1-(3-Chloro-2-pyridinyl)-4,5-dihydro-3-[(phenylsulphonyl)oxy]-1H-pyrazole-5-carboxylic 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:

March 2007 - June 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:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

adopted 29 December 1992

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

adopted 23 March 2006

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 3-[(benzenesulfonyl)oxy]-1-(3-chloropyridin-2-yl)-4,5-dihydro-1H-pyrazole-5-carboxylate

EC Number:

846-153-4

Cas Number:

653592-41-7

Molecular formula:

C17H16ClN3O5S

IUPAC Name:

ethyl 3-[(benzenesulfonyl)oxy]-1-(3-chloropyridin-2-yl)-4,5-dihydro-1H-pyrazole-5-carboxylate

Test material form:

solid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Samples of the test solution, negative control and positive control were collected at approximately 0 and 72 hours to measure concentrations of the test substance.

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
A primary stock solution was prepared by mixing 100 mg test item in 10 mL acetone. The primary stock solution was then diluted with algal inoculum and test medium to achieve a nominal concentration of 1.0 mg/L.
- Controls:
Negative control: 10.3 mL of the algal inoculum was made up to 1 litre using test medium
Vehicle control: 100 μL of acetone and 10.3 mL of the algal inoculum was made up to 1000 mL using test medium.

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: 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 four days prior to test initiation.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

22-24ºC

pH:

7.86-8.05

Nominal and measured concentrations:

Nominal concentration: 1.0 mg/L
Initial measured concentration: 0.71 mg/L
At the end of the exposure period there was 100% reduction of test item, therefore all results are reported based on the initial measured concentration.

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
- Aeration: No
- Initial cells density: 10000 cells/mL
- Control end cells density: 69300 cell/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6
- No. of vessels per positive control (replicates): 6

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 end 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: 7960-8080 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 initiation of the test using a haemocytometer.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.01, 0.1, 1.0 mg/L (nominal concentration)
- Results used to determine the conditions for the definitive study:
The vehicle, acetone, did not inhibit the algal cell concentration when compared to the negative control. The percent inhibition based on growth rate at 72 hours in the nominal tested concentrations of 0.01 and 0.1 mg/L was not significant when compared to the vehicle control. The percent inhibition based on growth rate at 72 hours in the nominal tested concentration of 1 mg/L was 7.04% when compared to the vehicle control. Based on this information, a nominal limit test concentration of 1.0 mg/L was selected for the definitive test (solubility limit).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (0.9 mg/L)

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.71 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

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 15.7 at the end of the test. The percent inhibition of biomass was 75.78 and the percent growth rate inhibition was 35.14 at the end of the test.

Any other information on results incl. tables

Table 1: 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	3.1	17.4	69.3
G2	Vehicle control	-	1.0	2.7	17.5	68.3
G3	1.0	0.71	1.0	2.9	16.0	62.3
G4	Positive control	-	1.0	1.5	5.9	15.7

 

Table 2: Definitive test, growth inhibition

Group	Nominal concentration (mg/L)	Initial measured concentration (mg/L)	% Growth inhibition
			Biomass (b)	Growth rate (r)	Yield (y)
G1	Negative control	-	-	-	-
G2	Vehicle control	-	1.04	0.17	0.61
G3	1.0	0.71	8.73	2.38	9.64
G4	Positive control	-	75.78	35.14	78.45

% growth inhibition for vehicle control and positive control were calculated against cell counts of negative control.
% growth inhibition for 1.0 mg/L was calculated against cell counts of vehicle control.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, ErC50 (72 h) > 0.71 mg/L based on initial measured concentrations.

Executive summary:

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

The algae were exposed to a nominal limit test concentration of 1.0 mg/L of the test item (limit test) along with a negative control, a vehicle control and a positive control (potassium dichromate). This concentration was chosen based on the results of a preliminary range finding experiment and solubility of the test item in the test media. The initial measured concentration was 0.71 mg/L for this nominal concentration. Since, there was a significant reduction in test item concentration compared to the nominal concentration at the end of the test all results are based on the initial measured concentration. The test conditions fulfilled all the validity criteria of the guideline. Hence, the results of the test were considered valid.
The test item at the tested concentration showed modest inhibitory effects on the growth and specific growth rate of Pseudokirchneriella subcapitata. The data showed a 2.38% inhibition in specific growth rate at 0.71 mg/L. The observed NOEC values for all three evaluated parameters (biomass, growth rate and yield) were less than 0.71 mg/L. The observed LOEC values for all three evaluated parameters (biomass, growth rate, and yield) were 0.71 mg/L.

Accordingly, the EC50 values for all three evaluated parameters (biomass, growth rate and yield) were >0.71 mg/L (initial measured concentration).