N-[4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]phenyl]acetamide

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:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experimental test result performed using standard OECD test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

no

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

The test solution was prepared by dissolving 500 mg of test chemical in 500 mL of OECD medium to get the final concentration of 1000 mg/L, which was then analytically determined. The final solubility value obtained after analytical detection is 0.55 mg/L. To get the maximum dissolution, stock solution was stirred for 96 hr. To have a better growth and visibility of cells, the initial cell count of the culture was kept 10,000 cells/ml. Test chemical concentrations used for the study were 0, 0.15, 0.19, 0.24, 0.31, 0.40 and 0.52 mg/L, respectively.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Length: 8 – 14 μm
- Weight: 2 - 3 μm
- Source: Sterile, unicellular, suspension cultures of algae were obtained from the laboratory for Biological Research in Aquatic Pollution (LABRAP) at the University of Ghent in Belgium and maintained in Laboratory.
- Method of cultivation: OECD medium

ACCLIMATION
- Culturing media and conditions (same as test or not): The medium to be used for the growth of algae was OECD medium. It is a medium composed of macronutrients, micronutrients, alkaline EDTA solution and Iron solution. Stock solution of each of these was prepared separately and then a complete medium was prepared and sterilized. De-ionized water was used to prepare the BBM
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21 to 24 ± 2°C

Nominal and measured concentrations:

Test chemical concentrations used for the study were 0, 0.15, 0.19, 0.24, 0.31, 0.40 and 0.52 mg/L, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flasks
- Material, size, headspace, fill volume: 100 ml conical flasks filled with 60 ml was used for the study.
- Initial cells density: 10000cells/ml
- No. of organisms per vessel: 10000cells/ml
- No. of vessels per concentration (replicates): Two replicates for each test concentration
- No. of vessels per control (replicates): Three replicates for Control

GROWTH MEDIUM
- Standard medium used: yes, OECD medium was used as a test medium in the study.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Yes
- Photoperiod: 16 Hour Light Period : 8 Hour Dark Period
- Light intensity and quality: continuous, uniform fluorescent illumination(3000 - 4000 Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell counts were measured using microscope.
- Chlorophyll measurement: No data
- Other: The cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to
observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer.

TEST CONCENTRATIONS
- Test concentrations: Six test concentration were: 0, 0.15, 0.19, 0.24, 0.31, 0.40 and 0.52 mg/L
- Results used to determine the conditions for the definitive study: Mortality of test organisms

Other:
Incubation :
1. The temperature of the orbital shaking incubator was kept constant throughout the period of exposure of the experiment. The temperature was maintained at 21 to 24 ± 2°C.
2. The test vessels were incubated with a continuous, uniform fluorescent illumination (3000 - 4000 Lux).
3. The pH of the control cultures needs to be noted during the study and the pH of the control medium should not increase by more than 1.5 units during the test.
4. The orbital shaking incubator was set at a speed of 120 revolutions per minute throughout the study period. This is to provide constant shaking to the algal cells to keep them in suspension and to ensure that they do not settle down on the bottom of the test vessel.
5. Study duration : The experimental phase of the study was lasted for a period of 72 hours.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7) was used as a reference substance.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.52 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.17 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Results with reference substance (positive control):

- Results with reference substance valid?
- EC50: 0.868 mg/l

Reported statistics and error estimates:

To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) was determined.

Table: Assessment of Dose Range concentrations

Sr. no.	Concentrations mg/L	Wavelength (nm)	Absorbance	Temperature (°C)
1	blank	584	0.00	25°C
2	0.04	584	0.000	25°C
3	0.07	584	0.001	25°C
4	0.12	584	0.001	25°C
5	0.21	584	0.003	25°C
6	0.37	584	0.005	25°C
7	0.66	584	0.010	25°C
8	0.77	584	0.010	25°C

 

The absorbance and concentrations were recorded at 584 nm.

 

Table: Concentration after Analytical Determination

Sr. no.	Concentrations mg/L	Absorbance (mean) (0 hours)	Analytical concentrations (0 hours)	Absorbance (mean) (72 hours)	Analytical concentrations (72 hours)
1	blank	0.000	0.000	0.000	0.01
2	0.15	0.003	0.22	0.004	0.26
3	0.19	0.003	0.23	0.014	0.89
4	0.24	0.005	0.30	0.011	0.66
5	0.31	0.006	0.39	0.001	0.10
6	0.40	0.011	0.68	0.006	0.41
7	0.52	0.012	0.72	-0.004	-0.23

 

Table: Cell count and percent inhibition

Experimental Flasks and Test Concentration(mg/L)	0 Hr Cell Count	24 Hr Cell Count	48 Hr Cell Count	72 Hr Cell Count	Avg Specific Growth Rate (μ)	Mean Avg Specific Growth Rate (μ)	Percent Inhibition(%)
Control	10000	30000	125000	230000	1.05	0.105	0
Control	10000	30000	110000	260000	1.07
Control	10000	45000	135000	225000	1.03
0.15 (R1)	10000	30000	100000	215000	1.02	1.102	2.86
0.15 (R2)	10000	25000	105000	210000	1.01
0.19 (R1)	10000	25000	95000	175000	0.95	0.96	8.52
0.19 (R2)	10000	25000	95000	180000	0.96
0.24 (R1)	10000	25000	90000	110000	0.80	0.81	22.86
0.24 (R2)	10000	20000	95000	115000	0.81
0.31 (R1)	10000	20000	85000	90000	0.73	0.72	31.43
0.31 (R2)	10000	20000	90000	85000	0.71
0.40 (R1)	10000	20000	70000	80000	0.69	0.70	33.33
0.40 (R2)	10000	15000	70000	85000	0.71
0.52 (R1)	10000	15000	55000	70000	0.65	0.66	37.14
0.52 (R2)	10000	15000	60000	75000	0.67

 

Table: pH and temperature

Test Concentration(mg/L)	Experimental Flasks	pH			Temperature (°C)
		0 hours	72 hours		0 hours	72 hours
control	R1	7.33	6.98		23.1	23.1
control	R2	7.55	7.01		23.1	23.1
control	R3	7.60	8.28		23.1	23.1
0.15	R1	7.52	7.26		23.1	23.1
0.15	R2	7.52	7.32		23.1	23.1
0.19	R1	7.26	7.27		23.1	23.1
0.19	R2	7.26	7.31		23.1	23.1
0.24	R1	7.22	7.17		23.1	23.1
0.24	R2	7.21	7.27		23.1	23.1
0.31	R1	7.14		7.10	23.1		23.1
0.31	R2	7.12		7.25	23.1		23.1
0.40	R1	7.11		7.22	23.1		23.1
0.40	R2	7.10		7.27	23.1		23.1
0.52	R1	7.02		7.16	23.1		23.1
0.52	R2	7.01		7.38	23.1		23.1

Validity criteria fulfilled:

yes

Conclusions:

Based on the growth inhibition of green alga Pseudokirchneriella subcapitata by the test chemical, the 72 hr EC10 and median effect concentration (EC50) was determined to be 0.17 and > 0.52 mg/l.

Executive summary:

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test solution was prepared by dissolving 500 mg of test chemical in 500 mL of OECD medium to get the final concentration of 1000 mg/L, which was then analytically determined by UV-VIS spectrophotometer. Green algae were exposed to nominal concentration of test chemical ( 0, 0.15, 0.19, 0.24, 0.31, 0.40 and 0.52 mg/l) in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 21 to 24 ± 2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation of specific growth rate was not exceeded 35%, thus fulfilling the validity criterion. As the concentration of the test chemical being tested has not been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on measured concentration. On the basis of growth rate of the test organism, the 72 hr EC10 and median effect concentration (EC50) was determined to be 0.17 and > 0.52 mg/l , respectively.

Description of key information

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test solution was prepared by dissolving 500 mg of test chemical in 500 mL of OECD medium to get the final concentration of 1000 mg/L, which was then analytically determined by UV-VIS spectrophotometer. Green algae were exposed to nominal concentration of test chemical ( 0, 0.15, 0.19, 0.24, 0.31, 0.40 and 0.52 mg/l) in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 21 to 24 ± 2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation of specific growth rate was not exceeded 35%, thus fulfilling the validity criterion. As the concentration of the test chemical being tested has not been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on measured concentration. On the basis of growth rate of the test organism, the 72 hr EC10 and median effect concentration (EC50) was determined to be 0.17 and > 0.52 mg/l , respectively.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.17 mg/L

Additional information

Experimental study and predicted data of the target chemical and various supporting weight of evidence studies for its read across substance was reviewed for toxicity to aquatic algae end point which are summarized as below:

 

In an experimental study from study report (2020), a freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test solution was prepared by dissolving 500 mg of test chemical in 500 mL of OECD medium to get the final concentration of 1000 mg/L, which was then analytically determined by UV-VIS spectrophotometer. Green algae were exposed to nominal concentration of test chemical ( 0, 0.15, 0.19, 0.24, 0.31, 0.40 and 0.52 mg/l) in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 21 to 24 ± 2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation of specific growth rate was not exceeded 35%, thus fulfilling the validity criterion. As the concentration of the test chemical being tested has not been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on measured concentration. On the basis of growth rate of the test organism, the 72 hr EC10 and median effect concentration (EC50) was determined to be 0.17 and > 0.52 mg/l , respectively.

In a prediction done using EPI suite ECOSAR version 1.11, toxicity of the test chemical to green algae was evaluated. On the basis of effect of test chemical observed in a static system on the growth rate of the test organism during the 96 hr exposure duration, the median effect concentration (EC50) for the test chemical was estimated to be 1.503 mg/l.

 

Another toxicity to aquatic algae study was carried out for 72 hrs for assessing the effect of test chemical on Selenastrum capricornutum (Secondary source, 1996). The study was performed in accordance with the OECD Guideline 201 (Alga, Growth Inhibition Test) in a static fresh water system. Selenastrum capricornutum ATCC 22662 (Green algae) was used as test organism. 8 different nominal concentrations of test chemical were used. Test chemical conc. ranges from 0.058 to 3.2 mg/l, respectively. Stock solution of test chemical was prepared with DMSO. No analytical monitoring of test chemical concentration were done. On the basis of effect of the test chemical on biomass of the test organism Selenastrum capricornutum, the 72 hr NOEC and EC50 value was determined to be 0.10 and 0.25 mg/l, respectively.

 

For the test chemical from secondary source (2017), toxicity to aquatic algae study was carried out for 72 hrs for assessing the effect of test chemical on Scenedesmus subspicatus. The study was performed in accordance with the OECD Guideline 201 (Alga, Growth Inhibition Test) in a static fresh water system. Scenedesmus subspicatus (Green algae) was used as test organism. On the basis of effect of the test chemical on growth rate of the test organism Scenedesmus subspicatus, the 72 hr ErC50 value was determined to be 0.6 mg/l.

Thus, based on the EC50 value, test chemical can be considered as toxic to aq. algae and thus can be considered to be classified in ‘Aquatic acute/chronic 1 category’ as per CLP classification criteria.

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