1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-coco acyl derivs., hydroxides, inner salts

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 February, 2018 - 10 August, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch: DSP-001.
- Expiration date of the lot: 15 January 2019.
- Purity test date: 1 May 2018.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilutions: 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L.

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours, from additional media ran alongside at 24 and 48 hours, and from the pooled replicates at 72 hours for immediate quantitative analysis.

Details on test solutions:

A nominal amount of test item (50 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 5 minutes and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0, 0.32, 0.10, 0.032 and 0.010 mg/L.
An aliquot (700 mL) of each of the 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L stock solutions was separately inoculated with algal suspension (4.5 mL) to give the required test concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Name: Pseudokirchneriella subcapitata.
- Strain: CCAP 278/4.
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C.

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 ±1 ºC

pH:

7.2-7.7

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flask, plugged with polyurethane foam bungs.
- Initial cells density: 5.00 x 10^3 cells per mL.
- Control end cells density: 1.70 x 10^6 cells per mL.
- No. of vessels per concentration (replicates): 3.
- No. of vessels per control (replicates): 3.

GROWTH MEDIUM
- Standard medium used: The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 with 0.1N NaOH or HCl.

OTHER TEST CONDITIONS
- Adjustment of pH: None.
- Photoperiod: Continuous illumination (intensity approximately 7000 lux).
- Light intensity and quality: Warm white lighting (380 to 730 nm).

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: One order of magnitude.
- Range finding study: 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/L for a period of 72 hours. Due to issues with the analytical methodology, the results obtained from the analysis of the range-finding test preparations were inconclusive and as such not reported. A preliminary stability trial was therefore conducted in order to determine the stability of the test item under the conditions of the test.

Reference substance (positive control):

yes

Remarks:

A positive control (Envigo study number RD71YQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.32 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.045 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.061 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.046 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.033 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

cell number

Details on results:

- Exponential growth in the control (for algal test): Cell concentration of the control cultures increased by a factor of 340 after 72 hours.
- Observation of abnormalities (for algal test): After 72 hours there were no abnormalities detected in the control or test cultures at 0.010, 0.032 and 0.10 mg/L, however cell debris was observed to be present in the test cultures at 0.32 and 1.0 mg/L.
- Colour differences: After the 72-Hour test period all control and 0.010, 0.032 and 0.10 mg/L test cultures were green dispersions. The 0.32 mg/L test replicates were observed to be either pale green or green dispersions whilst the 1.0 mg/L test replicates were observed to be clear colorless solutions, extremely pale green and pale green dispersions.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- ErC50 (72 hour): 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L.
- Other: EyC50 (72 hour): 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L.

Reported statistics and error estimates:

It was not possible to calculate 95% confidence limits for the test item ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.
EyC50 95% confidence limits: 0.043 - 0.049 mg/L.

Validity criteria fulfilled:

yes

Remarks:

All validity crtieria (increase in cell concentration of the control cultures, mean coefficient of variation of section by section and average specific growth rates for control cultures) were met.

Conclusions:

Based on the geometric mean measured test concentrations over the 72 hour exposure period, EC50 growth rate and yield for the test item were determined to be 0.32 and 0.046 mg/L, respectively. A ErC10 growth rate (72 hour) was also determined as 0.061 mg/L.

Executive summary:

A GLP compliant algal growth inhibition test was performed on the freshwater unicellular green alga Pseudokirchneriella subcapitata and the substance, in accordance with OECD Test Guideline 201.

Following an initial range-finding test, the test concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L were applied in the definitive test.

The test results are considered valid as the performance criteria for the control cultures were met and there were no deviations from the study plan.

A decline in measured test concentrations was observed at intervals of 24, 48 and 72 hours, therefore the geometric mean measured concentrations were used to calculate EC50 values.

The ErC50 (72 hour) was determined to be 0.32 mg/L and the EyC50 (72 hour) was 0.046 mg/L.

Growth rate NOEC and LOEC were determined as 0.045 and 0.29 mg/L, respectively.

Yield NOEC and LOEC were determined as 0.033 and 0.045 mg/L, respectively.

A growth rate ErC10 (72 hour) was also determined as 0.061 mg/L from the data based on geometric mean measured test concentrations - this value is used for classification purposes.

Based on these results, the test substance should be classified as Aquatic Acute 1; H400 in accordance with CLP Regulation (EC) 1272/2008, Annex I, Table 4.1.0, (a) and Aquatic Chronic 2; H411 in accordance with CLP Regulation (EC) 1272/2008, Annex I, Table 4.1.0, (b)(ii) as the substance is readily biodegradable.

Description of key information

A GLP compliant algal growth inhibition test was performed on the freshwater unicellular green alga Pseudokirchneriella subcapitata and the test item, in accordance with OECD Test Guideline 201.

The ErC50 (72 hour) was determined to be 0.32 mg/L and the EyC50 (72 hour) was 0.046 mg/L.

Growth rate NOEC and LOEC were determined as 0.045 and 0.29 mg/L, respectively.

Yield NOEC and LOEC were determined as 0.033 and 0.045 mg/L, respectively.

A growth rate ErC10 (72 hour) was also determined as 0.061 mg/L from the data based on geometric mean measured test concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.32 mg/L

EC10 or NOEC for freshwater algae:

0.061 mg/L