Benzeneacetic acid, 2-(chloromethyl)-.alpha.-(methoxymethylene)-, methyl ester

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

473-070-7

EC Name:

-

Cas Number:

117428-95-2

Molecular formula:

C12 H13 Cl O3

IUPAC Name:

methyl (2E)-2-[2-(chloromethyl)phenyl]-3-methoxyprop-2-enoate

Details on test material:

-Purity: not reported

Specific details on test material used for the study:

Substance name: Intermediate 5B
Lot #: 50216
Purity: Not reported

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

other: Activated sewage sludge

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A mixed population of activated sewage sludge micro-organisms was obtained from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
- Preparation of inoculum for exposure: The activated sewage sludge sample was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of 21°С and used on the day of collection.
Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper using a Buchner funnel. The filter paper was then dried in an oven at approximately 105°C for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids was equal to 2.9 g/L prior to use.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

10 mg/L

Based on:

IC (inorganic carbon)

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: Solution a: KH2PO4 (8.50 g/L), K2HPO4 (21.75 g/L), NA2HPO4.2H2O (33.40 g/L), NH4Cl (0.50 g/L); Solution b: CaCl2 (27.50 g/L); Solution c: MgSO4.7H2O (22.50 g/L); Solution d: FeCl3.6H2O (0.25 g/L). To 1 litre (final volume) of purified water 10 mL of Solution a, 1 mL each of Solution b, c, and d were added.
- Test temperature: 21°C
- pH: 7.4
- Aeration of dilution water: Continuous aeration
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: 5 litre glass culture vessels
- Number of culture flasks/concentration: Duplicate
- Method used to create aerobic conditions: The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of approximately 40 mL/minute and stirred continuously by magnetic stirrer. The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.
- Details of trap for CO2 and volatile organics if used: The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified de-gassed water.

CONTROL AND BLANK SYSTEM
- Inoculum blank: A control, in duplicate, consisting of inoculated culture medium.
- Standard control: The standard material (sodium benzoate), in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/L.
- Toxicity control: The test substance plus the standard material in inoculated culture medium to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Preliminary study:

Results obtained from the samples taken for DOC analysis from the preliminary investigational work indicated that the test substance did not adsorb to filter matrices or activated sewage sludge. For the purpose of the study the samples taken for DOC analysis could therefore be filtered to remove the suspended solids present without the loss of any test substance.

Test performance:

The total CO2 evolution in the control vessels on Day 28 was 22.54 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.

The IC/TC ratio of the test substance suspension in the mineral medium at the start of the test was below 5% and hence satisfied the validation criterion given in the OECD Test Guidelines.

The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satisfied the validation criterion given in the OECD Test Guidelines.

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

46

Sampling time:

28 d

Details on results:

The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels with the exception of standard material replicate R1 and test substance replicate R2. These increases were considered to be due to CO2 present in solution being driven off by the addition of hydrochloric acid on Day 28 and coupled with the decrease in inorganic carbon within test substance replicate R2 resulted in an increase in the percentage degradation value for the test substance from 46% on Day 28 to 47% on Day 29.

The toxicity control attained 58% degradation after 14 days and 67% degradation after 28 days thereby confirming that the test substance was not toxic to the sewage treatment micro-organisms used in the test. The increase in inorganic carbon in the first absorber vessel on Day 29 resulted in an increase in the percentage degradation value for the toxicity control from 67% on Day 28 to 68% on Day 29.

BOD5 / COD results

Results with reference substance:

Sodium benzoate attained 69% degradation after 14 days and 81% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

28 d CO2 evolution: 46% (Not readily biodegradable)

Executive summary:

A study was performed to assess the ready biodegradability of the test substance in an aerobic aqueous medium. The method followed that described in the OECD Guideline 301B referenced

as Method C.4-C of Commission Directive 92/69/EEC (which constitutes Annex V of Council

Directive 67/548/EEC), and US EPA OPPTS 835.3110.

 

The test substance, at a concentration of 10 mg Carbon/L, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21°С for 28 days.

 

The degradation of the test substance was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.

 

Sodium benzoate attained 69% degradation after 14 days and 81% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

 

The toxicity control attained 58% degradation after 14 days and 67% degradation after 28 days thereby confirming that the test substance was not toxic to the sewage treatment micro-organisms used in the test. The increase in inorganic carbon in the first absorber vessel on Day 29 resulted in an increase in the percentage degradation value for the toxicity control from 67% on Day 28 to 68% on Day 29.

 

The test material attained 46% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.