Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-10-20 to 2007-06-12
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study performed according to OECD Guideline 201 (Alga, Growth Inhibition Test) and EU method C.3 (Algal Inhibition Test) without deviations.
Reason / purpose for cross-reference:
reference to other study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 201 (Alga, Growth Inhibition Test)
The testing facility indicated that the protocol was followed without deviation.
according to guideline
EU Method C.3 (Algal Inhibition test)
The testing facility indicated that the protocol was followed without deviation.
GLP compliance:
yes (incl. QA statement)
Certificate given by the Department of Health of the Government of the United Kingdom

Test material

Details on test material:
- Name of test material (as cited in study report): VRT-753136 hcl
- Molecular formula: Not applicable
- Molecular weight: Not applicable
- Smiles notation: Not applicable
- InChl: Not applicable
- Structural formula attached as image file: Not applicable
- Substance type: No data
- Physical state: White powder
- Analytical purity: 100 area %
- Impurities: 0.03 area% of unknowns
- Composition of test material, percentage of components: No data
- Isomers composition: No data
- Purity test date: 2006-09-15
- Batch number 25515
- Lot No. AF6-001
- Expiration date of the lot/batch: 2008-05
- Stability under test conditions: No data
- Storage condition of test material: Room temperature
- Other: No data
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No data

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Concentrations: All concentrations
- Sampling method: ). At the start of the definitive test, four samples (10 mL) were taken from flasks containing the freshly-prepared control and test medium; after 72 hours, the contents of the replicate flasks for each group were pooled and further samples taken for analysis. Additional samples were also taken from four flasks containing VRT-753136 hcl at 100 mg/L but with no algal cells, in order to obtain information on the extent of adsorption/absorption of the test substance by the algal cells.
- Sample storage conditions before analysis: On each occasion, two of the samples were analyzed immediately; the remaining samples were stored frozen. The liquid slope cultures were stored in an illuminated refrigerator.

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance (100 mg) was dissolved in algal culture medium (1000 mL) in a volumetric flask. The contents of the flask were shaken and used directly as the test medium. An aliquot (3.53 mL) of the secondary algal inoculum was added to a portion (900 mL) of the test medium, to give an initial cell density of 1 x 104 cells/mL. An aliquot (100 mL) of the inoculated test medium was added to each of the test vessels.
- Eluate: Not applicable
- Differential loading: Not applicable
- Controls: Sterile algal nutrient medium
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Common name: Unicellular green algae
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Axenic, uni-cellular algae were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Dunstaffnage Marine Laboratory, Dunbeg, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation): At least a three-day-old secondary culture
- Method of cultivation: Liquid slope culture; transferred every 3 days

- Acclimation period: At least three days
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: No

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
Post exposure observation period:

Test conditions

No data
Test temperature:
The temperature ranged from 21.4 to 21.8 deg C at time 0 and 22.4 to 22.5 deg C at time 72 h.
The temperature of the incubator ranged between 23.0 and 23.9 deg C.
The pH ranged from 7.51 to 7.72 at time 0 and 8.29 to 8.68 at time 72 h.
Dissolved oxygen:
Not applicable
Not applicable
Nominal and measured concentrations:
Nominal-Control concentration: 100 mg/L
Measured- 95.6 mg/L
The measured concentrations of VRT-753136 hcl in samples of the test cultures ranged between 94 and 98% of its nominal value during the test, giving an overall mean measured level of 95.6 mg/L.
Details on test conditions:
- Test vessel:
- Type (delete if not applicable): Closed- stoppered with foam bungs covered with aluminum foil that was secured by autoclave tape.
- Material, size, headspace, fill volume: 250 mL conical flasks, 100 mL media
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Incubated without renewal of meduim
- Initial cells density: Estimated to be 1.07 x 104/mL
- Control end cells density: No data
- No. of organisms per vessel: Not applicable
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable

- Standard medium used: Yes
- Detailed composition if non-standard medium was used: Not applicable

- Source/preparation of dilution water: Four stock solutions were prepared using filtered, dechlorinated tap water which had been softened and treated by reverse osmosis, before microfiltration and purification (resistivity of 18 Megohm/cm). Aliquots of stock solutions 1-3 were further diluted with the same diluent and autoclaved again to produce the working strength nutrient medium. Stock solution 4 was added to the medium on the day of use. The pH of the medium after equilibration with air is approximately 8.
- Total organic carbon: No data
- Particulate matter: No data
- Metals: No data
- Pesticides: No data
- Chlorine: No data
- Alkalinity: No data
- Ca/mg ratio: No data
- Conductivity: No data
- Culture medium different from test medium: No
- Intervals of water quality measurement: Not applicable

- Sterile test conditions: Yes; Stock solutions were sterilised by autoclaving (solutions 1-3) or by membrane filtration (solution 4) before being stored at 4 deg C in the dark.
- Adjustment of pH: Yes
- Photoperiod: Contiunous light
- Light intensity and quality: approximately 6142 Lux (Day 0: 6160 Lux; Day 1: 6240 Lux; Day2: 6160 Lux; Day 3: 6006 Lux)
- Salinity (for marine algae): Not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Beckman Coulter Counter
- Chlorophyll measurement: Not applicable
- Other: No

- Spacing factor for test concentrations: Not applicable since is a single limit test
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study
- Test concentrations: Nominal test concentrations of 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):

Results and discussion

Effect concentrationsopen allclose all
72 h
Dose descriptor:
other: EbC50
Effect conc.:
> 95.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
Basis for effect:
other: Area under the growth curve
72 h
Dose descriptor:
other: ErC50
Effect conc.:
> 95.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
Basis for effect:
growth rate
72 h
Dose descriptor:
Effect conc.:
95.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
Details on results:
- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): No microscopic abnormalities of the cells were detected in the test group.
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any stimulation of growth found in any treatment: No
- Any observations that might cause a difference between measured and nominal values: After 72 hours, analysis of samples of medium containing VRT-753136 hcl at 100 mg/L, which had been incubated without algal cells gave similar results to test media incubated in the presence of algal cells. These results indicate that the presence of algal cells did not affect the stability of the test substance.
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
The data were compiled in an Excel spreadsheet and analysed using SAS 8.2 (SAS Institute 1999). The area under the curve was divided by initial count and total time to give AUCP (Area Under the Curve expressed as a Proportion of the initial cell count), where a value of 1 represents no growth and a value of 0 represents complete toxicity (all algae killed). In order to estimate the concentration at which 50% inhibition of growth occurred (EC50), sigmoidal curves were fitted to AUCP and growth rate. For both variables, 0% inhibition was defined as the control mean and 100% inhibition was defined as no growth. The minimum of the curve (for infinite concentration) was bounded between 0 and 1 for AUCP and between -1000 and 0 for growth rate. For AUC and growth rate, Williams’ test (1971, 1972) was also used to compare the treated group with control unless there was evidence of a non-monotonic dose-response relationship, in which case Dunnett’s test (1955, 1964) was used.

Any other information on results incl. tables

Not applicable

Applicant's summary and conclusion

After 72 hours of exposure to VRT-753136 hcl, neither the EbC50 nor the ErC50 could be determined but both must be >95.6 mg/L.
The “no observed effect concentration” (NOEC) was 95.6 mg/L.
After 72 hours, no significant inhibition of algal growth had been observed.
Executive summary:

Not applicable