1H-Pyrazolo[3,4-b]pyridine-3-carboximidamide, 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:

2019-05-21 to 2019-12-11

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)

Test guideline 2

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"

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

[amino({5-fluoro-1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl})methylidene]azanium chloride

EC Number:

811-931-4

Cas Number:

1350653-27-8

Molecular formula:

C14 H12 Cl F2 N5

IUPAC Name:

[amino({5-fluoro-1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl})methylidene]azanium chloride

Test material form:

solid

Sampling and analysis

Analytical monitoring:

yes

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 test start and test end without any sample treatment.

Test solutions

Vehicle:

no

Details on test solutions:

Test Concentrations: 50, 22.7, 10.3, 4.7 and 2.1 mg test item/L (spacing factor 2.2) and a control. Control: In the control, test water was used without addition of the test item.
Dosage of Test Item: The test medium of the highest test concentration of nominal 50 mg test item/L was prepared by dissolving 51.2 mg test item into 1024 mL test water by intense stirring for 10 minutes. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations.
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 5.5 to 21 hours old Origin: The daphnids introduced in the test were taken from ibacon's in house laboratory culture.
Breeding Conditions: The daphnids were bred in the laboratories of ibacon under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in Elendt M4 medium. The test organisms were not first brood progeny. The daphnids in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.
Acclimatisation: Was not necessary, since the test was performed in the same medium as the culturing.

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:

19.3 °C at test start; 20.1 °C at test end

pH:

7.8 to 7.9 at test start;
7.9 to 8.0 at test end; and thus the pH-value did not vary by more than 1.5 units

Dissolved oxygen:

8.9 mg/L at test start;
8.8 to 8.9 mg/L at test end

Nominal and measured concentrations:

50, 22.7, 10.3, 4.7 and 2.1 mg test item/L (spacing factor 2.2) and a control.

Details on 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: 19.3 °C at test start; 20.1 °C at test end
pH-Values: 7.8 to 7.9 at test start; 7.9 to 8.0 at test end; and thus the pH-value did not vary by more than 1.5 units
Dissolved Oxygen Concentration: 8.9 mg/L at test start;
8.8 to 8.9 mg/L at test end
Light Regime: 16 h light: 8 h dark
Light Intensity: The light intensity was 360 to 620 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

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

5.02 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

< 2.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

<= 2.1 mg/L

Nominal / measured:

nominal

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 in the lowest test item concentration of 2.1 mg test item/L one Daphnia was immobile. At the concentration of 4.7 mg test item/L, 8 Daphnia were immobile and 19 Daphnia were immobile at the concentration of 10.3 mg test item/L. At the higher concentrations of 22.7 and 50 mg test item/L all 20 introduced Daphnia 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 24-hour and 48-hour EC50, EC20 and EC10 and the 95 % confidence limits were calculated by probit analysis.
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 3.3.0, 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 8.8 mg O2/L in the control and test vessels at the end of the test.

Conclusions:

The toxic effect of the test item Fluoroamidin hydrochlorid to Daphnia magna was assessed in a static concentration-response test. The 48-hour NOEC was determined to be < 2.1 mg test item/L. The 48-hour LOEC was determined to be ≤ 2.1 mg test item/L and the 48-hour EC50 value was calculated to be 5.02 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 nominal values since the concentrations of the test item were within ± 20% of the nominal concentrations during the test.

Executive summary:

Title: Fluoroamidin hydrochlorid: 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 Fluoroamidin hydrochlorid 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

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: Fluoroamidin hydrochlorid

Test Species: Daphnia magna, clone 5; 5.5 to 21 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: 50, 22.7, 10.3, 4.7 and 2.1 mg test item/L (spacing factor 2.2) and a control.

Test Conditions: Water temperature: 19.3 to 20.1 °C; pH value: 7.8 to 8.0; dissolved oxygen concentration: 8.8 to 8.9 mg/L; photoperiod: 16 h light - 8 h dark; light intensity: 360 to 620 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 in the lowest test item concentration of 2.1 mg test item/L one Daphnia was immobile. At the concentration of 4.7 mg test item/L, 8 Daphnia were immobile and 19 Daphnia were immobile at the concentration of 10.3 mg test item/L. At the higher concentrations of 22.7 and 50 mg test item/L all 20 introduced Daphnia were immobile.

The 48-hour NOEC was determined to be < 2.1 mg test item/L. The 48-hour LOEC was determined to be ≤ 2.1 mg test item/L and the 48-hour EC50 value was calculated to be 5.02 mg test item/L.

 

Analytical test results:

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

At the start of the test 106 % of the nominal test concentrations were found (average of all test concentrations). After 48 hours test duration, 106 % of the nominal value was determined (average of all test concentrations). During the test the daphnids were exposed to a mean of 106 % of nominal.

 

Conclusion:

The toxic effect of the test item Fluoroamidin hydrochlorid to Daphnia magna was assessed in a static concentration-response test. The 48-hour NOEC was determined to be < 2.1 mg test item/L. The 48-hour LOEC was determined to be ≤ 2.1 mg test item/L and the 48-hour EC50 value was calculated to be 5.02 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 nominal values since the concentrations of the test item were within ± 20% of the nominal concentrations during the test. This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.