1H-Pyrazolo[3,4-b]pyridine-3-carbonitrile, 5-fluoro-1-[(2-fluorophenyl)methyl]-

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Mar - 26 Nov 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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

adopted April 13, 2004

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

dated August 24, 2009

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"

Version / remarks:

February 08, 2019

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

5-fluoro-1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridine-3-carbonitrile

EC Number:

811-889-7

Cas Number:

1350653-26-7

Molecular formula:

C14 H8 F2 N4

IUPAC Name:

5-fluoro-1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridine-3-carbonitrile

Test material form:

solid

Sampling and analysis

Analytical monitoring:

yes

Remarks:

LC-MS/MS

Details on sampling:

Duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control were collected at the end of the test (after 48 hours) by pouring together the contents of the test beakers of each treatment.
All samples were diluted by a factor of 2 with acetonitrile.
Additional samples of the control and the dilution solvent were taken at each sampling test start and test end without any sample treatment.

Test solutions

Vehicle:

no

Details on test solutions:

Test Concentrations:
Due to the low limit of water solubility of the test item, a filtrate of nominal 100 mg/L and dilutions of 1:2, 1:4, 1:8 and 1:16 of this filtrate were tested. Additionally, a control was tested in parallel.
The filtrate and its dilutions are corresponding to following arithmetic mean measured concentrations of the test item:
2.11, 1.02, 0.523, 0.280 and 0.137 mg test item/L, and a control.
Control:
In the control, test water was used without addition of the test item.
Dosage of Test Item:
The test item is not well soluble in test medium. To avoid physical effects of undissolved test item on the daphnids, no concentrations above the solubility limit of the test item in test water was tested. Therefore, a supersaturated stock solution of nominal 100 mg test item/L was prepared by suspending 100 mg test item in 1000 mL test water. The stock suspension was shaken overhead for 24 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 as the test medium of the highest test concentration and to prepare the desired 1:2, 1:4, 1:8 and 1:16 dilutions. The test media were prepared just before introduction of the daphnids (= start of the test).
Appearance of the Test Item in Test Medium: There were no remarkable observations.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Straus), clone 5
Age at Test Start: From 0.25 to 5.25 hours old
Sex: Female
Origin: The Daphnia introduced in the test were taken from ibacon's in house laboratory culture.
Breeding Conditions: The Daphnia were bred in the laboratories of ibacon under similar temperature and light conditions as used in the test. The cultivation of the parental Daphnia was performed in Elendt M4 medium. The test organisms were not first brood progeny. The Daphnia in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

Water Hardness: 2.5 mmol/L (= 250 mg/L) as Calcium Carbonate

Test temperature:

19 - 20 °C at test start;
20 °C at test end

pH:

8.2 at test start;
8.1 at test end; and thus the pH-value did not vary by more than 1.5 units

Dissolved oxygen:

8.2 to 9.2 mg/L at test start;
8.7 to 8.9 mg/L at test end

Nominal and measured concentrations:

Due to the low limit of water solubility of the test item, a filtrate of nominal 100 mg/L and dilutions of 1:2, 1:4, 1:8 and 1:16 of this filtrate were tested. Additionally, a control was tested in parallel.
The filtrate and its dilutions are corresponding to following arithmetic mean measured concentrations of the test item:
2.11, 1.02, 0.523, 0.280 and 0.137 mg test item/L, and a control.

Details on test conditions:

Type and Size:
Glass beakers of 100 mL volume containing approximately 60 mL of test medium. The test vessels were covered with a lid to reduce the loss of water due to evaporation and to avoid the entry of dust into the solutions.
Identification:
Each test unit was uniquely identified with serial number, treatment and replicate number.
Test Conditions
Test Environment:
Controlled environment room
Measurement of pH, Dissolved Oxygen and Water Temperature:
The water temperature, pH-values and dissolved oxygen concentrations were determined at test start and test end in each treatment group.
Water Temperature:
19.0 to 19.8 °C at test start; 19.8 to 20.0 °C at test end
pH-Values: 7.8 at test start; 7.7 to 7.8 at test end; and thus the pH-value did not vary by more than 1.5 units
Dissolved Oxygen Concentration:
8.2 to 9.2 mg/L at test start; 8.7 to 8.9 mg/L at test end
Light Regime: 16 h light : 8 h dark
Light Intensity: The light intensity was 300 to 730 lux (measured once during the test).
Recording: Test conditions were recorded with suitable instruments and documented in the raw data.

Reference substance (positive control):

yes

Remarks:

For the evaluation of the quality of the Daphnia clone and the consistency of the experimental conditions, the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 2.11 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 2.11 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 2.11 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to the test item concentration of 1.02 mg test item/L. At the concentration of 2.11 mg test item/L, the highest test item concentration, 3 daphnias’ were immobile.

Results with reference substance (positive control):

In the most recent test with the reference item potassium dichromate the EC50 after 24 hours was determined to be 0.932 mg test item/L, indicating that the sensitivity of the Daphnia was consistent with the level proposed by the OECD 202 guideline (EC50-24 h between 0.6 and 2.1 mg potassium dichromate/L).

Reported statistics and error estimates:

The 48-hour EC10 and the 95 % confidence limits were calculated by weibull 2 parametric analysis. The 48-hour EC50, EC20 and the 24-hour EC50, EC20 and EC10 could not be calculated due to the lack of and/or low toxicity of the test item and were determined directly from the raw data.
The NOEC and LOEC after 24 and 48 hours were determined directly from the raw data. The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Control Immobilisation Rate was 0 % and furthermore no daphnid showed signs of disease or stress. Dissolved Oxygen Concentration was above or equal 8.7 mg O2/L in the control and test vessels at the end of the test.

Conclusions:

The toxic effect of the test item Fluorpyridonitril to Daphnia magna was assessed in a static concentration-response test. The 48-hour NOEC was determined to be 1.02 mg test item/L. The 48-hour LOEC was determined to be 2.11 mg test item/L and the 48-hour EC50 value was > 2.11 mg test item/L.
The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to mean measured values since the concentrations of the test item were within ± 20 % of the measured initial concentrations during the test.

Executive summary:

Title:

Fluorpyridonitril: Acute Toxicity to Daphnia magna in a Static 48-hour Immobilisation Test

Purpose:

The purpose of this study was to determine the influence of the test item Fluorpyridonitril on the mobility of Daphnia magna.

For this purpose, young daphnids (< 24 hours old) were exposed in a static test to various concentrations under defined conditions for 48 hours. The recorded effects were the mobility of the daphnids after 24 and 48 hours. The test method of application and the test system are recommended by the test guidelines and Daphnia magna is the recommended test species. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

Guidelines:

OECD Guideline for Testing of Chemicals No. 202: "Daphnia sp., Acute Immobilisation Test" adopted April 13, 2004

OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals", 2nd Ed., February 08, 2019

Regulation (EC) No 761/2009 amending Regulation (EC) No 440/2008, Annex, Annex, Part C, C.2.: "Daphnia sp. Acute Immobilisation Test", Official Journal of the European Union (EN), dated August 24, 2009

SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414

Materials and Methods

Test Item:

Fluorpyridonitril

Test Species:

Daphnia magna, clone 5, 0.25 to 5.25 hours old

Source: The daphnids introduced in the test were taken from ibacon's in-house laboratory culture.

Test Design:

This study encompassed 6 treatment groups (5 dose rates of the test item and a control) each containing 20 individuals. The mobility of the daphnids was determined in a static 48-hour test by visual observation after 24 and 48 hours.

Endpoints:

Number of immobile organisms after 24 and 48 hours

Test Concentrations:

Due to the low limit of water solubility of the test item, a filtrate of nominal 100 mg/L and dilutions of 1:2, 1:4, 1:8 and 1:16 of this filtrate were tested. Additionally, a control was tested in parallel.

The filtrate and its dilutions are corresponding to following arithmetic mean measured concentrations of the test item:

2.11, 1.02, 0.523, 0.280 and 0.137 mg test item/L, and a control.

Test Conditions:

Water temperature: 19.0 to 20.0 °C; pH value: 7.7 to 7.8; dissolved oxygen concentration: 8.2 to 9.2 mg/L; photoperiod: 16 h light - 8 h dark; light intensity: 300 to 730 lux; and thus were within the ranges requested by guideline OECD 202

 

Results

Biological test results:

After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to the test item concentration of 1.02 mg test item/L. At the concentration of 2.11 mg test item/L, the highest test item concentration, 3 daphnias’ were immobile.

The 48-hour NOEC was determined to be 1.02 mg test item/L. The 48-hour LOEC was determined to be 2.11 mg test item/L and the 48-hour EC50 value was > 2.11 mg test item/L.

Analytical test results:

The quantification of the test item Fluorpyridonitril in the test samples was performed using liquid chromatography with MS/MS.

The concentrations of the test item were determined in the duplicate test media samples from all test concentrations (test item stock solution and its dilutions) and the duplicate control samples from all sampling times. The mean measured values are

2.11 mg test item/L in the filtrate

1.02 mg test item/L in the 1:2 dilution of filtrate

0.523 mg test item/L in the 1:4 dilution of filtrate

0.280 mg test item/L in the 1:8 dilution of filtrate

0.137 mg test item/L in the 1:16 dilution of filtrate

Conclusion:

The toxic effect of the test item Fluorpyridonitril to Daphnia magna was assessed in a static concentration-response test. The 48-hour NOEC was determined to be 1.02 mg test item/L. The 48-hour LOEC was determined to be 2.11 mg test item/L and the 48-hour EC50 value was > 2.11 mg test item/L.

The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to mean measured values since the concentrations of the test item were within ± 20 % of the measured initial concentrations during the test.

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.