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

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:

19 January 2018 to 07 March 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:

17 Jul 1992

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

Commission Regulation No. 440/2008

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
- Sampling method: Water samples were taken from the control and 100 mg/L loading rate WAF test vessel at 0, 24, 48 and 72 hours from fresh media and at 24, 48, 72 and 96 hours from old media for quantitative analysis. Duplicate samples at 0 and 72 (fresh media) and 24 and 96 hours (old media) were taken and for further analysis if necessary.
- Sample storage conditions before analysis: The samples were stored frozen prior to analysis.

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and a proportion of the incoming water softened (Elga Nimbus 1248D Duplex water softener) and then remixed with the main water supply. After dechlorination and softening, the water is then passed through a series of computer controlled heat exchangers to acheive the required temperature.
- Eluate: N/A
- Differential loading: N/A
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):
- Evidence of undissolved material (e.g. precipitate, surface film, etc.):

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 18 December 2017.
- Age at study initiation (mean and range, SD): Not reported
- Length at study initiation (length definition, mean, range and SD): 6.1 cm (standard deviation = 0.42)
- Weight at study initiation (mean and range, SD): 1.9 g (standard deviation = 0.35)
- 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 20 February 2018 to 27 February 2018
- Acclimation conditions (same as test or not): Yes
- Type and amount of food during acclimation: The stock fish were fed commercial trout pellets
- Feeding frequency during acclimation: Not reported
- Health during acclimation (any mortality observed):

QUARANTINE (wild caught)
N/A

FEEDING DURING TEST
N/A - not fed during testl

Test type:

semi-static

Water media type:

freshwater

Remarks:

Dechlorinated laboratory tap water

Limit test:

yes

Total exposure duration:

96 h

Remarks on exposure duration:

Standard guideline exposure duration

Post exposure observation period:

None

Hardness:

140 mg/L as CaCO3

Test temperature:

13 C to 14 C

pH:

7.3 to 8.0

Dissolved oxygen:

8.8 to 10.6 mg/L

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

Limit test at 100 mg/L (nominal - water accommodated fraction)

Details on test conditions:

TEST SYSTEM
- Test vessel: 30 litre glass vessels
- Type (delete if not applicable): Covered to reduce evaporation
- Material, size, headspace, fill volume: Glass
- Aeration: Yes - via narrow bore glass tubes
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): A semi static test regime was employed in the test involving a daily renewal of the test preparations
- 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: 0.665 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: DLaboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener)
- Culture medium different from test medium: No
- Intervals of water quality measurement: 24 h (including fresh and expired test medium)

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light, 8 h dark
- 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, 24, 49, 73 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: Not applicable, limit test
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study
- Test concentrations: 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

not specified

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities:
- Observations on body length and weight: Not reported
- Other biological observations: No sub-lethal effects observed
- Mortality of control: No
- Other adverse effects control: No
- Abnormal responses: None reported
- 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 effects were seen up to 100 mg/L (WAF), which is in excess of the test item water solubility.

Reported statistics and error estimates:

Not applicable, no statistics required.

Sublethal observations / clinical signs:

Observations on Test Item Solubility

At the start of the mixing period the 100 mg/L loading rate was observed to be a clear colorless water column with test item floating at the surface of the water.  After 23 hours stirring and a one hour standing period the 100 mg/L loading rate was observed to be a very slightly hazy water column with test item floating at the surface and dispersed throughout.  As visual examination of the WAF showed there to be dispersed test item present it was considered justifiable to remove the WAF by filtering through a glass wool plug (2 to 4 cm in length) and filter paper.  Microscopic examination after filtering showed the glass wool plug and filter paper had removed all of the dispersed test item.  

During the test the control and 100 mg/L loading rate WAF were observed to be clear, colorless solutions.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) was investigated and gave a 96 Hour LL50 value of > 100 mg/L loading rate WAF.

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.

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range finding test, seven fish were exposed to a WAF of the test item, at a single nominal loading rate of 100 mg/L for a period of 96 hours at a temperature of 13 C to 14 ºC under semi static test conditions.  The number of mortalities and any sub lethal effects of exposure in each test and control vessel were determined one and three hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Chemical analysis of the test preparations at 0, 24, 72 and 96 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.072 mg/L.  This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of rainbow trout to the test item gave LL50 values of greater than 100 mg/L loading rate WAF.  The No Observed Effect Loading (NOEL) Rate was 100 mg/L loading rate WAF.

Description of key information

96 h LL50 = > 100 mg/L (WAF); OECD 203; Squance, 2018

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.

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range finding test, seven fish were exposed to a WAF of the test item, at a single nominal loading rate of 100 mg/L for a period of 96 hours at a temperature of 13 ºC to 14 ºC under semi static test conditions.  The number of mortalities and any sub lethal effects of exposure in each test and control vessel were determined one and three hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Chemical analysis of the test preparations at 0, 24, 72 and 96 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.072 mg/L.  This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of rainbow trout to the test item gave LL50 values of greater than 100 mg/L loading rate WAF.  The No Observed Effect Loading (NOEL) Rate was 100 mg/L loading rate WAF.