4,5,6-Pyrimidinetriamine, 2-[5-fluoro-1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl]-

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 May - 27 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 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted 2006, corrected July 2011

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-{5-fluoro-1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl}pyrimidine-4,5,6-triamine

EC Number:

811-934-0

Cas Number:

1350653-30-3

Molecular formula:

C17 H14 F2 N8

IUPAC Name:

2-{5-fluoro-1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl}pyrimidine-4,5,6-triamine

Test material form:

solid

Sampling and analysis

Analytical monitoring:

yes

Remarks:

Samples were analysed by liquid chromatography with MS/MS detection (HPLC system from Agilent and MS/MS detector from AB Sciex)

Details on sampling:

Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from 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 (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment.
The samples were diluted by a factor of two with acetonitrile.
Additional samples of the control and of the dilution solvent were taken at each sampling 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 1:3, 1:9, 1:27 and 1:81 of this filtrate have been tested. Additionally, a control has been tested in parallel. The initial mean % of highest concentration (filtrate of 100 mg test item/L) was applied to the lower concentrations to calculate recoveries.
The tested concentrations corresponds to following initial mean extrapolated concentrations of the test item: 2.33, 0.776, 0.259, 0.0862 and 0.0287 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 was not well soluble in test water. In this study, only the inhibitory effects of dissolved test item was tested and thus no concentrations above the solubility limit of the test item in test water were tested. Therefore, a supersaturated stock solution of nominal 100 mg test item/L was prepared by suspending 70.1 mg test item in 701 mL test water for preparing the test concentrations. The stock suspension was ultrasonicated for 10 minutes and stirred 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 1:3, 1:9, 1:27 and 1:81 dilutions.
The test media were prepared just before introduction of the algae (= start of the test).
Appearance of the Test Item in Test Medium: There were no remarkable observations.

Test organisms

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata, Strain No. 61.81 SAG
Origin: The algae were supplied by the "Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen", 37073 Göttingen, Germany.
Breeding Conditions: The algae were cultivated in the laboratories of IBACON under standardised conditions according to the test guidelines.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

22.7 - 23 °C

pH:

8.0 to 8.1 at test start and 8.1 to 10.2 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 1:3, 1:9, 1:27 and 1:81 of this filtrate have been tested. Additionally, a control has been tested in parallel. The initial mean % of highest concentration (filtrate of 100 mg test item/L) was applied to the lower concentrations to calculate recoveries.
The tested concentrations corresponds to following initial mean extrapolated concentrations of the test item: 2.33, 0.776, 0.259, 0.0862 and 0.0287 mg test item/L, and a control.

Details on test conditions:

Water Temperature:
The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.

Light Regime: Continuous illumination
Light Intensity: The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media.
Mean light intensity: 5308 lux (range: 4620 to 5960 lux) Recording: Test conditions were recorded with suitable instruments and documented in the raw data.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.072 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 2.33 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Values refer to initial mean extrapolated test concentrations

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.028 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.086 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.125 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: Values refer to initial mean extrapolated test concentrations

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.029 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 7

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.086 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Results with reference substance (positive control):

Results with reference substance valid: yes
- EC50:
Yield [mg test item/L] 0.445
Growth rate [mg test item/L] 0.941
Biomass [mg test item/L] 0.494

Reported statistics and error estimates:

Based on the calculated cell densities, the 72 hour ErC50 and the 72 hour EyC50, the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by Probit analysis.
For the determination of the 72 hour LOEC and the 72 hour NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Williams t-test (yield) and Welch t-test after Bonferroni-Holm (growth rate).
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:

Cell Density Increase: 250.7-fold increase within 72 hours; Coefficient of Variation of Sectional (Daily) Growth Rates: 31.2 %; Coefficient of Variation of Average Growth: 0.7 %

Conclusions:

The influence of Fluorotrisamine on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyC50 was calculated to be 0.125 mg test item/L and the 72-hour ErC50 value was calculated to be > 2.33 mg test item/L (filtrate of the saturated stock solution with nominal 100 mg test item/L). The 72-hour NOEyC was determined to be 0.0287 mg test item/L and the associated 72-hour LOEyC was 0.0862 mg test item/L. The 72-hour NOErC was determined to be 0.0287 mg test item/L and the associated 72-hour LOErC was 0.0862 mg test item/L.
The initial concentrations and the maintenance of the exposure concentrations during the test were determined in the analytical part. All reported results refer to initial mean extrapolated values since the concentrations of the test item were decreasing to below the Limit of Detection.

Executive summary:

The purpose of this test was to determine the inhibitory effect of the test item Fluorotrisamine on the growth of the freshwater green algae Pseudokirchneriella subcapitata.

For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to various concentrations of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. The test method of application and the test system are recommended by the test guidelines and Pseudokirchneriella subcapitata is one of 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/Recommendations:

OECD Guidelines for the Testing of Chemicals, Section 2, No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", adopted March 23, 2006, corrected July 28, 2011

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: Fluorotrisamine

Test Species: Pseudokirchneriella subcapitata, Strain No. 61.81 SAG formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV).

Cultivated in the laboratories of ibacon; original source: "Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissen-schaften, Universität Göttingen", 37073 Göttingen, Germany.

Test Design: This study encompassed 6 treatment groups (5 dose rates of the test item and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test media were inoculated with approx. 5000  algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72  hours for determination of cell densities by spectrophotometrical measurement.

Endpoints: Yield and growth rate of the algae

Test Concentrations: Due to the low limit of water solubility of the test item, a filtrate of nominal 100 mg/L and dilutions of 1:3, 1:9, 1:27 and 1:81 of this filtrate have been tested. Additionally, a control has been tested in parallel. The initial mean % of highest concentration (filtrate of 100 mg test item/L) was applied to the lower concentrations to calculate recoveries.

The tested concentrations corresponds to following initial mean extrapolated concentrations of the test item: 2.33, 0.776, 0.259, 0.0862 and 0.0287 mg test item/L, and a control.

Test Conditions: Water temperature: 22.7 to 23.0 °C;

pH values in the control at test start: 8.1

pH values at test end: 10.3

pH values in the test item treatments at test start: 8.0 to 8.1,

pH values in the test item treatments at test end: 8.1 to 10.2;

continuous illumination; mean light intensity: 5308 lux (4620 to 5960 lux).

Results

Biological Results:

Values refer to initial mean extrapolated test concentrations:

Yield:

The 72-hour EC50 was calculated to be 0.125 mg test item/L. The 72-hour NOEC was determined to be 0.0287 mg test item/L and the associated 72-hour LOEC of 0.0862 mg test item/L.

Growth rate:

The 72-hour EC50 was calculated to be above 2.33 mg test item/L. The 72-hour NOEC was determined to be 0.0287 mg test item/L and the associated 72-hour LOEC of 0.0862 mg test item/L.

 

Analytical Results:

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

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 initial mean measured values are 2.3 % corresponding to 2.33 mg test item/L in the filtrate. This percentage was applied to the lower concentrations to extrapolate corresponding initial values. Over the course of the test a significant decrease of the test concentrations was determined.

 

Conclusion:

The influence of Fluorotrisamine on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyC50 was calculated to be 0.125 mg test item/L and the 72-hour ErC50 value was calculated to be > 2.33 mg test item/L (filtrate of the saturated stock solution with nominal 100 mg test item/L). The 72-hour NOEyC was determined to be 0.0287 mg test item/L and the associated 72-hour LOEyC was 0.0862 mg test item/L. The 72-hour NOErC was determined to be 0.0287 mg test item/L and the associated 72-hour LOErC was 0.0862 mg test item/L.

The initial concentrations and the maintenance of the exposure concentrations during the test were determined in the analytical part. All reported results refer to initial mean extrapolated values since the concentrations of the test item were decreasing to below the Limit of Detection.

This study is classified acceptable and satisfies the guideline requirements for growth inhibition algae studies.