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

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-10-20 to 2006-11-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed according to OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test) and EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution 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 301 B (Ready Biodegradability: CO2 Evolution 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.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution 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:

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-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 to off-white powder
- Analytical purity: 99.8 area % by High Performance Liquid Chromatography (HPLC)
- Impurities: chloride: <0.1%
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: 2006-02-02
- Lot/batch No.: 25719
- Expiration date of the lot/batch: February 2008
- Stability under test conditions: no data
- Storage condition of test material: at 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

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

other: activated sludge, domestic

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Worlingworth (UK) sewage treatment works, which treats primarily domestic sewage
- Laboratory culture: not applicable
- Method of cultivation: not applicable
- Storage conditions: aerated overnight with CO2 free air
- Storage length: overnight
- Preparation of inoculum for exposure: Aliquots of a homogenized sample were filtered through dried (approximately 105 deg C) and pre-weighed Whatman GF/C filter papers. The filters were dried for at least one hour, allowed to cool and re-weighed. The solids level in the sludge was determined and then an appropriate volume used to inoculate control and test vessels to give a final suspended solids concentration of 30 mg/L.
- Pretreatment: no data
- Concentration of sludge: 30 mg/L
- Initial cell/biomass concentration: no data
- Water filtered: yes-CO2-free ultrapure water
- Type and size of filter used, if any: Reversed osmosis

Duration of test (contact time):

29 d

Initial test substance concentration

Initial conc.:

10 other: mg C/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Remarks:

No identification/quantification of parent compound and/or transformation products

Details on study design:

TEST CONDITIONS
- Composition of medium: Stock 1: Potassium dihydrogen phosphate, di-Postassium hydrogen phosphate, di-Sodium monhydrogen phosphate dihydrate, and Ammonium chloride; Stock 2: Magnesium sulphate heptahydrate; Stock 3: Calcium chloride dihydrate; Stock 4: Iron (III) chloride hexahydrate. Medium: 10 mL stock 1/L + 1 mL stock 2 to 4/L
- Additional substrate: not applicable
- Solubilising agent (type and concentration if used): not applicable
- Test temperature: 21-23 deg C
- pH: in situ: 7.5-7.6
- pH adjusted: yes, 5N HCl
- CEC (meq/100 g): no data
- Aeration of dilution water: yes, continuously
- Suspended solids concentration: 30 mg/L
- Continuous darkness: no data; test performed in amber glass bottles

TEST SYSTEM
- Culturing apparatus: five-litre amber glass culture bottles
- Number of culture flasks/concentration: Total of 6: 1, 2 Controls - inoculated mineral salts medium; 3 Reference - inoculated mineral salts medium plus sodium benzoate (10 mgC/L); 4, 5 Test substance (10 mgC/L plus inoculated mineral salts medium; and 6 Sodium benzoate (10 mgC/L) plus test substance (10 mgC/L) plus inoculated mineral salts medium
- Method used to create aerobic conditions: Each vessel was fitted with a stopper holding an air inlet tube reaching approximately 10 cm below the liquid surface and an air outlet just below the stopper. Continuous flushing with CO2-free air (30-90 mL/min) for 29 days
- Method used to create anaerobic conditions: not applicable
- Measuring equipment: titration phenolphthalein indicator
- Test performed in closed vessels due to significant volatility of test substance: not applicable
- Test performed in open system: yes
- Details of trap for CO2 and volatile organics if used: Dreschel bottles containing barium hydroxide

SAMPLING
- Sampling frequency: 12 samples over test period
- Sampling method: Air outlet of each vessel was connected to three Dreschel bottles in series, each containing 0.025N, nominal barium hydroxide (100 mL). The residual concentration of barium hydroxide in the bottles nearest to the test vessels, were determined at intervals by duplicate titration of 20 mL samples with hydrochloric acid (0.05N), using phenolphthalein indicator. Following the removal of the first Dreschel bottle in a series, the second was connected to the test vessel, and a bottle containing fresh barium hydroxide was connected to the outlet of the bottle at the end of the series.
- Sterility check if applicable: not applicable
- Sample storage before analysis: no data

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes

STATISTICAL METHODS
-Theoretical carbon dioxide production (TCO2): TCO2/mg carbon = mw of carbon dioxide/atomic weight of carbon
-CO2 Production: mgCO2 = [Nx(Vs - Vt0}/2 x 100/V x mw(CO2)
-Percent Degradation: cumulative CO2 (mg)/TCO2 of test mixture x 100

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Preliminary study:

The test was preceded by solubility and formulation trials.

Test performance:

The results obtained for the degradation of sodium benzoate and cumulative CO2 production by the control mixtures, fulfill the validity criteria for the test.

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

43

Sampling time:

29 d

Details on results:

The degradation of sodium benzoate was also rapid in the presence of the test substance and had achieved 62% of its TCO2 after 6 days. This test vessel was terminated on day 6 of the test; the final pH of the culture was 7.6.
Mean cumulative CO2 production of the mixtures containing the test substance, was equivalent to 10% of the TCO2 after approximately 8 days and 43% by the end of the test on day 29.
The theoretical organic carbon content of the test substance was determined to be 66.2%.
Substances are considered to be readily biodegradable in this test if CO2 production is equal to or greater than 60% of the theoretical value within ten days of the level achieving 10%. The test substance cannot, therefore, be considered to be readily degradable. Cumulative CO2 production in the controls was within the acceptable range for this assay system. The degradation of sodium benzoate was rapid and had achieved 64% of its TCO2 after 6 days and 88% after 29 days.

Any other information on results incl. tables

Not applicable

Applicant's summary and conclusion

Conclusions:

Substances are considered to be readily biodegradable in this test, if CO2 production is equal to or greater than 60% of the theoretical value within ten days of the level achieving 10%. The test substance cannot, therefore, be considered to be readily biodegradable.

Executive summary:

Not applicable