally, two further control vessels were prepared.
The test was conducted under normal laboratory lighting in a temperature controlled room at measured temperatures of approximately 20 °C.
Reference substance (positive control):: yes
Remarks:: 3,5-Dichlorophenol (C6H3CI2.OH), Purity 97%
Duration:: 3 h
Dose descriptor:: EC50
Effect conc.:: 570 mg/L
Nominal / measured:: nominal
Conc. based on:: test mat.
Basis for effect:: inhibition of total respiration
Remarks:: respiration rate
Details on results:: Range-Finding Test
The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60% of the dissolved oxygen saturation level of 8.9 mg O2/L.
No statistically significant toxic effects were shown at the test concentrations of 10 and 100 mg/L, however statistically significant toxic effects were shown at the test concentration of 1000 mg/L.
Based on these results and after discussion with the Sponsor, it was considered necessary to perform a definitive test in order to obtain an EC50 value for the test item. Test concentrations of 100, 180, 320, 560 and 1000 mg/L were selected for the definitive test.

Validation Criteria
• The EC50 value (3-Hour contact time) for the reference item, 3,5-Dichlorophenol, was 7.9 mg/L.
• The specific respiration rate of the controls was 27.33 mg oxygen per gram dry weight of sludge per hour.
• The coefficient of variation of oxygen uptake in the control vessels was 11%.
All validation criteria for the study were therefore satisfied.

Inhibition of Respiration Rate:
In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7 mg O2/L and 2 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.
The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60% of the dissolved oxygen saturation level of 8.9 mg O2/L.
Results with reference substance (positive control):: 3,5-dichlorophenol EC50 (3 h): 7.9 mg/I
Validity criteria fulfilled:: yes
Conclusions:: The EC50 (respiration inhibition) values of the substance is on activated sewage sludge was 570 mg/l after 3 hours contact time.
Executive summary:: A study according to OECD 209 was performed to assess the inhibitory effect of Substance FRET 15-0735 on the respiration of activated sewage sludge. Cultures of activated sewage sludge were incubated with synthetic sewage under vigorous aeration and in the presence of the test substance. Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 100, 180, 320, 560 and 1000 mg/L (three replicates) for a period of 3 hours at measured temperatures of approximately 20 °C with the addition of a synthetic sewage as a respiratory substrate. A positive control i.e. 3,5-dichlorophenol was tested concurrently with the test substance in order to demonstrate the satisfactory performance of the procedure. The EC50 (respiration inhibition) values of the substance is on activated sewage sludge was 570 mg/l after 3 hours contact time.

Description of key information

 A study according to OECD 209 was performed to assess the inhibitory effect of Substance FRET 15-0735 on the respiration of activated sewage sludge. Cultures of activated sewage sludge were incubated with synthetic sewage under vigorous aeration and in the presence of the test substance. Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 100, 180, 320, 560 and 1000 mg/L (three replicates) for a period of 3 hours at measured temperatures of approximately 20 °C with the addition of a synthetic sewage as a respiratory substrate. A positive control i.e. 3,5-dichlorophenol was tested concurrently with the test substance in order to demonstrate the satisfactory performance of the procedure. The EC50 (respiration inhibition) values of the substance is on activated sewage sludge was 570 mg/l after 3 hours contact time.

Key value for chemical safety assessment

EC50 for microorganisms:: 570 mg/L

Additional information

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

Nominal Concentration (mg/L)		Dissolved Oxygen Concentration (mg O₂/L)	Dissolved Oxygen Saturation Level of 8.9 mg O₂/L (%)
Control	R₁	6.60	74
	R₂	6.53	73
	R₃	5.93	67
	R₄	5.96	67
Test Item	10	5.86	66
	100	5.82	65
	1000 R₁	6.34	71
	1000 R₂	7.10	80
	1000 R₃	7.14	80
3,5-Dichlorophenol	3.2	6.92	78
	10	7.36	83
	32	7.73	87

Nominal Concentration (mg/L)		Initial O₂Reading (mg O₂/L)	Measurement Period (minutes)	Final O₂Reading (mg O₂/L)	O₂Consumption Rates (mg O₂/L/hour)	Inhibition (%)
Control	R₁	5.0	4	2.0	45.00	-
	R₂	4.5	3	2.3	44.00	-
	R₃	4.4	3	2.2	44.00	-
	R₄	4.6	4	1.8	42.00	-
Test Item	10	3.8	3	1.5	46.00	[5]
	100	2.8	1	2.0	48.00	[10]
	1000 R₁	7.3	10	5.4	11.40	74
	1000 R₂	7.4	10	5.8	9.60	78
	1000 R₃	7.4	10	5.7	10.20	77
3,5-Dichlorophenol	3.2	5.9	6	2.4	35.00	20
	10	7.0	10	3.6	20.40	53
	32	7.8	10	6.8	6.00	86

Nominal Concentration (mg/L)		pH
Nominal Concentration (mg/L)		0 Hours	3 Hours
Control	R₁	7.7	7.3
	R₂	7.4	7.2
	R₃	7.5	7.2
	R₄	7.5	7.3
Test Item	10	7.4	7.2
	100	7.6	7.3
	1000 R₁	7.5	7.4
	1000 R₂	7.5	7.5
	1000 R₃	7.5	7.5
3,5-Dichlorophenol	3.2	7.6	7.7
	10	7.9	7.9
	32	7.6	7.6

Nominal Concentration (mg/L)		Observations on Test Preparations
Nominal Concentration (mg/L)		0 Hours*	30 Minutes Contact Time	3 Hours Contact Time
Control	R₁	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	R₂	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	R₃	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	R₄	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
Test Item	10	Pale yellow/brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
	100	Pale yellow/brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
	1000 R₁	Pale yellow/brown dispersion with oily globules of test item visible dispersed throughout, at the surface and settled on the bottom of the vessel	Dark brown dispersion with oily globules of test item visible dispersed throughout and at the surface	Dark brown dispersion with oily globules of test item visible dispersed throughout and at the surface
	1000 R₂	Pale yellow/brown dispersion with oily globules of test item visible dispersed throughout, at the surface and settled on the bottom of the vessel	Dark brown dispersion with oily globules of test item visible dispersed throughout and at the surface	Dark brown dispersion with oily globules of test item visible dispersed throughout and at the surface
	1000 R₃	Pale yellow/brown dispersion with oily globules of test item visible dispersed throughout, at the surface and settled on the bottom of the vessel	Dark brown dispersion with oily globules of test item visible dispersed throughout and at the surface	Dark brown dispersion with oily globules of test item visible dispersed throughout and at the surface
3,5-Dichlorophenol	3.2	Pale yellow/brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible
	10	Pale yellow/brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible
	32	Pale yellow/brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible

Nominal Concentration (mg/L)		pH
Nominal Concentration (mg/L)		Prior to Adjustment	After Adjustment
Test Item	100R₁	7.7	-
	100R₂	7.3	-
	100R₃	7.5	-
	180R₁	7.7	-
	180R₂	7.3	-
	180R₃	7.5	-
	320R₁	7.5	-
	320R₂	7.5	-
	320R₃	7.2	-
	560R₁	7.0	7.5
	560 R₂	6.3	7.2
	560 R₃	6.5	7.2
	1000 R₁	6.5	7.2
	1000 R₂	6.7	7.4
	1000 R₃	6.6	7.4

Nominal Concentration (mg/L)		Initial O₂Reading (mg O₂/L)	Measurement Period (minutes)	Final O₂Reading (mg O₂/L)	O₂Consumption Rates (mg O₂/L/hour)	Inhibition (%)
Control	R₁	3.1	2	1.9	36.00	-
	R₂	4.0	3	2.0	40.00	-
	R₃	4.8	5	1.8	36.00	-
	R₄	4.2	3	2.0	44.00	-
	R₅	4.4	3	2.3	42.00	-
	R₆	4.6	2	3.0	48.00	-
Test Item	100R₁	3.5	2	2.1	42.00	[2]
	100R₂	4.0	3	2.0	40.00	2
	100R₃	4.0	4	2.0	30.00	27*
	180R₁	4.2	4	2.0	33.00	20
	180R₂	5.7	4	3.8	28.50	30
	180R₃	3.3	2	2.8	15.00	63^*
	320R₁	3.7	3	2.1	32.00	22
	320R₂	3.7	2	2.9	24.00	41^*
	320R₃	4.7	5	2.0	32.40	21
	560R₁	5.5	9	2.2	22.00	46
	560 R₂	6.1	10	2.8	19.80	52
	560 R₃	6.0	10	2.5	21.00	49

Nominal Concentration (mg/L)		Initial O₂Reading (mg O₂/L	Measurement Period (minutes)	Final O₂Reading (mg O₂/L)	O₂Consumption Rates (mg O₂/L/hour)	Inhibition (%)
Test Item	1000R₁	7.5	10	6.0	9.00	78
	1000R₂	7.5	10	6.0	9.00	78
	1000R₃	7.3	10	5.8	9.00	78
3,5-Dichlorophenol	3.2	5.0	6	2.0	30.00	27
	10	6.2	10	3.2	18.00	56
	32	6.9	10	5.9	6.00	85

	FRET 15-0735		3,5-Dichlorophenol
	EC_x(3 Hours) (mg/L)	95% Confidence Limits (mg/L)	EC_x(3 Hours) (mg/L)	95% Confidence Limits (mg/L)
EC₁₀	140	-	1.6	-
EC₂₀	230	-	2.4	-
EC₅₀	570	500 - 640	7.9	6.1 - 10
EC₈₀	1000	-	26	-

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

Toxicity to microorganisms

Administrative data

Link to relevant study record(s)

Reference

Inhibition of Respiration Rate

Description of key information

Key value for chemical safety assessment

Additional information