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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2006 - April 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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

adopted 13 April 2004

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

adopted 29 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

Sampling and analysis

Analytical monitoring:

yes

Remarks:

HPLC/VW detector

Details on sampling:

- Sampling method: Samples from the test media of all test concentrations and the control were taken at the start of the test and at the end of the test.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 100 mg test item was mixed with a small volume of test medium in a beaker. This was stirred on a magnetic stirrer for 30 minutes. The volume was then made up to 100 mL with test medium in a volumetric flask (1000 mg/L). The stock solution was further diluted with test medium to prepare the test concentrations.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Age at study initiation: Less than 24 hours
- Source: Local pond at Peenya 2nd stage, Bangalore, INDIA. Bred at Department of Safety Assessment , Advinus Therapeutics Private Limited, Bangalore, INDIA (For ~10 years)
- Feeding during test: No

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Test temperature:

20.2-20.4ºC

pH:

7.59-7.93

Dissolved oxygen:

> 85% of saturation value

Nominal and measured concentrations:

Nominal concentrations: 0.20, 0.44, 0.97, 2.13, 4.69 and 10.31 mg/L
Mean measured concentrations: 0.17, 0.38, 0.82, 1.88, 4.45 and 9.83 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass beakers
- Fill volume: 150 mL
- Aeration: No
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per positive control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water prepared according to guideline
- Culture medium different from test medium: No
- Intervals of water quality measurement: Dissolved oxygen concentration and pH were measured at the start and end of the test. The measurements of the test solutions at the start were taken in the absence of Daphnia and the end measurements were made in the presence of Daphnia, after pooling the test solutions from 4 replicates of each group.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16/8 hours light/dark cycle
- Light intensity: 1030-1040 Lux

EFFECT PARAMETERS MEASURED: The immobility of daphnids was determined by visual controls after 24 and 48 hours. The number of immobile daphnids in each group was recorded.

RANGE-FINDING STUDY
- Test concentrations: 0 (negative control); 0.001, 0.01, 0.1, 1.0, 10.0, 100 mg/L
- Results used to determine the conditions for the definitive study: No immobility of the Daphnids was observed in the negative control at either 24 or 48 hours post exposure. The percent immobilization, for the test item concentrations of 0.001, 0.01, 0.1, 1.0, 10 and 100 mg/L was 0, 0, 0, 40, 90 and 100%, respectively, after 48 hours of exposure. Based on the range finding results, the definitive test was conducted using the nominal concentrations of 0.20, 0.44, 0.97, 2.13, 4.69 and 10.31 mg/L (factor 2.2).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (0.32 mg/L)

Results and discussion

Effect concentrations

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.22 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Results with reference substance (positive control):

- Results with reference substance valid? Yes
There were 25 and 75 percent immobilization of Daphnia in the positive control group at 24 and 48 hours post exposure, respectively, demonstrating the sensitivity of the test procedure.

Any other information on results incl. tables

Table 1: Definitive test - Immobilisation rates after 24 and 48 hours exposure

Total number of Daphnia in each replicate at start: 5

Group	Nominal concentration [mg/L]	Mean measured concentration [mg/L]	No. of Daphnia immobilised								% Immobilisation
			24 h				48 h				24 h	48 h
			R1	R2	R3	R4	R1	R2	R3	R4
G1	Negative control	-	0	0	0	0	0	0	0	0	0	0
G2	0.20	0.17	0	0	0	0	0	0	0	0	0	0
G3	0.44	0.38	0	0	1	0	2	1	2	0	5	25
G4	0.97	0.82	1	0	0	0	2	3	2	2	5	45
G5	2.13	1.88	1	0	1	0	3	2	3	3	10	55
G6	4.69	4.45	1	2	1	1	4	3	4	3	25	70
G7	10.31	9.83	3	2	3	3	5	5	5	5	55	100
G8	Positive control	-	2	1	1	1	4	5	3	3	25	75

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

There was no mortality in the negative control at the end of the test and the dissolved oxygen concentration of the test medium was greater than 85% of the air saturation value (6.6 mg/L).

Conclusions:

Under the test conditions, the calculated EC50 value for the test item at 48 hours was 1.22 mg/L based on the mean measured concentration and immobility with fiducial limits being 0.78 and 1.91 mg/L.

Executive summary:

The acute Toxicity of the test item to Daphnia magna was determined in a static 48-hour immobilisation test. The test was conducted in accordance with OECD Guideline 202 and EEC Guideline Part C.2.
The daphnids were exposed to six nominal concentrations of 0.20, 0.44, 0.97, 2.13, 4.69 and 10.31 mg/L (factor was 2.2) over a 48 hour period. The mean measured concentrations were 0.17, 0.38, 0.82, 1.88, 4.45 and 9.83 mg/L for these nominal concentrations. A concurrent negative control (dilution water only) and a positive control (potassium dichromate) were also tested. HPLC analysis of the test solutions showed a reduction in the measured concentration of the test item in the test media at the end of the treatment period compared to the nominal concentration. Hence, the results are reported based on the mean measured concentration of the test item.
The number of Daphnia immobilised was recorded at 24 and 48 hours after initiation. The exposure of daphnids to the mean measured concentrations of 0.17, 0.38, 0.82, 1.88, 4.45 and 9.83 mg/L resulted in 0, 25, 45, 55, 70 and 100% immobilization, respectively, at the end of 48 hours. No immobility was observed in the negative control.

The calculated EC50 value for the test item at 48 hours was 1.22 mg/L based on the mean measured concentration and immobility with fiducial limits being 0.78 and 1.91 mg/L