2(1H)-Pyridinone, 4-amino-5-methyl-

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:

2018-05-25 to 2018-09-20

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:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-amino-5-methyl-1,2-dihydropyridin-2-one

EC Number:

682-818-3

Cas Number:

95306-64-2

Molecular formula:

C6 H8 N2 O

IUPAC Name:

4-amino-5-methyl-1,2-dihydropyridin-2-one

Test material form:

solid

Sampling and analysis

Analytical monitoring:

yes

Remarks:

HPLC-UV

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: 100, 31.6, 10, 3.2 and 1.0 mg test item/L (spacing factor 3.16), and a control Control: In the control, test water was used without addition of the test item.
Dosage of Test Item: A stock solution of 100 mg test item/L was prepared by dissolving 100.3 mg test item into 1003 mL test water by intense stirring for 30 minutes. Adequate volumes of this stock solution were diluted with test water to prepare the test media of the desired test concentrations.
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:

21 to 24°C

pH:

7.8-8.0 at test start and 9.2-9.4 at test end

Nominal and measured concentrations:

100, 31.6, 10, 3.2 and 1.0 mg test item/L (spacing factor 3.16), 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.
Water temperature was: 21.3 to 24.0 °C
pH-Values: The pH was measured in all test item concentrations and the control at the start and the end of the test. pH was: 7.8-8.0 at test start and
9.2-9.4 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: 6023 lux (range: 5750 to 6540 lux)

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

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 highest concentration tested.
The 72-hour NOEC was determined to be at least 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 nominal 100 mg test item/L for both parameters.

Results with reference substance (positive control):

The influence of Potassium dichromate 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 0.402 mg test item/L, the 72-hour EbC50was calculated to be 0.414 mg test item/L and the 72-hour ErC50 value was calculated to be 0.963 mg test item/L. The 72-hour NOEyC was determined to be 0.094 mg test item/L and the associated 72-hour LOEyC was 0.21 mg test item/L. The 72-hour NOErC was determined to be 0.094 mg test item/L and the associated 72-hour LOErC was 0.21 mg test item/L. The 72-hour NOEbC was determined to be 0.094 mg test item/L and the associated 72-hour LOEbC was 0.21 mg test item/L.

Reported statistics and error estimates:

Based on the calculated cell densities, the 72 hours ErC50 and the 72 hours EyC50 (see Definitions) could not be calculated by Probit analysis due to the lack of effects and were therefore determined directly from the raw data.
For the determination of the 72 hours LOEC and the 72 hours NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Welch-t-test after Bonferroni Holm. No statistical significance was detected.
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:

Cell Density Increase: 163.0-fold increase within 72 hours; Coefficient of Variation of Sectional (Daily) Growth Rates: 4.4 %; Coefficient of Variation of Average Growth: 1.6 %

Conclusions:

The influence of 4-Amino-5-methylpyridinon on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour NOEC was determined to be at least 100 mg test item/L. The NOEC might even be higher than this concentration, but concentrations in excess of nominal 100 mg test item/L have not been tested according to the recommendations of the test guidelines. The LOEC was determined to be > 100 mg test item/L. The 72-hour EC50 were clearly higher than 100 mg test item/L. These values could not be quantified due to the absence of toxicity of 4-Amino-5-methylpyridinon up to the highest test item concentration of 100 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: 4-Amino-5-methylpyridinon: 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 4-Amino-5-methylpyridinon 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 concentration 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

-Commission Regulation (EC) No 761/2009, Annex, Part C, C.3.: "Freshwater Algae and Cyanobacteria, Growth Inhibition 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: 4-Amino-5-methylpyridinon; Batch No.: BXR7SWA; purity: 99.4 % (analytical) 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).

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 concentrations 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: 100, 31.6, 10, 3.2 and 1.0 mg test item/L (spacing factor 3.16), and a control

Test Conditions: Water temperature: 21.3-24.0 °C; pH values at test start 7.8-8.0, at the end of the test 9.2-9.4; continuous illumination; mean light intensity: 6023 lux (5750 to 6540 lux).

Results

The influence of 4-Amino-5-methylpyridinon on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour NOEC was determined to be at least 100 mg test item/L. The NOEC might even be higher than this concentration, but concentrations in excess of nominal 100 mg test item/L have not been tested according to the recommendations of the test guidelines. The LOEC was determined to be > 100 mg test item/L. The 72-hour EC50 were clearly higher than 100 mg test item/L. These values could not be quantified due to the absence of toxicity of 4-Amino-5-methylpyridinon up to the highest test item concentration of 100 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.

Analytical Results: The quantification of the test item 4-Amino-5-methylpyridinon in the test samples was performed using liquid chromatography with UV detection.

At the start of the test 100 % of the nominal test concentrations were found (average of all test concentrations). After 72 hours test duration, 100 % of the nominal value was determined (average of all test concentrations). During the test the algae were exposed to a mean of 100 % of nominal.

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