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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: 2-Propenoic acid, 2-[[(octadecylamino)carbonyl]oxy]ethyl ester
Physical state/Appearance: White solid
Purity: >98%
Expiry Date: 20 June 2017
Storage Conditions: Room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
The samples were stored frozen prior to analysis.

The test samples were thawed with the aid of a waterbath, and then diluted with tetrahydrofuran
Vehicle:
no
Details on test solutions:
A nominal amount of test item (1100 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm filter (first approximate 1 liter discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give stock solutions of 1.0, 3.2, 10, 32% v/v saturated solution. An aliquot (1000 mL) of each of the stock solutions was separately inoculated with 7.0 mL of algal suspension to give the required test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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).

Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.3). The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 2C.

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 104 - 105 cells/mL.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
Temperature was maintained at 24 ± 1 ºC throughout the test.
pH:
Please see pH table in other information on materual and method section (below).
Nominal and measured concentrations:
Following a preliminary range-finding test and initial experiments, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution.
Details on test conditions:
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.

Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions

A nominal amount of test item (1100 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm filter (first approximate 1 liter discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give stock solutions of 1.0, 3.2, 10, 32% v/v saturated solution. An aliquot (1000 mL) of each of the stock solutions was separately inoculated with 7.0 mL of algal suspension to give the required test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution.

The stock solutions and each of the prepared concentrations 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

Six flasks each containing 100 mL of solution were used for the control and three flasks each containing 100 mL were used for each 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 7.13 x 105 cells per mL. Inoculation of 1000 mL of test media with 7.0 mL of this algal suspension gave an initial nominal cell density of 5.00 x 103 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.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: % v/v SS
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: It was not possible to calculate an ErC50 value as no more than 50% inhibition of growth occurred at the maximum attainable test concentration of 100% v/v saturated solution
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
32 other: % v/v SS
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
100 other: % v/v SS
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
ErC10 (0 - 72 h): 86% v/v saturated solution
ErC20 (0 - 72 h): 97% v/v saturated solution
ErC50 (0 - 72 h): >100% v/v saturated solution

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

It was not possible to calculate an ErC50 value as no more than 50% inhibition of growth occurred at the maximum attainable test concentration of 100% v/v saturated solution.

All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 1.0, 3.2 and 10% v/v saturated solution. In the 32% v/v saturated solution test group, some slightly enlarged/misshapen cells were observed, and in the 100% v/v saturated solution test group some enlarged cells/cells clumped together were observed to be present.

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, 1.0, 3.2, 10 and 32% v/v saturated solution test cultures were observed to be pale green dispersions, whilst the 100% v/v saturated solution test cultures were observed to be extremely pale green dispersions.
Reported statistics and error estimates:
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences between the control and 1.0, 3.2, 10, 32% v/v saturated solution test concentration (P0.05), however the 100% v/v saturated solution test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 32% v/v saturated solution. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 100% v/v saturated solution.

Range Finding Results

The results showed no effect on growth at the test concentrations of 0.10, 1.0 and 10% v/v saturated solution.  However, growth was observed to be reduced at 100% v/v saturated solution.

Based on this information test concentrations of 10, 18, 32, 56, and 100% v/v saturated solution were selected for the initial experiment.

Chemical analysis of the test preparations at 0 and 72 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 0.018 mg/L. This does not infer that no test item was in solution, just that any dissolved test item present was at a concentration of less than the LOQ.

Validity criteria fulfilled:
yes
Conclusions:
The effect of 2-Propenoic acid, 2-[[(octadecylamino)carbonyl]oxy]ethyl ester on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on the nominal test concentrations gave the following results:

Growth rate:
EC50 (% v/v Saturated Solution): >100*
No Observed Effect Concentration (NOEC) (% v/v Saturated Solution): 32
Lowest Observed Effect Concentration (LOEC) (% v/v Saturated Solution): 100

Yield:
EC50 (% v/v Saturated Solution): 93
No Observed Effect Concentration (NOEC) (% v/v Saturated Solution): 32
Lowest Observed Effect Concentration (LOEC) (% v/v Saturated Solution): 100

Description of key information

In an OECD 201 study, under GLP conditions, the 72 hour EC50 (growth) of 2-Propenoic acid, 2-[[(octadecylamino)carbonyl]oxy]ethyl ester is >100* % v/v Saturated Solution. The NOEC is 32 % v/v Saturated Solution (Envigo 2016g).

 

* It was not possible to calculate an ErC50 value as no more than 50% inhibition of growth occurred at the maximum attainable test concentration of 100% v/v saturated solution

Key value for chemical safety assessment

EC50 for freshwater algae:
0.018 mg/L

Additional information