1-amino-4-hydroxy-2-(2-phenoxyethoxy)anthraquinone

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 June 2021 to 24 June 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD 23 (Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals)

Version / remarks:

2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

Concentrations were determined using the HPLC method.

Details on sampling:

For determination of the test material concentration in the test solutions and in the control samples all six test vessel were analysed at the start and at the end of the test. On the first and the second day of the test the sampling was performed from the extra replicate sample with six parallel samples.

Vehicle:

no

Details on test solutions:

The test solution used in the test was prepared by mechanical dispersion without using of any solubilising agent. As the test material is poorly soluble in deionized water as well in the test medium, preparation of test material solution was performed using the WAF method (according to OECD Series on Testing and Assessment No. 23). The test material solution of nominal 100 mg/L was prepared by suspending 0.0800 g of the test material in 800 mL OECD medium. This solution was shaken rigorously for a period of 72 hours to achieve an equilibrated concentration and then filtrated through a membrane filter (0.45 μm Nalgene®) to separate the possible non-dissolved test material. After the formulation procedure algal cells were immediately introduced into the test solutions.

The dilution water (OECD medium) without addition of test material was used as untreated control solution.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 61.81 SAG
- Source: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
- Breeding Conditions: The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardized conditions according to the test guidelines.

ACCLIMATION
- Pre-culturing: The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with OECD Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of 2-4 days. (The pre-culture was incubated for three days before this test.)
- Any deformed or abnormal cells observed: None

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

The temperature was between 22.8 and 22.9°C measured in the flask and in the range of 21.9 – 23.8°C measured within the climate chamber.

pH:

7.58 – 9.12

Nominal and measured concentrations:

During the experiment the test material was tested at aquatic saturation (equivalent to 100 mg/L nominal concentration).

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type: Closed (Test flasks were covered with air-permeable stoppers)
- Fill volume: Volumes of 100 mL algal suspension per replicate
- Aeration: No. However, flasks were continuously shaken by a laboratory orbital shaker.
- Renewal rate of test solution: none
- Initial cells density: 10^4 cells/mL
- No. of vessels per concentration (replicates): 6 (plus an additional replicate for the determination of the test material concentration)
- No. of vessels per control (replicates): 6 (plus an additional replicate for the determination of the test material concentration)

GROWTH / TEST MEDIUM
- Standard medium used: yes - Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water in the experiment.

OTHER TEST CONDITIONS
- Sterile test conditions: not specified
- Adjustment of pH: none
- Photoperiod: 72 h
- Light intensity and quality: The average light intensity measured at the position occupied by algal culture flasks at the start of the test was 6006 lux (SD: 244 lux), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.
- Measurement of environmental conditions:
Temperature: Culture temperature was checked at the beginning of the test and every 24 hours thereafter in a flask filled with water, in the climatic chamber. In addition ambient temperature was continuously measured (with a min/max thermometer) within the climate chamber.
pH: The pH was measured in each test group at the start (before test solutions had been distributed into the test vessels) and in each test vessel at the end of the test. The pH of the control medium was not increased by more than 1.5 units during the test.

EFFECT PARAMETERS MEASURED
The cell concentration was determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber. The cell morphology was examined in parallel.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 10, 50, 100 and 500 mg/L (nominal)
- Results used to determine the conditions for the definitive study: Yes. No toxic effects were observed during the 72 h preliminary test, therefore only one test concentration at saturation (equivalent to 100 mg/L nominal concentration) and one control group were tested in a limit test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 0.005 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: equivalent to > 100 mg/L nominal

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 0.005 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: equivalent to > 100 mg/L nominal

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.005 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: equivalent to > 100 mg/L nominal

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.005 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: equivalent to > 100 mg/L nominal

Details on results:

- Analytical Results
The mean measured and calculated concentration of the test material was 0.01461 mg/L at the start of the test, 0.00719 mg/L on day 1, 0.00250 on day 2 and 0.00323 mg/L at the end of the test. Exposure concentration of the test material was calculated as the geometric mean of the measured concentrations on day 0, day 1, day 2, day 3 and was determined to be 0.00540 mg/L. Test material was not detected in the control samples.

- Biological Results
Average Specific Growth Rate and Yield: No inhibition of algal growth was observed during the experiment (the growth inhibition calculated for the 72 hours exposure in treatment group compared to the control was -3.3 % for the average specific growth rate and -17.3 % for the yield). Based on the results of the statistical evaluation (Student-t Test; α = 0.05) neither the average specific growth rate nor the yield showed significant difference compared to the control. Accordingly, the 72-h NOEC based on growth rates and yield was determined as the saturation concentration (equivalent to 100 mg/L nominal concentration; measured as 0.00540 mg/L).

Morphological Changes of Algal Cells: No abnormal appearance of the algal cells was observed during the experiment.



VALIDITY CRITERIA
- The cell density in the untreated control cultures increased by a factor of 121.50 within 72 hours. This corresponds to a specific growth rate of 1.60 day^-1.
- The mean coefficient of variation (CV) for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72 h-tests) in the control cultures did not exceed 35 %, it was 17.64 %.
- The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the replicate control cultures did not exceed 7 %: it was 3.59 %.
All validity criteria were met, therefore the study can be considered as valid.

Results with reference substance (positive control):

For the evaluation of the reliability of the applied test system and the experimental conditions Potassium dichromate is tested at least twice a year.
In the latest study:
- Results with reference substance valid: Yes
- EC50: The 72h ErC50: 0.62 mg/L, (95 % confidence limits: 0.59 – 0.65 mg/L) and the 72h EyC50: 0.33 mg/L, (95 % confidence limits: 0.32 – 0.34 mg/L)

Reported statistics and error estimates:

Mean values and standard deviations were calculated for the test groups at each observation period using Microsoft Excel for Windows software.
For the determination of the LOEC and NOEC, the calculated mean growth rates and yield at the test concentration were tested on significant differences to the control values using Student-t Test (α = 0.05, one-sided smaller) by statistical software program ToxRatPro Version 3.3.0). EC10, EC20, EC50 values were determined directly from the raw data.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, 72-hour EC10, EC20, EC50, NOEC and LOEC were determined to be >0.00540 mg/L (geometric mean measured, equivalent to > 100 mg/L nominal) for both growth rate and yield.

Executive summary:

The growth inhibition activity of the test material to Raphidocelis subcapitata was assessed according to OECD Test Guideline 201 and EU Method C.3. and in compliance with GLP in a 72 hour static test.

In the test, exponentially-growing cultures of Raphidocelis subcapitata were exposed to the test material under defined conditions. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The alga cell concentration was determined by manual cell counting by microscope in each testing flask during the 72-hour test, in 24-hour intervals.

Under the conditions of this study, 72-hour EC10, EC20, EC50, NOEC and LOEC were determined to be >0.00540 mg/L (geometric mean measured, equivalent to > 100 mg/L nominal) for both growth rate and yield. Furthermore, the obtained results show that the test material has no toxic effect on the system at saturation.

Description of key information

Under the conditions of this study, 72-hour EC10, EC20, EC50, NOEC and LOEC were determined to be >0.00540 mg/L (geometric mean measured, equivalent to > 100 mg/L nominal) for both growth rate and yield.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The growth inhibition activity of the test material to Raphidocelis subcapitata was assessed according to OECD Test Guideline 201 and EU Method C.3. and in compliance with GLP in a 72 hour static test. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

In the test, exponentially-growing cultures of Raphidocelis subcapitata were exposed to the test material under defined conditions. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The alga cell concentration was determined by manual cell counting by microscope in each testing flask during the 72-hour test, in 24-hour intervals.

Under the conditions of this study, 72-hour EC10, EC20, EC50, NOEC and LOEC were determined to be >0.00540 mg/L (geometric mean measured, equivalent to > 100 mg/L nominal) for both growth rate and yield. Furthermore, the obtained results show that the test material has no toxic effect on the system at saturation.