(R)-2-(2,4-Difluorphenyl)-1,1-difluor-3-(tetrazol-1-yl)-1-{5-[4-(2,2,2-trifluorethoxy)-phenyl]-2-pyridyl}-2-propanol-(R,R)-tartrate

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:

05 May 2020 to 20 July 2020

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:

28 July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 10, 3.125, 0.98, 0.30, 0.095, 0.030, 0.009 mg/L
- Sampling method: One sample from the freshly prepared filtrate and duplicate samples from the freshly prepared test media of all test concentrations and the control (without algae) was taken at test start. Additionally, samples were taken after 24 and 48 hours by taking aliquots of each replicate which were pooled per treatment group.
All samples were diluted by a factor of two with acetonitrile.
Additional samples of the control and the dilution solvent were taken at each sampling without any sample treatment

- Sample storage conditions before analysis: All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed. Afterwards the samples were again stored deep frozen and were kept stored up to the date of the final report.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: a supersaturated stock solution of nominal 100 mg test item/L was prepared by suspending 100.0 mg test item in 1000 mL test water for preparing the test concentrations. The stock suspension was stirred for 47.5 hours at room temperature in the dark to dissolve as much test item as possible. Then, non-dissolved fractions of the test item were separated from the test medium by membrane filtration (0.45 µm cellulose acetate filter). The solution with dissolved test item was used to prepare the desired 1:10, 1:32, 1:102, 1:328, 1:1049, 1:3355 and 1:10737 dilutions.
- Controls: negative control (0 mg/L)

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (KORSHIKOV)
- Strain: strain No. 61.81 SAG
- Source: Originally supplied by Sammlung von Algenkulturen, Albrecht von Haller Institut für Pflanzenwissenschaften, Universität Göttingen, 37073 Göttingen, Germany
- Age of inoculum: exponentially growing culture set up 4 days prior to test start
- Method of cultivation: according to guideline (same as during test)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21.5 - 22.9

pH:

7.8 - 9.3

Nominal and measured concentrations:

Measured concentrations (geom. mean): 1.33, 4.13, 21.9, 96,4, 364, 1618, 6114 µg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 50ml Erlenmeyer flasks, loosely covered
- Initial cells density: 5000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 5045 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometric measurement

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3
- Range finding study: yes
- Test concentrations: 1, 10 and 100 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1 651 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

96.4 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

615 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

21.9 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: no
- Unusual cell shape: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 0.20 mg/L (yield), 0.20 mg/L (growth rate), 0.063 mg/L (biomass)

Validity criteria

Cell density increase in control cultures: 22.9-fold increase within 72 hours

Coefficient of Variation of sectional growth rates in control cultures: 6.4%

Coefficient of Variation of average growth between control replicates: 0.8%

Table 1: Influence of (R)-3 -Amino-2 -(2,4 -difluorophenyl)-1,1 -difluoro-1 -(5 -(4 -(2,2,2 -trifluoroethoxy)phenyl)pyridin-2 -yl)propan-2 -ol L tartrate on the growth of Pseudokirchneriella subcapitata

Parameter	Yield [µg test item/L]	Growth rate [µg test item/L]
72-hour EC50	615	1651
95% CI	543-711	1544-1767
72-hour EC20	343	932
95% CI	288-393	756-1061
72-hour EC10	253	691
95% CI	198-300	508-832
72-hour NOEC	21.9	96.4
72-hour LOEC	96.4	394

Table 2: Yield and percentage inhibition of yield during the test period

Geometric Mean Measured Concentrations [µg test item/L]	Yields y [10000 cells/ml] and % inhibition of y
	0-24 hours		0-48 hours		0-72 hours
	y	%	y	%	y	%
Control	2.261	-	18.377	-	110.930
1.33	2.537	-12.2	18.860	-2.6	118.284	-6.6
4.13	2.123	6.1	17.687	3.8	106.380	4.1
12.9	1.985	12.2*	18.446	-0.4	114.721	-3.4
96.4	1.985	12.2*	17.273	6.0*	100.997	9.0*
394	1.777	21.4*	14.237	22.5*	82.725	25.4*
1618	0.950	58.0*	2.369	87.1*	8.270	92.5*
6114	0.040	98.2*	0.031	99.8*	0.000	100.0*

negative values in % inhibition indicate an increase in growth relative to that of the control

* mean value significantly different from the control (tested with Williams t-test, a = 0.05, one sided smaller)

Table 3: Growth rates and percentage inhibition of growth rate during the test period

Geometric Mean Measured Concentrations [µg test item/L]	Growth rates µ [10000 cells/ml] and % inhibition of y
	0-24 hours		0-48 hours		0-72 hours
	µ	%	µ	%	µ	%
Control	1.707	-	1.815	-	1.802	-
1.33	1.802	-5.6	1.828	-0.7	1.823	-1.2
4.13	1.657	3.0	1.797	1.0	1.788	0.8
12.9	1.603	6.1	1.817	-0.1	1.813	-0.6
96.4	1.603	6.1	1.785	1.6	1.770	1.7
394	1.516	11.2	1.690	6.9	1.704	5.4
1618	1.058	38.1	0.866	52.3	0.937	48.0
6114	0.072	95.8	0.028	98.4	0.000	100.0

negative values in % inhibition indicate an increase in growth relative to that of the control

* mean value significantly different from the control (tested with Bonferroni-Welch t-test (24h), Williams t-test (48h) and Step down Jonckheere-Terprtra (72h), a = 0.05, one sided smaller)

Validity criteria fulfilled:

yes

Conclusions:

The influence of (R)-3-Amino-2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-(4-(2,2,2-trifluoroethoxy)phenyl)pyridin-2-yl)propan-2-ol L-tartrate on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyC50 was calculated to be 615 µg test item/L and the 72-hour ErC50 value was calculated to be 1651 µg test item/L. The 72-hour NOEyC was determined to be 21.9 µg test item/L and the associated 72-hour LOEyC was 96.4 µg test item/L. The 72-hour NOErC was determined to be 96.4 µg test item/L and the associated 72-hour LOErC was 394 µg test item/L.

Executive summary:

In a 72 hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata strain No. 61.81 SAG were exposed to (R)-3-Amino-2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-(4-(2,2,2-trifluoroethoxy)phenyl)pyridin-2-yl)propan-2-ol L-tartrate at measured concentrations of 1.33, 4.13, 21.9, 96,4, 364, 1618, 6114 µg/L (geom. mean) under static conditions in accordance with the OECD 202 guideline.  The NOEC and EC₅₀ values based on growth rate were 96.4 and 1651 µg/L, respectively.  The % growth inhibition in the treated algal culture as compared to the control ranged from -1.2 to 100 .

 

This toxicity study is classified as acceptable and satisfies the guideline requirements for green algae short-term toxicity study.

 

Results Synopsis

 

Test Organism: Pseudokirchneriella subcapitata

Test Type: static

 

72 hr EC₅₀ 1651 µg/L               95% C.I.:  1544 to 1767 mg/L

72 hr NOEC:  96.4.µg/L                  

Endpoint(s) Effected:  growth rate

Description of key information

In a 72 hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata strain No. 61.81 SAG were exposed to (R)-3-Amino-2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-(4-(2,2,2-trifluoroethoxy)phenyl)pyridin-2-yl)propan-2-ol L-tartrate at measured concentrations of 1.33, 4.13, 21.9, 96,4, 364, 1618, 6114 µg/L (geom. mean) under static conditions in accordance with the OECD 202 guideline.  The NOEC and EC₅₀values based on growth rate were 96.4 and 1651 µg/L, respectively.  The % growth inhibition in the treated algal culture as compared to the control ranged from -1.2 to 100 .

 

This toxicity study is classified as acceptable and satisfies the guideline requirements for green algae short-term toxicity study.

Key value for chemical safety assessment

EC50 for freshwater algae:

1 651 µg/L

EC10 or NOEC for freshwater algae:

96.4 µg/L