N-(2-hydroxyethyl)dodecanamide

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 November 2017 to 04 January 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 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
- Sampling method: Water samples were taken from the control and all surviving test groups at 0 and 72 hours from fresh media and at 24 and 96 hours from old media for quantitative analysis. Duplicate samples and samples at 24 hours (fresh media), 48 hours (old and fresh media) and 72 hours (old media) were taken for further analysis if necessary.
- 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: Nominal amounts of test item (2200, 2200 and 1100 mg) was dispersed in 22, 22 and 11 liters of test water respectively 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 filter (first approximate 1 liter discarded in order to pre-condition the filter). The replicate solutions were pooled to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test solutions of 10, 18, 32 and 56% v/v saturated solution. Each test solution was mixed with a flat bladed stirrer for 1 minute to ensure adequate mixing and homogeneity.
- Eluate: Laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener)
- Differential loading: No
- Controls: Yes, test water only
- 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.): The 0.65, 1.2, 2.0 and 3.5 mg/L test preparations were observed to be clear colourless solutions throughout the test. The 9.8 mg/L test preparation was observed to be a slightly white cloudy homogenous dispersion with a slight oily slick at the surface.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Strain: Not reported
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in-house since 12 September 2017
- Age at study initiation (mean and range, SD): Not reported
- Length at study termination (length definition, mean, range and SD): Mean standard length of 5.7 cm (standard deviation = 0.3)
- Weight at study termination (mean and range, SD): Mean body weight of 1.62 g (standard deviation = 0.28)
- Method of breeding: Not applicable
- Maintenance of the brood fish: Fish were maintained in a glass fiber tank with a "single pass" water renewal system.

ACCLIMATION
- Acclimation period: Fish were acclimatized to test conditions from 04 December 2017 to 11 December 2017.
- Acclimation conditions (same as test or not): Yes
- Type and amount of food during acclimation: The stock fish were fed commercial trout pellets
- Health during acclimation (any mortality observed): None reported

QUARANTINE (wild caught)
Not applicable

FEEDING DURING TEST
Not applicable

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

Standard guideline exposure duration

Hardness:

140 mg/L as CaCO3

Test temperature:

140 mg/L as CaCO3

pH:

6.8 - 7.8

Dissolved oxygen:

9.8 - 11.3 mg O2/L

Salinity:

Not applicable

Conductivity:

Not applicable

Nominal and measured concentrations:

Nominal Concentrations: 10, 18, 32, 56 and 100 % (v/v) of saturated solution
Geometric Mean Measured Concentrations: 0.65, 1.2, 2.0, 3.5 and 9.8 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Exposure Vessels
- Type (delete if not applicable): open (but covered to reduce evaporation)
- Material, size, headspace, fill volume: 25 to 30 L, filled with 20 L of test media
- Aeration: Not reported
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Daily (semi-static)
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: 0.57 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- 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 mortalities and sub-lethal effects of exposure were recorded at 1, 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Justification for using less concentrations than requested by guideline: 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):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

5.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % CL = 3.5 - 9.8

Details on results:

- Behavioural abnormalities:
- Observations on body length and weight: None
- Other biological observations: Sub-lethal effects of exposure were observed at test concentrations of 3.5 mg/L and above. These responses were sitting on the bottom of the tank and moribund. After approximately 6 and 48 hours exposure, 2 out of 7 and 5 out of 5 fish respectively at 9.8 mg/L were observed to be moribund. Due to animal welfare implications (The Animals (Scientific Procedures) Act 1986) these fish were killed and classed as mortalities for the following observational time point.
- Mortality of control: None
- Other adverse effects control: 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

Reported statistics and error estimates:

The 0.65, 1.2, 2.0 and 3.5 mg/L test preparations were observed to be clear colourless solutions throughout the test. The 9.8 mg/L test preparation was observed to be a slightly white cloudy homogenous dispersion with a slight oily slick at the surface.

Sublethal observations / clinical signs:

Table 1. Cumulative Mortality in the Definitive Test

	Cumulative mortality (initial population = 7)						Mortality (%)
Geometric mean measured concentration (mg/L)	3 h	6 h	24 h	48 h	72 h	96 h	96 h
Control	0	0	0	0	0	0	0
0.65	0	0	0	0	0	0	0
1.2	0	0	0	0	0	0	0
2.0	0	0	0	0	0	0	0
3.5	0	0	0	0	0	0	0
9.8	0	2	2	7	7	7	100

Media Preparation Trial

An 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 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
24 Hours Filtered ~ 2 liters discarded	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

It is evident from these results 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 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 (first approximate 1 liter discarded) to give a nominal test concentration of approximately 11 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of the study, the 96 h LC50 of the test item to rainbow trout (Oncorhyncus mykiss) was 5.9 mg/L (95 % CL = 3.5 - 9.8). The effect level ia based on geometric mean measured concentrations.

Executive summary:

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 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, fish were exposed, in groups of 7, to an aqueous solution of the test item at nominal concentrations of 10, 18, 32, 56 and 100% v/v saturated solution for a period of 96 hours at a temperature of 14 °C to 15 °C under semi-static test conditions. The test item solution was 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 (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. A series of dilutions were made from the 100% v/v saturated solution to give further test concentrations of 56, 32, 18 and 10% v/v saturated solution. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 1, 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Analysis of the freshly prepared test media at 0 and 72 hours showed measured test concentrations to range from 0.68 to 8.5 mg/L. Analysis of the old or expired test media at 24 and 96 hours showed measured test concentrations to range from 0.53 to 11 mg/L. Although an increase in measured concentration was seen in the 100% v/v saturated solution, the majority of all other test concentrations over each 24 hour sampling period showed a decline in measured concentration and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations only in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be 0.65, 1.2, 2.0, 3.5 and 9.8 mg/L.

Based on the results of the study, the 96 h LC50 of the test item to rainbow trout (Oncorhyncus mykiss) was 5.9 mg/L (95 % CL = 3.5 - 9.8).  The effect level ia based on geometric mean measured concentrations.

Description of key information

96 h LC50 = 5.9 mg/L (95 % CL: 3.5 - 9.8 mg/L); Rainbow trout (Oncorhynchus mykiss); OECD 203; Anon., 2018

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

5.9 mg/L

Additional information

A single key study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 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, fish were exposed, in groups of 7, to an aqueous solution of the test item at nominal concentrations of 10, 18, 32, 56 and 100% v/v saturated solution for a period of 96 hours at a temperature of 14 °C to 15 °C under semi-static test conditions. The test item solution was 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 (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. A series of dilutions were made from the 100% v/v saturated solution to give further test concentrations of 56, 32, 18 and 10% v/v saturated solution. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 1, 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Analysis of the freshly prepared test media at 0 and 72 hours showed measured test concentrations to range from 0.68 to 8.5 mg/L. Analysis of the old or expired test media at 24 and 96 hours showed measured test concentrations to range from 0.53 to 11 mg/L. Although an increase in measured concentration was seen in the 100% v/v saturated solution, the majority of all other test concentrations over each 24 hour sampling period showed a decline in measured concentration and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations only in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be 0.65, 1.2, 2.0, 3.5 and 9.8 mg/L.

Based on the results of the study, the 96 h LC50 of the test item to rainbow trout (Oncorhyncus mykiss) was 5.9 mg/L (95 % CL = 3.5 - 9.8).  The effect level is based on geometric mean measured concentrations.