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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:
31 August 2018 (Study Initiation) to 19 March 2019 (Study Completion)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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 (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Nitika Pharmaceutical Specialities, Pvt. Ltd.
- Lot/batch No.of test material: IRST7H112A
- Expiration date of the lot/batch: May 2023
- Purity test (release) date: June 2018 (CoA)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (ambient), in original container as asuuplied by the Sponsor. Container kept tightly closed in a dry, cool and well ventilated place
- Stability under test conditions: Assumed stable for the duration of the test
- Solubility and stability of the test substance in the solvent/vehicle: As the test item was not soluble in any suitable solvent, the test item was mixed with OECD test media and administered as a nominal concentration filtrate.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

Analytical monitoring:
no
Details on sampling:
- Concentrations: 0 (Control) and 100 mg/L (Fatty acids, C16-18 (even numbered), iron(III) salts)
- Sampling method: Not applicable. As the test item was determined as highly insoluble in water and below the limit of analytical detection, dose formulations were administered as a nominal concentration filtrate without determination of homogeneity, concentration and stability.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: As the test item was determined as highly insoluble in water and below the limit of analytical detection, dose formulations were administered as a nominal concentration filtrate without determination of homogeneity, concentration and stability. As dose formulations were prepared by single dilution and administered within two hours of preparation, this exception does not impact the outcome of the study or the interpretation of the results.
- Control: OECD test media
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None

- Other relevant information: None
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture collection, 10801, University of Boulevard, manassas, Virginia, 20110-2209, USA
- Age of inoculum (at test initiation): 2 days
- Method of cultivation: Static condition

ACCLIMATION
- Acclimation period: The pre-culture was prepared two days prior to the commencement of the study by transferring 8 mL from the latest sub culture into a new culture vessel.
- Culturing media and conditions (same as test or not): The pre-culture was incubated under the same conditions as those required for the test and were used once growing exponentially.
- Any deformed or abnormal cells observed: No
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Remarks on exposure duration:
Standard 72 h test sytem exposure in accordence with OECD 201
Hardness:
Not specified
Test temperature:
21 - 23°C
pH:
7.25 - 8.01
Dissolved oxygen:
Not specified
Conductivity:
Not specified
Nominal and measured concentrations:
Prelim test nominal concentratuons: (0.0 - Control) 1.0, 10.0, 50.0 and 100 mg Fatty acids, C16-18 (even numbered), iron(III) salts/L
Main test nominal concntrations: 0.0 - Control) 100 mg Fatty acids, C16-18 (even numbered), iron(III) salts/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: Conducted in 250 mL conical flasks
- Type: closed
- Material, size, headspace, fill volume: 120 mL
- Aeration: no

- Initial cells density: 5917 cells/mL at 0 h
- Control end cells density: 916667 cells/mL at 72 h
- No. of vessels per concentration (replicates): Six replicates in the limit study
- No. of vessels per control (replicates): Six replicates in the limit study

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
The culture medium was prepared as per OECD 201.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: 7.25 - 8.01
- Photoperiod: Not specified
- Light intensity and quality: 6713 - 6727 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: manual counting using a haemocytometer and a light microscope

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not applicable, limit test performed
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: Prelim test nominal concentratuons: (0.0 - Control) 1.0, 10.0, 50.0 and 100 mg Fatty acids, C16-18 (even numbered), iron(III) salts/L
- Main test nominal concntrations: 0.0 - Control) 100 mg Fatty acids, C16-18 (even numbered), iron(III) salts/L

- Results used to determine the conditions for the definitive study:
The cell concentration in control cultures should increase exponentially by a factor of at least 16 within the three-day test period.
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in the test with Pseudokirchneriella subcapitata.
The mean coefficient of variation for days 0-1, 1-2, and 2-3 in control cultures must not exceed 35%.

Reference substance (positive control):
yes
Remarks:
Potassium Dichromate as OECD recommended positive control
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
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

- Other:
- Any stimulation of growth found in any treatment: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No, stock filtered in preparation of nominal test item concentrations.
- Effect concentrations exceeding solubility of substance in test medium: None
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50 value for growth rate inhibition: 1.15 mg/L
Reported statistics and error estimates:
Calculation of NOEC and LOEC
The No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) was determined by a suitable statistical procedure for multisample comparison (e.g., Analysis of Variance, Dunnett’s Test and Student’s t-test) using the individual replicates values of the areas under the growth curves (A) or the specific growth rate (µ) with computer software. Mean values, Standard deviation and Coefficient of variation values presented.
Validity criteria fulfilled:
yes
Conclusions:
The EC50, NOEC and LOEC were all greater than 100.0 mg/l Fatty acids, C16-18 (even numbered), iron(III) salts/L (nominal concentration).

Description of key information

The ErC50, NOECand LOEC were all greater than 100 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L

Additional information