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 fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Jul 2006 to 27 Aug 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

Adopted 17 July 1992

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

Adopted 29 December 1992

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Version / remarks:

Public draft April 1996

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: JMAFF 2-7-1. Agchem Test Guidelines 12 Nousan No. 8147.

Version / remarks:

2001

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Analytical samples were taken from each test concentration and the relevant control at the start, after 48 hours and at the end of the test. On each sampling occasion, duplicate samples of approximately 100 mL were taken from the centre of each test vessel. One sample was analysed and the second sample was stored. Samples for the 54.5 and 120 µg/L exposure concentrations were taken after 24 hours, as all fish had died at these concentrations by that time point.

Vehicle:

yes

Remarks:

tetrahydrofuran (THF)

Details on test solutions:

- Preparation of the application solution: The application solution was prepared by adding 501.29 mg of the test substance to 250 mL of tetrahydrofuran (THF) and adding 250 mL of deionised water (1:1 v/v) to give a 1000 mg/L solution. The application solution was then diluted to provide the test concentration series, with the solvent control and test concentrations containing 60 µL of THF per litre of dilution water.
- Vehicle solution: The vehicle solution was 50% THF, 50% deionised water.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: carp
- Batch nr.: CP/2006/01
- Length at study initiation: 4.57 ± 0.34 cm
- Weight at study initiation: 1.89 ± 0.46 g
- Test species handling: A fine-mesh dip net was used to transfer the fish to minimise handling stress.

ACCLIMATION
- Acclimation period: 12 days
- Acclimation conditions: Test organisms were held in water of the same quality as used during the test. During acclimation, the temperature ranged from 23.1 - 24.9 ˚C, pH from 7.58 - 7.80, dissolved oxygen from 60 - 100% air saturation value, and water hardness was 17 1 mg CaCO3/L. A 16-hour light : 8-hour dark photoperiod was maintained with a 30 minute dawnldusk transition period.
- Type and amount of food during acclimation: The fish were fed a mixture of commercially available food; (Nishikoi carp pellets) and frozen Artemia.
- Feeding: Food was withheld for at least 48 hours before the start of the test. The fish were not fed throughout the 96 hour exposure period.
- Health during acclimation: No mortality observed

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

171 - 190 mg/L as CaCO3

Test temperature:

23.9 - 25.0 ˚C

pH:

7.11 - 7.48

Dissolved oxygen:

89 - 101% air saturation

Nominal and measured concentrations:

- Nominal concentrations: 0 (negative control), 0 (solvent control), 5.12, 11.3, 24.8, 54.5 and 120 µg/L
- Mean measured concentrations: 0 (negative control), 0 (solvent control), 4.73, 10.8, 25.3, 52.8 and 121 µg/L, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass tanks
- Type: Closed (covered with a lid)
- Fill volume: Approximately 15 L of test solution
- Type of flow-through: Proportional diluter
- Flow rate: Every diluter cycle delivered 0.5 L of dilution water or test solution to one or other set of replicate test vessels. The diluter was set to run a cycle every 4 minutes. The diluter alternately fed one of two replicates at each treatment each cycle, therefore each test vessel received a volume of approximately 90 L of water or test solution per 24 hours, giving approximately 6 replacements per day.
- No. of organisms per vessel: 7
- No. of vessels per concentration: 1
- No. of vessels per control: 1
- No. of vessels per vehicle control: 1
- Biomass loading rate: The fish loading was 0.88 g/L, based on the wet weight of a sample of fish taken from the stock tank during the test.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was prepared by mixing mains water with de-ionised water to give a total hardness of 100 - 250 mg CaCO3/L. The mixed water then passes through an activated carbon filter and a UV sterilizer to produce the dilution water. Analysis of representative samples of dilution water was conducted on at least a biannual basis for potential toxicants, pesticides, particulate matter, total organic carbon and metals.
- Intervals of water quality measurement: This study was carried out in a temperature controlled water bath. Temperature, pH and dissolved oxygen were also recorded on a daily basis in each test vessel. The temperature was monitored in the control vessel throughout the exposure period.

OTHER TEST CONDITIONS
- Photoperiod: A 16-hour light : 8-hour dark photoperiod was maintained with a 30 minute dawddusk transition period.

EFFECT PARAMETERS MEASURED
Observations of mortality and any unusual behaviour shown by surviving fish were made 3, 24, 48, 72 and 96 hours (± 1 hour) after the addition of fish to the test vessels. Fish were defined as dead when there was absence of respiratory movement and lack of response to physical stimulation of the caudal peduncle. Dead fish were removed when observed.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

25.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Details on results:

An overview of the results is provided in Table 1 – Table 3 in ‘Any other information on results incl. tables’
- Verification of test concentrations: All test concentrations appeared clear throughout the exposure period. The measured concentrations of the test substance at the start of exposure ranged from 95 to 108% of nominal, after 48 hours from 89 to 100% and from 89 to 97% of nominal at the end of exposure.
- Mortality: Mortality was observed at nominal concentrations of 25.3 μg/L and above. The NOEC based on mortality awas 10.8 μg/L. No mortality or symptoms of toxicity were observed in the controls.
- Sub-lethal effects: There were no sub-lethal effects observed in surviving fish at 96 hours. Sub-lethal effects observed at earlier time points included loss of equilibrium, unusual swimming and/or positioning in the tank, increased pigmentation and moribund fish.

Reported statistics and error estimates:

The LC50 was obtained by fitting a probit model to the respective set of data using the maximum likelihood method, in which the binomial distribution of the data was taken into account. The LC50 was obtained by fitting a probit model to the respective set of data using the maximum likelihood method, in which the binomial distribution of the data was taken into account. The 24-hour LC50 was calculated as the geometric mean of the concentrations causing 0% mortality (25.3 µg/L) and 100% mortality (52.8 µg/L). Statistical analyses to determine LC50 values for 48 - 96 hour timepoints were carried out using the Quantal Dose Response module in StEvE, version 1.1 (an in-house package that uses SAS for its internal computations).

Sublethal observations / clinical signs:

Table 1. Mortality

Mean meaured concentration (µg/L)	Cumulative number of dead fish (n = 7)
	3 h	24 h	48 h	72 h	96 h	Mortality % at 96 hours
Control	0	0	0	0	0	0
Solvent control	0	0	0	0	0	0
4.73	0	0	0	0	0	0
10.8	0	0	0	0	0	0
25.3	0	0		2	3	43
52.8	0	7	7	7	7	100
121	0	7	7	7	7	100

 

Table 2. Summary of analytical results

Occasion	Nominal concentration of the test substance (µg/L)	Measured concentration of the anti-isomer (µg/L) a	Measured concentration of the syn-isomer (µg/L) b	Equivalent concentration of the test substance (µg/L) c	% of nominal concentration
0 hours d	Control	ND	ND	-	-
	S. Control	ND	ND	-	-
	5.12	1.30	3.21	4.85	95
	11.3	3.14	7.73	l 1.7	104
	24.8	7.29	17.7	26.9	108
	54.5	15.6	37.1	56.8	104
	120	33.6	81.1	124	103
48 hours d	Control	ND	ND	-	-
	S. Control	ND	ND	-	-
	5.12	1.23	3.01	4.57	89
	11.3	2.83	6.91	10.5	93
	24.8	6.68	16.2	24.7	100
	54.5	13.1	32.2	48.8	90
	120	32.3	77.8	119	99
96 hours	Control	ND	ND	-	-
	S. Control	ND	ND	-	-
	5.12	1.26	3.15	4.75	93
	11.3	2.71	6.68	10.1	89
	24.8	6.48	15.9	24.1	97

a Corrected for mean recovery of 83%.

b Corrected for mean recovery of 80%

c Corrected for 92.8% purity

d Backup sample results after original sample analysis were unreportable due to method problems

ND none detected (LOD 0.5 µgL anti- and syn-isomer)

- not applicable

 

Table 3. Sub-lethal effects

Measured concentration of the test substance (µg/L)	Sub-lethal effects	Nr. of affected fish/number of surviving fish
		3h	24h	48h	72h	96h
Control	NAD	7/7	7/7	7/7	7/7	7/7
Solvent control	NAD	7/7	7/7	7/7	7/7	7/7
4.73	NAD	7/7	7/7	7/7	7/7	7/7
10.8	NAD	7/7	7/7	7/7	7/7	7/7
25.3	NAD	7/7	4/7	4/6	4/5	4/4
	IP	-	3/7	2/6	1/5	-
	US	-	3/7	2/6	1/5	-
52.8	LOE	7/7	AD	AD	AD	AD
	US	7/7	-	-	-	-
	UP	7/7	-	-	-	-
	IP	7/7	-	-	-	-
121	IP	7/7	AD	AD	AD	AD
	MD	7/7	-	-	-	-

NAD no adverse effects

MD moribund

UP unusual positioning

LOE loss of equilibrium

US unusual swimming

IP increased pigmentation

AD all dead

- not applicable

Validity criteria

All validity criteria were met. The mortality in the control at 96 hours was 0% (required ≤ 10%). The dissolved oxygen concentration ranged from 89 to 101 % of the air saturation value (required ≥ 60%). Thus, the study is considered valid.

Validity criteria fulfilled:

yes

Remarks:

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

Conclusions:

In an acute toxicity study on fish (Cyprinus carpio) performed in accordance with OECD TG 203, OPPTS 850.1075 and JMAFF2-7-1 guidelines, the 96-hour LC50 was determined to be 0.0258 mg/L.

Executive summary:

The toxicity of the test substance (mixture of syn- and anti- isomers) on carp (Cyprinus carpio) was investigated in a flow-through system. The study was performed following OECD TG 203, EPA OPPTS 850.1075 and JMAFF 2-7-1 guidelines and compliance with GLP criteria. The fish was exposed to the test substance at concentrations of 0 (control), 0 (vehicle control; Tetrahydrofuran), 5.12, 11.3, 24.8, 54.5 and 120 μg/L (measured concentrations: ND, ND, 4.73, 10.8, 15.3, 52.8 and 121 μg/L. respectively) for 96 hours. Seven fish were introduced into each of 1 replicate test vessel per treatment and controls. To check the concentrations of the test substance, water samples were taken at 0, 48 hours and 96 hours and analysed by using liquid chromatography with mass spectropic detection (LC/MS/MS). The toxic effects (mortality or unusual behaviour shown by surviving fish) on the test organisms were studied at 3, 24, 48, 72 and 96 hours (± 1 hour). The test conditions were as follows: 23.9 - 25.0 °C, pH 7.11 - 7.48, and dissolved oxygen in a range of 89 - 101 % air saturation level.

There were no sub-lethal effects observed in surviving fish at 96 hours. Sub-lethal effects observed at earlier time points included loss of equilibrium, unusual swimming and/or positioning in the tank, increased pigmentation and moribund fish. After 96 hours of exposure, no mortality was observed in the negative control, solvent control, or ≤ 10.8 µg/L treatments. 43%, 100% and 100% mortality were found in the 25.3, 52.8 and 121 µg/L treatments, respectively. The 96 h LC50 was determined to be 25.8 µg/L, based on mean measured concentrations.

Description of key information

All available data was assessed and the studies representing the worst-case effects are included here as key studies. Other studies are included as supporting information.

Freshwater, 96-h LC50 = 25.8 µg/L (based on mean measured concentrations), flow-through, Cyprinus carpio, mortality, OECD TG 203, EPA OPPTS 850.1075 and JMAFF 2-7-1, Sims, 2007a

Marine water, 96 -h LC50 = 31.4 µg/L (based on mean measured concentrations), flow-through, Cyprinodon variegatus, mortality, EPA OPPTS 850.1075, Palmer et al., 2007

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

25.8 µg/L

Marine water fish

Marine water fish

Dose descriptor:

LC50

Effect concentration:

314 µg/L

Additional information

There are eight standard guideline and GLP-compliant studies available for this endpoint. They all used flow-through system. The 96-h study (Sims 2007c) on carp (Cyprinus carpio) and 96-h study on sheepshead minnow (Cyprinodon variegatus) were selected as key studies, because they represent the worst-case effects (i.e. showed the lowest LC50 value) for freshwater and marine water conditions, respectively. The freshwater fish were exposed to nominal concentrations of 5.12, 11.3, 24.8, 54.5 and 120 µg/L test substance solution (mean measured concentrations: 4.73, 10.8, 25.3, 52.8 and 121 µg/L, respectively) in a flow-through system. The test conditions were as follows: 23.9 – 25.0 °C, pH 7.11 – 7.48, and dissolved oxygen in a range of 89 – 101% of saturation. The 96-hour LC50 was determined to be 0.0258 mg/L, based on the mean measured concentrations. The marine water fish were exposed to the test substance at nominal concentrations of 68, 135, 269, 538 and 1076 μg/L (mean measured concentrations: 67, 125, 247, 483, 916 μg/L). The test conditions were as follows: 21.7 – 22.1 °C, pH 7.9 – 8.1, salinity at 20 ‰, and dissolved oxygen in a range of 6.2 – 7.1 mg/L. The 96-h LC50 was determined to be 0.314 mg/L, based on mean measured concentrations (Palmer et al. 2007).

The other six studies on different freshwater species are included as supporting studies. Rainbow trout (Oncorhynchus mykiss) and fathead minnow (Pimephales promelas) were exposed to the test substance at nominal concentrations of 5.12, 11.3, 24.8, 54.5 and 120 µg/L (mean measured concentrations: 5.77, 13.5, 27.1, 72.3 and 120 µg/L and 4.73, 10.8, 24.8, 56.3 and 90.5 μg/L, respectively). The 96-h LC50 were determined to be 0.066 and 0.0263 mg/L for rainbow trout and fathead minnow, respectively, based on mean measured concentrations (Sims 2007a and Sims 2007d). These two species were also exposed to the test substance at nominal concentrations of 6.3, 13, 25, 50, 100 and 200 µg/L (mean measured concentrations: 7.2, 7.5, 14, 33, 68 and 151 µg/L and 6.4, 8.7, 17, 39, 67 and 162 µg/L, respectively). The 96-h LC50 was determined to be 0.063 and 0.034 mg/L for rainbow trout and fathead minnow, respectively, based on mean measured concentrations (Albuquerque 2005 and Albuquerque 2006). Bluegill sunfish (Lepomis macrochirus) and zebrafish (Danio rerio) were exposed to the test substance at nominal concentrations of 24.8, 54.5, 120, 264 and 581 µg/L (mean measured concentrations: 17.2, 45.4, 82.8, 185 and 399 μg/L and 19.6, 46.8, 98.9, 206 and 436 μg/L, respectively). The 96-h LC 50 was determined to be 0.181 and 0.300 mg/L for Bluegill sunfish and zebrafish, respectively, based on mean measured concentrations (Sims 2007b and Sims 2007e).