N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

Toxicity to microorganisms

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:

18 September 2017 to 19 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance with international guidelines and in accordance with GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Remarks:

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.

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 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 (3 replicates).
- Eluate: Deionised reverse osmosis water
- Differential loading: 10, 100 and 1000 mg/L
- Controls: Blank control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Yes, dispersion tested after maximising solubility

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: No
- Name and location of sewage treatment plant where inoculum was collected: Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK
- Method of cultivation: Not applicable
- Preparation of inoculum for exposure: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ℃ overnight prior to use in the test. On the day of collection the activated sewage sludge (7.5 liters) was fed synthetic sewage (375 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 3 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 ℃ for at least one 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.
- Pretreatment: Not applicable
- Initial biomass concentration: Not measured

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Remarks on exposure duration:

Standard guideline contact time.

Post exposure observation period:

Not applicable (respiration measurements only)

Test temperature:

20 °C

pH:

7.9 - 8.0 (measured)

Dissolved oxygen:

7.60 - 8.71 mg/L (measured)

Salinity:

Not measured

Conductivity:

Not measured

Nominal and measured concentrations:

Nominal concentrations: 10, 100 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL conical flask
- Type (delete if not applicable): open
- Aeration: Clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute
- No. of vessels per concentration (replicates): 3 (1000 mg/L), 1 replicate (10 and 100 mg/L)
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): Not applicable
- No. of vessels per abiotic control (replicates): Not applicable
- Sludge concentration (weight of dry solids per volume): 3.0 g/L
- Weight of dry solids per volume of reaction mixture per unit of time: Not applicable
- Nutrients provided for bacteria: Yes (synthitic sewage feed)
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
3g - Urea
0.7 g - NaCl
0.4 g - Cacl2.2H20
0.2 g - MgSO4.7H20
2.8 g - K2HPO4
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 was pH 7.4 and the pH of the synthetic sewage stock used for the test was pH 7.3. The pH values were measured using a Hach HQ40d Flexi handheld meter.
- Nitrification inhibitor used (delete if not applicable): none
- Biomass loading rate: Not applicable
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).
- Particulate matter: Not reported
OTHER TEST CONDITIONS
- Adjustment of pH: Not required
- Photoperiod: Not required
- Light intensity: Not reported
- Details on termination of incubation: Aeration stopped after 3 hours.
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
3 hour EC50, EC10 and NOEC values were planned to be estimated in the study:
The respiration rate, R, expressed in milligrams oxygen per liter per hour (mg O2/L/h), was calculated from the linear part of the recorded oxygen decrease graph according to the following equation:
R = [(Q1 -Q2)/Δt]x60
Where:
Q1= the oxygen concentration at the beginning of the selected section of the linear phase (mg/L);
Q2= the oxygen concentration at the end of the selected section of the linear phase (mg/L);
Δt= the time interval between the beginning and end of the selected section of the linear phase (min).
The specific respiration rate, RS, expressed as the amount of oxygen consumed per gram dry weight of sludge per hour (mg O2/g/h) was deduced according to the following equation:
RS= R/SS
Where:
SS= the concentration of suspended solids in the test mixture (g dry weight/L).
The percentage inhibition was calculated according to the following equation:
% inhibition = [1 – (R/Rbc)] x 100
Where:
Rbc = the mean respiration rate of the blank controls.
The percentage inhibition values were plotted against concentration for the reference item only, a line fitted using the Xlfit software package (IDBS) and the EC10, EC20, EC50 and EC80 values determined from the equation for the fitted line.
The EC10, EC20, EC50 and EC80 values for the test item were determined by inspection of the inhibition of respiration rate data.
95% confidence limits were calculated for the reference item EC50 value using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949).
In order to determine the NOEC, one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the oxygen consumption data after 3 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10 (range-finding limit test)
- Justification for using fewer concentrations than requested by guideline: Not applicable
- Range finding study
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: Not required, study terminated at range-finding limit test.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Remarks on result:

not determinable

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Remarks on result:

not determinable

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not applicable
- Effect concentrations exceeding solubility of substance in test medium: Not applicable
- Adsorption (e.g. of test material to the walls of the test container): Not applicable
- Blank controls oxygen uptake rate: 36.00 - 37.50 mg O2/L/hour
- Coefficient of variation of oxygen uptake rate in control replicates: Not reported

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: 3h EC50 = 6.7 mg/L
- Other: -

Reported statistics and error estimates:

Not required (no effect at 1000 mg/L limit concentration).

Table 1. pH values of the test item preparations after stirring and prior to the addition of inoculum in the range-finding test

Nominal Concentration (mg/L)		pH
		Prior to Adjustment	After Adjustment
Test Item	10	8.0	-
	100	8.0	-
	1000 R1	7.9	-
	1000 R2	8.0	-
	1000 R3	7.9	-

Table 2. Dissolved oxygen concentrations of the test preparations after 30 minute contact time in the range-finding test

Nominal Concentration (mg/L)		Dissolved Oxygen Concentration (mg/L)	Expressed as a Percentage of a Dissolved Oxygen Saturation Level of 7.9 mg/L
Control	R1	7.82	88
	R2	7.83	88
	R3	7.80	88
	R4	7.60	85
Test Item	10	7.76	87
	100	7.86	88
	1000 R1	7.59	85
	1000 R2	7.75	87
	1000 R3	8.02	90
3,5-dichlorophenol	3.2	7.90	89
	10	8.56	96
	32	8.71	98

Table 3. Oxygen consumption rates and percentage inhibition values after 3 hours contact time in range-finding test

Nominal Concentration (mg/L)		Initial O2 Reading (mg/L)	Measurement Period (minutes)	Final O2 Reading (mg/L)	O2 Consumption Rates (mg O2/L/hour)	% Inhibition
Control	R1	5.0	4	2.5	37.50	-
	R2	5.0	4	2.5	37.50	-
	R3	4.8	5	1.8	36.00	-
	R4	5.7	5	2.6	37.20	-
Test Item	10	4.9	4	2.3	39.00	[5]
	100	4.2	3	2.2	40.00	[8]
	1000 R1	4.8	4	2.2	39.00	[5]
	1000 R2	5.8	6	2.1	37.00	0
	1000 R3	5.0	5	1.5	42.00	[13]
3,5-dichlorophenol	3.2	5.8	7	3.0	24.00	35
	10	6.0	6	4.4	16.00	37
	32	7.7	10	6.8	5.40	85

Table 4. pH values of the test preparations at the start and end of the exposure period in the range-finding test

Nominal Concentration (mg/L)		pH
		0 Hours	3 Hours
Control	R1	7.2	7.6
	R2	7.3	7.5
	R3	7.4	7.7
	R4	7.2	7.7
Test Item	10	7.3	7.4
	100	7.2	7.3
	1000 R1	7.2	7.4
	1000 R2	7.3	7.5
	1000 R3	7.3	7.3
3,5-dichlorophenol	3.2	7.3	7.8
	10	7.4	7.7
	32	7.3	7.5

Table 5. Observations on the test preparations throughout the test period in the range-finding test

Nominal Concentration (mg/L)		Observation on Test Preparations
		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 with a few pieces of test item on the surface	Dark brown dispersion with a few pieces of test item visible dispersed throughout	Dark brown dispersion with a few pieces of test item visible dispersed throughout
	100	Pale yellow/brown dispersion with pieces of test item on the surface and dispersed throughout	Dark brown dispersion with pieces of test item visible on the surface and dispersed throughout	Dark brown dispersion with pieces of test item visible on the surface and dispersed throughout
	1000 R1	Pale yellow/brown cloudy dispersion with many pieces of test item on the surface and dispersed throughout	Dark brown dispersion with many pieces of test item visible on the surface and dispersed throughout	Dark brown dispersion with many pieces of test item visible on the surface and dispersed throughout
	1000 R2	Pale yellow/brown cloudy dispersion with many pieces of test item on the surface and dispersed throughout	Dark brown dispersion with many pieces of test item visible on the surface and dispersed throughout	Dark brown dispersion with many pieces of test item visible on the surface and dispersed throughout
	1000 R3	Pale yellow/brown cloudy dispersion with many pieces of test item on the surface and dispersed throughout	Dark brown dispersion with many pieces of test item visible on the surface and dispersed throughout	Dark brown dispersion with many pieces of test item visible on the surface and dispersed throughout
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

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 and EC10 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.

Executive summary:

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at measured temperatures of approximately 20°C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol.

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after
3 hours exposure was 1000 mg/L. The reference item gave a 3-Hour EC50value of 6.7 mg/L, 95% confidence limits 4.9 to 9.1 mg/L.

Description of key information

3h EC50 = >1000 mg/L, 3h EC10 = >1000 mg/L, 3h NOEC = ≥ 1000 mg/L; OECD 209; Best (2017)

Additional information

A single key study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at measured temperatures of approximately 20°C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol.

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after
3 hours exposure was 1000 mg/L. The reference item gave a 3-Hour EC50 value of 6.7 mg/L, 95% confidence limits 4.9 to 9.1 mg/L.

No hazard was therefore identified for this substance and it's exposure to sewage sludge microorganisms.