N-(2-hydroxyethyl)dodecanamide

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 November 2017 to 15 February 2018

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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
- Sampling method: Samples were taken from the control and each test group from the freshly prepared bulk test preparation at 0 and 24 hours and from the old or expired pooled replicates at 24 and 48 hours for quantitative analysis. Duplicate samples were taken at 0, 24 and 48 hours.
- Sample storage conditions before analysis: All samples were stored frozen prior to analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A nominal amount of test item (1100 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 1 liter discarded in order to pre condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 10, 18, 32 and 56% v/v saturated solution.
- Eluate: ELENDT M7 Medium
- Differential loading: No
- Controls: Test medium 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.): At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia
- Strain/clone: Not reported
- Justification for species other than prescribed by test guideline: Not applicable
- Source: In house culture
- Age of parental stock (mean and range, SD): Not reported
- Feeding during test
- Food type: Not applicable
- Amount: Not applicable
- Frequency: Not applicable

ACCLIMATION
- Acclimation period: In house culture
- Acclimation conditions (same as test or not): Yes
- Type and amount of food: Each culture was fed with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension.
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): Culture conditions ensured that reproduction was by parthenogenesis. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

QUARANTINE (wild caught)
- Duration: Not applicable
- Health/mortality: Not applicable

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

Standard guideline exposure duration

Hardness:

Not reported

Test temperature:

20 - 21

pH:

7.8 - 7.9

Dissolved oxygen:

8.5 - 9.2

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal Concentrations: 10, 18, 32 and 56% v/v saturated solution
Geometric Mean Measured Concentrations: 0.69, 0.81, 2.2, 2.9 and 7.1 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass jars
- Type (delete if not applicable): open (covered to limit evaporation)
- Material, size, headspace, fill volume: Glass, 150 mL, filled with 100 mL of test medium
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Semi static test conditions were employed in the test in an effort to maintain dissolved test item concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24 Hour old test media into the fresh test media. Concentrations at which 100% immobilization was observed after 24 hours exposure were not renewed.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: Not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Not reported
- Culture medium different from test medium: No
- Intervals of water quality measurement: Daily

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Light intensity: Not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilized if they were unable to swim within 15 seconds after gentle agitation.

VEHICLE CONTROL PERFORMED: Not applicable

RANGE-FINDING STUDY
- Test concentrations: 1, 10 and 100 % (v/v) saturated solution
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

4.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % CL not determinable

Details on results:

- Behavioural abnormalities:
- Observations on body length and weight: Not assessed
- Other biological observations: None
- Mortality of control: No
- Other adverse effects control: None
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid?
- Relevant effect levels: 24 and 48 h EC50
- Limit test: No
- Dose-response test: Yes
- ECx: 24 h EC50 = 0.79 mg/L (95 % CL: 0.73 - 0.86 mg/L); 48 h EC50 = 0.75 mg/L (95 % CL = 0.56 - 1.0 mg/L)
- Other: The results from the positive control with potassium dichromate were within the normal range for this reference item.

Reported statistics and error estimates:

The EC50 values and associated confidence limits at 0 and 48 hours and the slope of the response curve and its standard error were calculated by logit analysis using Linear Maximum Likelihood regression. The Lowest Observed Effect Concentration and the NOEC at 24 and 48 hours were calculated using the Fisher’s Exact Binomial Test with Bonferroni correction. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Table 1. Cumulative Immobilisation and Observations in the Definitive Test

	24 Hours
	Cumulative Immobilised Daphnids (Initial Population = 5)						Observations
Geometric Mean Measured Concentration (mg/L)	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.69	0	0	1	0	1	5	5 N	5 N	4 N	4 N
0.81	0	0	0	0	0	0	5 N	5 N	5 N	5 N
2.2	0	0	0	0	0	0	5 N	5 N	5 N	5 N
2.9	0	0	0	0	0	0	5 N	5 N	5 N	5 N
7.1	5	5	5	5	20	100	AI	AI	AI	AI

	48 Hours
	Cumulative Immobilised Daphnids (Initial Population = 5)						Observations
Geometric Mean Measured Concentration (mg/L)	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.69	0	0	1	0	1	5	5 N	5 N	4 N	5 N
0.81	0	0	0	0	0	0	5 N	5 N	5 N	5 N
2.2	0	0	0	0	0	0	5 N	5 N	5 N	5 N
2.9	0	0	0	1	1	5	5 N	5 N	5 N	4 N
7.1	5	5	5	5	20	100	AI	AI	AI	AI

R = Replicate

N = Normal

AI - All daphnia immobilized

Preliminary Medium Preparaion Trial

Saturated Solution Preparation

A nominal amount of test item (550 mg) was dispersed, in duplicate, in 11 liters of deionized reverse osmosis water with the aid of propeller stirring at approximately 1500 rpm for periods of either 24 or 48 hours.  After stirring samples were taken for chemical analysis after the following pre-treatments:

- Centrifugation at 10000 g for 30 minutes

- Centrifugation at 40000 g for 30 minutes

- Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 1 liter discarded in order to pre condition the filter)

- Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 2 liters discarded in order to pre condition the filter)

Results

Stirring Period and Treatment	Concentration Found (mg/L)
24 Hours Centrifuged 10000 g	10.4
24 Hours Centrifuged 40000 g	10.3
24 Hours Filtered ~ 1 liter discarded	10.7
24 Hours Filtered ~ 2 liters discarded	9.89
48 Hours Centrifuged 10000 g	11.2
48 Hours Centrifuged 40000 g	10.1
48 Hours Filtered ~ 1 liter discarded	9.59
48 Hours Filtered ~ 2 liters discarded	9.77

The results obtained indicated that there was no significant increase in the dissolved test item concentration obtained when the preparation period was extended beyond 24 hours.  Furthermore, there were no significant differences between the pre-treatment types.

Based on this information the test item was prepared using a saturated solution method of preparation at an initial loading rate of 100 mg/L, stirred for a period of 24 hours prior to the removal of any undissolved test item by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 1 liter discarded) to give a nominal test concentration of approximately 11 mg/L.  

Validity criteria fulfilled:

yes

Conclusions:

The 48 h EC50 of the test item to Daphnia Magna was 4.2 mg/L (95 % CL not determinable).

Executive summary:

A study was performed to assess the acute toxicity of the test item to Daphnia magna.  The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 11 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

Following a preliminary range finding test and initial experiment, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 10, 18, 32, 56 and 100% v/v saturated solution for 48 hours at a temperature of 20 C to 21 C under semi-static test conditions.  The test item solutions were prepared by stirring an excess (100 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours.  After the stirring period any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 1 liter discarded in order to pre condition the filter) to produce a 100% v/v saturated solution of the test item.  This saturated solution was then further diluted as necessary, to provide the remaining test concentrations.  Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the fresh test preparations at 0 and 24 hour showed measured test concentrations to range from 0.81 to 7.4 mg/L.  Analysis of the aged or expired test preparations at 24 and 48 hours showed measured test concentrations had declined, to between 0.25 and 6.9 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data.  The geometric mean measured test concentrations were determined to be 0.69, 0.81, 2.2, 2.9 and 7.1 mg/L.

The 48 h EC50 of the test item to Daphnia Magna was 4.2 mg/L (95 % CL not determinable).

Description of key information

48 h EC50 = 4.2 mg/L; Daphnia magna, OECD 202; Squance. 2018

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

4.2 mg/L

Additional information

A single key study was performed to assess the acute toxicity of the test item to Daphnia magna.  The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 11 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

Following a preliminary range finding test and initial experiment, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 10, 18, 32, 56 and 100% v/v saturated solution for 48 hours at a temperature of 20 ^oC to 21 ^oC under semi-static test conditions.  The test item solutions were prepared by stirring an excess (100 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours.  After the stirring period any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 1 liter discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item.  This saturated solution was then further diluted as necessary, to provide the remaining test concentrations.  Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the fresh test preparations at 0 and 24 hour showed measured test concentrations to range from 0.81 to 7.4 mg/L.  Analysis of the aged or expired test preparations at 24 and 48 hours showed measured test concentrations had declined, to between 0.25 and 6.9 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data.  The geometric mean measured test concentrations were determined to be 0.69, 0.81, 2.2, 2.9 and 7.1 mg/L.

The 48 h EC50 of the test item to Daphnia Magna was 4.2 mg/L (95 % CL not determinable).