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

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 15 November 2013 and 06 December 2013.

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition 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%
- 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:

Samples were taken from the control and the 100 mg ai/L test group (replicates R1 - R6 pooled) at 0 and 72 hours for quantitative analysis. All 0-Hour samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours 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. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg ai/L for a period of 72 hours. All test concentrations were corrected for a test item water content of 48.62%.


Definitive Test:
Based on the result 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 effect on algal growth was observed.

Experimental Preparation:
An amount of test item (195 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg ai/L stock solution. This stock solution was inoculated with algal suspension (13.9 mL) to give the required test concentration of 100 mg ai/L.

The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium . The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 °C.

Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4 - 10^5 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

Not stated.

Test temperature:

24

pH:

7.3 - 7.5

Dissolved oxygen:

no information.

Salinity:

Not applicable, freshwater study.

Nominal and measured concentrations:

Range-finder:
Nominal: 0.10, 1.0, 10 and 100 mg ai/L
Measured: 0 and 72 hoursshowed measured test concentrations of 134% and 25% of nominal respectively.

Definitive:
Nominal: 100 mg ai/L
Measured: 0 h - 93% of nomial; 72 h - 15% of nominal

Details on test conditions:

Definitive Test:
Exposure Conditions:
250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and 100 mg ai/L treatment group.

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

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 3.61 x 10^5 cells per mL. Inoculation of 1 liter of test medium with 13.9 mL of this algal suspension gave an initial nominal cell density of 5 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.

The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Evaluations:
Test Organism Observations:
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle counter.

To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Water Quality Criteria:
The pH of the control and 100 mg ai/L test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded daily.

Data analysis:
-Comparison of growth rates
-Comparison of yield
-Determination of ECx Values
-Statistical Analtysis
-Geometric Mean Measured Concentrations: The geometric mean measured test concentrations of the samples were calculated using the measured test concentrations of replicates R1 - R6 pooled


Validation Criteria:

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

The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.

The mean of the coefficients of variation of the section by section daily growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) must not exceed 35%.

The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: mg ai/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 37 other: mg ai/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 other: mg ai/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

37 other: mg ai/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Range-Finding Test:
The results showed no effect on growth at the test concentrations of 0.10, 1.0, 10 and 100 mg ai/L.

Based on this information a single test concentration of six 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 effect on growth was observed.

Chemical analysis of the 100 mg ai/L test preparations at 0 and 72 hours showed measured test concentrations of 134% and 25% of nominal respectively were obtained indicating that the test item was unstable over the test period.

Definitive Test:
Verification of Test Concentrations:
Analysis of the test preparation at 0 hours showed a measured test concentration of 93% of nominal was obtained. A decline in measured test concentration was observed at 72 hours to 15% of nominal indicating that the test item was unstable over the test period.

It was therefore considered justifiable to base the results on the geometric mean measured test concentration in order to give a “worst case” analysis of the data. The geometric mean measured test concentration was determined to be:
Nominal Test Concentration (mg ai/L): 100
Geometric Mean Measured Test Concentration (mg ai/L): 37

Growth Data:
From the data it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test item at a geometric mean measured test concentration of 37 mg ai/L over the 72-Hour exposure period.

Accordingly the following results were determined from the data:

Inhibition of Growth Rate
ErC10 (0 - 72 h): >37 mg ai/L
ErC20 (0 - 72 h): >37 mg ai/L
ErC50 (0 - 72 h): >37 mg ai/L

where ErCx is the test concentration that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and 37 mg ai/L test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981). There were no statistically significant differences (P≥0.05), between the control and 37 mg ai/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 37 mg ai/L.

Inhibition of Yield
EyC10 (0 - 72 h): >37 mg/L
EyC20 (0 - 72 h): >37 mg/L
EyC50 (0 - 72 h): >37 mg/L

Where:

EyCx is the test concentration that reduced yield by x%.

There were no statistically significant differences (P≥0.05), between the control and 37 mg ai/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 37 mg ai/L.

Validation Criteria:
The following data show that the cell concentration of the control cultures increased by a factor of 149 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Mean cell density of control at 0 hours : 4.33 x 10^3 cells per mL
Mean cell density of control at 72 hours : 6.45 x 10^5 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 14% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Observations on Cultures:
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Water Quality Criteria:
Temperature was maintained at 24 ± 1 ºC throughout the test.

The pH value of the control cultures (see Table 2) was observed to increase from pH 7.5 at 0 hours to pH 7.8 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Observations on Test Item Solubility:
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control and test cultures were observed to be green dispersions.

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h): 1.2 mg/L; 95% confidence limits 1.0 – 1.3 mg/L
EyC50 (0 – 72 h): 0.52 mg/L; 95% confidence limits 0.45 – 0.62 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L

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

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and based on nominal test concentrations gave EC50 values of greater than 100 mg ai/L. The No Observed Effect Concentration was 100 mg ai/L.

Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 values based on the geometric mean measured test concentration of greater than 37 mg ai/L. The No Observed Effect Concentration was 37 mg ai/L.

Executive summary:

Introduction:

A study was perford to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Methods:

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at a concentration of 100 mg active ingredient (ai)/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

 

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^®Multisizer Particle Counter.

Results:

Exposure of Pseudokirchneriella subcapitata to the test item gave EC₅₀values based on nominal test concentrations 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.

 

Analysis of the 100 mg ai/L test preparation at 0 hours showed a measured test concentration of 93% of nominal was obtained. A decline in measured test concentration was observed at 72 hours to 15% of nominal indicating that the test item was unstable over the test period.

 

Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data.

 

Exposure of Pseudokirchneriella subcapitata to the test item gave EC₅₀values based on the geometric mean measured test concentrations of greater than 37 mg ai/L. The No Observed Effect Concentration was 37 mg ai/L.