Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 10, 18, 32, 56 and 100 mg/L
- Sampling method: Measured at 0 and 72 hours using HPLC.
- Sample storage conditions before analysis: Not specified.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

A primary stock solution of the test substance with a nominal concentration of 100 mg/L was prepared. This stock was then stirred for 5 minutes and then ultrasonicated for 10 minutes after which it was observed to be a clear and colourless solution. this stock was then used directly as the 100 mg/L test solution and was serially diluted in culture medium to provide the other test solutions. All test solutions and the culture medium control were observed to be clear and colourless solutions.

The appropriate test solution (100 mL) was then dispensed to each test and blank vessel.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Sourced from laboratory cultures maintained under axenic conditions.
- Age of inoculum (at test initiation): 3 day old cuture
- Method of cultivation: Not specified

ACCLIMATION
- Acclimation period: Not specified
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: Not specified

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
Not specified
Test temperature:
22 +/- 2°C
pH:
pH values at 0 hours and 72 hours
Control: 7.1 to 7.5.
10 mg/L: 6.6 to 7.3
18 mg/L: 6.2 to 7.5
32 mg/L: 5.9 (6.9 after adjustment) to 7.5
56 mg/L: 5.5 (7.0 after adjustment) to 7.4
100 mg/L: 5.2 (7.0 after adjustment) to 7.4
Dissolved oxygen:
Not specified
Salinity:
Not specified
Conductivity:
Not specified
Nominal and measured concentrations:
Nominal: 0, 10, 18, 32, 56 and 100 mg/L.
Measured: 0, 10, 18, 32 57 and 100 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass conical flasks
- Type (delete if not applicable): open (stoppered with foam bungs)
- Material, size, headspace, fill volume: 250 mL volume filled with 100 mL of solutions
- Aeration: No
- Initial cells density: 5000 cells/mL
- Control end cells density: 634000 cells/mL
- No. of organisms per vessel: NA
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): NA

GROWTH MEDIUM
- Standard medium used: yes as per OECD guidelines

TEST MEDIUM / WATER PARAMETERS
- Standard

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH: Yes
- Photoperiod: Continuous
- Light intensity and quality: 6910 +/- 7% Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Cell density used to determine growth rate and yield with microscopic evaluation also performed.
- Determination of cell concentrations: Coulter counter counting at a lower threshold equivalent spherical diameter of approximately 2.3 µm.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Standard as per guidelines.
- Justification for using less concentrations than requested by guideline: NA
- Range finding study: Not specified
Reference substance (positive control):
not specified

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
56 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
56 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): None
- Unusual cell shape: None
- Colour differences: None
- Flocculation: None
- Adherence to test vessels: None
- Aggregation of algal cells: None
- Any stimulation of growth found in any treatment: Yes at 32 mg/L (104% of control)
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: None
Results with reference substance (positive control):
Not specified
Reported statistics and error estimates:
The data for cell counts obtained at 24, 48 and 72 hours was entered into electronic data files and statistically analysed using an in-house software package. Appropriate procedures were applied to test for significant differences (p < 0.05) between the control and test concentrations. Where possible, the EC50, EC20 and EC10 values and teh associated 95% confidence intervals were determined using the US EPA program ICPIN.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Validity criteria with regards to controls met
Conclusions:
Under the conditions of the study the 72 hour EC50 values for growth rate and yield were higher than the upper concentration tested (100 mg/L). Analytical verification was conducted providing near nominal results on all occassions.