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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 January 2008 - 30 January 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
March 23, 2006
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
December 1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
(2S)-2-(2-oxo-4R-propylpyrrolidin-1-yl)butanamide
Cas Number:
357336-20-0
IUPAC Name:
(2S)-2-(2-oxo-4R-propylpyrrolidin-1-yl)butanamide
Test material form:
solid: particulate/powder
Details on test material:
- Description: White to off-white powder
- Storage conditions: At room temperature protected from light

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: all test concentrations and the control, at t=0, t=24h and t=48h (1.5 mL)
- Sample storage conditions before analysis: in a freezer at the analytical laboratory of the Test Facility
- At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Preparation of test solutions started with a loading rate of 100 mg/L applying vigorous shaking to accelerate the dissolving of the test substance in the test medium. The lower test concentrations were prepared by subsequent dilutions of the loading rate in test medium. The final test solutions were all clear and colourless.
- After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: in-house laboratory culture
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (M1) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
- Age of inoculum (at test initiation): Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2) at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Study design

Test type:
static
Water media type:
freshwater
Total exposure duration:
48 h

Test conditions

Hardness:
24 mg CaCO3/L
Test temperature:
21.8-22.4°C
pH:
start: 8.2-8.3
end: 7.9
Nominal and measured concentrations:
- Nominal concentrations: 0.10, 1.0, 10 and 100 mg/L (combined limit/range-finding test)
- Measured concentrations: only samples taken from the control and the highest concentration, at t=0, t=24h and t=48h, were analysed. At test start and throughout the test the measured concentrations of the 100 mg/L test solution were at the level of nominal (98-113%). Therefore, the effect parameters were expressed based on the analytically confirmed nominal concentration.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Aeration: no
- Initial cells density: 1 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 6 replicates of the highest (limit) concentration; 3 replicates of the lower concentrations
- No. of vessels per control (replicates): 6

MEDIUM
- Standard medium used: yes (M2; according to OECD 201)

OTHER TEST CONDITIONS
- pH measurements: at the beginning and at the end of the test
- Temperature measurements: continuously in a temperature control vessel
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuously
- Light intensity and quality: TLD-lamps with a light intensity within the range of 83 to 87 µE.m-2.s-1.
- Incubation: vessels were distributed at random in the incubator. During incubation the algal cells were kept in suspension by continuous shaking.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): cell density (t=0, 24h and 48h)
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank. One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval.
- Quantification of cell densities was based on a calibration curve. Cell density was plotted versus extinction using spectrophotometric measurements of a minimum of six dilutions of an algal suspension with different cell densities. The calibration curve was composed using linear regression. The software automatically calculates the cell densities based on this curve for the spectrophotometric measurements at the various points in time during the test period.
- Other: Appearance of the cells: at the end of the final test microscopic observations were performed to verify a normal and healthy appearance of the inoculum culture and to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
Combined limit/range-finding test: 0.1, 1.0, 10 and 100 mg/L
Based on the results, a definitive study was not required.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (performed January 2008) (72h exposure)

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Nominal test concentration confirmed by analysis.
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Nominal test concentration confirmed by analysis.
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (unusual cell shape, colour differences, flocculation, adherence to test vessels, aggregation of algal cells): Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed algal cells when compared to the control.
- Other: No significant differences were recorded between the values for growth rate or yield at any of the test concentrations when compared to the control group.
- Water parameters (pH and temperature) remained within the limits prescribed by the test guidelines.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 72h-ErC50 = 1.0 mg/L (growth rate inhibition) and 72h-EyC50 = 0.43 mg/L (yield inhibition)
- These obtained results clearly show the sensitivity of the test system used by the test laboratory

Any other information on results incl. tables

Acceptability of the test:

- In the controls, cell density increased by an average factor of at least 16 during the whole test period of 2 days (54).

- The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (15%).

- The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (3%).

In addition:

- Constant test conditions (pH and temperature) were maintained throughout the study period.

- The concentration of the substance was satisfactorily maintained throughout the study period.

The study met all validity criteria and is therefore considered valid.

In accordance with the guideline, the test period may be shortened to at least 48 hours, provided that the validity criteria are met.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The 48h-ErC50 of the substance to the freshwater algae Pseudokirchneriella subcapitata was determined to be >100 mg/L, based on analytically confirmed nominal test concentrations (48h-NOErC: 100 mg/L).
Executive summary:

In a study performed in accordance with OECD 201 (2006) and according to GLP principles, the toxicity of the substance to the freshwater algae Pseudokirchneriella subcapitata was investigated.

In a combined limit/range-finding test, algae were exposed for 48 hours to an untreated control and nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L (control and 100 mg/L - 6 replicates; 0.10, 1.0 and 10 mg/L - 3 replicates).

As the substance was completely soluble in test medium up to 100 mg/L, preparation of test solutions started with a loading rate of 100 mg/L applying vigorous shaking to accelerate the dissolving of the test substance in the test medium. The lower test concentrations were prepared by subsequent dilutions of the loading rate in test medium. The final test solutions were all clear and colourless.

Samples for analytical confirmation of actual exposure concentrations were taken at t = 0, 24 and 48 hours, but only the control and the highest concentration samples from these sampling times were analysed. The measured concentration in the highest test concentration was found to be at the level of the nominal concentration throughout the test (98-113% of nominal). Since the test concentration remained stable during the test, the effect parameters were reported in terms of the analytically confirmed nominal concentration.

No significant differences in cell densities, and thus growth rate inhibition rates and yield inhibition, were recorded between the test concentrations and the control group. Therefore, the 48h-ErC50 of the substance to Pseudokirchneriella subcapitata is concluded to be >100 mg/L, based on analytically confirmed nominal test concentrations. The 48h-NOErC value is 100 mg/L.

The study met the OECD validity criteria.

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