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Diss Factsheets

Ecotoxicological information

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:
2019-08-19 to 2020-02-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline Study; EU method; GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
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

Constituent 1
Chemical structure
Reference substance name:
methyl {4,6-diamino-2-[5-fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-5-yl}carbamate compd with dimethylsulfoxide
EC Number:
818-416-3
Cas Number:
1438425-69-4
Molecular formula:
C19H16F2N8O2*2C2H6OS
IUPAC Name:
methyl {4,6-diamino-2-[5-fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-5-yl}carbamate compd with dimethylsulfoxide
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 (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 were tested, corresponding to following mean measured concentrations of the test item:
1.10, 0.355, 0.123, 0.0385 and 0.0129 mg test item. Additionally, a control was tested in parallel.
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 103.1 mg test item in 1031 mL test water for preparing the test concentrations. The stock suspension was 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).

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species: Pseudokirchneriella subcapitata (KORSHIKOV), Strain No. 61.81 SAG, formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV)
Origin: The algae were originally supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, 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 to 23°C
pH:
The pH was measured in all test item concentrations and the control at the start and the end of the test.
The pH in the control (untreated test water) was: 8.1 at test start and 9.1 at test end
The pH at different test item concentrations was: 8.0 at test start and 9.2 to 9.4 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 were tested, corresponding to following mean measured concentrations of the test item:
1.10, 0.355, 0.123, 0.0385 and 0.0129 mg test item. Additionally, a control was tested in parallel.
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.
Water temperature was:
21.8 to 22.7 °C
pH-Values:
The pH was measured in all test item concentrations and the control at the start and the end of the test.
The pH in the control (untreated test water) was:
8.1 at test start and 9.1 at test end
The pH at different test item concentrations was:
8.0 at test start and 9.2 to 9.4 at test end
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: 4723 lux (range: 4520 to 5210 lux) Recording: Test conditions were recorded with suitable instruments and documented in the raw data.
Reference substance (positive control):
yes
Remarks:
reference item potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 1.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 1.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The 72-hour EC50 and the 72-hour EC10 for the parameters growth rate and yield could not be calculated due to the low effects of the test item. Therefore the 72-hour EC50 and the 72-hour EC10 was determined to be higher than the limit of water solubility of the test item, represented by the filtrate of nominal 100 mg test item/L, the highest concentration tested.
The 72-hour NOEC was determined to be at least the filtrate of nominal 100 mg test item/L for the parameters growth rate and yield and the associated 72-hour LOEC was determined to be higher than the mean measured concentration of 1.10 mg test item/L for both parameters.
Signs of Phytotoxicity and Microscopic Observations:
The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to the mean measured test item concentration of 1.10 mg test item/L (filtrate of nominal 100 mg/L) and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested.
Results with reference substance (positive control):
Results with reference substance valid: yes
EC50:
In the reference test with the reference item Potassium dichromate, performed in July 2019, the EC50 for yield was 0.522 mg test item/L, the EC50 for growth rate was 1.34 mg test item/L and the EC50 for biomass was 0.551 mg test item/L.
Reported statistics and error estimates:
Based on the calculated cell densities, the 72 hour ErC50 and the 72 hour EyC50 (see Definitions), 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 Bonferroni-Welch t-test (yield) and Bonferroni-Holm t-test (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: 114.4-fold increase within 72 hours; Coefficient of Variation of Sectional (Daily) Growth Rates: 20.1 %; Coefficient of Variation of Average Growth: 5.1 %
Conclusions:
The influence of BAY 1021189 crude 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 > 1.10 mg test item/L and the 72-hour ErC50 value was calculated to be > 1.10 mg test item/L. The 72-hour NOEyC was determined to be 1.10 mg test item/L and the associated 72-hour LOEyC was > 1.10 mg test item/L. The 72-hour NOErC was determined to be 1.10 mg test item/L and the associated 72-hour LOErC was > 1.10 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 mean measured values since the concentrations of the test item were within ± 20 % of the measured initial concentrations during the test.
Executive summary:

Title:

BAY 1021189 crude: Toxicity to Pseudokirchneriella subcapitata in an Algal Growth Inhibition Test

Purpose:

The purpose of this test was to determine the inhibitory effect of the test item BAY 1021189 crude 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.mThe 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:

BAY 1021189 crude; Batch No.: FEY 7882-177-6; purity: 72.5 %, according to certificate of analysis.

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 were tested, corresponding to following mean measured concentrations of the test item:

1.10, 0.355, 0.123, 0.0385 and 0.0129 mg test item. Additionally, a control was tested in parallel.

Test Conditions:

Water temperature: 21.8 to 22.7 °C;

pH values in the control (untreated test water) at test start: 8.1

pH values in the control (untreated test water)at test end: 9.1

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

pH values in the test item treatments at test end: 9.2 to 9.4;

continuous illumination; mean light intensity: 4723 lux (4520 to 5210 lux).

Results

Biological Results:

Yield and Growth rate:

The 72-hour EC50 was calculated to be > 1.10 mg test item/L. The 72-hour NOEC was determined to be 1.1 mg test item/L and the associated 72-hour LOEC of > 1.10 mg test item/L.

Analytical Results:

The quantification of the test item BAY 1021189 crude 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 (filtrate of nominal 100 mg/L and its dilutions) and the duplicate control samples from all sampling times. The mean measured values are

1.10 mg test item/L in the filtrate of nominal 100 mg/L

0.355 mg test item/L in the 1:3 dilution of filtrate

0.123 mg test item/L in the 1:9 dilution of filtrate

0.0385 mg test item/L in the 1:27 dilution of filtrate

0.0129 mg test item/L in the 1:81 dilution of filtrate.

Conclusion:

The influence of BAY 1021189 crude 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 > 1.10 mg test item/L and the 72-hour ErC50 value was calculated to be > 1.10 mg test item/L. The 72-hour NOEyC was determined to be 1.10 mg test item/L and the associated 72-hour LOEyC was > 1.10 mg test item/L. The 72-hour NOErC was determined to be 1.10 mg test item/L and the associated 72-hour LOErC was > 1.10 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 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 acceptable and satisfies the guideline requirements for growth inhibition algae studies.