t-Butyl (2S,2aS,5aR)-1-azabicyclo[3.3.0] oct-2-carboxylate monooxalate salt

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:

2006-10-26 to 2007-03-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed according to OECD guideline 201 (Algal, Growth Inhibition Test) and EC 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:

Certificate from the 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-126028
- Molecular formula (if other than submission substance): C14H23NO6
- Molecular weight (if other than submission substance): 301.34
- 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: 99.3% area GC
- 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.: 25414
- 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):
Not applicable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentrations
- Sampling method: At the start of the definitive test, two samples (10 mL) were taken from the freshly-prepared control and test media; 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 a flask containing VRT-126028 at 6.25 and 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, one of the samples was analyzed immediately and the other was stored in a freezer 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: Dissolved directly into the medium
- Eluate: Not applicable
- Differential loading: Not applicable
- Controls: Sterile culture 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: Not applicable
- Strain: No. CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Dunstaffnage Marine Laboratory, Dunbeg, Oban, Argyll, Scotland and arrived on 13, February 2007
- 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
- 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:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

no data

Test temperature:

The temperature of the incubator ranged between 22.7 and 23.3 deg C.

pH:

The pH ranged from 7.18 to 9.01.

Dissolved oxygen:

Not applicable

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations: 6.25, 12.5, 25, 50, and 100 mg/L
Mean measured concentrations: 5.31, 10.1, 21.0, 39.9, and 82.6 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed- stoppered with foam plugs
- 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): not applicable
- Initial cells density: 10,000 cells/mL
- Control end cells density: 1959973 cells/mL
- 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: not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated tap water which had been softened and treated by reverse osmosis, before microfiltration and purification
- 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: 18 Megohn/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: Not applicable

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: Yes
- Photoperiod: Continuous light
- Light intensity and quality: 6161 lux generated by fluorescent tubes
- Salinity (for marine algae): Not applicable

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

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Spacing factor 2
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study
- Test concentrations: 1, 10, and 100 mg/L
- 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:

other: EbC50 (Area under growth curve)

Effect conc.:

30.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% confidence limits, 21.8 and 42.5 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

other: ErC50 (Average specific growth rate)

Effect conc.:

> 82.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10.1 mg/L

Nominal / measured:

meas. (arithm. mean)

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): No
- 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 (e.g. precipitation) that might cause a difference between measured and nominal values: One replicate flask from the control group and one in the test group at 100 mg/L (nominal) were eliminated from the statistical analysis as they were considered to be anomalous due to poor cell growth after 72 hours.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

The EC50 were calculated using the likelihood ratio method. For AUC and growth rate, William’s test was used to compare each treated group with the control, unless there was evidence of a non-monotonic dose-response relationship, in which case Dunnett's test was used.

Any other information on results incl. tables

Not applicable

Applicant's summary and conclusion

Conclusions:

This study was designed to assess the effect of the test substance on the growth of the unicellular green alga Pseudokircheriella subcapitata. After 72 hours of exposure to the test substance, the EbC50 was 30.5 mg/L (95% confidence limits 21.8 and 42.5 mg/L) and was calculated using the likelihood ratio method. The ErC50 could not be determined under the conditions of the test because growth was only inhibited by 38%, so the EC50 must be > 82.6 mg/L. The Williams test was used to compare each treated group with the control, unless there was evidence of a non-monotonic dose response relationship, in which case Dunnett's test was used. The NOEC for both area under the growth curve and growth rate was 10.1 mg/L.

Executive summary:

Not applicable