1,1,3-trimethylindan-4-amine

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:

2020-10-12 to 2021-01-20

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Due to their growth, algae extract CO2 from the test medium (biogenic decalcification), resulting in the pH increase

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,1,3-trimethyl-2,3-dihydro-1H-inden-4-amine

EC Number:

838-724-1

Cas Number:

94568-76-0

Molecular formula:

C12H17N

IUPAC Name:

1,1,3-trimethyl-2,3-dihydro-1H-inden-4-amine

Sampling and analysis

Analytical monitoring:

yes

Remarks:

Method for Determination: 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.

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 (≤ -20 °C) and were kept stored up to the date of the final report.

Test solutions

Vehicle:

no

Details on test solutions:

Dosage of Test Item: The test medium of the highest test concentration of nominal 50 mg test item/L was prepared by dissolving 51.0 mg test item into 1020 mL test water by intense stirring for 130 minutes and ultrasonic treatment for 30 minutes. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations. The test media were prepared just before introduction of the algae (= start of the test)
Test Concentrations: 50, 15.8, 5.0, 1.58 and 0.5 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.

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:

other: Reconstituted water (OECD Medium)

Remarks:

Analytical grade salts were added 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 to 22.3 °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 9.8 at test end.
The pH in the control increased by more than 1.5 units medium. However, this does not invalidate the tests since the validity criterion was met.
The pH at different test item concentrations was: 7.8 to 7.9 at test start and 7.8 to 9.8 at test end

Nominal and measured concentrations:

Nominal Test Concentrations:
50, 15.8, 5.0, 1.58 and 0.5 mg test item/L (spacing factor 3.16), and a control

Measured Concentration:
At the start of the test the mean recovery of the test item was 107% (n = 10, RSD 19%) of the expected nominal test concentrations.
After 72 hours test duration, the mean recovery was 99% (n = 10, RSD 10%).

All reported results are based on the nominal test item concentrations.

Details on test conditions:

Test Units: Erlenmeyer flasks of 50 mL volume with approximately 50 mL of test mediumcovered with air permeable glass dishes, stoppers or caps.

Introduction of Algae: 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 3 days prior to the test start under the same conditions as in the test.
Replicates: The test was performed with three replicates per test concentration and six replicates in the control.
Test Procedure: The test units were continuously stirred by magnetic stirrers.The flasks were covered with air permeable glass dishes and incubated in a water bath. The flasks were placed in a random order and were repositioned each day to minimize differences in test conditions.
Blanks: 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").
Exposure Time: 72 hours
Determination of the Cell Density: 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.

Water Temperature: The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.
pH-Values: The pH was measured in all test item concentrations and the control at the start and the end of the test.
Light Regime: Continuous illumination
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: 5270 lux (range: 5060 to 5520 lux)

Reference substance (positive control):

yes

Remarks:

For the evaluation of the quality of the algae and the experimental conditions the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.58 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.58 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.78 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 7

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

7.87 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 8

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

3.27 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 9

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.72 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 10

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.43 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

The 72-hour EyC50 was calculated to be 5.78 mg test item/L and the ErC50 14.5 mg test item/L. The 72-hour EyC10was calculated to be 2.43 mg test item/L and the ErC10 5.72 mg test item/L. The 72-hour NOEyC was determined to be 1.58 mg test item/Land the associated 72-hour LOEyC is 5 mg test item/L. The 72-hour NOErC was determined to be 1.58 mg test item/L and the associated 72-hour LOErC is 5 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 5.0 mg test item/L and the algal cells in the control. In the nominal concentration of 15.8 mg test item/L cells were deformed (straight, thicker) and no cells were visible after 72 hours in the highest concentration of 50 mg test item/L.

Analytical Results:
The concentrations of the test item were determined in the duplicate test media samples from all test concentrations and in the duplicate control samples from all sampling times.
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, apart from the lowest test concentration. This is regarded to have no impact on the outcome of this study, since the lowest test concentration is below the NOEC determined in this study.

Results with reference substance (positive control):

Results of the Most Recent Test with Pseudokirchneriella subcapitata performed with the Reference Item Potassium Dichromate in January 2020

72 h EC50 (yield): 0.470 mg/L
72 h EC50 (growth rate): 0.858 mg/L
72 h EC50 (biomass) 0.495 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 (yield and growth rate).Thesoftware 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:189.1-fold increase within 72 hours Coefficient of Variation of Sectional (Daily) Growth Rates in Control Cultures:15.7 % Coefficient of Variation of Average Growth between Control Replicates:0.8 %

Conclusions:

The influence of BCS-AF93710 on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyC50was calculated to be 5.78 mg test item/L and the 72-hour ErC50value was calculated to be 14.5 mg test item/L. The 72-hour NOEyC was determined to be 1.58 mg test item/L and the associated 72-hour LOEyC was 5 mg test item/L. The 72-hour NOErC was determined to be 1.58 mg test item/L and the associated 72-hour LOErC was 5 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 concentrationsduring the test, apart from the lowest test concentration. This is regarded to have no impact on the outcome of this study, since the lowest test concentration is below the NOEC determined in this study.

Executive summary:

Title:

BCS-AF93710: Toxicity to Pseudokirchneriella subcapitata in an Algal Growth Inhibition Test

Guidelines: OECD Guideline 201, 2006, corrected July 28, 2011

Purpose:

The purpose of this test was to determine the inhibitory effect of the test item BCS-AF93710 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 guidelines. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

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 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 Concentrations: 50, 15.8, 5.0, 1.58 and 0.5 mg test item/L (spacing factor 3.16), and a control

Results:

The quantification of the test item BCS-AF93710 in the test samples was performed using liquid chromatography with UV detection.

In case of the lowest test concentration of nominal 0.5 mg test item/L, at the start of the test, the recoveries varied between 138 and 153 % of the nominal test concentration. After 72 hours test duration, the recoveries of the nominal values varied between 112 and 124%. This is regarded to have no impact on the outcome of this study, since this treatment group is below the NOEC determined in this study.

In case of all other test concentrations (1.58, 5.0, 15,8 and 50 mg test item/L) at the start of the test the recoveries varied between 90 and 105 % of the nominal test concentration. After 72hours test duration, the recoveries of the nominal values varied between 92 and 98 %. The test item was dosed correctly and was stable during the run of the test.

The 72-hour EyC50 was calculated to be 5.78 mg test item/L and the ErC50 14.5 mg test item/L. The 72-hour EyC10was calculated to be 2.43 mg test item/L and the ErC10 5.72 mg test item/L. The 72-hour NOEyC was determined to be 1.58 mg test item/Land the associated 72-hour LOEyC is 5 mg test item/L. The 72-hour NOErC was determined to be 1.58 mg test item/L and the associated 72-hour LOErC is 5 mg test item/L.

Conclusion:

The influence of BCS-AF93710 on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyC50was calculated to be 5.78 mg test item/L and the 72-hour ErC50value was calculated to be 14.5 mg test item/L. The 72-hour NOEyC was determined to be 1.58 mg test item/L and the associated 72-hour LOEyC was 5 mg test item/L. The 72-hour NOErC was determined to be 1.58 mg test item/L and the associated 72-hour LOErC was 5 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, apart from the lowest test concentration. This is regarded to have no impact on the outcome of this study, since the lowest test concentration is below the NOEC determined in this study.