N-[[3,5-dichloro-2-fluoro-4-(1,1,2,3,3,3-hexafluoropropoxy)phenyl]carbamoyl]-2,6-difluorobenzamide

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:

01 May 2000 - 05 May 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 72-1 (Fish Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: U.S. EPA - FIFRA Standard Evaluation Procedure 540/9-85-006

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Test solutions were analysed on days 0, 2 and 4 of the study. Samples were obtained from each water control, solvent control and test solution test vessel on day 0, from each spent control, spent test, renewed control and renewed test solution test vessel on day 2 and from each spent control and test solution test vessel on day 4.
2.0 mL aliquots were collected from each solution. The aliquots were transferred to 4.0 mL autosampler vials and diluted with 2.0 mL acetonitrile, then were mixed thoroughly for analysis by HPLC/UV.
To define method precision, three additional samples were taken on day 0 from a representative 2.00 mg/L test solution (replicate A). These additional samples were collected, diluted, and analysed along with the other day 0 samples.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A test solution with a nominal concentration of 2.00 mg/L LDW was prepared in triplicate. A dosing stock solution with a concentration of 4.00 mg/mL acetone was prepared by adding approximately 80 mg of the test material (after adjustment for purity) into 20 mL of acetone. Five mL of the dosing stock was then added to each of 3 replicate test vessels filled with 10 L of LDW and stirred vigorously to prepare the 2.00 mg test solutions. In addition, three test aquaria each of control LDW and 0.5 mL acetone/L dosed control LDW (solvent control) were prepared.
Test solutions were renewed after 48 hours of exposure (day 2) and prepared at the same nominal concentrations as day 0 as described above.

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

TEST ORGANISM
- Common name: Bluegill sunfish
- Strain: Lepomis macrochirus Rafinesque
- Length at study initiation: Fish averaging 21 ± 2 mm in length (post-exposure) were used as test organisms. The length of the largest control fish was no more than twice that of the shortest fish.
- Weight at study initiation: Fish averaged 172 ± 68 mg in mass (post-exposure)
- Age at study initiation: Juvenile
- Feeding during test: No; fish were held without food for at least 48 h before testing

ACCLIMATION
- Acclimation period: At least 14 days before testing
- Acclimation conditions (same as test or not): All fish were held in a 230 L tank on a 16 h light / 8h dark transitional photoperiod and observed. The test lot of fish used for the definitive study was acclimated to 22 ± 1 °C for at least 7 days prior to test initiation. Temperature changes did not exceed 3 °C in the 72 hours prior to experimental start.
- Type and amount of food: Standard diet
- Feeding frequency: At least once daily
- Health during acclimation (any mortality observed): Mortality did not exceed 3 percent of the population in the 48 h period before testing.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

58 mg CaCO3/L

Test temperature:

21.2 - 22.4 °C

pH:

6.3 - 7.7

Dissolved oxygen:

3.5 - 8.3 mg/L. Percent oxygen saturation averaged 79 %.

Nominal and measured concentrations:

- Nominal: 2 mg/L
- Measured: 1.63 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Borosilicate glass beakers. Each beaker contained a screened insert.
- Type (delete if not applicable): Closed; each beaker was loosely covered with a glass plate.
- Fill volume: 12 L beakers. Each beaker/insert combination provided a test volume of approximately 10 L.
- Aeration: Dissolved oxygen levels averaged 79 % of saturation and remained greater than or equal to 41 % over the 96 hour exposure period. Dissolved oxygen levels decreased to 41 % of saturation on day 2 of the study prior to the renewal of the test solutions (no corrective action conducted). However, on day 3 (day after renewal of test solutions) of the study, the dissolved oxygen levels again decreased, and were as low as 47 % saturation. Therefore, aeration was necessary to maintain the required dissolved oxygen level for the remainder of the study and was provided via a glass capillary tube to each test vessel starting on day 3.
- Renewal rate of test solution (frequency/flow rate): Test solutions were renewed after 48 hours of exposure (day 2) at the same nominal concentrations as day 0. The screened inserts within each test vessel containing the fish were transferred from day 2 spent solutions to day 2 renewed solutions.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3
- Biomass loading rate: 0.8 mg fish/L of test solution

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The laboratory dilution water (LDW) was Lake Huron water supplied by the city Water Treatment Plant. The water was obtained from the upper Saginaw Bay of Lake Huron near Whitestone Point and was limed and flocculated with ferric chloride. The water was pumped to the laboratory prior to treatment for municipal use. Before use in the laboratory, the water was sand-filtered, pH-adjusted with gaseous CO₂, carbon-filtered, and UV-irradiated.
- Total organic carbon: 1633 ng/mL
- TSS: not detected at levels greater than 1000 ng/mL
- Chlorine: 30 ppb
- Alkalinity: 26 mg CaCO₃/L
- Conductivity: 101 µmho/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: Alkalinity, conductivity, pH, and hardness are monitored weekly. Total organic carbon (TOC), total suspended solids (TSS), and selected inorganic and organic compounds are monitored biannually.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light/8 h dark transitional photoperiod was provided using cool-white fluorescent lighting and an automatic timer.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
The fish were observed and mortality and sub-lethal effects recorded at 24, 48, 72, and 96 hours. Mortality was defined as a lack of response to prodding of the caudal peduncle accompanied by an absence of opercular movement. Behavioural effects were noted and reported, e.g., swimming at the surface, complete or partial loss of body equilibrium, lethargy, or hyperactivity. The presence of gross pathological conditions was also noted and reported, e.g., exophthalmia (bulging eyes), ascites (fluid accumulation in the abdomen), haemorrhaging (discharge of blood), excess mucus, sloughing of epidermis, and melanosis (increased amount of dark pigmentation). Dead fish were removed when observed.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: None (limit test)
- Range finding study: Yes
- Test concentrations: 0.785, 3.13, 12.5 and 25.0 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 1.63 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 1.63 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Details on results:

Mortality was observed in 3 % (1/30) of the fish in the water control vessels following 48 hours of exposure. Complete loss of equilibrium was observed in 3 % (1/30) of the fish at the 2.00 mg/L dose level following 48 hours of exposure and that fish was dead following 72 hours of exposure. No other mortalities or sub-lethal effects were observed during the conduct of the study.

Reported statistics and error estimates:

Based on the design of the study (limit test), no statistical analysis programs were used for the data analysis and the statistical determination of a NOEC value was not attempted.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 96 h LC50 value is >2.00 mg/L (equivalent to a mean measured concentration of 1.63 mg/L). The NOEC is taken to be ≥2.00 mg/L, nominal (≥1.63 mg/L, measured).

Executive summary:

The acute toxicity potential of the test material was investigated under GLP conditions and in accordance with the standardised guidelines OECD 201, EU method C.1, EPA OPP 72 -1 and U.S. EPA - FIFRA Standard Evaluation Procedure 540/9-85-006.

Bluegill sunfish, Lepomis macrochirus Rafinesque, were exposed to the test material over a 96 hour (4 day) period. The study was conducted as a limit test under static-renewal conditions with test solutions prepared on day 0 at a target concentration of 2.00 mg/L and renewed on day 2. Fish were also exposed to a water control and a 0.5 mL acetone/L dosed control Laboratory Dilution Water-solvent control.

Thirty fish were exposed per dose level in 3 replicates per concentration with 10 fish per replicate. Collected samples were analysed by high performance liquid chromatography with ultraviolet absorbance detection (HPLC/UV) on days 0, 2 (from spent and renewed solutions), and 4 of the study. The fish were observed and mortality and sub-lethal effects recorded at 24, 48, 72, and 96 hours of exposure.

Mortality was observed in 3 % (1/30) of the fish in the water control vessels following 48 hours of exposure. Complete loss of equilibrium was observed in 3 % (1/30) of the fish at the 2.00 mg/L dose level following 48 hours of exposure and that fish was dead following 72 hours of exposure. No other mortalities or sub-lethal effects were observed during the conduct of the study.

Under the conditions of this study, the 96 h LC50 value is >2.00 mg/L (equivalent to a mean measured concentration of 1.63 mg/L). The NOEC is taken to be ≥2.00 mg/L, nominal (≥1.63 mg/L, measured).

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 April 2000- 14 April 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 72-1 (Fish Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: U.S. EPA - FIFRA Standard Evaluation Procedure 540/9-85-006

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Test solutions were analysed on days 0, 2 and 4 of the study. Samples were obtained from each water control, solvent control and test solution test vessel on day 0, from each spent control, spent test, renewed control and renewed test solution test vessel on day 2 and from each spent control and test solution test vessel on day 4.
2.0 mL aliquots were collected from each solution. The aliquots were transferred to 4.0 mL autosampler vials and diluted with 2.0 mL acetonitrile, then were mixed thoroughly for analysis by HPLC/UV.
To define method precision, three additional samples were taken on day 0 from a representative 2.00 mg/L test solution (replicate A). These additional samples were collected, diluted, and analysed along with the other day 0 samples.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A test solution with a nominal concentration of 2.00 mg/L LDW was prepared in triplicate. A dosing stock solution with a concentration of 4.00 mg/mL acetone was prepared by adding approximately 80 mg of the test material (after adjustment for purity) into 20 mL of acetone. Five mL of the dosing stock was then added to each of 3 replicate test vessels filled with 10 L of LDW and stirred vigorously to prepare the 2.00 mg test solutions. In addition, three test aquaria each of control LDW and 0.5 mL acetone/L dosed control LDW (solvent control) were prepared.
Test solutions were renewed after 48 hours of exposure (day 2) and prepared at the same nominal concentrations as day 0 as described above.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Strain: Oncorhynchus mykiss Walbaum
- Length at study initiation: Fish averaging 33 ± 3 mm in length (post-exposure) were used as test organisms. The length of the largest control fish was no more than twice that of the shortest fish.
- Weight at study initiation: Fish averaged 500 ± 110 mg in mass (post-exposure)
- Method of breeding: The fish were obtained as “green” eggs and milt. Upon arrival, the eggs were fertilised according to the supplier’s directions and then placed in a flow-through hatcher in which they were incubated until emergent juveniles were transferred to a 230 L holding tank.
- Feeding during test: No; fish were held without food for at least 48 h before testing

ACCLIMATION
- Acclimation period: At least 14 days before testing
- Acclimation conditions (same as test or not): All fish were held on a 16 h light / 8h dark transitional photoperiod and observed. The test lot of fish used for the definitive study was acclimated to 12.0 ± 1 °C. Temperature changes did not exceed 3 °C in the 72 hours prior to experimental start.
- Type and amount of food: Standard diet
- Feeding frequency: At least once daily
- Health during acclimation (any mortality observed): Mortality did not exceed 3 percent of the population in the 48 h period before testing.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

62 mg CaCO3/L

Test temperature:

12.1 - 13.0 °C

pH:

6.6 - 7.3

Dissolved oxygen:

7.0 - 12.9 mg/L. Percent oxygen saturation averaged 91 %.

Nominal and measured concentrations:

- Nominal: 2 mg/L
- Measured: 1.77 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Borosilicate glass beakers. Each beaker contained a screened insert.
- Type (delete if not applicable): Closed; each beaker was loosely covered with a glass plate.
- Fill volume: 12 L beakers. Each beaker/insert combination provided a test volume of approximately 10 L.
- Aeration: The loading rate resulted in dissolved oxygen levels that were equal to or greater than 67 % of saturation throughout the exposure period; aeration was not required.
- Renewal rate of test solution (frequency/flow rate): Test solutions were renewed after 48 hours of exposure (day 2) at the same nominal concentrations as day 0. The screened inserts within each test vessel containing the fish were transferred from day 2 spent solutions to day 2 renewed solutions.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3
- Biomass loading rate: 0.8 mg fish/L of test solution

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The laboratory dilution water (LDW) was Lake Huron water supplied by the city Water Treatment Plant. The water was obtained from the upper Saginaw Bay of Lake Huron near Whitestone Point and was limed and flocculated with ferric chloride. The water was pumped to the laboratory prior to treatment for municipal use. Before use in the laboratory, the water was sand-filtered, pH-adjusted with gaseous CO₂, carbon-filtered, and UV-irradiated.
- Total organic carbon: 1633 ng/mL
- TSS: not detected at levels greater than 1000 ng/mL
- Chlorine: 17 ppb
- Alkalinity: 30 mg CaCO₃/L
- Conductivity: 127 µmho/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: Alkalinity, conductivity, pH, and hardness are monitored weekly. Total organic carbon (TOC), total suspended solids (TSS), and selected inorganic and organic compounds are monitored biannually.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light/8 h dark transitional photoperiod was provided using cool-white fluorescent lighting and an automatic timer.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
The fish were observed and mortality and sub-lethal effects recorded at 24, 48, 72, and 96 hours. Mortality was defined as a lack of response to prodding of the caudal peduncle accompanied by an absence of opercular movement. Behavioural effects were noted and reported, e.g., swimming at the surface, complete or partial loss of body equilibrium, lethargy, or hyperactivity. The presence of gross pathological conditions was also noted and reported, e.g., exophthalmia (bulging eyes), ascites (fluid accumulation in the abdomen), haemorrhaging (discharge of blood), excess mucus, sloughing of epidermis, and melanosis (increased amount of dark pigmentation). Dead fish were removed when observed.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: None (limit test)
- Range finding study: Yes
- Test concentrations: 0.785, 3.13, 12.5 and 25.0 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 1.77 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 1.77 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Details on results:

Mortality was observed in 3 % (1/30) of the fish at the 2.00 mg/L (1.77 mg/L, measured) dose level following 72 hours of exposure. Mortality was observed in 3 % (1/30) of the fish in one of the solvent control test vessels following 96 hours of exposure. No other mortalities were observed during the conduct of the study.
Lethargy was observed in 13 % (4/30), 7 % (2/30), and 20 % (6/30) of the fish at the LDW control, solvent control, and 2.00 mg/L (1.77 mg/L, measured) dose levels, respectively, following 96 hours of exposure; this was the only sub-lethal effect observed during the conduct of the study.

Reported statistics and error estimates:

Based on the design of the study (limit test), no statistical analysis programs were used for the data analysis and the statistical determination of a NOEC value was not attempted.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 96 h LC50 value is >2.00 mg/L (equivalent to a mean measured concentration of 1.77 mg/L). The NOEC is taken to be ≥2.00 mg/L, nominal (≥1.77 mg/L, measured).

Executive summary:

The acute toxicity of the test material to fish was investigated under GLP conditions and in accordance with the standardised guidelines OECD 201, EU method C.1, EPA OPP 72 -1 and U.S. EPA - FIFRA Standard Evaluation Procedure 540/9-85-006.

Rainbow trout, Oncorhynchus mykiss Walbaum, were exposed to the test material over a 96 hour (4 day) period. The study was conducted as a limit test under static-renewal conditions with test solutions prepared on day 0 at a target concentration of 2.00 mg/L and renewed on day 2. Fish were also exposed to a water control and a 0.5 mL acetone/L dosed control Laboratory Dilution Water-solvent control.

Thirty fish were exposed per dose level in 3 replicates per concentration with 10 fish per replicate. Collected samples were analysed by high performance liquid chromatography with ultraviolet absorbance detection (HPLC/UV) on days 0, 2 (from spent and renewed solutions), and 4 of the study. The fish were observed and mortality and sub-lethal effects recorded at 24, 48, 72, and 96 hours of exposure.

Mortality was observed in 3 % (1/30) of the fish at the 2.00 mg/L (1.77 mg/L, measured) dose level following 72 hours of exposure. Mortality was observed in 3 % (1/30) of the fish in one of the solvent control test vessels following 96 hours of exposure. No other mortalities were observed during the conduct of the study. Lethargy was observed in 13 % (4/30), 7 % (2/30), and 20 % (6/30) of the fish at the LDW control, solvent control, and 2.00 mg/L (1.77 mg/L, measured) dose levels, respectively, following 96 hours of exposure; this was the only sub-lethal effect observed.

Under the conditions of this study, the 96 h LC50 value is >2.00 mg/L (equivalent to a mean measured concentration of 1.77 mg/L). The NOEC is taken to be ≥2.00 mg/L, nominal (≥1.77 mg/L, measured).

Description of key information

In Lepomis macrochirus the 96 h LC50 value was >2.00 mg/L (equivalent to a mean measured concentration of 1.63 mg/L). The NOEC was taken to be ≥2.00 mg/L, nominal (≥1.63 mg/L, measured).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

1.63 mg/L

Additional information

Three studies are provided to address this endpoint, two key and one supporting. The two key studies were conducted in the standard species Oncorhynchus mykiss and Lepomis macrochirus. Both were awarded a reliability score of 1 in line with the criteria set forth by Klimisch et al. (1997). The supporting study was awarded a reliability score of 2 in accordance with the same criteria.

In the first key study, the acute toxicity potential of the test material was investigated under GLP conditions and in accordance with the standardised guidelines OECD 201, EU method C.1, EPA OPP 72 -1 and U.S. EPA - FIFRA Standard Evaluation Procedure 540/9-85-006.

Rainbow trout, Oncorhynchus mykiss Walbaum, were exposed to the test material over a 96 hour (4 day) period. The study was conducted as a limit test under static-renewal conditions with test solutions prepared on day 0 at a target concentration of 2.00 mg/L and renewed on day 2. Fish were also exposed to a water control and a 0.5 mL acetone/L dosed control Laboratory Dilution Water-solvent control. Thirty fish were exposed per dose level in 3 replicates per concentration with 10 fish per replicate. Collected samples were analysed by high performance liquid chromatography with ultraviolet absorbance detection (HPLC/UV) on days 0, 2 (from spent and renewed solutions), and 4 of the study. The fish were observed and mortality and sub-lethal effects recorded at 24, 48, 72, and 96 hours of exposure.

Mortality was observed in 3 % (1/30) of the fish at the 2.00 mg/L (1.77 mg/L, measured) dose level following 72 hours of exposure. Mortality was observed in 3 % (1/30) of the fish in one of the solvent control test vessels following 96 hours of exposure. No other mortalities were observed during the conduct of the study.

Lethargy was observed in 13 % (4/30), 7 % (2/30), and 20 % (6/30) of the fish at the LDW control, solvent control, and 2.00 mg/L (1.77 mg/L, measured) dose levels, respectively, following 96 hours of exposure; this was the only sub-lethal effect observed.

Under the conditions of this study, the 96 h LC50 value is >2.00 mg/L (equivalent to a mean measured concentration of 1.77 mg/L). The NOEC is taken to be ≥2.00 mg/L, nominal (≥1.77 mg/L, measured).

In the second key study, the acute toxicity potential of the test material was investigated under GLP conditions and in accordance with the standardised guidelines OECD 201, EU method C.1, EPA OPP 72 -1 and U.S. EPA - FIFRA Standard Evaluation Procedure 540/9-85-006.

Bluegill sunfish, Lepomis macrochirus Rafinesque, were exposed to the test material over a 96 hour (4 day) period. The study was conducted as a limit test under static-renewal conditions with test solutions prepared on day 0 at a target concentration of 2.00 mg/L and renewed on day 2. Fish were also exposed to a water control and a 0.5 mL acetone/L dosed control Laboratory Dilution Water-solvent control. Thirty fish were exposed per dose level in 3 replicates per concentration with 10 fish per replicate. Collected samples were analysed by high performance liquid chromatography with ultraviolet absorbance detection (HPLC/UV) on days 0, 2 (from spent and renewed solutions), and 4 of the study. The fish were observed and mortality and sub-lethal effects recorded at 24, 48, 72, and 96 hours of exposure.

Mortality was observed in 3 % (1/30) of the fish in the water control vessels following 48 hours of exposure. Complete loss of equilibrium was observed in 3 % (1/30) of the fish at the 2.00 mg/L dose level following 48 hours of exposure and that fish was dead following 72 hours of exposure. No other mortalities or sub-lethal effects were observed during the conduct of the study.

Under the conditions of this study, the 96 h LC50 value is >2.00 mg/L (equivalent to a mean measured concentration of 1.63 mg/L). The NOEC is taken to be ≥2.00 mg/L, nominal (≥1.63 mg/L, measured).

The value for the Bluegill sunfish was used as the key one to be taken forward for risk assessment purely on the basis that the measured concentration was slightly lower than that in the test with rainbow trout; this therefore represents the worst case scenario.

In the supporting study, the acute toxicity potential of the test material was investigated in accordance with a slightly modified method of the American Society for Testing and Materials, ASTM Standard E729-88a, Standard Guide For Conducting Acute Toxicity Tests With Fishes, Macroinvertebrates and Amphibians, Philadelphia, PA, 1989.

Rainbow trout, Oncorhynchus mykiss Walbaum, were exposed to the test material over a 96 hour period under static conditions at nominal concentrations of 0.5, 1, 5, 10, 50 and 100 mg/L. Fish were also exposed to a water control and a 0.5 mL acetone/L solvent control. 10 fish were exposed per dose level in 2 replicates per concentration with 5 fish per replicate. The fish were observed and mortality and sub-lethal effects recorded at 6, 24, 48, 72, and 96 hours of exposure.

At 72 hours, one control fish was found dead as were two fish in the 50 mg/L dose group. After 96 hours, one fish from the 10 mg/L dose group and one form the 100 mg/L dose group were also found to be dead.

Under the conditions of this study, the 96 hour LC50 value for the test material in rainbow trout was greater than the nominal concentration of 100 mg/L.