Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

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:

20 May 2005 to 29 Nov 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Version / remarks:

April 1996, "Public Draft"

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF Test Guideline, 2-7 -3

Version / remarks:

2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For the analysis of the actual test item concentrations (based on the quantification of the sum of the syn- and anti-epimer) the following samples were taken:
Just before the start of the test:
- one sample from the freshly prepared stock solution
- duplicate samples from each test medium (without algae)
- duplicate samples from the solvent control (without algae)
After 96 hours(stability samples):
- duplicate samples from each test medium (with algae)
- duplicate samples from the solvent control (with algae)

Two additional samples were taken from each of the two test media of the test item concentration of 3.2 and 10 mg/L at the start and at the end of the test. One of the additional samples from these two concentrations was centrifuged to determine the dissolved part of the test item in these test media.
For the 96-hour stability samples of nominal 3.2 and 10 mg/L additional flasks with algae were incubated separately under identical conditions as in the actual test.
All samples were deep-frozen (at about -20 ˚C) immediately after sampling. Based on preexperiments (without GLP) for investigation of the storage stability the test item is sufficiently stable in the test water under these storage conditions.

The concentration of the test item was analyzed in one of the duplicate test media samples from the test concentrations of nominal 0.32 - 10 mg/L from both samplircg times (0 and 96 hours). Additionally, the centrifuged samples with test item concentrations of 3.2 and 10 mg/L were analysed. The samples from the lowest test concentration of nominal 0.10 mg/L were not analyzed, since this concentration was below the determined 96-hour NOEC. From the solvent control samples only one of the duplicate samples was analysed from the corresponding sampling times.

Vehicle:

yes

Remarks:

N,N-dimethylformamide (DMF)

Details on test solutions:

A stock solution of nominal 100 mg/mL was prepared by dissolving 200.04 mg of the test item in 2 mL of DMF. The stock solution was diluted in a series of sequential dilutions with the solvent to add the same volumes of solvent (100 µL) to each test solution. The final nominal concentrations of the test substance in the test media were 0.10, 0.32, 1.0, 3.2 and 10 mg/L.
Additionally, a control (test water without addition of the test item) and a solvent control (100 µL DMF per litre test water) was tested in parallel. The test media were prepared just before the start of the test (= addition of algae).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: No. 61.81 SAG
- Method of cultivation: The algae are cultured in the test facility laboratories under standardized conditions according to the test guidelines.
- Pre-culture: The test cells were taken from an exponentially growing pre-culture, which was set up three days prior to the test under the same conditions as in the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

24 mg/L as CaCO3

Test temperature:

22 – 23 °C

pH:

- Test start: 7.9 to 8.3
- Test end: 9.0 to 9.1

Nominal and measured concentrations:

- Nominal concentrations: 0 (Solvent control), 0 (negative control), 0.10, 0.32, 1.0, 3.2 and 10 mg/L
- Measured concentrations: - (Solvent control), - (negative control), -, 0.31, 0.73, 1.80 and 4.00 mg/L, repsectively. See Table 1 in 'Any other information on materials and methods incl. tables'.

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flasks containing 15 mL of media
- Type: Closed (covered with glass dishes)
- Initial cells density: 1E+04 cell/mL
- No. of vessels per concentration: 3
- No. of vessels per control: 3
- No. of vessels per vehicle control: 6

GROWTH MEDIUM
- Standard medium used: Yes

WATER PARAMETERS
- Intervals of water quality measurement: The pH was measured and recorded in each test concentration and the controls at the start and at the end of the test. The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The appearance of the test media was also recorded daily.

OTHER TEST CONDITIONS
- Photoperiod: Continuous
- Light intensity and quality: 6510 - 7370 Lux
- Other: The test solutions were continuously stirred by magnetic stirrers.


EFFECT PARAMETERS MEASURED
- Sampling intervals: Small volumes of all test concentrations and the control were taken from all test flasks after 24, 48, 72 and 96 hours of exposure. In addition, after 96 hours exposure, a sample was taken from the control and from a test concentration with reduced algal growth (nominal 10 mg/L).
- Determination of cell concentrations and shape: The algal cell densities in the samples were determined by counting with an electronic particle counter. The shape of the algal cells was examined microscopically in these samples.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate is tested as a positive control at least once per year.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.31 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.31 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Details on results:

An overview of the results is provided in Table 2 – Table 5 in ‘Any other information on results inlc. tables’.
In the control the cell density increased from a nominal N = 1E+04 cells/mL at the start of the test (0 hours) to N = 1.51E+06 cells/mL (mean value) after 72 hours. In the solvent control the cell density increased from a nominal N = 1E+04 cells/mL at the start of the test (0 hours) to N = 1.62E+06 cells/mL cells/mL (mean value) after 72 hours. Thus, the algal growth in the control and in the solvent control was sufficiently high under the conditions of the test. Therefore, the validity criterion of increase of cell density by at least a factor of 16 over the duration of the study was fulfilled.
No remarkable observations were made concerning the appearance of the test media with the test concentrations of nominal 0.10 - 3.2 mg/L. All these test media were clear solutions throughout the test period. In the medium containing the highest test item concentration of nominal 10 mg/L, the test item was floating at the surface at the start of the test and was inhomogeneously distributed in the test medium at observations after 24 and 48 hours. Thereafter, the evaluation of the appearance of the test medium containing nominal 10 mg/L was no longer possible because of algae growth.

Based on the results, the 72-hour EbC50 was calculated to be 2.2 mg/L (95% confidence limits: 1.2 – 7.6 mg/L) and the 96-hour EbC50 was 2.8 mg/L (95% confidence limits: 1.6 – 12 mg/L). The 72-hour ErC50 was > 4.0 mg/L (95% confidence limits not determined) and the 96-hour ErC50 was > 4.0 mg/L (95% confidence limits not determined). The 72 & 96-h NOEC growth rate was 0.31 mg/L.

Results with reference substance (positive control):

The report of the test facility gives a 72 hour EC50 value of 1.70 mg/L, which is within the test facility's historical range of 0.71 to 1.74 mg/L.

Reported statistics and error estimates:

Dunnett’s test was used to identify significant differences in the calculated mean biomass and mean growth rate at the test concentrations compared to the control.

Table 2. Mean values at each concentration of test substance for the area under the growth curve at 72 and 96 hours for Pseudokirchneriella subcapitata

Test concentrations (mg/L)		Mean area under growth curve (0 – 72 hrs)	Percentage of control	Mean area under growth curve (0 – 96 hrs)	Percentage of inhibition compare to control
Nominal	Mean measured
Control	-	2836	0.0	8454	0.0
Solvent control	-	2665	6.0	8121	3.9
0.10	-	2933	-3.4	8565	-1.3
0.32	0.31	2806	1.0	8244	2.5
1.0	0.73	2445	13.8	7506	11.2
3.2	1.80	1173	58.6	4250	49.7
10	4.00	1083	61.8	3977	53.0

-% inhibition: increase in growth relative to that of solvent control

 

Table 3. Mean values at each concentration of the test substance for the growth rate at 72 and 96 hours for Pseudokirchneriella subcapitata 

Test concentrations (mg/L)		Mean growth rate (0 – 72 hrs)	Percentage of control	Mean growth rate (0 – 96 hrs)	Percentage of inhibition compare to control
Nominal	Mean measured
Control	-	1.70	0.0	1.43	0.0
Solvent control	-	1.67	1.3	1.42	0.2
0.10	-	1.71	-0.7	1.43	0.3
0.32	0.31	1.69	0.1	1.42	0.8
1.0	0.73	1.64	3.5	1.42	1.2
3.2	1.80	1.37	19.3	1.32	7.8
10	4.00	1.34	21.1	1.31	8.7

-% inhibition: increase in growth relative to that of solvent control

 

Table 4. Coefficients of variation for control growth

Coefficient of variation	0 – 72 hours	0 – 96 hours	Guideline validity criteria
section-by section specific growth rate	**%	**%	<35%
average specific growth rate	**%	**%	<10%

 

Table 5. Overview of effect concentrations

	after 72 h		after 96 h
Parameter	Biomass b (mg/L)	Growth rate r (mg/L)	Biomass b (mg/L)	Growth rate r (mg/L)
EC50 95%-confidence limits	2.2 1.2 - 7.6	> 4.0 n.d.	2.8 1.6-12	> 4.0. n.d.
EC10 95%-confidence limits	0.56 0.05-1.1	1.5 0.26-3.3	0.58 0.07 - 1.1	3.9 2.0-n.d.
EC90 95%-confidence limits	> 4.0 n.d.	> 4.0 n.d.	> 4.0 n.d.	> 4.0 n.d.
NOEC	0.31	0.31	0.31	0.31
LOEC	0.73	0.73	0.73	0.73

 

Validity criteria fulfilled:

yes

Conclusions:

In a toxicity study on green algae following OECD TG 201, OPPTS 850.5400 and EU Method C3 guidelines, the 72-h ErC50 and NOErC values were determined to be > 4 and 0.31 mg/L, respectively.

Executive summary:

This study was conducted to determine the toxicity of the test substance on green algae (Pseudokirchneriella subcapitata) according to OECD TG 201, OPPTS 850.5400, EU Method C3 and JMAFF test guideline, and in compliance with GLP principles. The algae were exposed for 96 hours in a static system at nominal test concentrations of 0 (solvent control, DMF), 0 (negative control, standard media), 0.10, 0.32, 1.0, 3.2 and 10 mg/L with an initial cell density of 1.0E+04 cells/mL. The test concentrations were analysed at 0 and 96 hours by using GC/MSD-detection. The mean measured concentrations for 0.32, 1.0, 3.2 and 10 mg/L were 0.31, 0.73, 1.80 and 4.00 mg/L, respectively. The test was carried out under the following conditions: 22 – 23 °C, continuous light (6510 - 7370 Lux), pH 7.9 – 8.3 at start and pH 9.0 – 9.1 at the end of the test. The EC and NOEC values were determined by using Dunnett-test.

The mean cell density after 72h exposure was 1.62E+06, 1.51E+06, 1.67E+06, 1.61E+06, 1.36E+06, 0.61E+06, 0.56E+06 in solvent control, negative control, 0.10, 0.32, 1.0, 3.2 and 10 mg/L treatments, respectively. Therefore, comparing to the original cell density, after 72 hours exposure, solvent control, negative control, 0.10, 0.32, 1.0, 3.2 and 10 mg/L treatments showed a growth inhibition of 0.0, 1.3, -0.7, 0.1, 3.5, 19.3 and 21.1%, respectively. Therefore, the 72-h ErC50 and NOErC values were determined to be > 4 and 0.31 mg/L, respectively, based on the mean measured concentrations.

Description of key information

Freshwater, 72-h NOErC = 0.31 mg/L (based on mean measured concentrations), Pseudokirchneriella subcapitata, OECD TG 201, OPPTS 850.5400, EU Method C3 and JMAFF test guideline, Volz 2005

Freshwater, 72-h ErC50 > 4.0 mg/L (based on mean measured concentrations), Pseudokirchneriella subcapitata, OECD TG 201, OPPTS 850.5400, EU Method C3 and JMAFF test guideline, Volz 2005

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.31 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, OPPTS 850.5400, EU Method C3 and JMAFF test guideline, and in compliance with GLP principles, green algae (Pseudokirchneriella subcapitata) were exposed to the test substance for 96 hours in a static system. The nominal test concentrations were 0 (solvent control, DMF), 0 (negative control, standard media), 0.10, 0.32, 1.0, 3.2 and 10 mg/L with an initial cell density of 1.0E+04 cells/mL. The mean measured concentrations for 0.32, 1.0, 3.2 and 10 mg/L were 0.31, 0.73, 1.80 and 4.00 mg/L, respectively. The test was carried out under the following conditions: 22 – 23 °C, continuous light (6510 - 7370 Lux), pH 7.9 – 8.3 at start and pH 9.0 – 9.1 at the end of the test. After 72 hours exposure, comparing to the original cell density, the solvent control, negative control, 0.10, 0.32, 1.0, 3.2 and 10 mg/L nominal treatments showed a growth inhibition of 0.0, 1.3, -0.7, 0.1, 3.5, 19.3 and 21.1%, respectively. Therefore, the 72-h ErC50 and NOErC values were determined to be > 4 and 0.31 mg/L, respectively, based on the mean measured concentrations.