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Ecotoxicological information

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)
GLP compliance:
yes
Analytical monitoring:
not specified
Details on sampling:
- Concentrations in mg/L: 0 (Blank control), 0.095, 0.305, 0.977, 3.13, and 10.0
- Sampling method: no data
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
A primary stock solution with a nominal concentration of 100 mg/L the test substance was prepared by dissolving approximately 10 mg of the test substance in 100 mL of filter-sterilized AAP nutrient medium. A secondary stock solution with a nominal concentration of 10 mg/L the test substance was prepared by diluting a 25 mL aliquot of the primary stock solution with filter-sterilized AAP nutrient medium to a final volume of 250 mL. Test solutions were prepared by adding the appropriate volume of the secondary stock solution to filter-sterilized AAP nutrient medium to make nominal concentrations of 0.095, 0.305, 0.977, and 3.13 mg/L the test substance, and stirring each for approximately 5 minutes. Aliquots of the secondary stock solution were used for the nominal 10.0 mg/L test concentration solution. Aliquots of the filter-sterilized AAP nutrient medium were used for the blank control solution. The blank control and test substance solutions were clear and colourless with no visible precipitate at test start.

AAP NUTRIENT MEDIUM PREPARATION:
To prepare one litre of AAP nutrient medium, 1 mL of each of the 6 macronutrient stock solutions and 1 mL of the micronutrient stock solution are added to approximately 800 mL of Milli-Q® (deionised) water with mixing after each addition. The final volume of the medium was brought to 1 litre with additional Milli-Q® water. The larger volume required for the definitive and recovery tests was prepared based on these proportions. The nutrient medium was adjusted to a pH of 7.43 and filter-sterilized through 0.22-μm cellulose acetate filters into sterile containers. The containers with the resulting filter-sterilized AAP nutrient medium were stored in the refrigerator in the dark at approximately 4°C and acclimated to ambient temperature prior to use.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: freshwater, unicellular, non-motile, green alga
- Age of inoculum (at test initiation): no data

ACCLIMATION PERIOD: 4 days
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
not reported
Test temperature:
23 ± 2°C
pH:
7.71 - 8.04
Dissolved oxygen:
not reported
Nominal and measured concentrations:
0, 0.095, 0.305, 0.977, 3.13, and 10.0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: sterilized 250-mL Erlenmeyer flasks containing approximately 50 mL of test solution. Flasks were fitted with sterilized foam stoppers to permit gas exchange.
- Initial cells density: 10000 cell/mL
- Control end cells density: 10000 mg/L
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: no data
- Light intensity and quality: 6340 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: haemocytometer and a compound microscope
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
other: EbC50
Effect conc.:
9.44 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 95% CL 2.43-16.4 mg/L
Duration:
72 h
Dose descriptor:
other: EyC50
Effect conc.:
9.41 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
(density)
Remarks on result:
other: 95% CL 2.40-16.4 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate; EC50 is actually an ErC50 value
Remarks on result:
other: EC50 is actually an ErC50 value
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none reported
Reported statistics and error estimates:
Analyses are reported based on nominal test substance concentrations and were conducted using SAS Version 8.2. The data for cell count, cell count yield, and growth rate based on cell count were determined to be non-normally distributed (Shapiro-Wilk test). Replicate 2 of the blank control and replicate 1 of the 0.305 mg/L test concentration were determined to be outliers by the Tukey outlier rule, and were excluded from the calculation of means and standard deviations and from the statistical analysis. After deletion of outliers, data for cell count, cell count yield, and growth rate based on cell count were determined to be normally distributed (Shapiro-Wilk test) with equal variances (Levene’s test). The Dunnett test was used to determine the LOEC and NOEC values from the data without outliers. All statistical tests were calculated at a significance level of p = 0.05. The EbC50, EyC50, and ErC50 values (and 95% confidence intervals) for cell count, cell count yield, and growth rate based on cell count were obtained by the OECD Model 2 inverse regression model.
Validity criteria fulfilled:
yes
Conclusions:
72-hour EbC50 = 9.44 mg/L
72-hour ErC50 = > 10.0 mg/L
Executive summary:

The toxicity of the test substance to the green algae, Pseudokirchneriella subcapitata, was determined in a 72-hour, static toxicity test. The study was conducted with a blank control and five nominal concentrations of the test substance at a mean lighting intensity of 6340 lux (range of 6030 to 6590 lux), a mean air temperature of 23°C, a liquid temperature range of 23°C, and a shaking speed of 102 rpm. Synthetic algal-assay-procedure (AAP) nutrient medium was used as the test diluent and blank (culture medium) control. Test solutions were not renewed. Three replicates were used per test concentration and six replicates were used for the blank control. Cell counts and growth rates were determined at 24-hour intervals over the 72-hour test.

Inhibition of growth expressed as cell count (density) of Pseudokirchneriella subcapitata exposed to nominal concentrations of 0.095, 0.305, 0.977, 3.13, and 10.0 mg/L the test substance for 72 hours was -8, -18, -15, -16, and 73%, respectively. Inhibition of cell density yield was -8, -19, -15, -16, and 74%, respectively. Inhibition of growth expressed as the average specific growth rate was -1, -3, -3, -3, and 25%, respectively.a Cell density increased in the blank control by at least a factor of 16 in 72 hours, the coefficient of variation of average specific growth rates during the whole test period (0-72 hour) in the blank control replicates did not exceed 7%, and the mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2, and 2-3) in the blank control replicates did not exceed 35%, thereby satisfying the appropriate test acceptance criteria. Replicate 2 of the blank control and replicate 1 of the 0.305 mg/L test concentration were determined to be outliers by the Tukey outlier rule, and were excluded from the calculation of means and standard deviations and from the statistical analysis. Nominal concentrations of the test substance were used for calculation of the EbC50 (72-hour), EyC50 (72-hour), ErC50 (72-hour), LOEC, and NOEC values.b The most sensitive parameter was cell density yield with a 72-hour EC50 of 9.41 mg/L.

 

aNegative values of inhibition indicate stimulation of growth.

bThe EbC50 (72-hour) is defined as the effect concentration producing a 50% inhibition of growth based on the 72-hour cell count (density) relative to the control. The EyC50 (72-hour) is defined as the effect concentration producing a 50% inhibition of growth based on the 72-hour cell count yield. The ErC50 (72-hour) is defined as the effect concentration producing a 50% inhibition of growth based on the 0-72 hour growth rate relative to the control. The LOEC is defined as the lowest concentration of test substance that had a significant effect on the measured parameter relative to the control. The NOEC is defined as the highest concentration of test substance that had no significant effect on the measured parameter relative to the control.

Description of key information

72-hour ErC50 > 10 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
10 mg/L

Additional information