N-(2-hydroxyethyl)dodecanamide

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:

17 - 18 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, dark
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: n/a - test item applied directly to test medium
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no - applied as supplied.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material) n/a

OTHER SPECIFICS: n/a

Analytical monitoring:

no

Details on sampling:

- Concentrations: 10, 100, 1000 mg/L
- Sampling method: As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between 7 mg O2/L and 2 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.
- Sample storage conditions before analysis: n/a -analysis conducted immediately after sampling.

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: A mixed population of activated sewage sludge micro-organisms was obtained on 17 July 2017 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage. 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 (5 liters) was fed synthetic sewage (250 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 ºC 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.At time "0", 16 ml synthetic sewage and the test item or reference compound were added to the test vessel and made up to 250 mL with chlorine-free tap water. After measurement and adjustment of pH, 250 mL of activated sludge (3 g/L dry solids) were added consequently obtaining a total volume of 500 ml with 1.5 g/L dry solids. Synthetic sewage sludge feed was prepared as follows: per 1 L demineralised water - 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.
- Differential loading: n/a
- Controls: For the controls the same procedure was made as with the test vessels, however without test or reference substance.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): n/a
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): n/a
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

sewage, domestic

Details on inoculum:

- Laboratory culture: No
- Name and location of sewage treatment plant where inoculum was collected: Activated sludge from Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK was used as test system (predominantly domestic waste).
- Method of cultivation: n/a
- Preparation of inoculum for exposure: The activated sludge was kept aerated until use at test temperature. Dry solid of the activated sludge was determined as 3.8 g/L (range finder test) and 3.2 g/L (limit test) by weight measurements before and after drying at 105°C (mean of triplicate measurements). The activated sludge was washed twice by settling the sludge, decanting the supernatant and re-suspending the sludge in chlorine-free tap water. Before using, the activated sludge was diluted to 3 g/L dry solids with chlorine-free tap water, in order to obtain a final concentration of 1.5 g/L dry solids in the test. At time "0", 16 ml synthetic sewage and the test item or reference compound were added to the test vessel and made up to 250 mL with chlorine-free tap water. After measurement and adjustment of pH, 250 mL of activated sludge (3 g/L dry solids) were added consequently obtaining a total volume of 500 ml with 1.5 g/L dry solids.
- Pretreatment: No
- Initial biomass concentration: 1.5 g/L dry solids

Test type:

static

Water media type:

other: Activated sewage sludge

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between 7 mg O2/L and 2 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.

Hardness:

not determined

Test temperature:

ca. 20 ºC

pH:

7.1- 7.8

Dissolved oxygen:

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.

Salinity:

n/a

Conductivity:

n/a

Nominal and measured concentrations:

10, 100 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flask/ BOD bottle
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: not reported
- Aeration: yes, 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.
- No. of vessels per concentration (replicates): 1 x 10 mg/L, 1 x 100 mg/L, 3 x 1000 mg/L
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): n/a
- No. of vessels per reference item control (replicates): 1 x 3.2 mg/L, 1 x 10 mg/L, 1 x 32 mg/L
- Sludge concentration (weight of dry solids per volume): 3.0 g/L in suspension and 1.5 g/L in test vessel
- Weight of dry solids per volume of reaction mixture per unit of time: not reported
- Nutrients provided for bacteria: On the day of collection, the activated sewage sludge (5 liters) was fed synthetic sewage (250 mL).
- Nitrification inhibitor used (delete if not applicable): none
- Biomass loading rate: not reported
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 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 (5 liters) was fed synthetic sewage (250 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 ºC 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. 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).
- Particulate matter: not reported
OTHER TEST CONDITIONS
- Adjustment of pH: Reference item stock solution was measured to be pH 6.1 and was adjusted to pH 7.1 using 1.0 M NaOH
- Photoperiod: shielded from light
- Light intensity: n/a
- Details on termination of incubation: As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between 7 mg O2/L and 2 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : O2 consumption measured after 3 h contact time.
TEST CONCENTRATIONS
- Spacing factor for test concentrations: as per OECD 209 (paragraph 40)
- Justification for using fewer concentrations than requested by guideline: n/a
- Range finding study
- Test concentrations: 10, 100, 1000 mg/L
- Results used to determine the conditions for the definitive study: no effect up to 1000 mg/L therefore definitive test not conducted.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorphenol

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 total respiration

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 total respiration

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not reported
- Effect concentrations exceeding solubility of substance in test medium: Not reported
- Adsorption (e.g. of test material to the walls of the test container): Not reported
- Blank controls oxygen uptake rate: The blank controls oxygen uptake rate was higher than 20 mg oxygen per one gram of activated sludge per hour in both tests
- Coefficient of variation of oxygen uptake rate in control replicates: The coefficient of variation (CV) of the blank respiration rates was less than 30% in both tests

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: The EC50 of the reference item was within the acceptable range of 2 to 25 mg/L
- Other: n/a

Reported statistics and error estimates:

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).

Table 1       Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time

 

Nominal Concentration (mg/L)		Initial O2 Reading (mg O2/L)	Measurement Period (minutes)	Final O2Reading (mg O2/L)	O2Consumption Rates (mg O2/L/hour)	% Inhibition
Control	R1	5.0	5	1.6	40.80	-
	R2	5.1	4	2.2	43.50	-
	R3	5.3	5	1.8	42.00	-
	R4	5.2	5	1.8	40.80	-
Test Item	10	5.4	5	1.9	42.00	[1]
	100	4.4	3	2.2	44.00	[5]
	1000 R1	4.3	3	2.2	42.00	[1]
	1000 R2	4.3	3	2.1	44.00	[5]
	1000 R3	4.3	3	2.2	42.00	[1]
3,5-dichlorophenol	3.2	6.7	10	2.2	27.00	35
	10	7.1	10	4.3	16.80	60
	32	8.1	10	7.3	4.80	89

values in parenthesis indicate an increase in respiration versus the control

Validity criteria fulfilled:

yes

Conclusions:

At a concentration of 1000 mg/L, no inhibition of the total respiration rate was observed. The resulting EC50 and NOEC values were > 1000 and 1000 mg/L, respectivey.

Executive summary:

OECD 209 (2017) - The inhibitory effects of N-(2-hydroxyethyl) dodecanamide on the respiration of activated sludge was assessed in an OECD 209 test.

The activated sludge (with sewage feed at a final concentration of 1.5 g/L dry solids) was exposed to the test item under aerobic conditions for a period of 3 hours, after which time the respiration rate of the microorganisms was measured (via oxygen consumption). Three test groups were prepared;

 

a) Blank control vessels (4) were prepared containing activated sludge and synthetic sewage feed.

b) Test item flasks (5) were prepared containing activated sludge, synthetic sewage feed and test item. The test item was added directly to the sludge to achieve nominal concentrations of 10 (1 flask), 100 (1 flask) and 1000 mg/L (3 flasks).

c) Reference item flasks (3) were prepared containing activated sludge, synthetic sewage feed and 3,5 -dichlorophenol, a known inhibitor of microbial respiration. The reference item was added directly to the sludge to achieve nominal concentration’s of 3.2, 10 and 32 mg/L (1 flask per concentration).

 

Upon preparation, the flasks were incubated aerobically for 3 h after which time the oxygen concentration in the solutions was measured for 3-5 minutes (10 minutes for low O₂consumption in reference item flasks). The respiration rate for each vessel was calculated and resulting inhibitory effects determined as a percentage versus the blank control vessels.

 

At a test concentration of 1000 mg/L, no inhibition of the total respiration rate was observed. The resulting EC₅₀and NOEC values were concluded to be > 1000 and 1000 mg/L, respectively.

 

The guideline validity criteria for the control and reference item vessels were satisfied. The study was therefore classified as acceptable and meets the requirement for Test Guideline OECD 209.

Description of key information

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

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

OECD 209 (2017) - The inhibitory effects of N-(2-hydroxyethyl) dodecanamide on the respiration of activated sludge was assessed in a single key study conducted in accordance with OECD 209. The activated sludge (with sewage feed at a final concentration of 1.5 g/L dry solids) was exposed to the test item under aerobic conditions for a period of 3 hours, after which time the respiration rate of the microorganisms was measured (via oxygen consumption). Three test groups were prepared;

 

a) Blank control vessels (4) were prepared containing activated sludge and synthetic sewage feed.

b) Test item flasks (5) were prepared containing activated sludge, synthetic sewage feed and test item. The test item was added directly to the sludge to achieve nominal concentrations of 10 (1 flask), 100 (1 flask) and 1000 mg/L (3 flasks).

c) Reference item flasks (3) were prepared containing activated sludge, synthetic sewage feed and 3,5 -dichlorophenol, a known inhibitor of microbial respiration. The reference item was added directly to the sludge to achieve nominal concentrations of 3.2, 10 and 32 mg/L (1 flask per concentration).

 

Upon preparation, the flasks were incubated aerobically for 3 h after which time the oxygen concentration in the solutions was measured for 3-5 minutes (10 minutes for low O₂consumption in reference item flasks). The respiration rate for each vessel was calculated and resulting inhibitory effects determined as a percentage versus the blank control vessels.

 

At a test concentration of 1000 mg/L, no inhibition of the total respiration rate was observed. The resulting EC₅₀and NOEC values were concluded to be > 1000 and 1000 mg/L, respectively.

 

The guideline validity criteria for the control and reference item vessels were satisfied. The study was therefore classified as acceptable and meets the requirement for Test Guideline OECD 209.