cyproconazole (ISO); (2RS,3RS;2RS,3SR)-2-(4-chlorophenyl)-3-cyclopropyl-1-(1H-1,2,4-triazol-1-yl)butan-2-ol

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Jan 1988 to 29 Jan 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EPA OPP 72-1 (Fish Acute Toxicity Test)

Version / remarks:

1982

Deviations:

yes

Remarks:

See Deviations in 'Any other information on materials and methods incl. tables'

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples were taken at 0 and 96 hours. 100 mL aliquots of test aquaria water were transferred to 250 mL separatory funnels. Fortifications of the quality control samples were prepared with the test substance at this time. The samples were extracted with two 50 mL portions of dichloromethane. The dichloromethane extracts were passed through powder funnels containing sodium sulphate (prewashed with dichloromethane) and collected in 250 mL flat bottom flasks. The combined extracts were evaporated by roto-evaporation to near dryness and reconstituted with toluene to appropriate volumes. The samples were diluted such that the concentration of the diluted sample would fall within the standard calibration range.

Vehicle:

yes

Remarks:

dimethylformamide (DMF)

Details on test solutions:

1.5 mL of dimethylformamide (DMF) was added to each sample weight to increase dispersion of the compound in the dilution water. Each sample weight and aliquot of solvent were added to an appropriately labelled 1 L jar containing 750 mL of test water. These solutions were sonicated about 15 minutes before being added to the appropriate test chamber containing the remaining 14.25 L of water. All test concentrations were corrected for sample purity. The solvent control chamber received a 1.5 mL aliquot of DMF, which was equivalent to the highest amount used in any test solution.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Length at study initiation: 33 ±1.4 mm
- Weight at study initiation: 0.48 ± 0.081 g
- Loading biomass: 0.32 g/L
- Feeding during test: No feed

CULTIVATION
- Cultivation period: 14 days
- Cultivation conditions: 16-hour daylight photoperiod
- Feeding during cultivation: Commercial fish food occasionally supplemented with brine shrimp nauplii (Artemia sp.) daily until 48 - 96 hours prior to testing at which time feeding was discontinued.

ACCLIMATION
- Acclimation period: 48 - 96 hours prior to testing
- Acclimation condition: Same as test temperature condition

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

Statistical analysis of the concentration vs. effect data (generally mortality) was obtained by employing a computerized LC50 program. This program calculated the LC statistic and its 95 percent confidence limits using the binomial, the moving average, and the probit tests. However, if no mortality occurred or if a dose response could not be demonstrated over a reasonable range (<37 to >63%) an LC50 and/or its 95 percent confidence limits could not be calculated. Three different methods of analysing the data were used since no one method of analysis is appropriate for all possible sets of data that may be obtained. The method of calculation selected for presentation in this report was that which gave the narrowest confidence limits for the LC50 although all three models are valid.

Test temperature:

12 - 13 ˚C

pH:

6.8 - 7.5

Dissolved oxygen:

4.4 - 9.3 mg O2/L (Dissolved oxygen saturation corrected for altitude at the test temperatures of 12 and 13°C is 10.3 and 10.1 mg/L, respectively.)

Conductivity:

500 – 600 μmhos/cm

Nominal and measured concentrations:

- Nominal concentrations: 0 (negative control), 0 (Solvent control), 1.0, 10, 18, 32, 56, 100 and 180 mg/L
- Measured concentrations: 0 (negative control), 0 (Solvent control), 0.98, 9.6, 18, 16, 9.4, 18 and 20 mg/L, respectively. See Analytical results and Table 1 in 'Any other information on matierlas and methods incl. tables'.

Details on test conditions:

TEST SYSTEM
- Test vessel: Five gallon glass vessels
- Fill volume: 15 L
- Aeration: No
- No. of organisms per vessel: 10
- No. of vessels per concentration: 1
- No. of vessels per control: 1
- No. of vessels per vehicle control: 1
- Others: The fish were added to the test chambers by random assignment within 30 minutes after addition of test material.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: This reconstituted water was prepared to yield a total hardness of 40 - 48 mg/L as CaCO3, a total alkalinity of 25 - 35 mg/L as CaCO3 and an initial pH of 7. 2 to 7. 6. The 0-hour measured control water parameters of this dilution water were dissolved oxygen 9.3 mg/L and pH 7.4. The well water source from which the dilution water was prepared had a conductivity ranging from 500 – 600 μmhos/cm.

- Water parameters: Water temperature, dissolved oxygen level and pH were measrued at 0, 48 and 96 hours

OTHER TEST CONDITIONS
- Photoperiod: 16/8 hour light/dark

EFFECT PARAMETERS MEASURED
All test organisms were observed once every 24 hours for mortality and abnormal (sub-lethal) effects. Any dead individuals were removed from the test chambers after each 24-hour observation.

RANGE FINDING STUDY
- Range finding study : Two range-finding tests of 96-hours duration were conducted to determine the concentration range for the definitive study.
- Test concentrations: The preliminary test concentrations were set at 1, 10 and 100 mg/L, and at 32 and 1000 mg/L.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

19 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

< 0.98 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

abnormality appearance (fish)

Details on results:

An overview of results is provided in Table 2 and Table 3 in 'Any other information on results incl. tables'

- Test substance solubility: The controls and 1 mg/L chambers were clear at 0-hour. The 10 and 18 mg/L chambers had a few particles of light brown undissolved compound that stuck to the glass. There was more undissolved compound in the 32 and 56 mg/L chambers (increasing with the concentration) and the compound was sticky and dark brown. There was a large amount of undissolved compound in the 100 and 180 mg/L chambers and the amount increased with the concentration. Solution observations during the remainder of the study from 24 to 96 hours were identical to those described at 0-hour regarding the amount of precipitate. However, the colour of the precipitate was noted to be light brown in colour for all chambers with precipitate. The 96-hour NOEC was estimated to be < 0.98 mg/L, the lowest concentration tested.

- Mortality: No mortality was observed in two control groups and 0.98 mg/L treatment group during the test. 10% mortality after 96 h exposure was shown in 9.6 and 18 mg/L treatment groups (measured concentrations). While, 60% mortality was found in the highest test concentration (20 mg/L) at 48 hours exposure time and the mortality increased to 100% after 96 hours exposure. The 24-, 48-, 72- and 96-hour LC50 values were > 20, > 18, 19 and 19 mg/L, respectively. All results were based on the measured concentrations of 0.98, 9.4, 9.6, 16, 18, 18 and 20 mg/L. These values correspond to the nominal test concentrations of 1.0, 56, 10, 32, 18, 100 and 180 mg/L, respectively.

- Abnormalities: Fish on the bottom of test chamber was the only abnormal effect observed in the 0.98 mg/L test concentration. The abnormal effects of loss of equilibrium, dark discoloration, quiescence and fish on the bottom of test chamber were observed in the 9.4, 9.6, 16, 20 and both 18 mg/L test concentrations. The additional abnormal effect of curved spine was noted in the 16 mg/L test level. Mortality was observed in the 9.6, 18 (100 mg/L nominal) and 20 mg/L test chambers. An examination of the fish culture and acclimation records for this test indicated that the fish were in good condition for testing.

Reported statistics and error estimates:

See Statistical analysis in 'Any other information on materials and methods inlc. tables'.

Sublethal observations / clinical signs:

Table 2. Percent Mortality

Nominal concentration (measured concentration) [mg/L]	Percentmortality
	24h	48h	96h
Control	0	0	0
Solvent Control	0	0	0
1.0 (0.98)	0	0	0
10 (9.6)	0	0	10
18 (18)	0	0	0
32 (16)	0	0	0
56 (9.4)	0	0	0
100 (18)	0	0	10
180 (20)	0	60	100

 

Table 3. Effect levels

Time	LC50 in mg/L
24h	> 20 a
48h	> 18 a
96h	19 b (18 - 20)c

a No mortality or insufficient mortality for the statistical calculation of a reliable LC50.

b LC50 calculated using Binomial Method

c 95% confidence limits.

Note. The 96-hour NOEC could be estimated at < 0.98 mg/L, the lowest concentration tested.

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the 96-h LC50 was determined to be 19 mg a.i./L with 95% confidence limits of 18 - 20 mg a.i./L.

Executive summary:

The acute toxicity of the test substance to rainbow trout (Oncorhynchus mykiss) was studied in a 96-hour static test. The study followed EPA guideline 72-1 and was in compliance with GLP criteria. The test organisms (10 fish/concentration) were exposed to nominal concentrations of 1, 10, 18, 32 , 56, 100 and 180 mg test substance/L (measured concentrations were 0.98, 9.6, 18, 16, 9.4, 18 and 20 mg/L, respectively). The effects were based on mean measured concentrations. Dimethylformamide (DMF) was used as solvent (vehicle) to prepare the solutions (0.1 ml/L max.). A solvent control and dilution water control were run in parallel with the test concentrations. All test fish were acclimatised to the dilution water and test temperature for 48 - 96 hours prior to test commencement. The assay was conducted in glass vessels containing 15 L of soft reconstituted water. The test was carried out under the following conditions: pH 6.8 - 7.5, 3.5 - 9.3 mg O2/L, 12 ± 1 °C and under artificial light with a 16/8 hour light/dark photoperiod. The average test chamber loading biomass was 0.32 g/L. Fish were observed for mortality and sub-lethal effects after 24, 48, 72, and 96 hours exposure. Dead fish were removed. The fish were not fed for the duration of the study and the test vessels were not aerated. LC50s were calculated by the binomial method.

 

No mortality was observed in two control groups and 0.98 mg/L treatment group during the test. 10% mortality after 96 h exposure was shown in 9.6 and 18 mg/L treatment groups (measured concentrations). While, 60% mortality was found in the highest test concentration (20 mg/L) at 48 hours exposure time and the mortality increased to 100% after 96 hours exposure. The fish exposed to 9.6, 18, 16, 9.4, 18 and 20 mg/L of the test substance showed loss of equilibrium, dark discolouration, quiescence and tended to stay at the bottom of the test chamber. In addition, curved spine (cramps) was noted in the 16 mg/L test level (32 mg/L nominal). The only effect noted at the lowest dose level (0.98 mg/L) was three fish on the bottom of the test chamber. After 96 hours of exposure, dissolved oxygen fell down below the saturation level recommended by the guidelines in the 10 mg/L and higher test systems. The control chambers remained above 78% saturation throughout the 96-hour study period. These conditions might have had an influence on mortality. A precipitate was also observed during the study in the 10 mg/L and higher test systems, which increased with concentration, indicating that the water solubility was likely to have been exceeded. Based on the findings, the 96-h LC50 was determined to be 19 mg/L.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Feb 1993 to 16 Feb 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EPA OPP 72-1 (Fish Acute Toxicity Test)

Version / remarks:

1982

Deviations:

yes

Remarks:

See Deviations in 'Any other information on materials and methods incl. tables'

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Duplicate 10 mL samples were collected from mid-depth of each test chamber on a daily basis (at 0, 24, 48, 72 and 96 hours) throughout the test to measure radioactivity and determine the equivalent test substance concentration. The samples were collected in 20 mL liquid scintillation vials and either prepared for liquid scintillation analysis immediately or stored at approximately 4˚C until prepared for analysis.

Vehicle:

yes

Remarks:

N,N-dimethyl formamide (DMF)

Details on test solutions:

A primary stock solution of the [14C]-labelled test substance was prepared by dissolving the radiolabelled test substance in 99 Mol % pure N,N-dimethyl formamide (DMF) at a concentration of 0.0598 mg/mL. A primary stock solution of the non-radiolabelled test substance was prepared by dissolving the technical material at a concentration of 150 mg/mL. One working stock solution then was prepared for each of the five test substance concentrations by combining aliquots of the two primary stocks and diluting with DMF when necessary to yield working stocks with concentrations of 0.016, 0.027, 0.045, 0.075, and 0.125 g/mL The five stocks were injected to the diluter mixing chambers where they were mixed with filtered seawater to achieve the desired test concentrations. The resultant test concentrations were not adjusted for purity of the active ingredient in the test substance. The concentration of DMF in the treatment and solvent control groups was approximately 0.4 mL/L.

Test organisms (species):

Cyprinodon variegatus

Details on test organisms:

TEST ORGANISM
- Common name: Sheepshead minnow
- Source: Cultures maintained by the test facility. All fish used in the test were from the same source and year class.
- Length at study initiation: The standard length of the longest fish was no more than twice the length of the shortest
- Length at study termination: The average length of 10 control fish measured at the end of the test was 20 mm with a range of 16 to 25 mm.
- Weight at study termination: The average weight of 10 control fish at the end of the test was 0.25 grams with a range of 0.13 to 0.46 grams.

HOLDING
- Holding period: 14 days
- Tmprature: 20.1 - 24.9 ˚C.
- Salinity: 22 - 24 ‰
- pH: 7.7 - 7.9

ACCLIMATION
- Acclimation period: Approximately 75 hours prior to the test
- Acclimation conditions (same as test or not): Yes, same as test
- Feeding during acclimation: Flaked fish food and live brine shrimp nauplii (Artemia sp.). The fish were not fed for approximately 75 hours prior to the test or during the test.
- Health condition: During acclimation, the organisms showed no signs of disease or stress. No mortalities occurred during acclimation.

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

21.8 - 22.1 ˚C

pH:

7.9 - 8.4

Dissolved oxygen:

6.6 - 7.8 mg O2/L

Salinity:

20‰

Nominal and measured concentrations:

- Nominal concentrations: 0 (negative control), 0 (solvent control), 6.5, 11, 18, 30 and 50 mg/L.
- Measuredconcentrations: < LOQ (negative control), < LOQ (solvent control), 6.7, 10.4, 17.0, 26.0 and 34.1 mg/L, respectively. See Table 1 in 'Any other information on materials and methods inlc. table'

Details on test conditions:

TEST SYSTEM
- Test vessel: 25 L polyethylene aquaria
- Filled volume: Approximately 15 L of test solution. The depth of the test water in each aquarium was approximately 18 cm.
- Type of flow-through: Proportional diluter
- Flow rate: Approximately 6 volume additions of test water every 24 hours
- No. of organisms per vessel: 10
- No. of vessels per concentration: 2
- No. of vessels per control: 2
- No. of vessels per vehicle control: 2
- Biomass loading rate: Loading, defined as the total wet weight of fish per liter of test water, was0.03 grams of fish per litre of solution that passed through the test chambers in 24 hours. Instantaneous loading was 0.17 grams of fish per liter of test solution present in the test chambers at any given time.
- Others: The delivery pump was calibrated before the test, and the delivery of test substance to test chambers was begun approximately 21 hours prior to test initiation in order to establish equilibrium concentrations of the test substance. The general operation of the diluter was checked visually at least two times per day during the test.

TEST MEDIUM
- Source/preparation of dilution water: The water used for holding, acclimation, and testing was natural seawater collected at Indian River Inlet, Delaware, and diluted to a salinity of approximately 20‰ with well water. The freshly collected seawater was passed through a sand filter to remove particles greater than approximately 25 μm, and pumped into a 37,800 L storage tank. The filtered seawater then was diluted with fresh water from a well on the test facility site and aerated with spray nozzles. Prior to delivery to the diluter system, the water again was filtered to remove microorganisms and particles.

WATER PARAMETERS
- Temperature: Temperature was measured continuously throughout the test period in one negative control replicate. Temperature also was measured in each test chamber at the beginning and end of the test.
- Salinity: The salinity of the water in the negative control was measured at the beginning of the test using a refractometer.
- Dissolved oxygen and pH: Dissolved oxygen and pH were measured in alternate test replicates of each treatment and control group at 24-hour intervals during the test.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours of light and 8 hours of darkness (A 30-minute transition period of low light intensity was provided as lights were turned on and off to avoid sudden changes in light intensity)
- Light intensity: Approximately 194 lux at the surface of the water

EFFECT PARAMETERS MEASURED
All organisms were observed to evaluate the numbers of mortalities and numbers of individuals exhibiting clinical signs of toxicity or abnormal behavior. Observations were made approximately 5.5, 24, 48, 72, and 96 hours after initiation of the test.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

21 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% C.L.: 17 - 26 mg/L

Details on results:

An overview of the results is provided in Table 2 – Table3 in ‘Any other information on results incl. tables’.
Sheepshead minnows in the negative control and solvent control groups appeared healthy and normal throughout the test. Sheepshead in the 6.7 and 10 mg/L treatment groups also appeared normal throughout the test with no mortalities or overt signs of toxicity. In the 17 mg/L treatment group, no mortalities were observed throughout the test. However, overt signs of toxicity were apparent at 24 hours in the 17 mg/L treatment. By the end of the test (96-hour), there was 100% mortality in the 26 mg/L treatment group. Mortality in the 34 mg/L treatment group, the highest concentration tested, was 95% within 24 hours of test initiation, and 100% by 48 hours.

Reported statistics and error estimates:

Where possible, LC50 values and 95% confidence intervals were calculated using the computer program. The program was designed to calculate the LC50 and the 95% confidence interval by probit analysis, the moving average method, or binomial probability. In this study, the 24-hour values were calculated using the moving average method while binomial probability was used to calculate values of 48, 72, and 96 hours. The no mortality concentration was determined by visually inspecting the mortality data.

Sublethal observations / clinical signs:

Table 2. Cumulative percent Mortality and Treatment-Related Effects

Measured concentration	Replication	5.5 hours		24 hours		48 hours		72 hours		96 hours
		Mortality	Effects	Mortality	Effects	Mortality	Effects	Mortality	Effects	Mortality	Effects
Negative Control	A	0% (0/10)	10 AN	0% (0/10)	10 AN	0% (0/10)	10 AN	0% (0/10)	10 AN	0% (0/10)	10 AN
	B	0% (0/10)	10 AN	0% (0/10)	10 AN	0% (0/10)	10 AN	0% (0/10)	10 AN	0% (0/10)	10 AN
Solvent control	A	0% (0/10)	10 AN	0% (0/10)	10 AN	0% (0/10)	10 AN	0% (0/10)	10 AN	0% (0/10)	10 AN
	B	0% (0/10)	10 AN	0% (0/10)	10 AN	0% (0/10)	10 AN	0% (0/10)	10 AN	0% (0/10)	10 AN
6.7	A	0% (0/10)	10 AN	0% (0/10)	10 AN	0% (0/10)	10 AN	0% (0/10)	10 AN	0% (0/10)	10 AN
	B	0% (0/10)	10 AN	0% (0/10)	10 AN	0% (0/10)	10 AN	0% (0/10)	10 AN	0% (0/10)	10 AN
10.4	A	0% (0/10)	10 AN	0% (0/10)	10 AN	0% (0/10)	10 AN	0% (0/10)	10 AN	0% (0/10)	10 AN
	B	0% (0/10)	10 AN	0% (0/10)	10 AN	0% (0/10)	10 AN	0% (0/10)	10 AN	0% (0/10)	10 AN
17.0	A	0% (0/10)	10 AN	0% (0/10)	5C;3C,N;2AN	0% (0/10)	1D,N;4N;5C	0% (0/10)	2E;8AN	0% (0/10)	2M;3N;5AN
	B	0% (0/10)	10 AN	0% (0/10)	6AN; 4C	0% (0/10)	1D;1N;8AN	0% (0/10)	1C;9AN	0% (0/10)	3M;3N;4AN
26.0	A	0% (0/10)	8C; 1C; N; 1N*	60% (6/10)	4C,N	90% (9/10)	1N,C	100% (10/10)	N/A	100% (10/10)	N/A
	B	0% (0/10)	5AN;4C;1C,N	80% (8/10)	2C,N	100% (10/10)	N/A	100% (10/10)	N/A	100% (10/10)	N/A
34.0	A	0% (0/10)	7C;2C,N;1N*	100% (10/10)	N/A	100% (10/10)	N/A	100% (10/10)	N/A	100% (10/10)	N/A
	B	0% (0/10)	2AN;5C;3C,N	90% (9/10)	1C,N	100% (10/10)	N/A	100% (10/10)	N/A	100% (10/10)	N/A

Observed Effects: A= Surfacing; C = Lethargy; D = Discoloration; E = Erratic Swimming; M = Moribund; N = Loss of Equilibrium;

AN = Appears Normal;*= Floating.

Table 3. Effect values

Time	LC50 (mg/L)	Lower 95% confidence limits	Upper 95% confidence limits	Statistical Method
24 hours	24	22	27	Probit
48 hours	22	17	26	Binomial
72 hours	21	17	26	Binomial
96 hours	21	17	26	Binomial

 

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the 96-hour LC50 value was determined to be 21 mg/L, with the 95% confidence limits of 17 - 26 mg/L.

Executive summary:

The acute toxicity of the test substance to Sheepshead minnow (Cyprinodon variegatus) was studied in a 96-hour flow-through test. The test was conducted according to EPA 72-1 and was in compliance with GLP criteria. The nominal concentrations of 6.5, 11, 18, 30, and 50 mg test substance/L. Dimethylformamide (DMF; 0.4 ml/L) was used as solvent to prepare the solutions. Two additional groups were included as seawater and solvent controls. [14C]-labelled test substance was added at a nominal radioactive concentration of 20 dpm/mL to allow for radiochemical determination of test substance equivalent concentrations. All test fish were acclimatised to the test conditions for 75 hours prior to test commencement and were not fed for the duration of the acclimatisation or study period. Twenty fish per concentration (10 fish/chamber) were used in the study. The average test chamber loading biomass was 0.17 g/L. Delivery of the test substance was initiated approximately 21 hours prior to the introduction of the test organisms to the test water in order to achieve equilibrium. The assay was conducted in 25 L polyethylene aquaria vessels containing 15 L of test solution The test conditions were as following: salinity 20‰, 22 ± 1 °C, pH 7.9 - 8.4, 16/8 hour light/dark light cycle (194 lux), and dissolved oxygen concentrations > 60% saturation. Fish were observed for mortality and sub-lethal effects after 5.5, 24, 48, 72, and 96 hours exposure. Dead fish were removed. LC50 values were calculated by the moving average method (24 hrs) or by the binomial method.

 

Precipitate was observed in the mixing chambers and increased from low to high concentrations. This precipitate was the most probable explanation for the decrease in percent of nominal with increasing concentration. At 24 hours, 14/20 fish at 26 mg/L and 19/20 fish at 34.1 mg/L test substance had died. All 20 fish at 26 mg/L had died by 72 hours, whereas all of the fish at the top dose level (34.1 mg/L) were dead by 48 hours. No mortalities occurred at any of the lower dose levels, though signs of intoxication (including lethargy and loss of equilibrium, discoloration, erratic swimming and moribund fish) were observed at 17 mg/L at various stages, from 24 hours onwards. Fish in the 6.7 and 10.4 mg/L treatment groups appeared normal throughout the test with no mortalities or overt signs of toxicity. Based on the findings, the 96-hour LC50 value was determined to be 21 mg/L, with the 95% confidence limits of 17 - 26 mg/L.

Description of key information

All available data was assessed and the studies representing the worst-case effects are included here as key. The results can be considered worst-case and are selected for the CSA.

Freshwater, 96-h LC50 = 19 mg/L, static, Oncorhynchus mykiss, mortality, EPA 72 -1, Bowman 1988

Marine water, 96-h LC50 = 21 mg/L, flow-through, Cyprinodon variegatus, mortality, EPA 72 -1, Drottar 1993

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

19 mg/L

Marine water fish

Marine water fish

Dose descriptor:

LC50

Effect concentration:

21 mg/L

Additional information

There are three standard guideline followed or equivalent to standard guideline and GLP compliant studies available for freshwater fish species. The 96-h study on rainbow trout (Oncorhynchus mykiss) is selected to be the key study, because it represents the worst-case effects (i.e. showed the lowest effect value) for freshwater conditions. 10 fish/concentration were exposed to nominal concentrations of 0 (water control), 0 (solvent control), 1, 10, 18, 32 , 56, 100 and 180 mg test substance/L (measured concentrations were < 0.010, < 0.010, 0.98, 9.6, 18, 16, 9.4, 18 and 20 mg/L, respectively) under static conditions. Fish were observed for mortality and sub-lethal effects after 24, 48, 72, and 96 hours of exposure.

No mortality was observed in the two control groups and the 0.98 mg/L treatment group during the test. 10% mortality after 96 h exposure was shown in the 9.6 and 18 mg/L treatment groups. While, 60% mortality was found in the highest test concentration (20 mg/L) at 48 hours exposure time and the mortality increased to 100% after 96 hours exposure. A precipitate was observed during the study in the nominally 10 mg/L and higher test systems, which increased with concentration, indicating that the water solubility was likely to have been exceeded. Based on the  measured concentrations, the 96-h LC50 was determined to be 19 mg/L (Bowman 1988, EPA 72-1, Reliability 2).

The other two freshwater studies are supporting studies. The first supporting study was performed with bluegill sunfish (Lepomis macrochirus). The fish were exposed to nominal test substance concentrations of 0 (blank control), 0 (solvent control), 5.6, 10, 18, 32, 56 and 100 mg/L (mean measured: < 0.010, < 0.010, 4.8, 9.2, 18, 24, 16 and 17 mg/L). A precipitate was observed during the study in the top four concentration levels (18, 32, 56 and 100 mg/L  nominal), which increased with concentration, indicating that the water solubility may have been exceeded. Based on the mean measured concentrations, the 96-hour LC50 was determined to be 21 mg/L (Bowman 1988, EPA 72-1, Reliability 1). The second supporting study was performed with common carp (Cyprinus carpio). The fish were exposed to nominal test substance concentrations of 0 (solvent control), 10, 12.5, 16, 20, 25 and 32 mg/L (mean measured: NM, 9.1, 11.9, 14.8, 18.7, 24.6 and 29.1, respectively). The 96-hour LC50 was determined to be 18.9 mg/L (Hamburger 1985, no guideline but similar to OECD TG 203, Reliability 1).

 

There is one standard guideline followed and GLP compliant study available for marine water fish species, which was selected as key study. The acute toxicity of the test substance to Sheepshead minnow (Cyprinodon variegatus) was studied in a 96-hour flow-through test. The nominal concentrations  were 0 (seawater control), 0 (solvent control), 6.5, 11, 18, 30, and 50 mg test substance/L (mean measured: < LOQ, < LOQ, 6.7, 10.4, 17.0, 26.0 and 34.1 mg/L, respectively. [14C]-labelled test substance was added at a nominal radioactive concentration of 20 dpm/mL to allow for radiochemical determination of test substance equivalent concentrations. Twenty fish per concentration (10 fish/chamber) were used in the study. Fish were observed for mortality and sub-lethal effects after 5.5, 24, 48, 72, and 96 hours exposure.

Precipitate was observed in the mixing chambers and increased from low to high concentrations. This precipitate was the most probable explanation for the decrease in percent of nominal with increasing concentration. At 24 hours, 14/20 fish at 26 mg/L and 19/20 fish at 34.1 mg/L test substance had died. All 20 fish at 26 mg/L had died by 72 hours, whereas all of the fish at the top dose level (34.1 mg/L) were dead by 48 hours. No mortalities occurred at any of the lower dose levels. Based on the  mean measured concentrations, the 96-hour LC50 value was determined to be 21 mg/L (Drottar 1993, EPA 72-1, Reliability 2).