3-(N-p-methoxy cinnamidopropyl-N,N-dimethyl ammonium)-2-hydroxypropane-1-sulphonate

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 09 October 2013 and 07 January 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxy-3-({3-[(2E)-3-(4-methoxyphenyl)prop-2-enamido]propyl}dimethylazaniumyl)propane-1-sulfonate

EC Number:

695-243-8

Cas Number:

500731-87-3

Molecular formula:

C18H28N2O6S

IUPAC Name:

2-hydroxy-3-({3-[(2E)-3-(4-methoxyphenyl)prop-2-enamido]propyl}dimethylazaniumyl)propane-1-sulfonate

Test material form:

other: liquid

Details on test material:

- Name of test material: Galaxy SunBeat
- Physical state: extremely pale yellow liquid
- Analytical purity: 51.38% solid content (i.e., 48.62% water content)
- Lot/batch No.: 07
- Expiration date of the lot/batch: 01 January 2015
- Storage condition of test material: room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control and the 100 mg ai/L test group (replicates R1 - R4 pooled) at 0 and 48 hours for quantitative analysis. Samples were stored frozen prior to analysis.

Duplicate samples were taken and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

Range-finding test:
The test concentration to be used in the definitive test was determined by a preliminary range-finding test.

In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg ai/L. All concentrations were corrected for a test item water content of 48.62%.

An amount of test item (195 mg) was dissolved in test water with the aid of ultrasonication for approximately 6 minutes and the volume adjusted to 1 liter to give the 100 mg ai/L test concentration (corrected for 48.62% water content) from which a series of dilutions was made to give the remaining test concentrations of 0.10, 1.0 and 10 mg ai/L.

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

Definitive test:
Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100 mg ai/L to confirm that at the maximum concentration given in the OECD/EC Test Guidelines, no immobilization or adverse reactions to exposure were observed.

Experimental Preparation:
An amount of test item (195 mg) was dissolved in test water and the volume adjusted to 1 liter to give the 100 mg ai/L test concentration.

The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

The concentration and stability of the test item in the test preparation were verified by chemical analysis at 0 and 48 hours

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.

Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. 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. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

Not recorded.

Test temperature:

20 - 21 Deg C

pH:

8

Dissolved oxygen:

8.6 - 9 mg O2/L

Salinity:

Not applicable, freshwater study.

Nominal and measured concentrations:

Definitve Test:
Nominal concentration: 100 mg ai/L
Measured concentration: 90% to 93% of nominal

Details on test conditions:

Exposure Conditions:
250 mL glass jars containing approximately 200 mL of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at approximately 20 °C with a photoperiod of 16 hours light (955 to 996 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

The control group was maintained under identical conditions but not exposed to the test item.

The test preparations were not renewed during the exposure period.

Evaluations:
Test Organism Observations:
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 for approximately 15 seconds after gentle agitation.

Water Quality Criteria:
Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

Statistical Analysis:
An estimate of the EC50 values was given by inspection of the immobilization data.

Validation Criteria:
The results of the test are considered valid if the following performance criteria are met:

•No more than 10% of the control daphnids show immobilization or other signs of disease or stress (e.g. discoloration or unusual behavior such as trapping at the surface water).

•The dissolved oxygen concentration at the end of the test is ≥3 mg/L in the control and test vessels.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: mg ai/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 other: mg ai/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Range-Finding Results:
No immobilization was observed at the test concentrations of 0.10, 1.0, 10 and 100 mg ai/L.

Chemical analysis of the 100 mg ai/L test preparation at 0 and 48 hours showed measured concentrations of 92 and 89 mg/L respectively indicating that the test item was stable under test conditions.

Based on this information, a single test concentration of four replicates, of 100 mg ai/L was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the maximum test concentration given in the OECD/EC Test Guidelines, no immobilization or adverse reactions to exposure were observed.

Definitive Test:
Verification of Test Concentrations
Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 90% to 93% of nominal value and so it was considered justifiable to estimate the EC50 values in terms of the nominal test concentrations only.

Immobilization Data:
There was no immobilization in 20 daphnids exposed to a test concentration of 100 mg ai/L for a period of 48 hours. Inspection of the immobilization data gave the following results:

Time (h) EC50 (mg ai/L)
24 >100
48 >100

The No Observed Effect Concentration after 24 and 48 hours exposure was 100 mg ai/L.

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

Validation Criteria:
The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was ≥3 mg/L in the control and test vessels.

Water Quality Criteria:
Temperature was maintained at approximately 20 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Observations on Test Item Solubility
The 100 mg ai/L test preparation was observed to be a clear colorless solution for the duration of the test.

Results with reference substance (positive control):

A positive control 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 by the trimmed Spearman-Karber method (Hamilton et al 1977 ) at 24 and 48 hours based on the nominal test concentrations gave the following results:
24 h EC50: 1.0
48 h EC50: 0.71
24 h NOEC 0.56
48 h NOEC: 0.32

The No Observed Effect Concentration is based upon zero immobilization at this concentration.

The results from the positive control with potassium dichromate were within the normal range for this reference item.

Applicant's summary and conclusion

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 EC50 value of greater than 100 mg ai/L. The No Observed Effect Concentration was 100 mg ai/L.

Executive summary:

Introduction:

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.

Methods:

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a concentration of 100 mg active ingredient (ai)/L for 48 hours at a temperature of approximately 20°C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results:

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 90% to 93% of nominal value and so the results are based on nominal test concentrations only.

Exposure of Daphnia magna to the test item gave EC₅₀ values of greater than 100 mg ai/L. The No Observed Effect Concentration was 100 mg ai/L.

 

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