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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

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/control:
The study was performed with five test item concentrations at 6.25 - 12.50 - 25.00 - 50.00 - 100.00 mg/L of test item in reconstituted test water (ISO 6341).

Sampling method:
20 daphnids, divided in 4 replicates of 5 daphnids, were used for each group.
The test organisms were exposed to the test solutions for 48 hours, with renewal of the test solutions after 24 hours. The number of mobile and immobile daphnids was counted at 24 and 48 hours after the beginning of the test.

Sample analysis:
The samples to be analysed were collected as follows:
- 6 samples at 0 hours, from the fresh solutions: 1 untreated control, 5 concentrations of test item.
- 6 samples at 48 hours, from the aged solutions: 1 untreated control, 5 concentrations of test item.
The samples were diluted to the calibration range with ISO medium before the analysis.

Vehicle:

no

Details on test solutions:

At the start of the test, a stock solution of 100 mg test item/L (SsA) was 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. This solution was treated with ultrasound for 15 minutes and shaking for 3 minutes until completely dissolved.
The solution was directly used as treatment group C5. Remaining test solutions (C1-C4) were prepared by dilution of the stock solution SsA with ISO medium.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
Species: Daphnia magna
- code of the culture: Dm 29/02/20.
- Age: < 24 hours old.
- Diet: No feeding during the test.
- Housing (before the test): Under breeding conditions, the test organisms were fed with algae suspension (Pseudokirchneriella subcapitata) and were maintained in reconstituted water.
- pH of medium: 6 - 9.
- dissolved oxygen: ≥ 3 mg/L.
- maintained in thermostatic chamber at the temperature range of 20±2°C, with a photoperiod of 16 hours light and 8 hours dark.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

between 140 and 250 mg/L (as CaCO3)

Test temperature:

20.0 - 20.1°C (temperature of the test medium).
19.3 - 20.5°C (temperature of the thermostatic chamber).

pH:

in the range of 6-9.

Dissolved oxygen:

≥ 3 mg/L.

Nominal and measured concentrations:

Nominal test item concentration [mg/L]: 6.25 - 12.50 - 25.00 - 50.00 - 100.00 mg/L.

Details on test conditions:

The test was conducted in a thermostatic chamber designed to maintain the test conditions specified in the protocol and in agreement with OECD 202 (2004). The temperature and light intensity values in the chamber were measured continuously during the test using a data-logger system.
During the exposure period, the temperature measured in the medium was in the range of 20.0 - 20.1 °C, the temperature measured in the thermostatic chamber was in the range of 19.3 - 20.5 °C, the light intensity was at maximum 868 Lux and a photoperiod of 16 hours light and 8 hours dark was applied.
Dissolved oxygen concentration and pH values were measured in the treated and untreated solutions, at the beginning and at the end of the test. The hardness of the medium was measured at the start of the test.
The test solutions were not aerated and the daphnids have not been fed during the test.
During the test, the vessel position was modified daily and recorded in the study notebook; the vessels were positioned without a predetermined order, in order to minimise the influence of spatial differences of incubation conditions.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

59.51 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

No other effects (e.g. behavioural effects, discoloration or trapping) were observed on the exposed organisms.

Reported statistics and error estimates:

The assessment of the effects was based on the nominal concentrations of test item.

Percent immobilisation after 24 and 48 hours of exposure of Daphnia magna to the test item:

Code	Nominal test item conc. [mg/L]	Number of exposed daphnids	Response at 24 h		Response at 48 h
			Number of immobilised	%I	Number of immobilised	%I
CTRL	0.00	20	0	0	0	0
C1	6.25	20	0	0	0	0
C2	12.50	20	0	0	0	0
C3	25.00	20	0	0	1	5
C4	50.00	20	0	0	2	10
C5	100.00	20	0	0	3	15

According to OECD 202 (2004), the validity criteria were evaluated: Validity criteria results of the immobilisation test with Daphnia magna:

Validity criteria	Threshold	Observed
Immobilised daphnids in the untreated group, at the end of the test:	< 10%	0%
Dissolved oxygen concentration at the end of the test in treated and untreated groups:	≥ 3 mg/L	7.95 - 8.12 mg/L

Accordingly, all validity criteria are met.

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

Code	Nominal test item conc. [mg/L]	Nominal conc. [mg a.i./L]	Mean of measured conc. [mg a.i./L]	Mean Recovery (%)
CTRL	0.00	0.00	< LOD *	n.d.
C1	6.25	6.03	5.64	93.52
C2	12.50	12.06	11.44	94.90
C3	25.0	24.12	21.66	89.82
C4	50.0	48.24	46.26	95.89
C5	100	96.48	95.04	98.51

* Parameter from the validated analytical method: LOD 0.4969 μg/L; LOQ 1.0 μg/L;

n.d. = not determinable

The analytical determination of the content of test item in the test solutions showed recovery values within the range 80-120% of the nominal values (with a mean recovery of 92.15% in the fresh samples and 92.51% in the aged samples), therefore the data evaluation was performed using the nominal concentrations.

 

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

 

Effect concentrations of the Test item on Daphnia magna after 48 h exposure:

Endpoint (48 hours)	Test item concentration [mg/L]	Confidence limits
		Lower	Upper
EC10	59.51	20.82	> 100
EC20	> 100	n.d.	n.d.
EC50	> 100	n.d.	n.d.
LOEC	50.00	-	-
NOEC	25.00	-	-

Validity criteria fulfilled:

yes

Conclusions:

For 3-Bromo-1-(3-chloro-2-pyridinyl)-1H-pyrazole-5-carboxylic acid the water hazard on Daphnia magna after 48 hours of exposure was determined with the following results:
-EC10 59.51 mg/L; EC20 > 100 mg/L; EC50 > 100 mg/L (value higher than the maximum concentration tested).
- Corresponding LOEC 50.00 mg/L.
- Corresponding NOEC 25.00 mg/L.
Additionally no other effects were observed on the exposed organisms.

No classification as hazardous according to EU regulation 1272/2008 (CLP) is required.

Executive summary:

The acute immobilisation test was performed, under static conditions, to assess the effects of test. item 3-Bromo-1-(3-chloro-2-pyridinyl)-1H-pyrazole-5-carboxylic acid on Daphnia magna after 48 hours of exposure.

20 daphnids, less than 24 hours old, were exposed to 6.25 - 12.50 - 25.00 - 50.00 - 100.00 mg/L of test item in ISO medium plus an untreated control group.

The analytical determination of the active ingredient content was performed by UHPLC-MS/MS. The

analytical method was validated according to SANCO/3029/99 rev.4 in a separate GLP study (BT013/20). The analytical determination of the active ingredient content showed a mean recovery of 94.24% in the fresh samples and 94.81% in the aged samples.

Accordingly, the results are presented based on nominal concentrations of test item.

After 48 hours, the endpoints based on the test item nominal concentrations were estimated to be the following:

- EC10 59.51 mg/L; EC20 > 100 mg/L; EC50 > 100 mg/L (value higher than the maximum concentration tested).

- Corresponding LOEC: 50.00 mg/L.

- Corresponding NOEC: 25.00 mg/L.

No other effects were observed on the exposed organisms.

The validity criteria of OECD 202 (2004) were fulfilled.

Description of key information

Short-term toxicity to aquatic invertebrates according OECD 202:

After 48 hours, the endpoints based on the test item nominal concentrations were estimated to be the following:

- EC₁₀ 59.51 mg/L; EC₂₀ > 100mg/L; EC₅₀ > 100 mg/L.

- Corresponding LOEC: 50.00 mg/L.

- Corresponding NOEC: 25.00 mg/L.

No other effects were observed on the exposed organisms.

The validity criteria of OECD 202 (2004) were fulfilled.

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