1H-Pyrazole-5-carboxylic acid, 3-bromo-1-(3-chloro-2-pyridinyl)-

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:

16 March 2020 to 16 April 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Storage: Dark, room temperature (15-25°C), < 70 % relative humidity, keep container tightly closed in a well-ventilated place.

Analytical monitoring:

yes

Details on sampling:

- Concentration nominal: 100 mg/L; Active ingredient conc.: 96.48 mg/L.
- Sampling method: six replicates for the treated (C1) and untreated (CTRL: medium without test item) groups; each replicate was prepared with 100 mL of solution.

- The samples to be analysed were collected as follows:
- 2 samples at 0 hours, from the fresh solutions: 1 untreated control, 1 concentration of test item.
- 2 samples at 72 hours, from the aged solutions: 1 untreated control, 1 concentration of test item.
The samples were diluted to the calibration range with EPA medium before the analysis.

Each sample was measured by double injection and the content of active ingredient in the test samples was calculated based on the calibration curve equation obtained in the same chromatographic session.

The biomass (cell density) in each test vessel was evaluated after 24, 48 and 72 hours of exposure.

Vehicle:

no

Details on test solutions:

The limit concentration of 100 mg test item/L in test medium (SsA) was be prepared as follows: 100.0 mg test item were added to a 1 L volumetric flask containing 500 mL test medium and made up to approx. 700 mL with test medium.
The pH of the limit concentration test solution was corrected within the range of pH 7.5±0.1 (required for the control group) with NaOH solution as described in the OECD Guidance No. 23 (2019).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (new name: Raphidocelis subcapitata).
- Strain: SAG 61.81.
- Original source of cultured organisms: SAG Culture collection of algae (Göttingen).
Pseudokirchneriella subcapitata is maintained in an incubator, at a temperature in the range of 23±2°C and with continuous light (intensity in the range of 4440-8880 Lux), in AAP medium (US EPA, OECD 201).
The algae cultures are periodically prepared by inoculating the fresh growth medium with algal cells (104 cells/mL) from older cultures.
The culture utilized for the inoculums of this test was prepared 4 days before the test beginning (code of the culture: Ps 19/03/20).

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

21.8 - 24.0 °C

pH:

In the range of 7.50 - 7.89 in the untreated control group and 7.50 - 7.75 in the treatment group.

Nominal and measured concentrations:

Nominal concentrations: 100.00 mg test item/L.

Details on test conditions:

TEST SYSTEM
- thermostatic chamber
- Initial cells density: 104 cells/mL.
- Control end cells density:
- No. of vessels per concentration (replicates): six replicates for the treated groups.
- No. of vessels per control (replicates): six replicates for the untreated control groups.

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: measured at the beginning and at the end of the study, were in the range of 7.50 - 7.89 in the untreated control group and 7.50 - 7.75 in the treatment group.
- Light intensity and quality: continuous and in the range of 6391 - 6848 Lux.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: The biomass in each flask and the appearance of the cells were assessed daily during the test period, by electronic particle counter (each sample was evaluated with three counts and the cell density was expressed as mean of the three obtained values).
Microscopic observations were performed in order to assess any abnormal appearance of the algal cells that may have been caused by exposure to the test item.

TEST CONCENTRATIONS
- Test concentrations: 100.00 mg test item/L.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

EyC10/20/50

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: no inhibitory effect

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

ErC10/20/50

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: no inhibitory effect

Key result

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

growth and yield

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: no effects were observed

Details on results:

- Exponential growth in the control (for algal test): yes.
- Observation of abnormalities (for algal test): no.

Reported statistics and error estimates:

The assessment of the effects was based on the nominal concentration of test item.
The data of growth were elaborated by statistical program to compare the responses obtained in control group and in treated group.
According to the results of Student t-test, there was no statistically significant difference between the control and the treated group. Accordingly, the EyC10/20/50 and ErC10/20/50 values are greater than 100.0 mg test item/L (equivalent to 96.48 mg a.i./L ) and the NOEC for both growth and yield is at least this concentration.

The biomass (cell density) in each test vessel was evaluated after 24, 48 and 72 hours of exposure and the results are reported in the following Table. The initial cell density was 10⁴ to the 4th cells/mL.

For the calculation of effect concentrations below, the limit treatment group was compared with the control.

 

Cell density (×10⁴cells/mL) after 24, 48 and 72 hours of exposure of Pseudokirchneriella subcapitata to the Test item

Nominal test item conc. [mg/L]	Code	After 24 hours		After 48 hours		After 72 hours
		Mean value* (replicate)	Mean value (group)	Mean value* (replicate)	Mean value (group)	Mean value* (replicate)	Mean value (group)
0.00	CTRL-A	4.68	4.09	25.07	30.81	180.03	180.88
	CTRL-B	4.04		25.47		176.95
	CTRL-C	3.54		34.32		181.51
	CTRL-D	3.65		33.21		185.22
	CTRL-E	3.44		33.36		179.04
	CTRL-F	3.76		33.40		182.52
100.00	C1-A	4.21	3.93	24.37	30.83	207.27	180.45
	C1-B	3.69		25.49		166.62
	C1-C	3.88		34.27		164.95
	C1-D	3.53		33.23		161.85
	C1-E	4.12		34.32		181.16
	C1-F	3.58		33.32		200.87

* each sample was counted three times

 

 

Section-by-section growth rate (μ) during the exposure of Pseudokirchneriella subcapitata to the Test item:

Test item conc. [mg/L]	Code	μ 0-24 hours		μ 24-48 hours		μ 48-72 hours
		Mean value* (replicate)	Mean value (group)	Mean value* (replicate)	Mean value (group)	Mean value* (replicate)	Mean value (group)
0.00	CTRL-A	1.543	1.344	1.679	2.076	1.971	1.779
	CTRL-B	1.397		1.841		1.938
	CTRL-C	1.264		2.272		1.665
	CTRL-D	1.296		2.207		1.719
	CTRL-E	1.236		2.271		1.681
	CTRL-F	1.325		2.183		1.699
100.00	C1-A	1.437	1.342	1.756	2.077	2.141	1.773
	C1-B	1.304		1.934		1.878
	C1-C	1.357		2.177		1.572
	C1-D	1.261		2.242		1.584
	C1-E	1.415		2.121		1.663
	C1-F	1.276		2.230		1.797

* each sample was counted three times

 

The yield values (y) during 72 hours of exposure and the percentage of inhibition (%Iy) are reported in the following tables. There is no statistically significant difference between the Control and the limit concentration of 100 mg/L.

 

Yield value (×10⁴cells/mL) and %Iy after 72 hours of exposure of Pseudokirchneriella subcapitata to the Test item:

Test item conc. [mg/L]	Code	N°cells 0 hours	N°cells 72 hours	Yield 0-72 hours	Yield mean value	Std. Dev.	CV%	%Iy	%Iy mean value
0.00	CTRL-A	1	180.03	179.03	179.88	2.88	1.60	-	-
	CTRL-B	1	176.95	175.95
	CTRL-C	1	181.51	180.51
	CTRL-D	1	185.22	184.22
	CTRL-E	1	179.04	178.04
	CTRL-F	1	182.52	181.52
100.00	C1-A	1	207.27	206.27	179.45	19.57	10.91	-14.67	0.24
	C1-B	1	166.62	165.62				7.93
	C1-C	1	164.95	163.95				8.86
	C1-D	1	161.85	160.85				10.58
	C1-E	1	181.16	180.16				-0.16
	C1-F	1	200.87	199.87				-11.11

Section-by-section yield during the exposure of Pseudokirchneriella subcapitata to the Test item:

Test item conc. [mg/L]	Code	μ 0-24 hours		μ 24-48 hours		μ 48-72 hours
		Mean value* (replicate)	Mean value (group)	Mean value* (replicate)	Mean value (group)	Mean value* (replicate)	Mean value (group)
0.00	CTRL-A	3.68	2.85	20.39	26.95	154.96	150.07
	CTRL-B	3.04		21.43		151.48
	CTRL-C	2.54		30.78		147.19
	CTRL-D	2.65		29.56		152.01
	CTRL-E	2.44		29.92		145.68
	CTRL-F	2.76		29.64		149.12
100.00	C1-A	3.21	2.84	20.16	27.00	182.90	149.62
	C1-B	2.69		21.80		141.13
	C1-C	2.88		30.39		130.68
	C1-D	2.53		29.70		128.62
	C1-E	3.12		30.20		146.84
	C1-F	2.58		29.74		167.55

* each sample was counted three times

 

During the test, no abnormal cells were observed.

Validity criteria results of the growth inhibition test with Pseudokirchneriella subcapitata:

VALIDITY CRITERIA	Threshold	Observed in the solvent control
Cells concentration in the control cultures within 3 days:	Increased by a factor of at least 16 (specific growth rate at least 0.92 day^-1)	Factor 180.9 (mean specific growth rate 1.733 day^-1)
Mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures:	Not exceed 35%	23.3%
Coefficient of variation of average specific growth rates during the whole test period in the replicate of the control group:	Not exceed 7%	0.32%

Accordingly, all validity criteria are met.

Validity criteria fulfilled:

yes

Conclusions:

The growth inhibition test was performed as limit test, to demonstrate that the test item 3-Bromo-1-(3-chloro-2-pyridinyl)-1H-pyrazole-5-carboxylic acid has no inhibitory effect on the green alga Pseudokirchneriella subcapitata, after 72 hours of exposure at the concentration of 100.0 mg/L.

The validity criteria of the biological test were satisfied.

The analytical determination of the content of 3-Bromo-1-(3-chloro-2-pyridinyl)-1H-pyrazole-5-carboxylic acid showed a recovery of 91.79 % in the fresh sample and 92.49 % in the spent sample.
Therefore, the assessment of the effects was based on the nominal concentration.

For 3-Bromo-1-(3-chloro-2-pyridinyl)-1H-pyrazole-5-carboxylic acid, no effects were observed on the growth of P. subcapitata cultures exposed at 100.0 mg test item/L (equivalent to 96.480 mg a.i./L).

Accordingly, the EyC10/20/50 and ErC10/20/50 values are greater than 96.480 mg a.s./L and the NOEC for both growth and yield is at least this concentration.

During the test, no abnormal cells were observed.

The validity criteria of OECD 201 (2011) were fulfilled, thus the study is valid.

Based on EU regulation 1272/2008 it is proposed classification is not required.

Executive summary:

The growth inhibition test was performed as limit test, in order to demonstrate that the test item has no inhibitory effect on the algal growth at the concentration of 100 mg/L.

Algal cultures were exposed to 100 mg/L of 3-Bromo-1-(3-chloro-2-pyridinyl)-1Hpyrazole-5-carboxylic acid for a period of 72 hours and no effects were observed on the exposed organisms.

At the start of the test, the pH of the limit concentration test solution was corrected within the range of pH 7.5±0.1 required for the control group with NaOH solution.

The analytical determination of the active ingredient content showed a mean recovery of 91.79 % in the fresh samples and 92.49 % in the aged samples.

Accordingly, the assessment of the effects was based on the nominal concentration of the test item.

 

For the test item, no effects were observed on the growth of P. subcapitata cultures exposed at 100.0 mg test item/L (equivalent to 96.480 mg a.i./L). Accordingly, the EyC10/20/50 and ErC10/20/50 values are greater than 100 mg test item/L, corresponding to 96.480 mg a.s./L and the NOEC for both growth and yield is at least this concentration.

 

After 72 hours exposure, no cells with abnormal appearance were observed in the treatment group.

 

The validity criteria of OECD 201 (2011) were fulfilled, thus the study is valid. 

Description of key information

Toxicity to aquatic algae according OECD 201:

The growth inhibition test was performed as limit test, to demonstrate that the test item 3-Bromo-1-(3-chloro-2-pyridinyl)-1H-pyrazole-5-carboxylic acid has no inhibitory effect on the green alga Pseudokirchneriella subcapitata, after 72 hours of exposure at the concentration of 100.0 mg/L.

In accordance with the provisions of regulation 1272/2008, Annex I, 4.1, it is proposed classification is not required.