2-(3-Chloro-2-pyridinyl)-5-oxo-3-pyrazolidinecarboxylic acid ethyl ester

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:

July 2006 - January 2007

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:

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

Version / remarks:

adopted 17th July 1992

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 29th December 1992

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 2-(3-chloropyridin-2-yl)-5-oxopyrazolidine-3-carboxylate

EC Number:

610-490-3

Cas Number:

500011-88-1

Molecular formula:

C11H12ClN3O3

IUPAC Name:

ethyl 2-(3-chloropyridin-2-yl)-5-oxopyrazolidine-3-carboxylate

Test material form:

solid

Study design

Inoculum or test system:

sewage, predominantly domestic, non-adapted

Details on inoculum:

The inoculum was secondary effluent collected from a treatment plant in Bangalore, India, receiving predominantly domestic sewage. A fresh sample of effluent was collected from the treatment plant and was kept aerobic during transport. This effluent was allowed to settle for one hour and decanted. The decanted effluent was preconditioned by aerating for 5 days at 20 to 23°C.
The bacterial population in the inoculum was determined as colony forming units (CFU/mL) by diluting the inoculum up to 10e-6 dilution and then plating on nutrient agar plates.

Duration of test (contact time):

29 d

Initial test substance concentration

Initial conc.:

20 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: According to OECD 301 guideline
- Additional substrate: no
- Solubilising agent: no
- Test temperature: 20-23ºC
- pH: 7.9-8.1
- Aeration of dilution water: Mineral medium was aerated before use
- Continuous darkness: no, diffuse light

TEST SYSTEM
- Culturing apparatus: Conical flasks – 5 L capacity each fitted with an aeration tube reaching nearly to the bottom of the flask and an outlet. Test volume was 3 L.
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Carbon dioxide free air was passed through the vessels continuously throughout study
- Measuring equipment: Residual hydroxide in the absorber traps was determined by titration with 0.05 M HCl
- Details of trap for CO2: The outlet of each test flask was connected to the inlet of three gas absorption bottles in series, each containing 100 mL of 0.0125 M barium hydroxide solution. The outlet of the last absorption bottle was left open.

SAMPLING
- Sampling frequency: On day 3, 6, 9, 11, 14, 17, 21, 25, and 29
- Sampling method: The barium hydroxide gas absorber bottle closest to the test vessel was disconnected. The residual concentration of barium hydroxide was determined by titration with HCl. Following the removal of the first gas absorption bottle, 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.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2
- Toxicity control: 1
- Reference control: 1

Reference substance

Reference substance:

other: Sodium acetate

Results and discussion

Preliminary study:

Initially the test was conducted using a test concentration of 33.33 mg/L of test medium. As the test item was found to be toxic to the inoculum, the experiment was discontinued. A second experiment was set up using a test concentration of 20 mg/L.

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

11.87

Sampling time:

29 d

BOD5 / COD results

Results with reference substance:

The reference material met the validity criteria for the test, greater than 60% biodegradation within 14 d, and within the 10 d window (Day 2 = 19%, Day 11 = 74%, Day 29 = 99% ThCO2).

Any other information on results incl. tables

Table 1: Degradation of test/reference item (% ThCO2)

Day	Test item			Reference	Test item + reference
	Flask 1	Flask 2	Mean	Control	Toxicity control
3	1,07	0,66	0,87	18,78	12,75
6	-0,06	-0,57	-0,32	38,61	23,52
9	0,86	0,45	0,66	56,61	41,94
11	2,03	1,01	1,52	74,23	54,13
14	3,15	2,23	2,69	80,55	61,22
17	5,09	3,56	4,33	83,67	65,21
21	7,17	5,34	6,26	87,17	68,51
25	9,42	8,09	8,76	98,98	74,15
29	13,09	10,64	11,87	98,85	74,71

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the test conditions, the test material undergoes 12% degradation within the 28 d period and therefore can be considered as not readily biodegradable.

Executive summary:

The ready biodegradability of the test item was determined according to OECD Guideline 301 B (CO₂ Evolution Test).

The test item (20 mg/L), blank control, reference control (sodium acetate) and toxicity control (test item + sodium acetate) were incubated with inoculum from a secondary effluent treatment plant receiving predominantly domestic sewage and observed for degradation by measurement of the theoretical amount of carbon dioxide (ThCO₂) over a 28 d period.
Sodium acetate had biodegraded by 18.78% at day 3 and 80.55% by day 14 in the absence of test material meeting the validity criteria of the test. Test mixture containing sodium acetate and test material had biodegraded by 61.22% at day 14 showing that the test item was not inhibitory at this concentration.
The cumulative CO₂ production by mixtures containing only test item had achieved 11.87% degradation of test item during the treatment period. The test item had biodegraded by 0.87% by day 3 and by 2.69% at day 14. Based on the pass levels (60% bio-degradation in the 10-day window period), the test item cannot be considered as readily biodegradable, since only 11.87% degradation was achieved during the test period of 28 days. However, it may be considered biodegradable, as there was 11.87% degradation during the treatment period.