Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Apr 2005 to 28 Oct 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

adopted 13 April 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling intervals: Duplicate samples were taken for analysis from each test concentration and the control at the start and end of the test.
- Sampling procedures and sample preparations: At the start of the test, samples were taken from the excess test solutions after filling the test vessels (one sample 'A' to be analysed, and the second sample 'B' to be stored as a backup). At the end of the test, the first three replicates at each test concentration and controls were combined and the required sample amount poured into the sample vessels 'A' and 'B'. Samples of approximately 100 mL were transferred to plastic bottles. The 0 hour samples were stored at a temperature < 7 °C for up to 2 days and the 48 hour samples were processed on their day of receipt. Prior to analysis the samples were allowed to warm to room temperature and shaken to thoroughly mix the contents. Two procedural recovery samples were prepared and processed alongside the samples as a check for method performance.

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

- Method of preparation: A stock solution was prepared by dissolving 80.49 mg of the test item in 50 mL acetone. The appearance of this stock solution was clear and colourless. A series of stock solutions was produced in acetone by serial dilution. The appearance of all the stock solutions was clear and colourless. To produce the test solutions, 100 μL of the appropriate stock solution was spiked into 1000 mL dilution water. The solvent control was produced by spiking 100 μL of acetone into 1000 mL dilution water. All test solutions were clear and colourless. The negative control test vessels were set up with dilution water alone.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water fleas
- Strain/clone: Straus 1820, Clone 5
- Justification for species: Characteristics that make this test organism suitable for aquatic toxicity testing are its ease of culturing, its sensitivity to a variety of chemicals and the extensive database that exists for this common freshwater invertebrate species.
- Age at study initiation: First instar Daphnia (≤24 hours old)
- Source: In-house cultures (Daphnia magna for the start of the cultures were received on 15 September 2005)
- Culture condition: The Daphnia were cultured in the same water and similar environment conditions to those used in the test. At test initiation, the animals were not from the first progeny.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

256 - 257 mg/L as CaCO3

Test temperature:

20.4 - 21.0 °C

pH:

8.19 - 8.33

Dissolved oxygen:

99 - 102 % air saturation

Nominal and measured concentrations:

- Nominal concentrations: 0 (negative control), 0 (solvent control), 5, 10, 20, 40, 80, 160 μg/L.
- Measured concentrations: ND (negative control), ND (solvent control), 4.71, 10.3, 22.6, 37.3, 82.0, 177 μg/L, respectively. See Table 1 in 'Any other information on materials and methods incl. tables'.

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL glass beakers
- Type: Closed (covered with watch glasses to reduce evaporation and entry of dust into the solutions)
- Volume of solution: 100 mL test solution
- No. of organisms per vessel: 5 (without conscious bias)
- No. of vessels per concentration: 4
- No. of vessels per control: 4
- No. of vessels per vehicle control : 4

WATER PARAMETERS
Temperature, pH and dissolved oxygen were also recorded in the test solutions and control at the start and the end of the study. Hardness of the dilution water was measured prior to use in the test. Light intensity was measured at the start of the test with a lux meter

OTHER TEST CONDITIONS
- Temperature: This study was carried out in a temperature-controlled room. Throughout the exposure period room temperature was monitored by means of a data logger in a vessel containing deionised water.
- Photoperiod: 16-hour light:8-hour dark photoperiod with 30 minute dawn and dusk transition periods
- Light intensity: 739 - 901 Lux

EFFECT PARAMETERS MEASURED
Observations of immobility (defined as animals that do not swim within 15 seconds of applying a gentle stimulus) were made approximately 24 and 48 hours (± 1 hour) after the addition of organisms to the test vessels. Details of any other observed toxic effects were also recorded.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

44 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

20 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

An overview of the results is provided in Table 2 - Table 3 in 'Any other information on results incl. tables'.
After 48 hours exposure, no immobility of daphnia was observed in the controls or ≤ 20 µg/L treatments either. After 24 hours exposure 10%, 60% and 85% of immobility were found in 40, 80 and 160 µg/L treatments, respectively. After 48 hours exposure, the immobility increased to 50%, 90% and 100% in 40, 80 and 160 µg/L treatments, respectively. Therefore, the 48 hour EC50 was 44 μg/L (95% confidence limits: 36 - 52 μg/L) based on nominal concentrations of formulation.

Reported statistics and error estimates:

- EC50:The median effect concentration (EC50) was determined at 24 and 48 hours. The EC50 is defined as the test concentration that results in 50% immobility of the test population. At each assessment time the EC50 was obtained by fitting a probit model to the respective set of data. The probit model assumes a straight-line relationship between the probit transformation of the proportion immobile and log10 concentration. The probit model was fitted using maximum likelihood, in which the binomial distribution of the data was taken into account. Confidence intervals for the 24 and 48-hour EC50 estimate were also determined, using Fieller's method. Statistical analyses to determine the 24 and 48-hour EC50s were carried out using the Quantal Dose Response module in StEvE, version 1.1.
- NOEC: The NOEC was determined at 24 and 48-hours. The NOEC is defined as the highest concentration tested which did not produce an adverse effect when compared to the control. The NOEC was obtained by comparing the percentage immobility at each test concentration with the control by visual inspection of the data.

Table 2. Immobility of Daphnia magna after 24 and 48 hours of exposure to the test substance 

Treatment (µg/L)	Rep.	No. introduced	No. immobile 24 hours	No. immobile 48 hours	% immobile per treatment at 48 hours
Control	1	5	0	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0
Solvent control	1	5	0	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0
5	1	5	0	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0
10	1	5	0	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0
20	1	5	0	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0
40	1	5	0	1	50
	2	5	0	2
	3	5	2	5
	4	5	0	2
80	1	5	2	3	90
	2	5	2	5
	3	5	5	5
	4	5	3	5
160	1	5	4	5	100
	2	5	4	5
	3	5	5	5
	4	5	4	5

 

Table 3. Summary of effect values

Time point (hours)	EC50 value (µg/L)	95% Confidence limits (µg/L)	NOEC(µg/L)
24	79	63 - 99	20
48	44	36 - 52	20

 

Validity Criteria

The study is considered valid as all validity criteria were met.

- Control immobility at 48 hours was 0% (required ≤10%).

-The dissolved oxygen concentration ranged from 99 to 102% air saturation (required ≥ 60%).

Validity criteria fulfilled:

yes

Remarks:

See validity criteria in 'Any other information on results incl. tables'

Conclusions:

In a 48-h toxicity study on Daphnia magna, following OECD TG 202 and under GLP, the EC50 was determined to be 44 µg/L, based on nominal concentrations.

Executive summary:

The toxicity of the test substance (mixture of syn- and anti- isomers) on Daphnia magna was investigated in a static study following OECD TG 202 and in compliance with GLP criteria. Daphnia magna was exposed to the test substance at concentrations of 0 (control), 0 (vehicle control; acetone), 5, 10, 20, 40, 80, and 160 μg/L (measured concentrations: ND, ND,4.71, 10.3, 22.6, 37.3, 82.0 and 177 μg/L, respectively) for 48 hours. Twenty Daphnia was introduced into each of 4 replicate test vessels per treatment and control. To check the concentrations of the test substance, water samples were taken at 0 and 48 hours and analysed by using liquid chromatography with mass spectropic detection (LC/MS/MS). Daphnia magna were assessed for toxic effects at 3, 6, 24 and 48 hours. The test conditions were as follows: 20.4 - 21.0 °C, pH 8.19 - 8.33 and dissolved oxygen in a range of 99 - 102 % air saturation. Observations of immobility were made approximately 24 and 48 hours (± 1 hour) after the addition of organisms to the test vessels.

After 48 hours of exposure, no immobility of daphnia was observed in the controls or ≤ 20 µg/L treatments. After 48 hours of exposure, the immobility was 50%, 90% and 100% in 40, 80 and 160 µg/L treatments, respectively. Based on the findings, the EC50 of the test substance was determined to be 44 µg/L (based on nominal concentration).

 

Description of key information

All available data was assessed. The study representing the worst-case effects was included here and its effect value was used as the key value. Other studies are included as supporting information.

Freshwater, 48-h EC50 = 44 µg/L (based on nominal concentrations), static, Daphnia magna, immobility, OECD TG 202, Benyon & Richardson, 2007

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

44 µg/L

Additional information

There are three standard guideline and GLP-compliant studies available for this endpoint. Water fleas (Daphnia magna) were exposed to the test substance under static condition at nominal concentrations of 5, 10, 20, 40, 80 and 160 μg/L (measured concentrations: ND, ND, 4.71, 10.3, 22.6, 37.3, 82.0 and 177 μg/L, respectively) for 48 hours. One blank control group was exposed to dilution water only and one vehicle control group was exposed to the solvent (acetone). Twenty Daphnia were introduced into each of 4 replicate test vessels per treatment and control. The test organisms were assessed for toxic effects at 3, 6, 24 and 48 hours. The test conditions were as follows: 20.4 - 21.0 °C, pH 8.19 - 8.33 and dissolved oxygen in a range of 99 - 102 % air saturation. The 48-hour EC50 was determined to be 0.044 mg/L, based on nominal concentrations (Benyon & Richardson, 2007). This study represents the worst-case with regards to aquatic invertebrate toxicity of the substance and was selected as key study.

In a supporting study, Daphnia magna was exposed to the test substance under static condition at nominal concentrations of 2.5, 25, 50, 100, 200 and 400 μg/L (mean measured concentrations: 8.35, 17.9, 39.4, 62.1, 138 and 251 μg/L). The 48-hour EC50 was determined to be 0.130 mg/L, based on mean measured concentrations (Benyon & Ramsay 2007). In a specific project, a range of aquatic invertebrate species were exposed to the test substance under static condition at nominal concentrations of each 62.5, 125, 250, 500 and 1000 μg/L. The 48-h EC50 was determined to be > 1.000 mg/L for Coenagrionidae, Cloeon sp., Ostracoda, and Brachionus calyciflorus. The 48 -h EC50 was > 0.955, > 0.900, > 0.775, > 0.750, > 0.740 and > 0.730 mg/L for Lumbriculus variegatus, Lymnaea sp., Asellus aquaticus, Planariidae, Crangonyx pseudogracilis and Chaoborussp., respectively, based on nominal concentrations (Ashwell & Langridge 2007).