3,5-bis(difluoromethyl)-1H-pyrazole

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2019 - Oct. 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

July 2011

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

January 2012

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3,5-bis(difluoromethyl)-1H-pyrazole

EC Number:

814-072-3

Cas Number:

77614-79-0

Molecular formula:

C5H4F4N2

IUPAC Name:

3,5-bis(difluoromethyl)-1H-pyrazole

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100, 32, 10, 3.2 and 1.0 mg test item/L
- Sampling method: Duplicate samples from all test concentrations and the control at the start and every 24 hours until the end of the experiment.
- Sample storage conditions before analysis: Storage in a freezer (<= -20°C) in the dark until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of a 100 mg/L stock solution in test medium. Lower concentrations were prepared by dillution of the stock solution.
- Test medium: Reconstituted Water (OECD Medium)
- Controls: Yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: Strain No. 61.81 SAG,
- Source (laboratory, culture collection): in-House Breeding, originally supplied by Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen"

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test conditions

Hardness:

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

22.2 - 23.5 °C

pH:

The pH value of untreated test water was checked and in range of 8.1 ± 0.1 at test start.
The pH in the control was: 8.0 at test start, 9.1 after 72 hours of exposure and 10.0 at test end The pH in the control medium increased by 1.1 units over the first 72 h. Over the entire test duration of 96 h, an increase of 2.0 units was observed in the control media.
The pH at different test item concentrations was: 7.8 to 7.9 at test start, 7.0 to 9.1 after 72 hours of exposure and 4.7 to 10.0 at test end

Nominal and measured concentrations:

Nominal concentrations: 100, 32, 10, 3.2 and 1.0 mg test item/L and a control
Measured concentrations (geometric mean): 72 hours: 66.0, 15.5, 2.65, 0.595 and 0.195 mg test item/L and a control


The initial concentrations and the maintenance of the exposure concentrations during the test were determined in the analytical part. All reported results refer to geometric mean measured values since the concentrations of the test item were not within ± 20 % of the measured initial concentrations during the test.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): open covered with air permeable glass dishes
- Material, size, headspace, fill volume: Size of 200 mL volume filled with approximately 100 mL of test medium (≤ 50% of flask volume)
- Aeration: none
- Initial cells density: 10'000 cell/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water (OECD Medium)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Continuous illumination
- Light intensity: 4691 lx (range: 4450 to 5060 lx)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer (at 0, 24, 48, and 72 h)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

47.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % conf. interval

Remarks:

45.6 - 49.7 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.65 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

11.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % conf. interval

Remarks:

10.6 - 12.9 mg/L

Results with reference substance (positive control):

72-h EC50 (Growth rate) Potassium dichromate = 0.941 mg/L
72-h EC50 (Biomass) Potassium dichromate = 0.494 mg/L
72-h EC50 (Yield) Potassium dichromate = 0.445 mg/L

Any other information on results incl. tables

Table 1: Growth Rates µ and Percentage Inhibition of µ during the Test Period

Nominal test concentration [mg test item/L]	Growth rate (1/day) 0 - 24 h	Inhibition growth rate 0 - 24 h in %	Significance	Growth rate (1/day) 24 - 48 h	Inhibition growth rate 24 - 48 h in %	Significance	Growth rate (1/day) 0 - 24 h	Inhibition growth rate 48 - 72 h in %	Significance
Control	1.823	-		1.837	-		1.675	-
1	1.757	3.7		1.812	1.4		1.659	1
3.2	1.805	1		1.837	0		1.682	-0.4
10	1.807	0.9		1.797	2.1		1.668	0.4
32	1.694	7.1	*	1.615	12.1	*	1.422	15.1	*
100	1.277	30	*	0.93	49.4	*	0.64	61.8	*

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, α = 0.05, one-sided)

Table 2: Validity criteria for OECD 201

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	153	Yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	17.1 %	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus.	1.7 %	Yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.