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 aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Dec 1992 to 22 Dec 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 72-3 (Estuarine/Marine Fish, Mollusk, or Shrimp Acute Toxicity Test)

Version / remarks:

October 1992

Deviations:

not specified

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Duplicate 10 mL samples were collected from mid-depth of each test chamber on a daily basis throughout the test to measure radioactivity and determine the equivalent test substance concentration.

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 test substance was prepared by dissolving the technical material in N,N-dimethyl formamide at a concentration of 100 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 0.0026, 0.0043, 0.0072, 0.012, and 0.020 mg/mL. The five stocks were injected to the diluter mixing chambers where they were mixed with unfiltered saltwater to achieve the desired test concentrations. The concentration of DMF in the treatment and solvent control groups was approximately 0.3 ml/L. All stock solutions were held in amber bottles at the diluter.

Test organisms (species):

other aquatic mollusc: Crassostrea virginica

Details on test organisms:

TEST ORGANISM
- Common name: Eastern oyster
- Length at study initiation: 28 mm (with a range of 25 - 33 mm)
- Feeding during test : Same as acclimation period

ACCLIMATION
- Acclimation period: 10 days
- Acclimation conditions: The oysters were supplied unfiltered saltwater during the holding period and showed no signs of disease or stress.
- Photoperiod: 16 hours of light and 8 hours of dark, with a 30-minute transition period of low light intensity as lights were turned on and off.
- Temperature: 19.5 - 23.0 ˚C
- Salinity: 18 - 20‰
- pH: 8.0 - 8.6
- Dissolved xygen cencentration: 7.8 - 8.0 mg O2/L
- Type and amount of food: To supplement the diet of the oysters and enhance their condition and growth, the oysters were provided with an algal suspension of Thalassiosira sp. during holding and throughout the test.

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

22 ± 1 ˚C

pH:

8.0 - 8.4

Dissolved oxygen:

- 6.8 - 7.2 mg O2/L
- > 60% saturation

Salinity:

17 - 21‰

Nominal and measured concentrations:

- Nominal concentrations: 0 (negative control), 0 (solvent control), 0.80, 1.3, 2.2, 3.6, and 6.0 mg/L
- Measured concentrations:

Details on test conditions:

TEST SYSTEM
- Test vessel: 56 L polyethylene aquaria
- Filled volume: Approximately 13 L of test solution
- Depth: The depth of the water in each aquarium was approximately 7.4 cm.
- Type of flow-through: Peristaltic diluter
- Flow rate: 1 L of water/oyster/hour (equivalent to 334 mL/minute/chamber);
The delivery pump was calibrated before the test, and the delivery of test substance to test chambers was begun approximately 25 hours prior to test initiation in order to establish equilibrium concentrations of test substance. Immediately prior to test initiation, all new shell growth was removed from each oyster using a motorized grinder.
- No. of organisms per vessel: 20
- No. of vessels per concentration: 1
- No. of vessels per control: 1
- No. of vessels per vehicle control: 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used for holding, acclimation and testing of oysters was collected from the Indian River Inlet, Delaware. The seawater was stored in a 19,000 L tank, where it was aerated, and diluted with well water from the test facility to a salinity of approximately 20‰. All aspects of holding, acclimation and testing of oysters were conducted with approximately 20‰ salinity saltwater.
- Temperature: Temperature was measured continuously throughout the test period in the negative control. Temperature measurements
in each test chamber were made at the beginning and end of the test using a calibrated hand-held thermometer.
- Dissolved oxygen and pH: Measured in each test chamber at the beginning, midpoint and end of the test.
- Salinity: Salinity was measured in the negative control test chamber at the beginning, midpoint and end of the test.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours of light and 8 hours of dark, with a 30-minute transition period of low light intensity as lights were turned on and off.
- Light intensity: Approximately 258 lux at the surface of the water

EFFECT PARAMETERS MEASURED
Oysters were observed daily during the test for mortality and clinical signs of toxicity. At the end of the test, the longest finger of new shell growth on each oyster was measured to the nearest 0.05 mm.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

2.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: growth

Remarks on result:

other: 95% C.L: 19. - 3.0 mg/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: growth

Details on results:

An overview of the results is provided in Table 2 in 'Any other information on results incl. tables'.
- Mortality: No mortalities occurred during the test, and all oysters appeared normal.
- Shell growth: Oyster shell growth in the negative control averaged 4.4 mm over the 96-hour test period, while oyster shell growth in the solvent control group averaged 3.5 mm. A t-test comparison of the mean shell growth from the two control groups revealed a statistical difference (p > 0.05). Therefore, the measurements of the solvent control group were used to analyse the growth inhibition of the treatment groups. Oyster shell growth in the 0.66, 1.2, 1.9, 3.0, and 5.4 mg/L treatment groups averaged 3.5, 3.7, 3.9, 0.7, and 0.0 mm, respectively. Statistically significant shell growth inhibition was observed in the 3.0 and 5.4 mg/L treatment group, while no significant differences were observed in the 0.66, 1.9 and 1.2 mg/L treatment groups compared with the solvent control. Although slight inhibition was observed in the 0.66 mg/L treatment group, the effect was not considered to be treatment related.

Reported statistics and error estimates:

New shell growth was measured and the mean growth was calculated for each treatment group. The shell deposition measured in the negative and solvent control groups - were compared by a t-test, and found to be significantly different (p > 0.05). Due to statistical difference, the percent inhibition in shell growth was calculated for each treatment group as the percent reduction in shell growth relative to the growth found in the solvent control group.
A 96-hour EC50 value and 95% confidence limits were calculated using the computer program. The program was designed to calculate the EC50 and the 95% confidence limits by probit analysis, the moving average method, or binomial probability. In this study, the EC50 value was determined using the binomial probability method. The no observed effect concentration was determined by visual inspection of the growth data.

Table 2. 96-Hour Shell Deposition and Shell Growth Inhibition

Measured concentration (mg/L)	Shell Deposition (mm) Mean ± SD1	Shell Growth Inhibition, %^2
Negative control	4.445 ± 1.476	- 25.7
Solvent control	3.540 ± 1.222	N/A^3
0.66	3.533 ± 1.036	0.3
1.2	3. 723 ± 1.423	0.0
1.9	3 . 900 ± 1.309	0.0
3.0	0.715 ± 1.010	79.7
5.4	0.000 ± 0.000	100.0
96 hours EC50 = 2.6 mg/L

1 Standard Deviation; n = 20 oysters.

2 Shell growth inhibition compared to solvent controls.

3 N/A = Not Applicable.

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the 96-hour EC50 value for eastern oysters exposed to the test substance was determined to be 2.6 mg a.i./L, with 95% confidence limits of 1.9 - 3.0 mg a.i./L. The NOEC was 1.9 mg a.i./L.

Executive summary:

To determine the 96-hour acute toxicity of the test substance to the eastern oyster (Crassostrea virginica), the exposure was conducted under a flow through system following EPA 72-3 guideline. The study conducted in compliance with GLP criteria. The oysters were exposed to the test substance at nominal concentrations of 0.8, 1.3, 2.2, 3.6, and 6.0 mg a.i./L (mean measured concentration as 0.66, 1.2, 1.9, 3.0 and 5.4 mg a.i./L, respectively) for 96 hours. In addition, a negative control and solvent control (DMF 0.3 mL/L) were included in the test. One test chamber was maintained for each treatment level each containing 20 oysters. Prior to adding the oysters to the test chambers, all new shell growth was removed using a motorized grinder. The flow of unfiltered saltwater into each test chamber was approximately 1 L water/oyster/hour. Algal cells (Thalassiosira sp.) were provided to supplement naturally occurring algae, and to maximise oyster growth rates. Test chambers were held in a temperature-controlled bath at 22 ± 1 °C. Photoperiod was 16/8 light/dark with a 30-minute transition period (258 lux). During the test, the salinity, pH and dissolved oxygen remained at 17 - 21‰, 8.0 - 8.4 and > 60% saturation, respectively. Oysters were observed daily during the test for mortality and clinical signs of toxicity. At the end of the test the longest finger of new shell growth on each oyster was measured to the nearest 0.05 mm.

No mortalities occurred during the test, and all oysters appeared normal. Oyster shell growth in the negative control averaged 4.4 mm over the 96-hour test period, while oyster shell growth in the solvent control group averaged 3.5 mm. A t-test comparison of the mean shell growth from the two control groups revealed a statistical difference (p > 0.05). Therefore, the measurements of the solvent control group were used to analyse the growth inhibition of the treatment groups. Oyster shell growth in the 0.66, 1.2, 1.9, 3.0, and 5.4 mg/L treatment groups averaged 3.5, 3.7, 3.9, 0.7, and 0.0 mm, respectively. Statistically significant shell growth inhibition was observed in the 3.0 and 5.4 mg/L treatment group (79.7 and 100% inhibition, respectively), while no significant differences were observed in the 0.66, 1.9 and 1.2 mg/L treatment groups compared with the solvent control. Although slight inhibition was observed in the 0.66 mg/L treatment group (0.3%), the effect was not considered to be treatment related. Based on the findings, the 96-hour EC50 value for eastern oysters exposed to the test substance was determined to be 2.6 mg a.i./L. The NOEC was 1.9 mg a.i./L.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Jan to 14 Jan 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EPA OPP 72-2 (Aquatic Invertebrate Acute Toxicity Test)

Version / remarks:

October 1982

Deviations:

not specified

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Water samples were taken at 0-hour and 48-hour of the test for analysing the concentration of the test substance. The test substance primary stock standards of 1.04 mg/mL and 5.10 mg/mL in acetone were prepared, respectively and stored in a refrigerator. Subsequent dilutions of the 1.04 mg/mL stock solution were prepared in toluene for GLC chromatography standards. Both test substance primary stock standards were used as spiking solutions for the fortification of the quality control samples during the study. All standard preparations and dilutions were recorded.

Vehicle:

yes

Remarks:

Dimethylformamide (DMF)

Details on test solutions:

Test concentrations (prepared as serial dilutions) were corrected for sample purity. Dimethylformamide (DMF) was used in the preparation of primary (technical) standards used to spike working (test) standards. Daphnid test water was used for dilutions. The solvent control received an aliquot (0.50 mL/L rate) of DMF, equivalent to the solvent concentration of the highest test level. The 0.01 mg/mL working standard was prepared and alternatively stirred and sonicated for 26 hours until used in the preparation of test concentrations.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Resource: Cultured in well water at the test facility
- Feeding before test: Adult daphnids were fed algae (Selenastrum capricornutum) at least every three days prior to testing and supplemented with a suspension of cereal leaves, vitamin solution and yeast.
- Age at study initiation: First instar < 24 hours old

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

206 - 275 ppm as CaCO3

Test temperature:

20 ± 2 °C

pH:

8. 0 - 8. 3

Dissolved oxygen:

- 8.0 - 9.0 mg O2/L
- 92% - 102% at 20 °C

Conductivity:

500 - 650 µmhos/cm

Nominal and measured concentrations:

- Nominal concentrations: 0 (Negative control), 0 (solvent control), 5.6, 10,18, 32, 56 and 100 mg/L
- Measured concentrations: 0 (Negative control), 0 (solvent control), 4.6, 8.5, 15, 27, 44 and 77 mg/L, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass beakers
- Type: Closed (covered with a loose-fit ting petri dish cover to minimize evaporation and prevent contamination during the study)
- Fill volume: 200 mL of daphnid culture/test water
- 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

TEST MEDIUM / WATER PARAMETERS
- Intervals of water quality measurement: Water chemistry parameters of temperature, dissolved oxygen and pH were measured in replicate "A" of the control, solvent control, low, middle, and high concentrations at 0-hour and in replicate "B" in the controls and all treated concentrations at 48-hours.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness (with 30-minute simulated dawn and dusk periods)
- Light intensity: 60 - 70 footcandles

EFFECT PARAMETERS MEASURED
All concentrations were observed at 4 hours and once every 24 hours for immobility and abnormal effects such as surfacing, clumping of the daphnids together and daphnids tending to the bottom of test chambers.

RANGE-FINDING STUDY
- Comdition: Using five daphnids each in two exposure concentration series
- Test concentration series: 0.10, 1.0 and 10 mg/L, and 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: 5.6, 10,18, 32, 56 and 100 mg/L were selected as the test concentration for the definitive study.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

26 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

All results were based on the mean measured concentrations of 4.6, 8.5, 15, 27, 44 and 77 mg/L. An overview of the results is provided in Table 2 in 'Any other information on results incl. tables'.
- Test substance solubility: At 0 hour the 77 mg/L test concentration was observed to be a light tan-coloured solution. At 4. 24 and 48 hours this solution appeared clear. At 4 hours a slight precipitate was observed in the 77 mg/L test concentration. The precipitate was also observed in the 44 and 77 mg/L test concentrations at 24 hours and in all of the treated test concentrations at 48 hours.

- Immobility and abnormal effects: After 48 hours exposure, all test organisms in the negative control, solvent control and 4.6 mg/L treatment groups were able to move. The immobilisation of the test organism in 8.5, 15, 27, 44 and 77 mg/L treatments were 5, 20, 30, 90 and 100%, respectively. In addition, abnormal effects of immobility and/or daphnids tending to the bottom of test vessels were observed in the 8.5. 15. 27. 44 and 77 mg/L test concentrations. The NOEC based on the absence of immobility and abnormal effects was 4.6 mg/L after 48 hours. The 4-, 24- and 48-hour EC50 values were 62, 35 and 26 mg/L, respectively.

Reported statistics and error estimates:

Statistical analysis of the concentration vs. effect data (immobility) was obtained by employing a computerized LC50 (EC50) program. This program calculated the EC50 statistic and its 95% confidence limits using the binominal, the moving average and the probit tests if data permitted. 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 EC50 although all three models are valid. However, if no immobility occurred or if a dose response could not be demonstrated over a reasonable range (< 37 to > 63%) an Ec50 and/or its 95% confidence limits could not be calculated.

 Table 2. Percent Immobilization and Water Quality Measurements During the Acute Toxicity Test of the test substance to Daphnia magna

Mean measured concentration (mg/L)	Percent immobility (hours)			0 Hours (Replicate “A”)			48 hours (replicate “B”)
	4	24	48	Temperature (˚C)	Dissolved oxygen (mg O2/L)	pH	Temperature (˚C)	Dissolved oxygen (mg O2/L)	pH
Control	0	0	0	20	8.9	8.0	20	8.4	8.2
Solvent Control	0	0	0	20	9.0	8.1	20	8.3	8.3
4.6	0	0	0	20	8.8	8.1	20	8.3	8.3
8.5	0	0	5				20	8.4	8.3
15	0	0	20				20	8.3	8.3
27	0	20	30	20	8.5	8.2	20	8.4	8.2
44	0	75	90				20	8.3	8.2
17	90	100	100	20	8.0	8.1	20	8.4	8.2

 

 

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the 48-hour EC50 value was determined to be 26 mg/L.

Executive summary:

The acute toxicity of the test substance to < 24 hours old daphnids (Daphnia magna) was studied in a 48-hour static test. The test was performed in accordance with EPA 72-2 guideline and was in compliance with GLP criteria. 20 daphnids per test concentration (10 per vessel were exposed to mean measured test concentrations of 4.6, 8.5, 15, 27, 44 and 77 mg/L (measured by GLC; the nominal concentrations were 5.6, 10,18, 32, 56 and 100 mg/L, respectively). In addition, a negative and a solvent (0.50 mL/L rate DMF) control groups were tested. The test conditions were: pH 8.0 - 8.3, dissolved oxygen concentration 8.0 – 9.0 mg/L and 20 ± 2°C. All water parameters in this study were measured at 0-hour and 48-hour of the test. All concentrations were observed at 4 hours and once every 24 hours for immobility and abnormal effects such as surfacing, clumping of the daphnids together and daphnids tending to the bottom of test chambers.

After 48 hours of exposure, all test organisms in the negative control, solvent control and 4.6 mg/L treatment groups were able to move. The immobilisation of the test organism in 8.5, 15, 27, 44 and 77 mg/L treatments were 5, 20, 30, 90 and 100%, respectively. In addition, abnormal effects of immobility and/or daphnids tending to the bottom of test vessels were observed in the 8.5. 15. 27. 44 and 77 mg/L test concentrations. Based on these findings, the 48-h EC50 was determined to be 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. Other studies are included as supporting information.

Freshwater 48-h EC50 = 26 mg/L, static, Daphnia magna, immobility, EPA 72 -2; Frazier 1988

Marine water, 96-h LC50 = 2.6 mg/L, flow-through, Crassostrea virginica, growth, EPA 72-3, Sved et al. 1993

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

26 mg/L

Marine water invertebrates

Marine water invertebrates

Dose descriptor:

LC50

Effect concentration:

2.6 mg/L

Additional information

Freshwater:

In the freshwater key study, the acute toxicity of the test substance to < 24 hours old daphnids (Daphnia magna) was studied in a 48-hour static test. The test was performed in accordance with EPA 72-2 guideline and was in compliance with GLP criteria. 20 daphnids per test concentration (10 per vessel were exposed to mean measured test concentrations of 4.6, 8.5, 15, 27, 44 and 77 mg/L (measured by GLC; the nominal concentrations were 5.6, 10,18, 32, 56 and 100 mg/L, respectively). In addition, a negative and a solvent (0.50 mL/L rate DMF) control groups were tested. The test conditions were: pH 8.0 - 8.3, dissolved oxygen concentration 8.0 – 9.0 mg/L and 20 ± 2°C. All water parameters in this study were measured at 0-hour and 48-hour of the test. All concentrations were observed at 4 hours and once every 24 hours for immobility and abnormal effects such as surfacing, clumping of the daphnids together and daphnids tending to the bottom of test chambers.

After 48 hours of exposure, all test organisms in the negative control, solvent control and 4.6 mg/L treatment groups were able to move. The immobilisation of the test organism in 8.5, 15, 27, 44 and 77 mg/L treatments were 5, 20, 30, 90 and 100%, respectively. In addition, abnormal effects of immobility and/or daphnids tending to the bottom of test vessels were observed in the 8.5. 15. 27. 44 and 77 mg/L test concentrations. Based on these findings, the 48-h EC50 was determined to be 26 mg/L.

Marine water:

To determine the 96-hour acute toxicity of the test substance to the eastern oyster (Crassostrea virginica), the exposure was conducted under a flow through system following EPA 72-3 guideline. The study conducted in compliance with GLP criteria. The oysters were exposed to the test substance at nominal concentrations of 0.8, 1.3, 2.2, 3.6, and 6.0 mg a.i./L (mean measured concentration as 0.66, 1.2, 1.9, 3.0 and 5.4 mg a.i./L, respectively) for 96 hours. In addition, a negative control and solvent control (DMF 0.3 mL/L) were included in the test. One test chamber was maintained for each treatment level each containing 20 oysters. Prior to adding the oysters to the test chambers, all new shell growth was removed using a motorized grinder. The flow of unfiltered saltwater into each test chamber was approximately 1 L water/oyster/hour. Algal cells (Thalassiosira sp.) were provided to supplement naturally occurring algae, and to maximise oyster growth rates. Test chambers were held in a temperature-controlled bath at 22 ± 1 °C. Photoperiod was 16/8 light/dark with a 30-minute transition period (258 lux). During the test, the salinity, pH and dissolved oxygen remained at 17 - 21‰, 8.0 - 8.4 and > 60% saturation, respectively. Oysters were observed daily during the test for mortality and clinical signs of toxicity. At the end of the test the longest finger of new shell growth on each oyster was measured to the nearest 0.05 mm.

No mortalities occurred during the test, and all oysters appeared normal. Oyster shell growth in the negative control averaged 4.4 mm over the 96-hour test period, while oyster shell growth in the solvent control group averaged 3.5 mm. A t-test comparison of the mean shell growth from the two control groups revealed a statistical difference (p > 0.05). Therefore, the measurements of the solvent control group were used to analyse the growth inhibition of the treatment groups. Oyster shell growth in the 0.66, 1.2, 1.9, 3.0, and 5.4 mg/L treatment groups averaged 3.5, 3.7, 3.9, 0.7, and 0.0 mm, respectively. Statistically significant shell growth inhibition was observed in the 3.0 and 5.4 mg/L treatment group (79.7 and 100% inhibition, respectively), while no significant differences were observed in the 0.66, 1.9 and 1.2 mg/L treatment groups compared with the solvent control. Although slight inhibition was observed in the 0.66 mg/L treatment group (0.3%), the effect was not considered to be treatment related. Based on the findings, the 96-hour EC50 value for eastern oysters exposed to the test substance was determined to be 2.6 mg a.i./L. The NOEC was 1.9 mg a.i./L.