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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Jan 1989 to 22 Mar 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples for analysis were taken at the start and at the end of the exposure.

Vehicle:

yes

Remarks:

1-methyl-2-pyrrolidon

Details on test solutions:

- Test substance treated groups: Calculated amounts of stock solution to produce the desired test concentrations were given into the water and were homogeneously distributed. The test substance appeared homogeneously distributed in the test vessels.
- Stock solution: 2.5 g test substance were mixed with and made up to 10 mL with 1-methyl-2-pyrrolidon NMP. This solution was diluted to 50 mg/L with water.
- Blank: Water
- Vehicle: 61.8 mg/L1-methyl-2-pyrrolidon in the concentration used for the highest test concentration

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: Cambridge 276/20
- Preculture period: 3 days under test conditions
- Culture condition: Accodring to OECD TG 021 guideline

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 ± 2 ˚C

pH:

- At the start of the test: 7.4
- At the end of the test: 8.1 - 10.0

Nominal and measured concentrations:

- Nominal concentrations: 0.074, 0.22, 0.67, 2.0, 6.0, 18 mg/L
- Measured concentrations (0 h): n.d., 0.281, 0.709, 2.12, 6.54, 19.4 mg/L, respectively (See Table 1 in 'Any other information on materials and methods incl. tables')
- Measured concentrations (72 h): n.d., 0.195, 0.542, 1.71, 5.79 and 17.9 mg/L, respectively

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL erlenmeyer flask
- Type (delete if not applicable): Closed (stoppered with aluminium caps)
- Filled volume: 50 mL test solution
- Aeration: None
- Initial cells density: 1.2* 10000 cells/mL
- Control end cells density: Daily on TOA cell counter
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Others: On lab-shaker

OTHER TEST CONDITIONS
- Light source: Cold white fluorescent light
- Photoperiod: Continuous
- Light intensity and quality: 120 µE/m2sec ± 20%

WATER PARAMETER MEASUREMENT INTERVALS
- Temperature: Continuous
- pH : At start and end of exposure

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Daily on TOA cell counter

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Recalculated outside of the original study with a Probit model

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.67 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: reported in original study

Details on results:

An overview of the results is provided in Table 2 - 5 in ‘Any other information on results incl. tables’.
The mean cell density after 72h exposure was 1.55E+06, 1.68E+06, 1.98E+06, 1.70E+06, 1.36E+06, 9.17E+05, 9.83E+05 and 4.35E+05 cells/mL in blank control, vehicle control, 0.074, 0.22, 0.67, 2.0, 6.0 and 18.0 mg/L treatment, respectively. Therefore, comparing to the original cell density, after 72 hours exposure, the blank control, vehicle control, 0.074, 0.22, 0.67, 2.0, 6.0 and 18.0 mg/L treatments showed a growth inhibition of 0, 0, 0, 0, 12.5, 41.9, 38.2 and 56.7%, respectively.
The 72-hour EC50 of the test substance to was determined to be 10 mg/L. The NOEC was 0.67 mg/L. The raw data of this study were used in a Probit model to estimate a quantal dose response using SAS 9.1 outside of this original study. In this model. an EC50 value of 8.8 mg/L was determined

Reported statistics and error estimates:

In the original report: The EC50 values were calculated according to BERKSON, JASA 48 (1953), 569-599. EC-values were graphically determined on gausso-logarithmic probability paper. The NOEC was calculated according to DUNNETT, C.W., JASA 50 (1955), 1096-1121, Biometrics 20 (1964), 482-491, and modified by MARCUS et al., Biometrika 63 (1976), 655-660.
For recalculating the EC50, a Probit model was used to estimate a quantal dose response, using SAS 9.1. The model was fitted using the method of maximum likelihood.

Table 2. Toxicity of the test substance to Scendesmus subspicatus - percentage growth inhibition

Nominal concentration (mg/L)	Mean % inhibition (72 hours)
Control	0
Vehicle control	0
0.074	0
0.22	0
0.67	12.5
2.0	41.9
6.0	38.2
18	56.7

Table 3. Cell densities after 24h exposure

Conc. Nominal	Dilut. Factor	Cell densities/sample/mL*10000						Mean Dens. N 24h Cells/mL*10000
mg/L	1:	1	2	3	4	5	6
Blank	10	3.2	3.3	3.3	2.8	3.7	4.3	3.42
Vehicle	10	3.3	3.5	3.6				3.45
0.074	10	4.2	3.8	4.1				4.00
0.22	10	3.8	3.8	3.6				3.72
0.67	10	4.0	4.1	4.0				4.03
2.0	10	4.0	4.0	3.4				3.78
6.0	10	4.0	4.3	3.6				3.95
18	10	5.0	5.2	4.8				4.98

Table 4. Cell densities after 48h exposure

Conc. Nominal	Dilut. Factor	Cell densities/sample/mL*10000						Mean Dens. N 48h Cells/mL*10000
mg/L	1:	1	2	3	4	5	6
Blank	10	24.0	26.0	28.3	28.6	36.9	36.9	30.09
Vehicle	10	35.7	33.5	40.5				36.57
0.074	10	36.6	37.1	35.1				36.23
0.22	10	29.5	26.7	28.9				28.35
0.67	10	24.3	27.8	25.0				25.65
2.0	10	18.6	18.3	11.5				16.10
6.0	10	15.1	19.5	15.4				16.62
18	10	23.0	21.8	24.4				23.05

Table 5. Cell densities after 72h exposure

Conc. Nominal	Dilut. Factor	Cell densities/sample/mL*10000						Mean Dens. N 72h Cells/mL*10000
mg/L	1:	1	2	3	4	5	6
Blank	100	151	169	172	136	143.5	158	154.92
Vehicle	100	161.5	157.5	184				167.67
0.074	100	215	188	191.5				198.17
0.22	100	177.5	173.5	159.5				170.17
0.67	100	128.5	147.5	130.5				135.5
2.0	100	95	86.5	93.5				91.67
6.0	100	129	105	61				98.33
18	100	44.5	42.5	43.5				43.5

Validity criteria fulfilled:

yes

Conclusions:

In an OECD TG 201 followed toxicity study on green algae, the EC50 and NOEC values of the test substance were determined to be 10 and 0.67 mg/L, respectively. The EC50 was later recalculated in a Probit model and was determined to be 8.8 mg/L.

Executive summary:

This study was conducted to determine the acute toxicity of the test substance on green algae (Scenedesmus subspicatus) according to OECD TG 201 and GLP principles. The algae were exposed for 72 hours at the following nominal test concentrations: 0.074, 0.22, 0.67, 2.0, 6.0 and 18 mg/L with an initial cell density of 1.2E+04 cells/mL. The test substance appeared homogeneously distributed in the test vessels. Samples for analysis were taken at the start and at the end of the exposure. The test substance was dissolved in 1-methyl-2-pyrrolidon which was used as vehicle. The test was carried out under the following conditions: 24 ± 2 °C, Continuous light (8000 lux), pH 7.4 at start and pH 8.1 -10.0 at the end of the test. The EC values were graphically determined in this study.

The mean cell density after 72h exposure was 1.55E+06, 1.68E+06, 1.98E+06, 1.70E+06, 1.36E+06, 9.17E+05, 9.83E+05 and 4.35E+05 cells/mL in blank control, vehicle control, 0.074, 0.22, 0.67, 2.0, 6.0 and 18.0 mg/L treatment, respectively. Therefore, comparing to the original cell density, after 72 hours exposure, the blank control, vehicle control, 0.074, 0.22, 0.67, 2.0, 6.0 and 18.0 mg/L treatments showed a growth inhibition of 0, 0, 0, 0, 12.5, 41.9, 38.2 and 56.7%, respectively. Based on the results, the EC50 and NOEC values were determined to be 10 and 0.67 mg/L, respectively. The EC50 was later recalculated in a Probit model and was determined to be 8.8 mg/L.

Description of key information

Freshwater, 72-h NOEbC = 0.67 mg/L, Scenedesmus subspicatus, OECD TG 201, Rufli 1986

Freshwater, 72-h EbC50 = 8.8 mg/L, Scenedesmus subspicatus, OECD TG 201, Rufli 1986

Key value for chemical safety assessment

EC50 for freshwater algae:

8.8 mg/L

EC10 or NOEC for freshwater algae:

0.67 mg/L

Additional information

One study is available for this endpoint, which was selected as key study. In this study performed according to OECD TG 201 and in compliance with GLP, green algae (Scenedesmus subspicatus) was exposed to the test substance at concentrations of 0 (control), 0 (vehicle control; 1-methyl-2-pyrrolidon), 0.074, 0.22, 0.67, 2.0, 6.0 and 18 mg/L with an initial cell density of 1.2E+04 cells/mL. The test was carried out under the following conditions: 24 ± 2 °C, continuous light (8000 lux), pH 7.4 at start and pH 8.1 -10.0 at the end of the test. Comparing to the original cell density, after 72 hours exposure, the blank control, vehicle control, 0.074, 0.22, 0.67, 2.0, 6.0 and 18.0 mg/L treatments showed a growth inhibition of 0, 0, 0, 0, 12.5, 41.9, 38.2 and 56.7%, respectively. Based on the results, the EC50 and NOEC values were graphically determined to be 10 and 0.67 mg/L, respectively. The EC50 was later recalculated in a Probit model and was determined to be 8.8 mg/L.

Metabolites - available information

In addition to the study with the test substance, three toxicity studies on algae are available with the major degradation products (i.e. M4, M6 and M7), all performed in accordance with OECD TG 201 or EPA OPPTS 850.5400 guideline, and in compliance with GLP requirements. As the dossier has been prepared to address the test substance itself, these studies are not summarized as endpoint study records but are briefly discussed here. In the study on Pseudokirchneriella subcapitata with M4, the 72-h ErC 50 and NOErC values were determined to be 0.495 and 0.051 mg/L, respectively (Swarbrick and Woodyer 2001). In the study on Scenedesmus subspicatus with M6, the 72-h EbC 50 and NOEbC values were determined to be 0.017 and 0.00123 mg/L, respectively (Grade 1994). In the study with M7, which used the same algae species as in the study with M6, the 72-h EbC 50 and NOEbC values were determined to be > 100 and 100 mg/L, respectively (Rufli 1989). The results indicate that for the studied metabolites of the substance, M6 is the most toxic one for algae, while M7 is the least toxic one.