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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 November 2000 - 17 November 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: U.S. EPA - FIFRA Standard Evaluation Procedure 540/9-85-005

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

All test and control solutions were sampled and analysed to determine test material concentrations on days 0 and 2 of the study by extracting the aqueous solutions with toluene (containing an internal standard to facilitate quantitation).
40 mL aliquots were collected from each test vessel following a diluter recycle. The aliquots were transferred to 11 dram vials and acidified with 40 µL acetic acid. The samples were extracted with 1 mL of toluene containing 504 µg/L of the internal standard. After the addition of the extraction solvent, the samples were shaken for 1 hour on a mechanical flatbed shaker at low speed (~180 oscillations per minute). The samples were then centrifuged for 5 minutes to produce a phase separation of the organic and aqueous layers. Then, the organic (top) layer of each sample was transferred to a high-recovery autosampler vial and evaporated to dryness under a stream of nitrogen. The samples were then reconstituted with 250 µL of 50/50 acetonitrile/water, vortexed for 30 seconds, and analysed by LC/NESI-MS.
On day 0 of the study, three additional aliquots were collected from test vessels #1A (800 ng/L target level) and #5A (104 ng/L target level). Those additional samples were collected, extracted, and analysed along with the other day 0 samples in order to determine method precision.
The contents of the mixing chamber were also analysed on days 0 and 2 by taking a 40 mL sample at the end of the mixing cycle as the contents of the chamber was being dumped to the series of cascading diluter vessels.
Aliquots (20 µL) of the primary dosing feedstock supplying the diluter (prepared at a nominal concentration of 9495 ng test material/mL of acetone) were sampled on days 0 and 2 of the study, diluted with extraction solvent (500:1), evaporated to dryness, reconstituted with 50/50 acetonitrile/water and analysed along with the other samples.

Vehicle:

yes

Details on test solutions:

The test beakers were replenished on a regular intermittent cycle with test solutions delivered from a flow-through proportional diluter, which was fortified from a primary feedstock prepared at a nominal concentration of 9495 ng test material/mL of acetone.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain: Daphnia magna Straus
- Source: In-house cultures
- Age at study initiation: Daphnia instars less than 24-hours old from a laboratory-reared culture were used as the test organism. The day before instars were needed for testing, stock tanks with daphnids greater than 14 days old which had had at least four broods were removed from the incubator. The instars were separated from adults by gently lifting the screened insert from the 2 L stock tank, releasing instars through the nylon mesh screen while retaining the adult daphnids. The screened insert, with adult daphnids, was then placed in another stock tank that contained daphnid water. The original solution with instars was poured through a metal sieve into another stock tank. The instars collected on the sieve were discarded and the original solution was poured back into the initial stock tank and the corresponding screened insert holding adult daphnids was put back in place. This procedure was repeated the day the study was set to cull <24-hour old instars for use in the study.
- Feeding during test: No

ACCLIMATION
- Acclimation conditions (same as test or not): Yes. Rearing conditions were as follows: illumination (cool-white fluorescent) 2050 ± 350 lux; 16 hour light/8 hour dark photoperiod; temperature 20 ±2 °C.
- Type and amount of food: A mixed diet of Selenastrum capricornutum (algae) and YCT (yeast-ceraphyll trout) trout chow
- Feeding frequency: Five times weekly

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

62 mg CaCO3/L

Test temperature:

19.4 - 20.1 °C

pH:

6.9 - 7.2

Dissolved oxygen:

8.5 - 9.7 mg/L. Percent oxygen saturation averaged 102 %.

Nominal and measured concentrations:

- Nominal: 62.2, 104, 173, 288, 480, and 800 ng/L
- Measured: 78.6, 120, 175, 239, 443, and 884 ng/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers with 7.9 x 14 cm glass inserts. The inserts were open on the upper end and equipped with nylon screens on the lower end such that the screens would rest approximately 1 cm above the bottom of the beaker upon installation of the inserts. The pore size of the mesh screens (80 µm) was small enough to prevent passage of the daphnids.
- Volume: Vessel volume 600 mL; fill volume 450 mL
- Type of flow-through (e.g. peristaltic or proportional diluter): An intermittent-flow proportional diluter system was used to maintain constant exposure concentrations during the 2 day study period. The system is designed to deliver up to six test concentrations, a vehicle control and a water control.
- Renewal rate of test solution (frequency/flow rate): Average cycles per day: 52; average volume per cycle: 100 mL; average turnovers per day: 11.6.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2

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 municipal treatment for human use. Before use in the laboratory, the water was sand-filtered, pH-adjusted with gaseous CO₂, carbon-filtered, and UV-irradiated.
- Total organic carbon: <1000 ng/mL
- TSS: not detected at levels greater than 1000 ng/mL
- Chlorine: 30 ppb
- Alkalinity: 30 mg CaCO₃/L
- Conductivity: 66.3 µmho/cm
- Culture medium different from test medium: Yes. Daphnid culturing water was prepared by adjusting laboratory water to a hardness of about 170 mg/L as CaCO3 before autoclaving. After adjusting hardness, the water was autoclaved at 250 °C and 18 psi for 30 minutes, cooled, and aerated for approximately 24 hours before use.
- 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 at the test lab biannually.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Diluter and laboratory lighting provided a 16-hour light/8-hour dark transitional photoperiod during testing.
- Light intensity: 1258 - 1491 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
Daphnids were observed for immobility (inability to swim within 15 seconds after gentle agitation of the test container) after 24 and 48 hours of exposure. Dissolved oxygen, pH, and temperature data were recorded from each bulk-dose solution at test initiation (0 hours) and from all test vessels (spent test solutions) at 48 hours

TEST CONCENTRATIONS
- Spacing factor for test concentrations: The diluter was calibrated so that the concentration of the test material in each treatment below the high concentration was approximately 60 percent of that in the next higher treatment level.
- Range finding study: Yes
- Test concentrations: 38.9, 64.8, 108, 180, 300, and 500 ng/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

311 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % CI 242 to 400 ng/L

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

175 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Following 24 hours of exposure, immobility was observed in 30 % (6/20) and 60 % (12/20) of the daphnids at the 443 and 884 ng/L exposure levels, respectively. Following 48-hours of exposure, immobility was observed in 10 % (2/20), 5 % (1/20), 10 % (2/20), 10 % (2/20), 60 % (12/20), 70 % (14/20), and 90 % (18/20) of the daphnids at the solvent control, 78.6, 120, 175, 239, 443, and 884 ng/L exposure levels, respectively.
No daphnid immobility was observed in the water control vessels at any time during the conduct of this study.
The 24-hour EC50 value was 708 ng/L, with a 95 % confidence interval of 570 to 983 ng/L and a slope of 4.1 (95 % confidence interval of 2.4 – 5.8). The 48-hour EC50 value was 311 ng/L, with a 95 % confidence interval of 242 to 400 ng/L and a slope of 3.3 (95 % confidence interval of 2.1 – 4.5).
The no-observed effect concentration or NOEC was calculated using the Fishers Exact Probability Test and determined to be 175 ng/L. When compared to controls, no statistically significant (α = 0.01) effects were observed in this study up through the 175 ng/L dose level.

Reported statistics and error estimates:

The U.S. EPA Probit Program, Version 1.5, was used to calculate the 24- and 48-hour EC50 values and confidence intervals. The NOEC was calculated using the Fishers Exact Probability Test (α = 0.01). All values were obtained using analytically verified concentrations of the test material.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 48-hour EC50 value was 311 ng/L (95 % confidence interval of 242 to 400 ng/L). The no-observed effect concentration or NOEC was determined to be 175 ng/L.

Executive summary:

A study was conducted to evaluate the acute toxicity potential of the test material to the invertebrate Daphnia magna Straus. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 202, EU Method C.2, EPA OPP 72-2 and U.S. EPA - FIFRA Standard Evaluation Procedure 540/9-85-005.

The study was conducted as a 48-hour flow-through test. Twenty daphnids (10 individuals/replicate, 2 replicates per dose level) were exposed to nominal concentrations of 0 (Laboratory Dilution Water control), 0 (84 µL/L acetone-dosed LDW-solvent control), 62.2, 104, 173, 288, 480, and 800 ng/L-LDW, over a 48-hour exposure period at 20 ± 1 °C. The test solutions were sampled and analysed to determine concentrations on days 0 and 2 of the study by liquid chromatography with negative electrospray ionisation-mass spectrometry (LC/NESI-MS). This resulted in mean measured concentrations of less than 50 ng/L, which was the lowest level quantified (LLQ), for the water and solvent controls, 78.6, 120, 175, 239, 443, and 884 ng/L.

Following 24 hours of exposure, immobility was observed in 30 % (6/20) and 60 % (12/20) of the daphnids at the 443 and 884 ng/L exposure levels, respectively. Following 48-hours of exposure, immobility was observed in 10 % (2/20), 5 % (1/20), 10 % (2/20), 10 % (2/20), 60 % (12/20), 70 % (14/20), and 90 % (18/20) of the daphnids at the solvent control, 78.6, 120, 175, 239, 443, and 884 ng/L exposure levels, respectively. No daphnid immobility was observed in the water control vessels at any time during the conduct of this study.

Under the conditions of this study, the 48-hour EC50 value was 311 ng/L (95 % confidence interval of 242 to 400 ng/L). The no-observed effect concentration or NOEC was determined to be 175 ng/L.

Description of key information

In Daphnia magna, the 48 h EC50 value was 311 ng/L (95 % confidence interval of 242 to 400 ng/L). The NOEC was determined to be 175 ng/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

311 ng/L

Additional information

Two studies are provided to address this endpoint. The key study was awarded a reliability score of 1 in line with the criteria set forth by Klimisch et al. (1997) and the supporting study was awarded a reliability score of 2 in line with the same criteria.

The key study was conducted to evaluate the acute toxicity potential of the test material to the invertebrate Daphnia magna Straus. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 202, EU Method C.2, EPA OPP 72-2 and U.S. EPA - FIFRA Standard Evaluation Procedure 540/9-85-005.

The study was conducted as a 48-hour flow-through test. Twenty daphnids (10 individuals/replicate, 2 replicates per dose level) were exposed to nominal concentrations of 0 (Laboratory Dilution Water control), 0 (84 µL/L acetone-dosed LDW-solvent control), 62.2, 104, 173, 288, 480, and 800 ng/L-LDW, over a 48-hour exposure period at 20 ± 1 °C. The test solutions were sampled and analysed to determine concentrations on days 0 and 2 of the study by liquid chromatography with negative electrospray ionisation-mass spectrometry (LC/NESI-MS). This resulted in mean measured concentrations of less than 50 ng/L, which was the lowest level quantified (LLQ), for the water and solvent controls, 78.6, 120, 175, 239, 443, and 884 ng/L.

Following 24 hours of exposure, immobility was observed in 30 % (6/20) and 60 % (12/20) of the daphnids at the 443 and 884 ng/L exposure levels, respectively. Following 48-hours of exposure, immobility was observed in 10 % (2/20), 5 % (1/20), 10 % (2/20), 10 % (2/20), 60 % (12/20), 70 % (14/20), and 90 % (18/20) of the daphnids at the solvent control, 78.6, 120, 175, 239, 443, and 884 ng/L exposure levels, respectively. No daphnid immobility was observed in the water control vessels at any time during the conduct of this study.

Under the conditions of this study, the 48-hour EC50 value was 311 ng/L (95 % confidence interval of 242 to 400 ng/L). The no-observed effect concentration or NOEC was determined to be 175 ng/L.

In the supporting study, the short-term toxicity of the test material to daphnia magna was investigated in a study conducted in accordance with the American Society for Testing and Materials (ASTM), Standard Guide For Conducting Acute Toxicity Tests With Fishes, Macroinvertebrates and Amphibians, E729-88A, Philadelphia, PA, 1989.

Daphnids were exposed to the test material at concentrations of 0.5, 1, 5, 10, 50 and 100 µg/L. Five organisms per vessel were used, with 4 replicate vessels per concentration. Observations were made after 6, 24 and 48 hours of exposure.

5/20 daphnids were immobile at the end of the 48 hour exposure period in the 0.5 µg/L dosage group, with 10/20 immobile in the 1 µg/L group. The 48h EC50 to Daphnia magna was 1 µg/L (nominal) based on immobility.