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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 June 2003 - 02 July 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 72-4 (Fish Early Life-Stage and Aquatic Invertebrate Life-Cycle Studies)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Replicate K of each dose level was sampled immediately following preparation and was used to provide analytical confirmation of the renewed solutions. The renewed solution samples, collected from an actual test vessel (replicate K) as opposed to the bulk dose solutions, afforded the most representative account of the exposure concentrations allowing for potential loss due to adsorption prior to sample collection. One representative spent test solution was sampled at the termination of each exposure period (days 2, 4, 6, 8, 10, 12, 14, 16, 20, and 21), with the exception of day 18, whereupon all remaining spent test solutions were sampled for analytical verification. The test substance concentration of the acetone-based dose stock solutions was confirmed at study initiation and termination.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Bulk test solutions at each of six target exposure concentrations were prepared every 48 hours from acetone-based dosing stock solutions (nominal concentrations of 80, 160, 320, 640, 1280 and 2560 ng/mL acetone). Eleven replicate test vessels (125-mL glass beakers uniquely identified as A - K) were prepared from each bulk dose solution in addition to eleven test vessels each of control daphnid dilution water (DDW) and of acetone solvent control.
Test solutions for the definitive study were prepared in bulk on day 0 and renewed every second day, as described above. Nominal concentrations for this study were 0 (water control), 0 (acetone control), 8.00, 16.0, 32.0, 64.0, 128, and 256 ng/L. Test solutions were spiked with solutions of test material solubilised in acetone; these acetone dosing stocks were prepared on test day -1 and used throughout the study.
A working stock solution with a nominal concentration of 709.8 µg/mL was prepared by adding 14.50 mg of test material (97.9 % purity) to 20 mL of acetone. One mL of this solution was then added to 19 mL of acetone to generate a stock solution of 35.49 µg/mL (35490 ng/mL). A 1.44-mL aliquot of this 35490 ng/L was then transferred into 18.56 mL of acetone to generate the highest dosing stock, with a concentration of 2560 µg/mL. The next five acetone dosing stocks were prepared by serial dilution of this 2560 µg/mL solution.
Bulk test solutions were prepared in 1-L volumetric flasks by injecting acetone stocks into DDW at a constant ratio of 0.100 mL acetone/L of water. Ten mL of Selenastrum capricornutum suspension (3.0 x 10⁷ cells/mL) and 5 mL of YCT suspension (1840 mg solids/L) were added per litre of solution to each flask as a daphnid food source before the solutions were brought to volume with DDW. Feeding was performed on non-renewal days by adding 0.5 mL of the Selenastrum capricornutum suspension to each test vessel.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Freshwater water flea
- Strain/clone: Daphnia magna Straus
- Source: In-house cultures
- Age of parental stock (mean and range, SD): Adult daphnids producing the neonates were 26 - 29 days old on test day 0.
- Feeding during test: Yes
- Food type: mixed diet of Selenastrum capricornutum (a green alga) and YCT (yeast, Cerophyll, and trout chow suspension)
- Frequency: 5 times per week

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Young Daphnia were removed from mass cultures the day before test initiation (day -1) by sieving with a 300-µm mesh screen that retains mature individuals, while allowing immature daphnids to pass through the mesh. Cultures were sieved again on day 0 and neonates < 24-hours old were collected for testing.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

120 - 170 mg CaCO₃/L

Test temperature:

Range: 19.9 - 21.3 °C
Mean ± SD: 20.6 ± 0.5

pH:

Range: 7.1 - 7.8
Mean ± SD: 7.6 ± 0.2

Dissolved oxygen:

Range: 3.0 - 9.1 (bulk and spent solutions)
Mean ± SD: 6.1 ± 2.3

Salinity:

N/A

Nominal and measured concentrations:

- Nominal: 8.00, 16.0, 32.0, 64.0, 128, 256 ng/L
- Mean measured: 4.55, 9.39, 18.3, 38.7, 68.2, 141 ng/L

Details on test conditions:

TEST SYSTEM
- Test Vessel: The test was conducted in 120-mL glass jars containing 90 mL of solution. Vessels were covered with a sheet of Plexiglas to reduce evaporation. The test vessels were held in an incubator at 20 ± 1 °C.
- Renewal rate of test solution (frequency/flow rate): every second day
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
The laboratory water was Lake Huron water supplied to the testing facility by the City of Midland Water Treatment Plant. The water was obtained from the upper Saginaw Bay of Lake Huron off Whitestone Point, and was limed and flocculated with ferric chloride. The water was pumped to the laboratory prior to municipal treatment for human consumption. Before use in the laboratory, the water was sand-filtered, pH adjusted with carbon dioxide, carbon-filtered, and UV-irradiated. DDW was prepared by adjusting this laboratory water to a hardness of approximately 170 mg/L as CaCO₃. After adjusting hardness, the water was autoclaved at 250 F and 18 psi for 30 minutes and cooled before use. Both laboratory and DDW were monitored weekly for pH, alkalinity, conductivity, and hardness, and twice yearly for total organic carbon (TOC), total suspended solids (TSS), and selected inorganic and organic compounds.

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Light intensity: 858 ± 103 lux

OBSERVATIONS
Observations were made and the number of surviving daphnids recorded daily. Mortality was defined as an inability to swim within 15 seconds after gentle agitation of the test vessel. For the purposes of this study, immobile organisms were recorded as dead.
Reproduction was evaluated by counting surviving and dead (if present) Daphnia magna neonates in each test vessel on renewal days (test days 2, 4, 6, 8, 10, 12, 14, 16, 18, 20), and at test termination (test day 21). This was performed at the same time as test solution renewal by first transferring the adult daphnid into a new test vessel, followed by sieving of the spent solution from the old test vessel.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY: Yes
- Test concentrations: replicate vessels, each containing a single daphnid, and nominal test concentrations were 0 (water control), 0 (acetone control), 3.07, 7.68, 19.2, 48.0, 120, and 300 ng/L of test solution.
- Results used to determine the conditions for the definitive study: Effects on mortality and reproduction above 48.0 ng/L were indicated.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

68.2 ng/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

141 ng/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

18.3 ng/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

38.7 ng/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

Survival during the test was 80, 90, 100, 90, 90, 80, 90 and 50 % for the water controls, solvent controls, 8.00, 16.0, 32.0, 64.0, 128, and 256 ng/L nominal treatments, respectively. Survival results based on TWM measured concentrations were thus 80, 90, 100, 90, 90, 80, 90, and 50 % for the 0 (water control), 0 (acetone control), 4.55, 9.39, 18.3, 38.7, 68.2, and 141 ng/L treatments, respectively. At 50 %, survival in the 141 ng/L treatment was significantly different from solvent controls at an alpha level of 0.0704 (Fisher’s Exact Test). This is slightly greater than the alpha = 0.05 value that is frequently used to identify significant effects. However, biological interpretation of the data suggests that 50 % mortality is significant. The NOEC for survival is thus set at 68.2 ng/L, and the LOEC for survival is 141 ng/L (TWM measured concentrations).
Survival in all treatments was ¬ 80 % with the exception of the 141 ng/L treatment, which had 50 % survival. The survival EC50 for adults is empirically estimated to be 141 ng/L, TWM concentration.
The mean number of live young produced per surviving adult was 154.5, 192.8, 193.1, 186.2, 164.4, 44.4, 39.2, and 28.8 for the 0 (water control), 0 (acetone control), 4.55, 9.39, 18.3, 38.7, 68.2, and 141 ng/L treatments, respectively. Over the course of the test, no dead young were found in the controls, the 4.44 ng/L or the 9.39 ng/L TWM measured concentration treatments. The mean number of dead young produced per surviving adult (and percent of young found dead) in the 18.3, 38.7, 68.2 and 141 ng/L (TWM measured concentration) treatments was 0.9 (0.5 %), 65.0 (59.4 %), 32.9 (45.6 %), and 3.8 (11.7 %), respectively.
The number of progeny per surviving female adult met the normality and homogeneity of variance assumptions, and the NOEC for reproduction was determined using Dunnett’s test. This procedure maintained alpha = 0.05 while making multiple comparisons to the control group. Significant differences in reproduction were found between the negative (water) control and the solvent control, and solvent control results were used for statistical analyses. The statistically-derived NOEC, LOEC, and MATC for reproduction, calculated using mean live progeny per surviving adult, were 18.3, 38.7, and 26.6 ng/L (TWM measured concentration). The coefficient of variation for solvent control reproduction was 16.0 %.

CHEMICAL ANALYSIS
Since the test material concentration decreased between the renewal periods, time-weighted mean (TWM) values were calculated for each dose level to derive a better estimate of the concentration to which the daphnia were exposed.
Each test solution was sampled at the beginning and termination of the renewal cycle, thus enabling a TWM concentration of the test material to be calculated for each exposure period of each dose solution. The TWM concentrations were calculated using a standard equation from the OECD Guideline 211 which accounts for the concentration of an analyte over a renewal period for solutions in which the analyte concentration declines exponentially over the duration of the exposure period.
TWM (as ng/L) = (Conc 0 - Conc 1) / [Ln(Conc 0) - Ln(Conc 1)]
where:
Conc 0 represents the measured concentration of the test material in the test solution at the start of an exposure period
Conc 1 represents the measured concentration of the test material in the test solution at the end of an exposure period

For each of the three exposure weeks, a weekly time-weighted mean concentration was calculated. For weeks 1 and 2, in which each of the four exposure periods were of equal duration (48 hours), the weekly TWM’s of each dose level were calculated by averaging the TWM’s of the four individual exposure periods. However, for week 3, the exposure periods were of unequal duration (two 48-hour periods, and one 24-hour period). Therefore, it was necessary to weight the TWM of each exposure period by the duration of the exposure prior to calculating the week 3 TWM. Similar weighting was done to account for the discrepancy between the number of days in the three consecutive weeks used in the calculations (8 days for week 1 and 2; 5 days in week 3).
Due to the low concentrations involved in this study (ppt levels), measured values for nominal dose levels of 16.0 and 32.0 ng /L were sometimes less than the lowest level quantified (LLQ) of 5.88 ng/L DDW. Therefore, for calculation purposes, a value of 2.94 ng test material/L (equivalent to ½ LLQ) was used as a conservative estimate of concentration where a measured value was reported as Study Time Weighted Mean values for the individual dose levels that were generally above LLQ ranged from 53.3 - 60.5 % of target, with an overall study TWM of 56.9 ± 2.8 % of target concentration. Most spent solutions for the lowest dose level, 8.00 ng/L, were below LLQ. The TWM concentration for this treatment was estimated by multiplying the nominal concentration by the overall study TWM of 56.9 %: (8.00 mg/L x 56.9 % = 4.55 mg/L). The calculated TWM measured test concentrations for the 8.00, 16.0, 32.0, 64.0, 128, and 256 ng/L treatments were thus 4.55, 9.39, 18.3, 38.7, 68.2, and 141 ng/L, respectively. TWM concentrations deviated greater than ± 20 % of target, and statistical results are reported based on TWM concentrations.
Test material concentrations in the water and acetone controls were in most cases less than 5.88 ng/L, the lowest level quantified (LLQ). Test material was detected in isolated control samples. This contamination is due to the difficulties involved with the low concentrations used in this study.
The overall percent of target values for the quantifiable dose levels ranged from 53.3 to 60.5 %, with a grand study mean of 56.9 %.
The dosing stock solutions ranged from 104 to 127 % of target on day 0 and 81.4 to 119 % on day 21. Study average percent of target values (based on the average of day 0 and 21 measured concentrations) for the individual dose stock solutions ranged from 97.2 to 116 % with an overall average for the entire study of 106 ± 7.33 %. The analysed concentrations of the dosing stock solutions provide further indication that the test solutions were dosed at their intended concentrations.

Reported statistics and error estimates:

Survival and reproduction were evaluated using appropriate statistical procedures. In order to calculate NOEC and LOEC values for reproduction, the raw data were first tested for normality using the Shapiro-Wilk's test at a type I error rate of 0.01. If the data were not normally distributed, the logarithmic, inverse, and square root transformations were tested sequentially to search for a normalising transformation. Next, the data and the same transformed variables were tested for homogeneity of variance using Bartlett's test at a type I error rate of 0.01. If the raw data, or a transformed variable, were both normal and homogenous, a parametric analysis was conducted using a Dunnett's test to compare each treated group with the control. A one-tailed Dunnett's test, looking for a significant decrease from the control group, was conducted at a type I error rate of 0.05. The no-observed-effect-concentration (NOEC) was defined as the highest dose group not significantly different compared to the control.

Data that were not normally distributed and/or not homogenous were analysed non-parametrically with a Steel's Many-One Rank Test if the number of replicates in each treatment group were the same. A Kruskal-Wallis test was utilised if the number of replicates was different. Steel's Many-One Rank Test is one-sided and the Kruskal-Wallis test is two-sided; both have a type I error rate of 0.05. A significant result in the Kruskal-Wallis test leads to a pairwise comparison of each treatment with the control using the Wilcoxon procedure having a type I error rate of 0.01 (one-sided). Both the Steel's test and the Wilcoxon test lead to the determination of a NOEC.

NOEC and LOEC values for survival were calculated using Fisher’s Exact Test with a type I error rate of 0.05.

Table 1: Survival and Progeny of Daphnids

Nominal Concentration (ng/L)	TWM Measured Concentration (ng/L)	Percent Survival (%)	Average Progeny Per Surviving Female Adult (mean ± SD)
0 (water control)	0	80	154.5 ± 75.2
0 (solvent control)	0	90	192.8 ± 30.9
8.00	4.55	100	193.1 ± 24.9
16.0	9.39	90	186.2 ± 18.2
32.0	18.3	90	164.4 ± 46.6
64.0	38.7	80	44.4 ± 21.0
128	68.2	90	39.2 ± 20.0
256	141	50	28.8 ± 19.3
Survival NOEC (ng/L)		68.2
Survival LOEC (ng/L)		141
Survival MATC (ng/L)		98.1
Survival EC₅₀ (ng/L)		141
Reproduction NOEC (ng/L)		18.3
Reproduction LOEC (ng/L)		38.7
Reproduction MATC (ng/L)		26.6
Reproduction EC₅₀ (ng/L) Slope of EC₅₀ Standard Error of Slope		31.4 -136.97 10.86

Validity criteria fulfilled:

yes

Conclusions:

The NOEC and the LOEC for survival were 68.2 and 141 ng/L, respectively. The statistically-derived NOEC and LOEC for reproduction, calculated using mean live progeny per surviving adult, were 18.3 and 38.7 ng/L, respectively.

Executive summary:

A study was conducted to assess the chronic toxicity of the test material to the daphnid, Daphnia magna Straus, in a 21-day static-renewal life-cycle test. The study was conducted in accordance with the standardised guidelines OECD 211 and EPA OPP 72-4 under GLP conditions.

The study was conducted with ten daphnids (one individual per replicate with ten replicates per dose level) exposed to nominal test concentrations of 0 (water control), 0 (acetone control), 8.00, 16.0, 32.0, 64.0, 128, and 256 ng/L. Test solutions were renewed every second day over the course of this 21-day exposure. The concentration in the test solutions was confirmed by analysing samples from the freshly-prepared bulk test solutions and from spent test solution on each renewal day. The test material concentration of the acetone-based dosing stock solutions was confirmed at study initiation and termination and recoveries ranged from 81 - 127 % of target values (mean = 106 % ± 7.4), indicating that solutions were prepared correctly. The calculated Time-Weighted Mean measured test concentrations for the 8.00, 16.0, 32.0, 64.0, 128, and 256 ng/L treatments were 4.55, 9.39, 18.3, 38.7, 68.2, and 141 ng/L, respectively. TWM measured concentrations deviated greater than ± 20 % of target and statistical results are reported based on TWM concentrations.

Survival during the test was 80, 90, 100, 90, 90, 80, 90, and 50 % for the 0 (water control), 0 (acetone control), 4.55, 9.39, 18.3, 38.7, 68.2, and 141 ng/L treatments, respectively. The mean number of live young produced per surviving adult was 154.5, 192.8, 193.1, 186.2, 164.4, 44.4, 39.2, and 28.8 for the 0 (water control), 0 (acetone control), 4.55, 9.39, 18.3, 38.7, 68.2, and 141 ng/L treatments, respectively. Over the course of the test, no dead neonate daphnids were found in the controls, the 8 or the 16 ng/L treatments. The mean number of dead neonates produced per surviving adult (and percent of young found dead) in the water control, solvent control, 4.55, 9.39, 18.3, 38.7, 68.2, and 141 ng/L treatments was 0 (0 %), 0 (0 %), 0 (0 %), 0 (0 %), 0.9 (0.5 %), 65.0 (59.4 %), 32.9 (45.6 %), and 3.8 (11.7 %), respectively.

Significant differences in reproduction were found between the negative (water) control and the solvent control, and solvent control results were used for statistical analyses.

Under the conditions of this study, the NOEC and the LOEC for survival were 68.2 and 141 ng/L, respectively. The statistically-derived NOEC and LOEC for reproduction, calculated using mean live progeny per surviving adult, were 18.3 and 38.7 ng/L, respectively.

Description of key information

In a 21-day static-renewal life-cycle test in Daphnia magna Straus the NOEC and the LOEC for survival were 68.2 and 141 ng/L, respectively. The statistically-derived NOEC and LOEC for reproduction were 18.3 and 38.7 ng/L, respectively. OECD 211, US OPPS 72-4, Marino et al. 2003.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

68.2 ng/L

Additional information

A study was conducted to assess the chronic toxicity of the test material to the daphnid, Daphnia magna Straus, in a 21-day static-renewal life-cycle test. The study was conducted in accordance with the standardised guidelines OECD 211 and EPA OPP 72-4 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set for by Klimisch et al. (1997).

The study was conducted with ten daphnids (one individual per replicate with ten replicates per dose level) exposed to nominal test concentrations of 0 (water control), 0 (acetone control), 8.00, 16.0, 32.0, 64.0, 128, and 256 ng/L. Test solutions were renewed every second day over the course of this 21-day exposure. The concentration in the test solutions was confirmed by analysing samples from the freshly-prepared bulk test solutions and from spent test solutions on each renewal day. The test material concentration of the acetone-based dosing stock solutions was confirmed at study initiation and termination and recoveries ranged from 81 - 127 % of target values (mean = 106 % ± 7.4), indicating that solutions were prepared correctly. The calculated Time-Weighted Mean measured test concentrations for the 8.00, 16.0, 32.0, 64.0, 128, and 256 ng/L treatments were 4.55, 9.39, 18.3, 38.7, 68.2, and 141 ng/L, respectively. TWM measured concentrations deviated greater than ± 20 % of target and statistical results are reported based on TWM concentrations.

Survival during the test was 80, 90, 100, 90, 90, 80, 90, and 50 % for the 0 (water control), 0 (acetone control), 4.55, 9.39, 18.3, 38.7, 68.2, and 141 ng/L treatments, respectively. The mean number of live young produced per surviving adult was 154.5, 192.8, 193.1, 186.2, 164.4, 44.4, 39.2, and 28.8 for the 0 (water control), 0 (acetone control), 4.55, 9.39, 18.3, 38.7, 68.2, and 141 ng/L treatments, respectively. Over the course of the test, no dead neonate daphnids were found in the controls, the 8 or the 16 ng/L treatments. The mean number of dead neonates produced per surviving adult (and percent of young found dead) in the water control, solvent control, 4.55, 9.39, 18.3, 38.7, 68.2, and 141 ng/L treatments was 0 (0 %), 0 (0 %), 0 (0 %), 0 (0 %), 0.9 (0.5 %), 65.0 (59.4 %), 32.9 (45.6 %), and 3.8 (11.7 %), respectively.

Significant differences in reproduction were found between the negative (water) control and the solvent control, and solvent control results were used for statistical analyses.

Under the conditions of this study, the NOEC and the LOEC for survival were 68.2 and 141 ng/L, respectively. The statistically-derived NOEC and LOEC for reproduction, calculated using mean live progeny per surviving adult, were 18.3 and 38.7 ng/L, respectively.