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

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:

19 January 2018 to 04 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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

Commission Regulation (EC) No. 440/2008

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
- Sampling method: Samples were taken from the control and 100 mg/L loading rate WAF test group from the freshly prepared bulk test preparation at 0 and 24 hours and from the old or expired pooled replicates (R1 to R4) at 24 and 48 hours for quantitative analysis.
- 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: Water accommodated fraction (WAF)
- Eluate: ELENDT M7 medium
- Differential loading: Yes
- Controls: Yes, Elendt M7 medium 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.): Microscopic observations of the WAF were performed after filtering and showed no test item.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Waterflea
- Strain/clone: Straus (defined by supplier as clone 5)
- Justification for species other than prescribed by test guideline: N/A
- Source: University of Sheffield (UK), bred at Envigo Research Limited
- Age of parental stock (mean and range, SD):
- Feeding during test: None
- Food type:
- Amount:
- Frequency:

ACCLIMATION
- Acclimation period:
- Acclimation conditions (same as test or not):
- Type and amount of food:
- Feeding frequency:
- Health during acclimation (any mortality observed):

QUARANTINE (wild caught) N/A

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

Guideline standard exposure duration

Hardness:

Not measured

Test temperature:

18 - 22 °C

pH:

7.9 - 8.4

Dissolved oxygen:

7.8 - 8.9

Salinity:

Not measured

Conductivity:

Not measured

Nominal and measured concentrations:

Nominal loading rates (WAF): 100 mg/L (limit test)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: Glass
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel:
- No. of vessels per concentration (replicates):
- No. of vessels per control (replicates):
- No. of vessels per vehicle control (replicates):
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Salinity:
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: 16 h light : 8 h dark
- Light intensity:

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

VEHICLE CONTROL PERFORMED: yes/no

RANGE-FINDING STUDY
- Test concentrations:
- Results used to determine the conditions for the definitive study:TEST SYSTEM
- Test vessel: 150 mL glass beaker
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 150 mL glass beaker, containing 100 mL test solution
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): esmi-static, renewal after 24 h
- 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:
ELENDT M7 medium, prepared according to OECD 202 guideline. The pH of the prepared media was 7.9 ±0.3 and stored at approximately 21 ºC.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light; 8 h dark (with 20 minute dusk and dawn transition periods)
- Light intensity: 200 to 1200 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

VEHICLE CONTROL PERFORMED: No

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):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

water accommodated fraction

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: None
- Observations on body length and weight: Not applicable
- Other biological observations: None
- Mortality of control: No
- Other adverse effects control: No
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not applicable (WAF)
- 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.

Results with reference substance (positive control):

- Results with reference substance valid?
- Relevant effect levels: EC50
- Limit test: No
- Dose-response test: Yes
- ECx: 24 h EC50 = 0.79 mg/L; 48 h EC50 = 0.75 mg/L
- Other:
A positive control (Envigo study number LK67NP) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Exposure conditions for the positive control were similar to those in the definitive test.
Analysis of the immobilization data was carried out using the Binomial Distribution method at 24 hours and the Trimmed Spearman-Karber method at 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations. The results from the positive control with potassium dichromate were within the normal range for this reference item.

Reported statistics and error estimates:

Not applicable, no statistics performed in this study.

Chemical Analysis of Test Loading Rates

Chemical analysis of the test preparations at 0, 24 and 48 hours showed measured test concentrations of less than the LOQ of the analytical method employed were obtained which was determined to be 0.019 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48 Hour EL50 value of greater than 100 mg/L loading rate WAF.

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.

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, 20 daphnids (4 replicates of 5 animals) were exposed to a WAF of the test item at a nominal loading rate of 100 mg/L for 48 hours at a temperature of 20 C to 22 C under semi‑static test conditions.  Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the test preparations at 0, 24 and 48 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 0.019 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 Daphnia magna to the test item gave EL50 values of greater than 100 mg/L loading rate WAF.  The No Observed Effect Loading (NOEL) rate was 100 mg/L loading rate WAF.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Description of key information

48 h EL50 = > 100 mg/L; OECD 202; Squance, 2018

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.

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, 20 daphnids (4 replicates of 5 animals) were exposed to a WAF of the test item at a nominal loading rate of 100 mg/L for 48 hours at a temperature of 20 C to 22 C under semi‑static test conditions.  Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the test preparations at 0, 24 and 48 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 0.019 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 Daphnia magna to the test item gave EL50 values of greater than 100 mg/L loading rate WAF.  The No Observed Effect Loading (NOEL) rate was 100 mg/L loading rate WAF.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.