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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
Study period:
2000-05-30 to 2000-06-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor and R90076
- Expiration date of the lot/batch: 2000-06-31

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark.
- Solubility and stability of the test substance in the solvent/vehicle: The predictied solubilty is up to 75 ug/L

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dissolution into a stock solution of DMF to a concentration of 1.0 mg/ml
- Final dilution of a dissolved solid, stock liquid or gel: The stock solution was addded to dilutent water

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Nominal Concentration: 100 ug test substance/L test solution
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100 ug/L
- Sampling method: At study intiation and after 72 hours, dupliace medium samples of 200 ml were taken from the solvent control and test sotck solutions for analysis. The samples were not filtered to remove algal cells before analysis. Additional samples were also taken from flasks containing the "no algae" cultures at 72 hours with replicates pooled.
- Sample storage conditions before analysis: Not specified
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance was dissolved in dimethylformamide (DMF) to give a primary stock solution with a nominal concentration of 10 mg/mL. This solution was then further diluted with DMF to give a working stock solution of 1.0 mg/mL. An 0.2 mL aliquot of the working stock solution was added to diluent water to give a nominal test concentration of I 00 µg/L. This provided an excess of test material in diluent water, and was stirred with a magnetic stirrer for approximately 30 minutes to give a saturated solution.
- Controls: A negative control and solvent control were used.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Unicellular green algae
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Algal cultures were obtained from the Culture Collection of Algae & Protozoa, Institute of Freshwater Ecology, Cumbria, UK.
- Age of inoculum (at test initiation): Old enough to be in log phase growth
- Method of cultivation: Continuous illumination (6000 - 10000 lux) in an orbital incubator at 23 ± 2°C

ACCLIMATION
- Acclimation period: None
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
Not specified
Test temperature:
23
pH:
7.6-8.2 over the duration of the test.
Dissolved oxygen:
Not specified
Salinity:
Not specified
Conductivity:
Not specified
Nominal and measured concentrations:
Nominal Concentrations: 100 ug/L
Measured Concentrations: 90 ug/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Conical flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Glass, 250 ml, 100 ml, 150 ml
- Aeration: No
- Initial cells density: ~10,970 cells/ml
- Control end cells density: ~1,500,000 cells/ml
- No. of organisms per vessel: ~1.5*10^8 cells
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Elendt M4 medium prepared using analytical grade reagents and deionized water.
NAME mg/L
1. Trace elements:
H3BO3 2.86
MnCl2.4H2O 0.36
LiCl 0.31
RbCI 0.071
SrCl2.6H2O 0.152
NaBr 0.016
Na2MoO4.2H2O 0.063
CuCl2.2H2O 0.017
ZnCl2 0.013
CoCl2.6H2O 0.010
KI 0.0033
Na2.SeO3 0.0022
NH4VO3 0.00058
Fe-EDTA solution 3.50
2. Macro nutrients:
CaCl,.2H2O 294
MgSO4.7H2O 123
KCl 5.80
NaHCO3 64.8
3. Buffer nutrients:
Na2.SiO3.9H2O 10.0
NaNO3 0.274
KH2PO4 0.143
K2HPO4 0.184
4. Vitamins:
Thiamine hydrochloride 0.075
Cyanocobalamine (Vitamin Bl2) 0.0010
Biotin (Vitamin H) 0.00075
The above analytical grade reagents are dissolved in reverse osmosis deionized water.
- Culture medium different from test medium: No
- Intervals of water quality measurement: Immediately prior to testing

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: Not necessary
- Photoperiod: Constant lighting was applied
- Light intensity and quality: 7000-8000 lux using fluorescent tubes

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter Multisizer II particle counter


TEST CONCENTRATIONS
- Spacing factor for test concentrations: The maximum soluble concentration was tested.
- Justification for using less concentrations than requested by guideline: The protocol states that a limit test will be performed at either: (i) 100-150 mg a.i./1, (ii) a concentration equal to the limit of solubility of the test substance or (iii) the maximum concentration forming a stable dispersion. However, the limit test for this study was conducted at a concentration greater than the limit of solubility of the test substance to ensure saturation of test medium. This method was approved by the HSA.
The protocol states that a range finding test will be conducted before the definitive test. An initial range finding test was performed, using a different preparation method to that subsequently used in the definitive test. Following the decision to perform the study at a concentration greater than the limit of solubility, the range finding was not repeated, because the test compound was expected to have little, if any, toxicity. Therefore the definitive test was conducted immediately as a limit test.

RANGE-FINDING STUDY
- Test concentrations: Not specified
- Results used to determine the conditions for the definitive study: A preliminary study was conducted to determine the solubilty of the test substance in water. The study found that the solubility of the test substance in water was maximized by using a cofactor solvent. The maximum solubility that was able to be achieved with this method was ~61 ug/L, so a nominal concentration of 100 ug/L was chosen to ensure the solution was saturated.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 90 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 90 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): None
- Effect concentrations exceeding solubility of substance in test medium: Mean measured concentrations at 0 and 72 hours remained above the estimated limit of solubility of BMS 233101-01 in algal medium; 61 µg/L.
Results with reference substance (positive control):
No reference substance was used.
Validity criteria fulfilled:
yes
Conclusions:
This study validly assessed the EC50 and NOEC of the test substance according to the specified guideline.

Description of key information

BMS 233101-01 did not inhibit the growth of Selenastrum capricornutum, Strain No. CCAP 278/4 at the limit of solubility in algal medium under the conditions of this test (mean measured concentration of 90 µg/L).The EC50. (72 hours) and the EyC50 (0 - 72 hours) were greater than the limit of solubility of BMS 233101-01 in algal medium (mean measured concentration of 90 µg/L).

Key value for chemical safety assessment

EC50 for freshwater algae:
90 µg/L

Additional information