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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 October 2002 - 25 October 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: FIFRA Guideline 123-2

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

At test initiation and termination, one sample was removed from each test solution and the controls for analysis of test material concentrations. Samples analysed at 0 hour were removed from the test and control solutions prior to division into the replicate test vessels.
Since the results of two of three quality control samples associated with the 0-hour samples were outside of the established acceptable range, archived 0-hour test solution samples were removed from frozen storage and analysed on the following day. Samples analysed at 96 hours were removed from individually composited replicate solutions of the treatment levels and controls. At 96 hours of exposure, a sample was also removed from the replicate flask of the 0.25 mg a.i./L test concentration which did not contain algae and composited. The results of this analysis were compared with that obtained for the 96-hour analysis of the 0.25 mg a.i./L solution containing algae to assess the impact that algae had on the test material concentration.

Three quality control (QC) samples were prepared at each analytical interval (0-hour, 0-hour reanalysis and 96-hour) and remained with the appropriate set of exposure solution samples throughout the analytical process. The QC samples were prepared in dilution water at nominal concentrations which approximated the test concentration range. The results of the analyses of the QC samples were used to judge the precision and the quality control maintained during the analytical process.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A 10 mg a.i./mL stock solution was prepared by placing 0.2554 g (0.2500 g as active ingredient) of the test material in a 25 mL volumetric flask and diluting to volume with dimethylformamide (DMF). The resulting stock solution was observed to be clear and colourless, with no visible undissolved test material. Nominal test concentrations were prepared from secondary dilutions of the 10 mg a.i./mL stock solution.
Immediately following preparation, each test solution was observed to contain undissolved test substance. After being subjected to two minutes of ultrasonciation, only the 0.50 and 1.0 mg a.i./L test solutions contained undissolved test material.
Additional untreated AAP medium was used to prepare the control. A 0.1 mL/L solvent control solution was prepared by diluting 0.1 mL of DMF with 1000 mL of AAP medium.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Strain: 1648, Class Chlorophyceae
- Source (laboratory, culture collection): University of Texas, Austin, Texas
- Age of inoculum (at test initiation): The inoculum used to initiate the toxicity test was taken from a stock culture that had been transferred to fresh medium three days before testing.
- Method of cultivation: The stock cultures were maintained within the following conditions for at least one transfer before testing: a shaking rate of 100 ± 10 rpm, a temperature of 24 ± 1 °C and continuous illumination at the surface of the medium with an intensity of 300 to 500 footcandles (3200 to 5400 lux). Lighting was supplied by Duro-Test® Vita-Lite® fluorescent bulbs. Culture flasks were agitated continuously on an orbital shaker. Temperature was controlled using an environmental chamber.

ACCLIMATION
- Culturing media and conditions (same as test or not): The culture medium used was Algal Assay Procedure (AAP) medium prepared with sterile, deionised water. If necessary, the pH of the culture medium was adjusted to pH 7.5 ± 0.1 with either 0.1 N hydrochloric acid or 0.1 N sodium hydroxide. Stock cultures were grown in 125-mL glass flasks each containing 50 mL of medium. The flasks were covered with stainless steel caps which permitted gas exchange.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

23 - 24 °C

pH:

7.2 - 7.3 at test initiation; 8.2 - 8.4 after 96 hours of exposure.

Nominal and measured concentrations:

- Nominal Test Concentrations: 0.063, 0.13, 0.25, 0.50 and 1.0 mg a.i./L
- Initial Measured Concentrations: 0.064, 0.12, 0.24, 0.45 and 0.75 mg a.i./L

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile 250-mL Erlenmeyer flasks
- Type: closed. All test vessels were fitted with stainless steel caps which permit gas exchange
- Material, size, headspace, fill volume: One hundred mL of the appropriate exposure or control solution was placed in each replicate flask
- Aeration: Continuous shaking. The shaking rate was maintained at a constant rate of 100 rpm throughout the exposure.
- Initial cell density: Approximately 3 hours after the test solutions were added to the test flasks (100 mL per flask), a 0.191 mL inoculum of Pseudokirchneriella subcapitata cells, at a density of approximately 525 x 10⁴ cells/mL, was aseptically introduced into each flask. This inoculum provided the required initial (0 hour) cell density of approximately 1.0 x 10⁴ cells/mL.
- Control end cells density: 106 x 10⁴ cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
Representative samples of the dilution water source used in the preparation of the culture medium were analysed periodically for the presence of pesticides, PCBs and toxic metals. None of these compounds have been detected at concentrations that are considered toxic in any of the samples analysed in agreement with ASTM standard practices (ASTM, 2002). In addition, a representative sample of AAP medium was analysed monthly for total organic carbon (TOC) concentration. The TOC concentration of the sample collected in October 2002 was 0.56 mg/L.
Conductivity of the exposure and control solutions measured at test initiation and termination ranged from 80 to 90 µmhos/cm.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: If necessary, the pH of the culture medium was adjusted to pH 7.5 ± 0.1 with either 0.1 N hydrochloric acid or 0.1 N sodium hydroxide
-The test was conducted in an environmental chamber designed to maintain the test conditions specified: a temperature of 24 ± 1 °C, and continuous light intensity of 400 to 500 footcandles (4300 to 5400 lux). An orbital shaker table provided a shaking rate of 100 ± 10 rpm.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: At each subsequent 24-hour interval, cell counts were conducted on each replicate vessel of each treatment, the control and the solvent control solutions using a hemacytometer (Neubauer Improved) and compound microscope. The cell density in each test flask was calculated for each daily interval by dividing the number of cells counted by the number of fields examined. Means and standard deviations for cell density for the treatments and the controls were calculated from individual replicate values.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Test concentrations: 0.0010, 0.010, 0.10 and 1.0 mg a.i./L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 0.75 mg/L

Nominal / measured:

estimated

Conc. based on:

act. ingr.

Basis for effect:

cell number

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.12 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.75 mg/L

Nominal / measured:

estimated

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

75 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.75 mg/L

Nominal / measured:

estimated

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.75 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Details on results:

At 96 hours of exposure, cells exposed to all the treatment levels tested and the controls were observed to be normal. The 96-hour cell density in the control and solvent control averaged 106 and 87 x 10⁴ cells/mL, respectively. Cell densities in the 0.064, 0.12, 0.24, 0.45 and 0.75 mg a.i./L treatment levels averaged 101, 82, 69, 69 and 86 x 10⁴ cells/mL, respectively.
Statistical analysis (Williams’ Test), determined a significant reduction in cell density in the 0.24, 0.45 and 0.75 mg a.i./L treatment level as compared to the pooled control (pooled control = 97 x 10⁴ cells/mL).

The 0- to 72-hour biomass in the control and solvent control averaged 22.7 and 15.0 x 10⁴ cells.days/mL, respectively. Biomass in the 0.064, 0.12, 0.24, 0.45 and 0.75 mg a.i./L treatment levels averaged 21.8, 16.3, 17.4, 17.4 and 20.4 x 10⁴ cells.days/mL, respectively.
Statistical analysis (Williams’ Test) determined there was no significant difference between pooled control data (18.8 x 10⁴ cells.days/mL) and the treatment data.

Growth rates are presented in Table 1. The 0- to 72-hour growth rate in the control and solvent control averaged 1.11 and 0.94 days^-1, respectively. The 0- to 72-hour growth rate in the 0.064, 0.12, 0.24, 0.45 and 0.75 mg a.i./L treatment levels averaged 1.08, 0.85, 1.03, 0.95 and 1.09 days^-1, respectively.
Statistical analysis (Williams’ Test) determined there was no significant difference between the pooled control (1.03 days^-1) data and treatment data.

Additional testing to further define the toxicity of the test material was not performed due to the fact that the highest nominal concentration tested exceeded the water solubility limit (<1.0 mg a.i./L) of the test material.

The following acceptance criteria were required by the protocol: the cell density in the control must increase by more than 16 times after 72 hours of growth, and the pH of the control solution should not increase by more than 1.5 units from test initiation.
During this test, the mean control 72-hour cell density (30 x 10⁴ cells/mL) exceeded the requirement of a 16-fold increase from the initial density of 1.0 x 10⁴ cells/mL. The 72-hour pH of the control solution was 7.3, which was within the allowed 1.5 pH unit increase from the initial value (pH = 7.3).

Reported statistics and error estimates:

A t-test was used to compare the biomass and growth rate of the control to that of the solvent control. If no significant difference was determined, data were pooled for further statistical analysis to determine treatment level effects. If a significant difference was detected, the treatment data were compared to the solvent control data.
If a sufficient response was observed, the EC25 and EC50 values were calculated after 24, 48, 72 and 96 hours of exposure. The EC50 values for total biomass and growth rate, denoted as EbC50 and ErC50, respectively, were calculated after 72 hours of exposure. When less than the designated percent inhibition was observed for the noted parameter, the EC value was empirically estimated to be greater than the highest concentration tested.
Where required, the EC values and their 95 % confidence limits were determined by linear regression of response (percent reduction of cell density, biomass or growth rate as compared with the appropriate control) versus initial measured exposure concentration. A computer program, TOXSTAT®, was used to calculate the EC values and 95 % confidence limits.

Based on the results of statistical analysis performed for 96-hour cell density and 72-hour total biomass and average growth rate data, the No-Observed-Effect Concentration (NOEC), the highest test concentration which demonstrated no statistically adverse effect (p<0.05) for each parameter when compared to the appropriate control data, was determined. The data were first checked for normality using Shapiro-Wilks' Test and for homogeneity of variance using Bartlett's Test. If the data set passed the tests for homogeneity and normality, Williams’ Test was used to determine the NOEC. If the data did not pass the tests for homogeneity and normality, then Kruskal-Wallis’ Test was used to determine the NOEC. All statistical determinations were made at the 95 % level of certainty (except Shapiro-Wilks' and Bartlett's Tests, 99 % level of certainty applied).

Table 1: Mean Calculated Growth Rate (days^-1)

Initial measured Concentration (mg a.i./L)	Observation Interval (hours)			Percent Inhibition*
	0 - 24	0 - 48	0 - 72
Control	1.17	0.80	1.11	NA
Solvent Control	1.14	0.77	0.94	NA
Pooled Control	-	-	1.03	NA
0.064	1.51	0.85	1.08	-5
0.12	1.43	0.90	0.85	17
0.24	1.06	0.79	1.03	0
0.45	1.30	0.91	0.95	8
0.75	1.21	0.66	1.09	-6

*Relative to the pooled control

NA = Not applicable

Validity criteria fulfilled:

yes

Conclusions:

The 72-hour ErC50 was empirically estimated to be >0.75 mg a.i./L, the highest concentration tested. The 72-hour NOEC was determined to be 0.75 mg a.i./L.

Executive summary:

A study was conducted to determine the effect of the test material on the growth of the freshwater green alga Pseudokirchneriella subcapitata. The study was carried out in accordance with the standardised guidelines OECD 201, EU Method C.3 and FIFRA Guideline 123-2 under GLP conditions.

Pseudokirchneriella subcapitata, in Algal Assay Procedure (AAP) medium, was exposed to the test material for 96 hours at nominal concentrations of 0.063, 0.13, 0.25, 0.50 and 1.0 mg a.i./L. The conditions of exposure were 24 ± 1 °C, continuous illumination at 350 to 425 footcandles (3800 to 4600 lux) and a shaking rate of 100 rpm. Inhibition of 24-, 48-, 72- and 96-hour cell density, 0- to 72-hour biomass and 0 to 72-hour growth rate relative to the performance of the control was determined.

The 72-hour ErC50 was empirically estimated to be >0.75 mg a.i./L, the highest concentration tested. Based on the results of Williams’ Test, the 72-hour NOEC was determined to be 0.75 mg a.i./L. Additional testing to further define the toxicity of the tets material was not performed since the highest nominal concentration tested exceeded the water solubility limit (<1.0 mg a.i./L) of the test material.

For the cell density, the 96-hour EC25 and EC50 were empirically estimated to be >0.75 mg a.i./L. The 96-hour No-Observed-Effect Concentration (NOEC) was determined to be 0.12 mg a.i./L based on Williams’ Test.

The 72-hour EbC50 was empirically estimated to be >0.75 mg a.i./L. Based on the results of Williams’ Test, the 72-hour NOEC was determined to be to be 0.75 mg a.i./L.

Description of key information

The 72 h ErC50 was empirically estimated to be >0.75 mg a.i./L;  the 72 h NOEC was determined to be 0.75 mg a.i./L.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.75 mg/L

EC10 or NOEC for freshwater algae:

0.75 mg/L

Additional information

A study was conducted to determine the effect of the test material on the growth of the freshwater green alga Pseudokirchneriella subcapitata. The study was carried out in accordance with the standardised guidelines OECD 201, EU Method C.3 and FIFRA Guideline 123-2 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Pseudokirchneriella subcapitata, in Algal Assay Procedure (AAP) medium, was exposed to the test material for 96 hours at nominal concentrations of 0.063, 0.13, 0.25, 0.50 and 1.0 mg a.i./L. The conditions of exposure were 24 ± 1 °C, continuous illumination at 350 to 425 footcandles (3800 to 4600 lux) and a shaking rate of 100 rpm.

Inhibition of 24-, 48-, 72- and 96-hour cell density, 0- to 72-hour biomass and 0 to 72-hour growth rate relative to the performance of the control was determined.

The 72-hour ErC50 was empirically estimated to be >0.75 mg a.i./L, the highest concentration tested. Based on the results of Williams’ Test, the 72-hour NOEC was determined to be 0.75 mg a.i./L. Additional testing to further define the toxicity of the tets material was not performed since the highest nominal concentration tested exceeded the water solubility limit (<1.0 mg a.i./L) of the test material.

For the cell density, the 96-hour EC25 and EC50 were empirically estimated to be >0.75 mg a.i./L. The 96-hour No-Observed-Effect Concentration (NOEC) was determined to be 0.12 mg a.i./L based on Williams’ Test.

The 72-hour EbC50 was empirically estimated to be >0.75 mg a.i./L. Based on the results of Williams’ Test, the 72-hour NOEC was determined to be to be 0.75 mg a.i./L.