3-bromo-1-(3-chloropyrid-2-yl)-5-methyl-1H-pyrazole

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:

13 March 2020 to 6 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:

adopted 2006, corrected 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Storage Condition:
Storage Temperature: Room temperature (15 to 30 °C).
Storage Condition: Dark, room temperature (15-25°C), < 70 % relative humidity.
Storage Container: In original container as supplied by the Sponsor.
Storage Location: Test Item Control Office, JRF.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 4.27, 9.39, 20.66, 45.45 and 100.00 mg test item/L in EPA’s AAP growth medium containing 0.01 % acetone as solvent. Two untreated control groups (with and without solvent).

- Sampling method: Three replicates were prepared for the treated groups and six replicates for the control groups, each replicate containing 100 mL of solution. The samples to be analysed were collected as follows: at the start of the test from fresh test solutions (T0) and at the end of the test from the aged test solutions (T72, collected after pooling of the replicates). The samples to be analysed were collected as follows:
- 7 samples at 0 hours, from the fresh solutions: 2 untreated controls, 5 concentrations of test item;
- 7 samples at 72 hours, from the aged solutions: 2 untreated controls, 5 concentrations of test item.
The samples were stored deep-frozen at ≤ -18°C prior to analysis. Analysis was performed within one week after termination of the biological part of the experiment.

Vehicle:

no

Details on test solutions:

Test solutions were prepared in EPA medium containing 100 μL/L solvent (Acetone, 0.01%).

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata (strain SAG 61.81).
Source: In-house culturing at the Test Facility, originating from SAG Culture collection of algae (Germany).
Housing (before the test):
Culturing conditions: the test system was maintained in AAP medium, in an incubator with orbital shaker at the temperature range of 23 ± 2°C and with continuous light intensity in the range of 4440-8880 Lux.

Test type:

not specified

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

23 ± 2 °C.

pH:

7.41 - 7.55 in the untreated control groups.
7.19 - 7.38 in the treatment groups.

Nominal and measured concentrations:

Nominal Test item concentration (mg/L): 4.27, 9.39, 20.66, 45.45, 100.00.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

EyC50

Effect conc.:

30.05 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Growth inhibition based on yield

Key result

Duration:

72 h

Dose descriptor:

LOEC

Remarks:

LOEyC

Effect conc.:

20.66 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Growth inhibition based on yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

NOEyC

Effect conc.:

9.39 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Growth inhibition based on yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

ErC50

Effect conc.:

66.46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Growth inhibition based on growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Remarks:

LOErC

Effect conc.:

20.66 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Growth inhibition based on growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

NOErC

Effect conc.:

9.39 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Growth inhibition based on growth rate

Details on results:

- Exponential growth in the control (for algal test): Yes.
- Observation of abnormalities (for algal test): No.
- Unusual cell shape: No.
- Colour differences: No.
- Aggregation of algal cells: No.
- Any stimulation of growth found in any treatment: No.

Reported statistics and error estimates:

There were no significant statistical differences between control and solvent control, therefore the treated groups were compared with the solvent control.
The assessment of the effects was based on the nominal concentrations of test item. The ECx endpoints, with 95% confidence limits, the lowest observed effect concentrations (LOEC) and the no observed effect concentrations (NOEC).

The percentage of inhibition of growth based on growth rate (%Ir) and on yield (%Iy) for each test concentration and any significant inhibition compared to the control (after 72 hours) are reported in the Table with a summary of the analytical results for the biological samples from the current study:

 

Summary of the analytical results for fresh and aged media samples from the biological part of the study with Pseudokirchneriella subcapitata:

Code	Nominal conc. [mg a.i./L]	Mean of measured conc. [mg a.i./L]	Mean Recovery (%)
CTRL	0.00	< LOD *	n.d.
CT SOLV	0.00	< LOD *	n.d.
C1	4.16	4.41	106.10
C2	9.15	8.58	93.77
C3	20.14	19.69	97.78
C4	44.31	41.18	92.94
C5	97.49	103.58	106.25

* Parameter from the validated analytical method analytical method: LOD 0.5308 μg/L; LOQ 1.0 μg/L;

n.d. = not determinable.

The analytical determination of the content of active ingredient in the test solutions showed recovery values within the range 80-120% of the nominal values (with a mean recovery of 101.42% in the fresh samples and 97.32% in the aged samples), therefore the data evaluation was performed using the nominal concentrations.

For yield, data was normally distributed (Shapiro-Wilk’s Test) but variance homogeneity was not given (Levene’s Test), thus a Multiple Sequentially-rejective Welsh-t-test after Bonferroni-Holm (α = 0.05, one-sided smaller) was applied.

For growth rate, data was normally distributed (Shapiro-Wilk’s Test) and variance homogeneity given (Levene’s Test) a Williams Multiple Sequential t-test was applied (α = 0.05, one-sided smaller).

Inhibition of growth of Pseudokirchneriella subcapitata based on growth rate (Ir) and yield (Iy) in percent after 72 h-exposure to the Test item, in relation to the control:

Code	Test item conc. [mg/L]	Nominal 3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole conc. [mg a.i./L]	%Ir^A	%Iy^B
C1	4.27	4.16	0.33	1.61
C2	9.39	9.15	1.90	8.71
C3	20.66	20.14	11.55^+	41.52^+
C4	45.45	44.31	17.85^+	56.42^+
C5	100.00	97.49	83.53^+	98.82^+

A⁺indicates a significant difference compared to the untreated control (Williams Multiple Sequential t-test, α = 0.05, one-sided smaller).

B⁺indicates a significant difference compared to the untreated control (Multiple Sequentially-rejective Welsh-t-test after Bonferroni-Holm, α = 0.05, one-sided smaller).

Effect concentrations as calculated by Probit analysis are presented in the following table.

 

Effect concentrations of the Test item for growth rate and yield of Pseudokirchneriella subcapitata after 72 h exposure:

Endpoint (72 hours)	Test item concentration [mg/L]	Confidence limits		Endpoint (72 hours)	Test item concentration [mg/L]	Confidence limits
		Lower	Upper			Lower	Upper
EyC10	8.81	5.56	11.78	ErC10	38.46	32.52		43.33
EyC20	13.43	9.62	16.78	ErC20	46.40	40.80		51.14
EyC50	30.05	25.26	35.80	ErC50	66.46	61.14		72.30
LOEyC	20.66	-	-	LOErC	20.66	-		-
NOEyC	9.39	-	-	NOErC	9.39	-		-

Validity criteria fulfilled:

yes

Conclusions:

The growth inhibition test was performed in order to evaluate the effects of the test item 3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole on the green alga Pseudokirchneriella subcapitata, after 72 hours of exposure to a geometric series of test item concentrations.

Calculated endpoints based on the nominal concentrations of test item are shown in the following:
- Growth inhibition based on yield: EyC50 = 30.05 mg/L (95 % C.L. 25.26 - 35.80 mg/L);
- Corresponding LOEyC = 20.66 mg/L and NOEyC = 9.39 mg/L;
- Growth inhibition based on growth rate: ErC50 = 66.46 mg/L (95 % C.L. 61.14 - 72.30 mg/L);
- Corresponding LOErC = 20.66 mg/L and NOErC = 9.39 mg/L.
No cells with abnormal appearance were observed in any of the control or test item treatment groups.
The validity criteria of OECD 201 (2011) were fulfilled, thus the study is valid.
Based on EU regulation 1272/2008 the results lead to classification as hazardous to aquatic environment, chronic category 3 (H412: Harmful to aquatic life with long lasting effects.).

Executive summary:

The growth inhibition test was performed according OECD 201 in order to evaluate the effects of the test item 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole on green alga Pseudokirchneriella subcapitata, after 72 hours of exposure.

The cultures were exposed to 4.27, 9.39, 20.66, 45.45 and 100.00 mg test item/L, plus two untreated control groups (with and without solvent).

The analytical determination of the active ingredient content showed a mean recovery of 101.42% in the fresh samples and 97.32% in the aged samples. Accordingly, the assessment of the effects was based on the nominal concentrations of the test item.

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

 

The endpoints, based on the nominal concentrations of test item, were estimated to be the following:

- Growth inhibition based on yield: E_yC₅₀= 30.05 mg/L(95 % C.L. 25.26 - 35.80 mg/L);

- Corresponding LOE_yC = 20.66 mg/L and NOE_yC = 9.39 mg/L;

- Growth inhibition based on growth rate: E_rC₅₀= 66.46 mg/L(95 % C.L. 61.14 - 72.30 mg/L);

- Corresponding LOE_rC = 20.66 mg/L and NOE_rC = 9.39 mg/L.

 

The validity criteria of OECD 201 (2011) were fulfilled.

Description of key information

Growth inhibition test according OECD 201 with green alga Pseudokirchneriella subcapitata (72 h):

After 72 hours exposure, no cells with abnormal appearance were observed in any of the treatment groups. The endpoints, based on the nominal concentrations of test item, 3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole, were estimated to be the following:

- Growth inhibition based on yield:EyC50 = 30.05 mg/L (95 % C.L. 25.26 - 35.80 mg/L);

- Corresponding LOEyC = 20.66 mg/L and NOEyC = 9.39 mg/L;

- Growth inhibition based on growth rate: ErC50 = 66.46 mg/L (95 % C.L. 61.14 - 72.30 mg/L);

- Corresponding LOErC = 20.66 mg/L and NOErC = 9.39 mg/L.

The validity criteria of OECD 201 (2011) were fulfilled.

Based on EU regulation 1272/2008 the results lead to classification as hazardous to aquatic environment, chronic category 3 (H412: Harmful to aquatic life with long lasting effects.).

Key value for chemical safety assessment

EC50 for freshwater algae:

30.05 mg/L