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Toxicity to aquatic algae and cyanobacteria

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toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 Apr. 2018 to 24 May 2018
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
Adopted Mar. 2006, revised Jul. 2011
according to guideline
EU Method C.3 (Algal Inhibition test)
Version / remarks:
GLP compliance:
Specific details on test material used for the study:
- Lot number: 70900202
Analytical monitoring:
Details on sampling:
Samples of the test solutions were collected to measure concentrations of the test substance at 0 h and 72 h from the control and the treatment groups. At each sampling interval, approximately 30-35 mL of test solution was added to 50 mL plastic centrifuge tubes. The samples collected from the dimethylformamide stocks consisted of 5.0 mL of stock added to a 20mL glass scintillation vial.
Details on test solutions:
The primary stock solution was prepared by mixing 0.0100 g of the test substance in 100 mL of N,N-dimethylformamide (DMF), to achieve a nominal concentration of 100 μg/mL. The mass of test substance was weighed in a tared 30 mL glass beaker on an analytical balance and quantitatively transferred to a 100 mL glass volumetric flask. The 100 mL volumetric flask was brought to volume with DMF. The primary stock was inverted at least 20 times. The primary stock appeared clear and yellow-green in colour.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
- The freshwater alga, Raphidocelis subcapitata, was selected as the test species for this study. The species is representative of an important group of freshwater algae, and was selected for use in the test based upon a past history of use, and ease of culturing in the laboratory.
- Original algal cultures were obtained from the University of Texas at Austin, and have been maintained in culture medium at the test facility since June 2017.
- Algal cells used in this test were obtained from the test facility cultures that had been actively growing in culture medium under similar environmental conditions as used in this test for at least two weeks prior to test initiation. Algal cells for this study were taken from a culture that had been transferred to fresh medium four days prior to test initiation.
Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
Test temperature:
23.87 - 24.43 C
7.4 - 9.7
Nominal and measured concentrations:
Nominal test concentrations: 0 (control), 0.63, 1.3, 2.5, 5.0, 10 μg/L
Measured test concentrations: < LOD (control), < LOD, < LOD, 2.37, 9.93, 1.83, 10.4 μg/L.
Details on test conditions:
- Test vessel: Test chambers were sterile, 250-mL glass Erlenmeyer flasks plugged with sterile foam stoppers, and contained 100 mL of test or control medium.
- Initial cells density: approx. 8100 cells/mL
- Control end cells density: 2.44E+06 - 2.63E+06 cells/mL.
- No. of vessels per concentration: 3
- No. of vessels per control: 6
- No. of vessels per vehicle control: 6

- Standard medium used: yes (freshwater AAP medium)

- Sterile test conditions: Yes
- Adjustment of pH: The pH of the medium was adjusted to 7.5 ± 0.1 with 10% HCl, as necessary.
- Photoperiod: Continuous
- Light intensity and quality: Fluorescent lighting at an intensity of 6.198 ± 273 lux.

- Temperature, pH and light intensity.

EFFECT PARAMETERS MEASURED: cell density, cell morphology
- The toxicity of the test substance to the test organism was determined by evaluating changes in cell density over a 72-hour exposure period. One test medium sample was collected from each replicate of the treatment and control groups at each sampling interval for the determination of algal cell densities. Samples were collected at approximately 24-hour intervals during the 72-hour exposure and were held in the dark for a maximum of six days under refrigerated conditions sufficient to inhibit growth until cell counts could be performed. Prior to counting, the sample solutions were sonicated for 0.5 minutes and swirled to ensure the cells within each sample were homogeneously distributed. Cell counts were performed using a hemacytometer and microscope. A small amount of each sample was loaded onto a hemacytometer and 10 grids were counted. The mean number of cells per grid was calculated and this value was used to calculate the cell density of the sample. Using this technique, the minimum quantifiable cell density was 1,000 cells/mL. Samples were diluted with Isoton®, if necessary, to facilitate counting.

- At the end of the exposure period, samples of test solution were collected from each of the replicates per treatment and control group, pooled within their respective treatments, and subsamples were removed and examined microscopically for atypical cell morphology (e.g., changes in cell shape, size or color). Cells in the replicate test chambers also were assessed for aggregation or flocculation of cells, and adherence of the cells to the test chamber.
Reference substance (positive control):
Key result
72 h
Dose descriptor:
Effect conc.:
10 µg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
72 h
Dose descriptor:
Effect conc.:
> 10 µg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Test substance solubility: There was no evidence of surface slicks or precipitation at test initiation or at exposure termination.
- Cell density: after 72 hours of exposure, inhibition of cell density in the 6.3, 1.3, 2.5, 5.0, and 10 μg/L treatment groups was -16, -3, -11, -10, and -11%, respectively, relative to the negative control.
- Yield: inhibition of yield in the 6.3, 1.3, 2.5, 5.0, and 10 μg/L treatment groups was -16, -3, -11, -10, and -11%, respectively relative to the negative control.
- Goth rate: inhibition of growth rate in the 6.3, 1.3, 2.5, 5.0, and 10 μg/L treatment groups was -3, -1, -2, -2, and -2%, respectively, relative to the negative control.
- Appearance and morphology: After 72 hours of exposure, aggregation, flocculation, or adherence to the test chambers was not observed in the controls or in any treatment groups. There were no noticeable changes in cell morphology in any of the treatment groups when compared to the control replicates during the microscopic examinations of the cells.
Reported statistics and error estimates:
- The 72-hour cell density, growth rate, and yield data for the negative and solvent control groups were compared using a t-test (α = 0.05). The data from the two control groups were determined to be significantly different (p > 0.01). The mean responses of each treatment group were compared to the negative control response based on U.S. EPA guidance.
- EC50 values in this study represent the theoretical test concentrations that would produce a 50% reduction in a variable of interest relative to the pooled controls. EC50, ErC50, and EyC50 values and their corresponding 95% confidence intervals were calculated, when possible, using non-linear regression with replicate data (cell density, growth rate, and yield, respectively) and nominal test concentrations.
- The 72-hour cell density, growth rate, and yield data were evaluated for normality and homogeneity of variance (α = 0.01) using Shapiro-Wilk’s and Levene’s tests, respectively. All data met assumptions of normality and homogeneity of variance. The responses of the treatment groups were compared to the negative control response using Dunnett’s test (α = 0.05). The results of the statistical analyses of the cell density, growth rate, and yield data, as well as an evaluation of the concentration-response pattern, were used to determine the NOEC relative to each parameter at 72 hours.
Validity criteria fulfilled:
See 'Any other information on materials and methods incl. tables'.

Description of key information

72-h ErC50 > 10 µg/L (Pseudokirchneriella subcapitata)

72-h NOErC = 10 µg/L (Pseudokirchneriella subcapitata)

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
10 µg/L

Additional information

The toxicity towards freshwater algae was determined in a study according to OECD TG 201 and in compliance with GLP criteria (EAG, 2019). In this study, exponentially growing freshwater alga (Pseudokirchneriella subcapitata) were exposed to solutions of the test substance of 0, (control),0.63, 1.3, 2.5, 5.0, 10 μg/L (nominal concentrations) for 72 hours under static conditions. The test was performed in three replicates per test concentration and six for the control groups. The initial concentrations and the maintenance of the exposure concentrations during the test were analytically determined. Cell density was measured after 24, 48 and 72 hours exposure and the growth rate, yield and biomass per time point were determined. Throughout the test, no effects on the growth rate and yield were observed. Based on these findings, the 72-h EC50 values for growth rate and yield/biomass are determined to be >10 μg/L (highest concentration). The NOEC was also determined to be equal to 10 μg/L.