(2S)-[[(benzyloxy)carbonyl]amino](cyclohexyl)acetic acid

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

Study period:

2006-11-13 to 2006-12-05

Reliability:

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Remarks:

The testing facility indicated that the protocol was followed without deviation.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Remarks:

The testing facility indicated that the protocol was followed without deviation.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Department of Health of the Government of the United Kingdom

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): VRT-126016
- Molecular formula (if other than submission substance): Not applicable
- Molecular weight (if other than submission substance): Not applicable
- Smiles notation (if other than submission substance): Not applicable
- InChl (if other than submission substance): Not applicable
- Structural formula attached as image file (if other than submission substance): Not applicable
- Substance type: No data
- Physical state: White powder
- Analytical purity: 99.8 area %
- Impurities (identity and concentrations): No data
- Composition of test material, percentage of components: No data
- Isomers composition: No data
- Purity test date: No data
- Lot/batch No.: 25719
- Expiration date of the lot/batch: 2008-02-01
- 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):
Not applicable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentrations
- Sampling method: At the start of the test, 2 10 mL samples were taken from an additional flask containing freshly prepared control and test media; after 72 h the contents of the flasks for each group were pooled and further samples taken for analysis. Additional samples were taken from a flask containing test substance at 6.25 and 100 mg/L, but without algae to obtain information on the extent of adsorption/absorbtion of the test substance by the algal cells.
- Sample storage conditions before analysis: On each ocassion, one of the samples was immediately prepared for analysis and retained frozen until analysed, and the other was stored refrigerated in case further analysis was required.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test media were prepared from an aqueous dilution in which 200 mg of the test substance was added directly to 2 L of algal culture medium. This solution was treated with ultrasound for 40 min and stirred for approximatelt 3 h before being used directly, or diluted further for use. 3.2 mL of the secondary algal inoculum was added to 800 mL of the test medium, to give an initial cell density of 10,000 cells/mL. An aliquot of the appropriate inoculated test medium was added to each test vessel.
- Eluate: not applicable
- Differential loading: Not applicable
- Controls: Sterile algal nutrient test 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): None

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): The Culture Collection of Algae and Protazoa, SAMS Research Services Ltd., Dunstaffnage Marine Laboratory, Dunbeg, Argyll, Scotland.
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: axenic liquid slope cultures, tranferred at 3 days to secondary cultures.

ACCLIMATION
- Acclimation period: 3 days of growth of secondary culture
- Culturing media and conditions (same as test or not): Yes
- Any deformed or abnormal cells observed: No data

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Test conditions

Hardness:

No data

Test temperature:

22.7-23.3 deg C at 0h
22.5-23.3 deg C at 72h

pH:

7.13-7.66 at 0h
7.9-7.25 at 72h

Dissolved oxygen:

Not applicable

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal- Control, 6.25, 12.5, 25, 50, and 100 mg/L.
Measured- Control, 5.22, 10.3, 21.0, 44.0, and 95.9 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed-foam stoppers
- Material, size, headspace, fill volume: 250 mL conical flasks, 100 mL test solution
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): not applicable
- Initial cells density: 940 cells/mL estimate in each test flask
- Control end cells density:
- 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

GROWTH MEDIUM
- Standard medium used: Yes
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: No data
- 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 data
- Intervals of water quality measurement: No data

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH: No data
- Photoperiod: No data
- Light intensity and quality: 7250-7650 Lux
- Salinity (for marine algae): No data

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Beckman Coulter Counter
- Chlorophyll measurement: No Data
- Other: No data

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Range finding test
- Justification for using less concentrations than requested by guideline: Not Applicable
- Range finding study Yes
- Test concentrations: 1, 10 and 100 mg/kg
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

44 mg/L

Nominal / measured:

meas. (not specified)

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 95.9 mg/L

Nominal / measured:

meas. (not specified)

Basis for effect:

growth rate

Remarks on result:

other: 24% inhibition

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 95.9 mg/L

Nominal / measured:

meas. (not specified)

Basis for effect:

growth rate

Remarks on result:

other: 6% inhibition

Details on results:

- Exponential growth in the control (for algal test): yes/no
- Observation of abnormalities (for algal test): No microscopic the abnormalities of the cells were observed.
- Unusual cell shape: Not applicable
- Colour differences: Not applicable
- Flocculation: No data
- Adherence to test vessels: No data
- Aggregation of algal cells: No data
- Other: No data
- Any stimulation of growth found in any treatment: No data
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No data
- Effect concentrations exceeding solubility of substance in test medium: No data

Results with reference substance (positive control):

Not applicable

Any other information on results incl. tables

Not applicable

Applicant's summary and conclusion

Validity criteria fulfilled:

not applicable

Conclusions:

After 72h of exposure to VRT-126016, the
Growth curve EC50 >95.9 mg/L (24% inhibition); Growth rate ECr50 > 95.9 mg/L (6% inhibition); NOEC = 44mg/L

Executive summary:

Not applicable