Ethyl 2-chloroacetoacetate

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:

10 March 2010 - 8 April 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 2-chloroacetoacetate

EC Number:

210-180-4

EC Name:

Ethyl 2-chloroacetoacetate

Cas Number:

609-15-4

Molecular formula:

C6H9ClO3

IUPAC Name:

ethyl 2-chloro-3-oxobutanoate

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Source:
The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap de Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.

Treatment:
The freshly obtained sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was 4.8 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant). Before use, the sludge was allowed to settle (75 minutes) and the liquid was decanted for use as inoculum at the amount of 10 ml/l of mineral medium.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

27.5 mg/L

Based on:

test mat.

Initial test substance concentration 2

Initial conc.:

12 mg/L

Based on:

other: Total Organic Carbon (TOC)

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: 1 litre mineral medium contains: 10 ml of solution (A), 1 ml of solutions (B) to (D) and Milli-RO water
Stock solutions of mineral components
A) 8.50 g KH2PO4; 21.75 g K2HPO4; 67.20 g Na2HPO4.12H2O; 0.50 g NH4Cl; dissolved in Milli-Q water and made up to 1 litre, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli-Q water and made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli-Q water and made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli-Q water and made up to 1 litre.

- Test temperature: between 21.8 and 22.5°C.

- pH:
At t=0 d: 7.6
At t=28 d: 7.5 - 8.0
- pH adjusted:no
- Aeration of dilution water: Not before the test, the test is aerated continously
- Suspended solids concentration: The concentration of suspended solids was 4.8 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant).
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 2 litre all-glass brown coloured bottles
- Number of culture flasks/concentration:
Test suspension: containing test substance and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Positive control: containing reference substance and inoculum (1 bottle).
Toxicity control: containing test substance, reference substance and inoculum (1 bottle).
- Method used to create aerobic conditions:
Synthetic air (a mixture of oxygen (ca. 20%) and nitrogen (ca. 80%)) was sparged through the solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 ml/min).
- Test performed in open system: yes
- Details of trap for CO2 and volatile organics if used:
CO2 was trapped in barium hydroxide solution. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampul). Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day, for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made at least 14 days.


SAMPLING
- Sampling frequency: Titration were made on day: 2, 5, 7, 13, 14, 19, 23, 27 and 29
- Sampling method: Titration of the whole volume of CO2-absorber

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

Reference substance

Reference substance:

acetic acid, sodium salt

Results and discussion

% Degradation

% Degradation 1

Parameter:

% degradation (CO2 evolution)

Value:

99

Sampling time:

29 d

Remarks on result:

other: HCl added on the 28th day (last CO2 measurement on the 29th day)

% Degradation 2

Parameter:

% degradation (CO2 evolution)

Value:

84

Sampling time:

29 d

Remarks on result:

other: HCl added on the 28th day (last CO2 measurement on the 29th day)

Details on results:

In the toxicity control more than 25% biodegradation occurred within 14 days (70%, based on ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity.

BOD5 / COD results

Results with reference substance:

The positive control substance was biodegraded by at least 60% (76%) within 14 days.

Any other information on results incl. tables

-

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Chloracetessigester was readily biodegradable under the conditions of the modified Sturm test presently performed.

Executive summary:

The relative biodegradation values calculated from the measurements performed during the test period revealed 99 and 84% biodegradation of Chloracetessigester, for A and B, respectively.

Furthermore, biodegradation of Chloracetessigester of at least 60% was reached within a

10-day window(the criterion for Ready biodegradability).

In the toxicity control, Chloracetessigester was found not to inhibit microbial activity.

 

Since all criteria for acceptability of the test were met, this study was considered to be valid.

 

In conclusion, Chloracetessigester is designated as readily biodegradable.