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

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-11-10 to 2006-12-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed according to OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test) and EU Method C.11 (Biodegradation-Activated Sludge Respiration Inhibition Test) without deviations.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

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 209 (Activated Sludge, Respiration 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.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

Remarks:

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

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)

Deviations:

no

Remarks:

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

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 to off white solid
- Analytical purity: 99.3% area by 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: May 2008
- 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:

no

Details on sampling:

Not applicable

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Appropriate weights were added to 1 L test beakers, 16 mL synthetic sewage feed, 284 mL dechlorinated tap water, and 200 mL microbial inoculum.
- Eluate: Not applicable
- Differential loading: Not applicable
- Controls: 16 mL synthetic sewage feed, 284 mL water, and 200 mL microbial inoculum, no test substance
- 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): Results of preliminary test showed the test substance insufficiently soluble to allow a suitable stock solution. Once prepared, the mixtures were treated with ultrasound for fifteen minutes in order to form dispersions.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: Sample of activated sludge was obtained the day before the start of the test from the aeration tank at Worlington Sewage Treatment Works, which treats predominantly domestic sewage.
- Method of cultivation: In the lab, the samples were maintained under aerobic conditions until required.
- Preparation of inoculum for exposure: The concentration of suspended solids in a homogenised sample was determined on the day of collection and immediately before the start of the tests. On the day of collection, aliquots (25 mL) of the activated sludge were filtered through dried and pre-weighed Whatman GF/C filter papers which were then dried again at approximately 105 degrees C for at least one hour, allowed to cool in a desiccator and re-weighed. The mixed liquor suspended solids (MLSS) content of the activated sludge was then calculated and adjusted to 4 g/L by the removal of supernatant.
- Pretreatment: Synthetic sewage (50 mL/L) was added to each stock of activated sludge and these were aerated overnight. On the day of the test, the MLSS content of the sludge was determined and adjusted to 4 g/L by the addition of dechlorinated tap water.
- Initial biomass concentration: 4 g/L

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

none

Test conditions

Hardness:

200-250 mg/L as CaCO3

Test temperature:

19.6-19.8 degrees C

pH:

The pH ranged from 7.4-8.2 at the beginning of the test, and from 7.3 to 8.2 at the end of the test.

Dissolved oxygen:

Initial DO 7.3-9.2 mg/L

Salinity:

Not applicable

Nominal and measured concentrations:

Preliminary: Nominal - Control, 10, 100, and 1000 mg/L; 3,5-DCP- 2 Controls, 3, 10, 32 mg/L.
Definitive: Nominal - Control, 500, 1000, 2000, 4000 and 8000 mg/L; 3,5 - DCP - 2 Controls, 3, 10, 32 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 1 L test beaker
- Aeration: yes, with oil-free compressed air
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate):Not applicable
- No. of organisms per vessel: Not applicable
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1 in definitive test, 2 for reference substance
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: 4 g/L


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated and purified tap water
- 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: Not applicable
- Intervals of water quality measurement: Not applicable


OTHER TEST CONDITIONS
- Adjustment of pH: yes, the pH of the mixtures was determined and adjusted to approximately 7 with 5N NaOH, where required.
- Photoperiod: Not applicable
- Light intensity: Not applicable


EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
Not applicable

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study
- Test concentrations: 10, 100, and 1000 (3) mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol

Results and discussion

Effect concentrations

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

1 540 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% confidence limits 545-3330 mg/L

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Concentrations were not measured.
- Effect concentrations exceeding solubility of substance in test medium: Not applicable

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: 3 h EC50 10.0 mg/L in the preliminary test, 15.8 mg/L in the definitive test
- Other: acceptable range 5-30 mg/L.

Reported statistics and error estimates:

The EC50 and confidence limits were calculated using the SAFEStat curvefit program.

Any other information on results incl. tables

Table 1. Definitive test respiration rate and % inhibition.
Test Mixture (mg/L)	Respiration Rate (mg O2/g/h)	% inhibition
Control	27.8	- -
500	18.8	34
1000	19.2	32
2000	14.8	48
4000	3.0	89
8000	0.8	97
3,5-DCP
3	24.2	15
10	16.0	44
32	10.5	63
Control (2)	28.8	- -

 

Applicant's summary and conclusion

Validity criteria fulfilled:

not applicable

Conclusions:

The 3 hour EC50 of the test substance to activated sludge was found to be 1540 mg/L, with 95% confidence limits of 545-3330 mg/L.

Executive summary:

Not applicable