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

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted 7 June 1984

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

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

Details on sampling:

Sampling method: Samples of the test solutions were collected at approximately 0 and 72 hours to measure concentrations of the test substance.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A primary stock solution was prepared by mixing 250 mg of test item with a small volume of test medium and mixing using a magnetic stirrer for 30 minutes. The volume was brought to 250 mL using the test medium (1000 mg/L). The primary stock solution was then proportionally diluted with test medium and algal inoculum to prepare five test solutions.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: NIVA-CHL 1
- Source (laboratory, culture collection): Norwegian Institute for Water Research, P.O. Box 173, Kjelsas, N-0411, Oslo, NORWAY
- Age of inoculum (at test initiation): The culture was last transferred to fresh medium two days prior to test initiation.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22 - 24ºC

pH:

7.9 - 8.0

Nominal and measured concentrations:

Nominal concentrations: 7.5, 15, 30, 60 and 120 mg/L + negative control
Measured concentrations: 8.22, 16.61, 34.45, 67.06 and 132.3 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical flasks
Flasks were manually shaken at least 3 times a day during the treatment period.
- Fill volume: 150 mL
- Initial cells density: 10000 cells/mL
- Control end cells density: 715000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per negative control (replicates): 6
- No. of vessels per positive control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes, freshwater medium according to OECD TG 201

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD TG 201 medium according to guideline
- Intervals of water quality measurement: The pH was measured at the start and finish of the definitive test. The temperature inside the environmental chamber was recorded once daily.

OTHER TEST CONDITIONS
- Photoperiod: Continuous cool-white fluorescent light
- Light intensity and quality: 7800 to 8020 Lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: The algal cell density in each flask was determined at 24, 48 and 72 hours after the start of the test using a haemocytometer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
Range finding study:
- Test concentrations: 0.001, 0.01, 0.1, 1.0, 10.0 and 100.0 mg/L in test medium.
- Results used to determine the conditions for the definitive study:
The results showed no or very limited effect on growth at the nominal tested concentrations 0.001, 0.01, 0.1, 1, 10 mg/L. The percent inhibition based on growth rate at 72 hours in the nominal tested concentrations of 0.001, 0.01, 0.1, 1, 10 and 100 mg/L was -1.17, -0.33, 0.17, 0.33, -0.17 and 100, respectively over the negative control. Based on this information the nominal concentrations 7.5, 15, 30, 60 and 120 mg/L were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (0.9 mg/L)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

31.27 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

45.66 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

27.94 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Results with reference substance (positive control):

- Results with reference substance valid? yes
There was an increase in the cell biomass of the positive control culture by a factor of 16.8 at the end of the test. The percent inhibition of biomass was 75.63 and the per cent growth rate inhibition was 33.9 at the end of the test.

Any other information on results incl. tables

Table 1: Definitive test, meaured concentrations of test item in test solution

Nominal concentration (mg/L)	Start		End
	Measured Mean Concentration (mg/L)	% of nominal concentration	Measured Mean Concentration (mg/L)	% of nominal concentration
Negative control	-	-	-	-
7,5	8,22	110	4,09	55
15	16,61	111	6,18	41
30	34,45	115	2,64	9
60	67,06	112	5,29	9
120	132,3	110	20,12	17
120 (*)	142,29	119	6,43	5

(*) Additional test item concentration prepared to check the stability of test item in the test medium.

 

Table 2: Definitive test, average cell counts at different time interval and growth inhibition

Group	Nominal concentration (mg/L)	Initial measured concentration (mg/L)	Average cell counts (x 10e4 cells/mL)
			0 h	24 h	48 h	72 h
G1	Negative control	-	1,0	4,5	19,7	71,5
G2	7,5	8,22	1,0	4,8	19,8	70,5
G3	15	16,61	1,0	4,6	15,4	64,9
G4	30	34,45	1,0	4,1	10,2	21,5
G5	60	67,06	1,0	3,2	3,9	2,6
G6	120	132,3	1,0	0	0	0
G7	Positive control	-	1,0	2,7	5,4	16,8

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Executive summary:

The effect of the test item on growth and growth rate of the single cell green alga Pseudokirchneriella subcapitata was investigated according to OECD Guideline 201 and EEC, Guideline, Part C.3.

The alga was exposed to the test item at nominal concentrations of 7.5, 15, 30, 60 and 120 mg/L (the serial dilution factor was 2) along with a negative control and a positive control (potassium dichromate). The initial measured concentrations were 8.22, 16.61, 34.45, 67.06 and 132.3 mg/L for these nominal concentrations. There was a significant reduction in test item concentration compared to the nominal concentration at the end of the test, therefore all results are based on the initial measured concentrations. The cell growth was measured at 24, 48 and 72 hours after initiation of the test. The inhibition of growth was determined by calculating the EbC50, ErC50 and EyC50. The test conditions fulfilled all the validity criteria of the guideline. Hence, the results of the test were considered valid.

The results of the study show that the test item at various concentrations has inhibitory effects on the growth and specific growth rate of Selenastrum capricornutum.

 

NOEC (biomass, growth rate and yield)  < 8.22 mg/L based on initial measured concentrations.

LOEC (biomass, the growth rate and the yield) = 8.22 mg/L, based on initial measured concentrations.

EbC50 (72 h) = 31.27 mg/L based on initial measured concentrations.

ErC50 (72 h) = 45.66 mg/L based on initial measured concentrations.

EyC50 (72 h) = 27.94 mg/L based on initial measured concentrations.