1,4-bis(isopropylamino)anthraquinone

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:

28 MAY 20191to 14 JUNE 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EU) No. 2016/266 amending Regulation (EC) No.
440/2008, Annex IV, Method C.3: “FRESHWATER ALGAE AND CYANOBACTERIA,
GROWTH INHIBITION TEST,” adopted 07. December 2015

Deviations:

yes

Remarks:

The temperature in the experiment was in the range 19.6 – 21.8 °C The temperature during pre-culture incubation was in the range 19.8 – 21.6 °C The stock solutions to be autoclaved were autoclaved for 20 minutes instead of the required 15 minutes.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guideline for the Testing of Chemicals No. 201, adopted 23. Mar. 2006, Annex 5 corrected: 28 July 2011 “Freshwater Alga and Cyanobacteria, Growth Inhibition
Test”

Deviations:

yes

Remarks:

The temperature in the experiment was in the range 19.6 – 21.8 °C The temperature during pre-culture incubation was in the range 19.8 – 21.6 °C The stock solutions to be autoclaved were autoclaved for 20 minutes instead of the required 15 minutes.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,4-bis(isopropylamino)anthraquinone

EC Number:

238-101-9

EC Name:

1,4-bis(isopropylamino)anthraquinone

Cas Number:

14233-37-5

Molecular formula:

C20H22N2O2

IUPAC Name:

1,4-bis[(propan-2-yl)amino]-9,10-dihydroanthracene-9,10-dione

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: cell concentration of 2.3 *10^3 cells/mL. 6 replicates.
- Sampling method: 45 ±1 mL test solution and algae
- Sample storage conditions before analysis:

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The solubility of the test item is < 1 µg/L (see validation report AB18062904G926). Therefore, this study was performed as a limit test at 1 µg/L.
Acetone was used to spike the test solution. A stock solution containing 10.0 mg/L in acetone was prepared.
The concentration of the stock solution is 1000-fold higher than the nominal concentration
in the treatment (1 µg/L). For the solvent control, acetone in a concentration of 100 µL/L was used.
- Eluate:
- Differential loading:
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):
- Evidence of undissolved material (e.g. precipitate, surface film, etc.):
- Other relevant information:

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: 86.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen)
- Age of inoculum (at test initiation): 3.5 years
- Method of cultivation: solid agar at 2 - 8 °C

ACCLIMATION
- Acclimation period: 96 hours
- Culturing media and conditions (same as test or not): The algal medium corresponds to the
OECD TG 201 medium.
- Any deformed or abnormal cells observed: none

Study design

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

No significant inhibition of algal growth was observed. Algal growth in the treatment was
slightly higher than in the control. Therefore, no statistical evaluation was performed.

Post exposure observation period:

No significant inhibition of algal growth was observed. Algal growth in the treatment was
slightly higher than in the control. Therefore, no statistical evaluation was performed.

Test conditions

Hardness:

59.5 mg/L

Test temperature:

19.6 – 21.8 °C

pH:

7.8 (0h) - 8.7 (72h)

Nominal and measured concentrations:

1 µg/L of test item.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass flasks total volume 65 mL
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: 2.3 *10^3 cells/mL
- Control end cells density: 517387 cells/mL
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: H2O deionised
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH:
- Photoperiod:72h
- Light intensity and quality: within the specified range (4440 – 8880 Lux)
- Salinity (for marine algae):

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter
- Chlorophyll measurement:
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline: The solubility of the test item in algal test medium was determined to be below the lowest calibration level 1 µg/L
- Range finding study
- Test concentrations: <1 µg/L of test item
- Results used to determine the conditions for the definitive study: <1 µg/L of test item

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a separate reference test.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: No significant inhibition of algal growth was observed. Algal growth in the treatment was slightly higher than in the control. Therefore, no statistical evaluation was performed

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

The solubility of the test item in algal test medium was determined to be below the lowest calibration level 1 µg/L. No effects have been detected.

Basis for effect:

growth rate

Remarks on result:

other: No significant inhibition of algal growth was observed. Algal growth in the treatment was slightly higher than in the control. Therefore, no statistical evaluation was performed

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other:
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: The 72h-EC50 values of potassium dichromate were determined in a separate reference
test. For the estimation of the 72h-EC50 values of the positive control, the fits showed sufficient statistical correspondence of the data with the dose-response-equation. The values
were within the range of the laboratory
- Other:

Reported statistics and error estimates:

At the beginning and at the end of the test, the content of the test item in the test solutions was determined using LC/MS-MS-determination. All measured values are below the LOQ (1 µg/L). At the beginning of the test, however, the measured raw data (area) clearly indicate that the test item is present in the solution.
In this study, the lowest calibration level was given as LOQ / LOD. Whereas in the validation report, the proposed LOQ, according to the SANCO/3029/99 rev. 4 , is not necessarily the lowest concentration the analytical instrument is able to detect, but the concentration where the validity criteria of the SANCO/3029/99 rev. 4 is fulfilled.
Therefore, the measured initial value is used for evaluation, even if it is below the LOQ.
In addition, the results are displayed in relation to the nominal values.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

All validity criteria were met. No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.

Conclusions:

No significant inhibition of algal growth was observed. Algal growth in the treatment was
slightly higher than in the control.