2,4,6-trimethylpyridine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 24, 2020 to August 20, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

July 28, 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

The quantification of the test item 2,4,6-Collidine in the test samples was performed using liquid chromatography with UV detection.

Details on sampling:

- Concentrations: 0, 0.5, 1.6, 5, 16, 50 mg test item/L
- Sampling method: 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. All 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.
- Sample storage conditions before analysis: All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed. Afterwards the samples were again stored deep frozen (≤ -20 °C) and were kept stored up to the date of the final report.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.
The test medium of the highest test concentration of nominal 50 mg test item/L was prepared by dissolving 50.4 mg test item into 1008 mL test water by intense stirring for 20 minutes. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations (50, 16, 5, 1.6 and 0.5 mg test item/L.)
The test media were prepared just before introduction of the algae (= start of the test).
- Controls: In the control, test water was used without addition of the test item.
- Test concentration separation factor: 3.16

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: 61.81 SAG
- Source: Cultivated in the laboratories of ibacon; original source: "Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissen-schaften, Universität Göttingen", 37073 Göttingen, Germany.
- Age of inoculum (at test initiation): The cells were taken from an exponentially growing pre-culture, which was set up 4 days prior to the test start under the same conditions as in the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.5 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.
pH value in the control at test start: 8.2
pH value in the control at test end: 9.7
pH values in the test item treatments at test start: 8.3 to 8.4
pH values in the test item treatments at test end: 8.6 to 9.8

Nominal and measured concentrations:

The concentrations of the test item were within +/- 20 % of the nominal concentrations (50, 16, 5.0, 1.6 and 0.5 and 0 mg test item/L) during the test.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks of 50 mL volume
- Type: closed
- Initial cells density: nominal 5000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: mean light intensity: 6618 lux

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: spectrophotometer (24, 48, 72 h)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6.89 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

10.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

23.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.54 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

5.78 mol/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

175.1-fold increase of cell density within 72 hours; CV of sectional (daily) growth rate of control: 32.3 %; CV of average growth of control replicates: 0.8 %; and thus, the validity criteria were met.

Please refer to the attached document.

Validity criteria fulfilled:

yes

Conclusions:

The 72-hour EyC50 was calculated to be 9.16 mg test item/L and the ErC50 23.6 mg test item/L. The 72-hour EyC10 was calculated to be 4.54 mg test item/L and the ErC10 6.89 mg test item/L. The 72-hour NOEyC was determined to be 1.6 mg test item/L and the associated 72-hour LOEyC of 5.0 mg test item/L. The 72-hour NOErC was determined to be 1.6 mg test item/L and the associated 72-hour LOErC is 5.0 mg test item/L.

Executive summary:

The purpose of this test was to determine the inhibitory effect of the test item 2,4,6-Collidine 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 and the test species Pseudokirchneriella subcapitata are recommended by the test. This study encompassed 6 treatment groups (5 dose rates of the test item: 50, 16, 5.0, 1.6 and 0.5 mg test item/L), 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 nominal 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 spectrophotometric measurement. The cell density on each observation time was determined by spectrophotometric measurement. Based on the counted cell densities and the absorption from an algal suspension and its dilutions, a linear regression was performed for the calculation of the cell densities of the replicates during the test. As a result, the 72-hour EyC50 was calculated to be 9.16 mg test item/L and the ErC50 23.6 mg test item/L. The 72-hour EyC10 was calculated to be 4.54 mg test item/L and the ErC10 6.89 mg test item/L. The 72-hour NOEyC was determined to be 1.6 mg test item/L and the associated 72-hour LOEyC of 5.0 mg test item/L. The 72-hour NOErC was determined to be 1.6 mg test item/L and the associated 72-hour LOErC is 5.0 mg test item/L.

Description of key information

The 72-hour EyC50 was calculated to be 9.16 mg test item/L and the ErC50 23.6 mg test item/L. The 72-hour EyC10 was calculated to be 4.54 mg test item/L and the ErC10 6.89 mg test item/L. The 72-hour NOEyC was determined to be 1.6 mg test item/L and the associated 72-hour LOEyC of 5.0 mg test item/L. The 72-hour NOErC was determined to be 1.6 mg test item/L and the associated 72-hour LOErC is 5.0 mg test item/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

23.6 mg/L

EC10 or NOEC for freshwater algae:

6.89 mg/L

Additional information

The purpose of the study was to determine the inhibitory effect of the test item 2,4,6-Collidine 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 and the test species Pseudokirchneriella subcapitata are recommended by the test. This study encompassed 6 treatment groups (5 dose rates of the test item: 50, 16, 5.0, 1.6 and 0.5 mg test item/L), 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 nominal 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 spectrophotometric measurement. The cell density on each observation time was determined by spectrophotometric measurement. Based on the counted cell densities and the absorption from an algal suspension and its dilutions, a linear regression was performed for the calculation of the cell densities of the replicates during the test. As a result, the 72-hour EyC50 was calculated to be 9.16 mg test item/L and the ErC50 23.6 mg test item/L. The 72-hour EyC10 was calculated to be 4.54 mg test item/L and the ErC10 6.89 mg test item/L. The 72-hour NOEyC was determined to be 1.6 mg test item/L and the associated 72-hour LOEyC of 5.0 mg test item/L. The 72-hour NOErC was determined to be 1.6 mg test item/L and the associated 72-hour LOErC is 5.0 mg test item/L.