1-(2,2,4-trimethyl-1-quinolyl)ethanone

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:

October 29, 2020 to March 12, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals"

Version / remarks:

2nd Ed., February 08, 2019

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

adopted March 23, 2006, corrected July 28, 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-(2,2,4-trimethyl-1,2-dihydroquinolin-1-yl)ethan-1-one

EC Number:

665-911-3

Cas Number:

5855-23-2

Molecular formula:

C14 H17 N O

IUPAC Name:

1-(2,2,4-trimethyl-1,2-dihydroquinolin-1-yl)ethan-1-one

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Sampling: The duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the controlswere 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 controls (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 weretaken at each sampling without any sample treatment.
Storage: All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed. Afterwards the samples were again stored deep frozen and will be kept stored up to the date of the final report.
Analyses: The concentrations of the test item were analysed in the duplicate test media samples from all test concentrations, and in the duplicate control samples and the additional control and solvent samples, from both sampling times (0 and 72 hours).

Test solutions

Vehicle:

no

Details on test solutions:

The highest test item concentration of 100 mg test item/L was prepared by mixing 82.9 mg test item into 829 mL test water. After cessation of stirring for 24 hours and a following period (0.5 hours) of settling to allow phase separation, the aqueous phase, i.e. the water soluble fraction, was drawn off carefully and used as the test medium of the highest test concentration, and mixed into test water to obtain the desired dilutions of 1:3.1, 1:10, 1:31 and 1:100, corresponding to nominal concentrations of 100, 32, 10, 3.2 and 1.0 mg test item/L. Thetest media were prepared just before introduction of the algae (=start of the test).

In the control, test water was used without addition of the test item.

Test organisms

Test organisms (species):

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

Details on test organisms:

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:

other: re-consituted water (OECD Medium)

Remarks:

Analytical grade salts were added at the following nominal concentrations in deionised water.

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:

Water temperature was:22.4 to 22.5 °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 was:8.0 at test start and 9.5 at test end
The pH at different test item concentrations was: 7.6 to 7.8 at test start and 7.8 to 9.6 at test end

Nominal and measured concentrations:

Water Soluble Fraction (WSF) of nominal 100, 32, 10, 3.2 and 1.0 mg test item/L (spacing factor 3.16), and a control, corresponding to geometric mean measured concentrations of 85.9, 23.8, 6.90, 2.10 and 0.591 mg test item/L.

Details on test conditions:

Test vessel: Erlenmeyer flasks of 50 mL volume with approximately 50 mL of test medium covered with watch glasses.
The test was started (0 hours) by inoculation of a biomass of nominal 5000 algal cells per mL test medium. These 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.
The test was performed with three replicates per test concentration and six replicates in the control.
Additionally, one replicate of each test concentration and of the control was prepared without algae to provide a "blank" for the spectrophotometric measurements. The additional replicates were incubated under the same conditions as described above. The blank values were subtracted from the absorption measured in the samples containing algae in order to eliminate absorption caused by the test item (see also "Determination of the Cell Density").

Growth medium: standard medium (OECD medium)
Test medium: standard medium (OECD medium) prepared according to SOP
Culture medium different from test medium: no

Sterile test conditions: no
Adjustment of pH: no
Photoperiod: 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: 4787 lux (range: 4530 to 5060 lux)


Parameter measured (with observation intervals if applicable) :
Determination of cell concentrations:
The cell density on each observation time was determined by spectrophotometric measurement. Therefore, defined volumes of the algal suspensions from all replicates and from the blanks were sampled after 24, 48 and 72 hours of exposure, and were not replaced. The algal cell densities were calculated by subtracting the absorption of the blanks, from each of the measured absorption of the test media (with algae). 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.

Test concentrations
Spacing factor for test concentrations: spacing factor 3.16
Range finding study: Pre-experiments were performed to establish suitable methods to prepare the test solutions.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate, at least once a year

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

4.26 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.36 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

6.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: yield

Effect concentrations 7

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

2.43 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: yield

Effect concentrations 8

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.03 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: yield

Effect concentrations 9

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

6.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: yield

Effect concentrations 10

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: yield

Details on results:

The 72-hour EyC50 was calculated to be 3.45 mg test item/L and the ErC50 6.70 mg test item/L. The 72-hour EyC10 was calculated to be 2.03 mg test item/L and the ErC10 3.36 mg test item/L. The 72-hour NOEyC was determined to be 2.10 mg test item/Land the associated 72-hour LOEyC of 6.90 mg test item/L. The 72-hour NOErC was determined to be 2.10 mg test item/L and the associated 72-hour LOErC is 6.90 mg test item/L.

The microscopic examination of the shape of the algal cells after 72hours of test duration did not show any difference between the algae that had been growing up to a nominal test concentration of 100 mg test item/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 Potassium Dichromate:
72-hour EC50 (yield): 0.402 mg/L
72-hour EC50 (growth rate): 0.878 mg/L
72-hour EC50 (biomass): 0.449 mg/L

Reported statistics and error estimates:

Based on the calculated cell densities, the 72 hour ErC50and the 72 hour EyC50 (see Definitions), the corresponding EC20 and EC10values 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.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 DensityIncrease in Control Cultures:298.3-fold increase within 72 h CV of Sectional (Daily) Growth Rates in Control Cultures:13.0 % CV of Average Growth between Control Replicates:1.3 %

Conclusions:

The influence of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitatawas assessed in a static concentration-response test. The 72-hour EyC50 was calculated to be 3.45 mg test item/L and the 72-hour ErC50 value was calculated to be 6.70 mg test item/L. The 72-hour NOEyC was determined to be 2.10 mg test item/L and the associated 72-hour LOEyC was 6.90 mg test item/L. The 72-hour NOErC was determined to be 2.10 mg test item/L and the associated 72-hour LOErC was 6.90 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 geometric mean concentrations, since the test item concentrations were not within ± 20 % of the nominal concentrations during the test.

Executive summary:

Title: [trade name]: Toxicity to Pseudokirchneriella subcapitatain an Algal Growth Inhibition Test

Purpose: The purpose of this test was to determine the inhibitory effect of the test item 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 subcapitatais 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.

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 Pflanzenwissenschaften, 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 mediawere inoculated with nominal 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 for determination of cell densities by spectrophotometrical measurement.

Endpoints: Yield and growth rate of the algae

Test Concentration: Water Soluble Fraction (WSF) of nominal 100, 32, 10, 3.2 and 1.0 mg test item/L (spacing factor 3.16), and a control, corresponding to geometric mean measured concentrations of 85.9, 23.8, 6.90, 2.10 and 0.591 mg test item/L.

Test Conditions: Water temperature: 22.4 to 22.5 °C; pH values in the control at test start: 8.0pH values at test end: 9.5pH values in the test item treatments at test start: 7.6 to 7.8, pH values in the test item treatments at test end: 7.8 to 9.6; continuous illumination; mean light intensity: 4787 lux (4530 to 5060 lux).

The quantification of the test item in the test samples was performed using liquid chromatography with UV detection. The test item concentrations decreased during the 72 hours of the biological test.

Result and Conclusion: The influence of 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 3.45 mg test item/L and the 72-hour ErC50value was calculated to be 6.70 mg test item/L. The 72-hour NOEyC was determined to be 2.10 mg test item/L and the associated 72-hour LOEyC was 6.90 mg test item/L. The 72-hour NOErC was determined to be 2.10 mg test item/L and the associated 72-hour LOErC was 6.90 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 geometric mean concentrations, since the test item concentrations were not within ± 20 % of the nominal concentrations during the test.