2'-acetonaphthone

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:

key study

Study period:

Study Initiation Date: November 24, 2020 to Study Completion Date: May 20, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.8, 1.6, 3.2, 6.4, 12.8 mg/L
- Sampling method: Test item formulation prepared in volumetric flask as stock, then respective volumes of solution were transferred from flask to measuring cylinder to obtain the required concentrations for the study.

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 12.5 mg of test item was dissolve in 50 µL acetone and made up to 500 mL in a volumetric flask using OECD medium (stock). 10 mL, 21.67 mL, 48.33 mL, 106.67 mL, 235 mL from stock solution were transferred into 500 mL measuring cylinder and made up to 35 mL using OECD medium. Hence concentrations of 0.8, 1.6, 3.2, 6.4, 12.8 mg/L were achieved.
- Controls: OECD medium
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.1ml/L

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: ATCC
- Source (laboratory, culture collection): Initially procured from “American Type Culture Collection”, Chromachemie Laboratory Private Limited, later test system was sub-cultured in the test facility.
- Age of inoculum (at test initiation): 3-4 days before start of the test.
- Method of cultivation:The required volume of OECD growth medium was prepared as per OECD 201, test system was sub-cultured using OECD medium.

ACCLIMATION
- Culturing media and conditions (same as test or not):Yes
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21.7 – 23.9 °C

pH:

7.6-8.1

Nominal and measured concentrations:

Nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical flasks
- Type (delete if not applicable): open / closed Sterilized, flasks were covered with cap
- Material, size, headspace, fill volume: Conical flasks were made of glass. Volume capacity 250 mL.
- Aeration: no aeration
- Initial cells density: 10000 cells/ ml
- Control end cells density: 10000 cells/ml
- No. of organisms per vessel: 10000 cells/mL
- No. of vessels per concentration (replicates): Range finding study: 3 replicates/test item concentration
Main Study: 3 replicates/test item concentration
- No. of vessels per control (replicates): 6 replicates for control group during range finding and main study.
- No. of vessels per vehicle control (replicates):6 replicates for control group during range finding and main study.

GROWTH MEDIUM
- Standard medium used: yes Yes (OECD Medium as per OECD 201)


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Culture medium different from test medium: NO
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH:no
- Photoperiod:Continuous light for test system
- Light intensity and quality:Average: 6766-6794 Lux
- Salinity (for marine algae):
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cells were counted using Haemocytometer under illumination of the microscope at 24, 48, and 72 h after inoculation.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study Yes
- Test concentrations: 0 (control), 0 (vehicle control), 1.5, 3, 6, 12, 24 mg/L
- Results used to determine the conditions for the definitive study: Based on the results of range finding test following concentrations of 0 (control), 0 (vehicle control), 0.8, 1.6, 3.2, 6.4, 12.8 mg/L were selected for the main study as significant changes (inhibition algal growth rate) were observed in treatment groups during 72h test period.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.19 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 5.43 mg/L and 6.95 mg/L.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.73 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CI 2.42 mg/L and 3.04 mg/L

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes Yes (64.25, 62.58 times of the initial cell count in control and vehicle control, respectively)

Results with reference substance (positive control):

- Results with reference substance valid Yes
- EC50:
ErC50 (72h) = 0.620 mg/L (growth rate)
EyC50 (72h) = 0.334 mg/L (yield)

Reported statistics and error estimates:

72h EC50 value with 95% confidence limits (upper limit, lower limit) were calculated by probit analysis using NCSS Software 2019, version 19.0.3.
The NOEC and LOEC value were calculated using the One-way Analysis of Variance - Kruskal - Wallis - Comparison Z-Value test.

Treatments (mg/L)	R. No		A	B	ln A	ln B	(ln B - ln A)/Ocassion	Inhibition growth rate (%Ir)			Yield        B - A	Inhibition  yield (%Iy)
Control	CR1		10000	27500	9.2103	10.2219	0.04	0.00			17500	0.00
	CR2		10000	27500	9.2103	10.2219	0.04	0.00			17500	0.00
	CR3		10000	25000	9.2103	10.1266	0.04	0.00			15000	0.00
	CR4		10000	25000	9.2103	10.1266	0.04	0.00			15000	0.00
	CR5		10000	30000	9.2103	10.3090	0.05	0.00			20000	0.00
	CR6		10000	32500	9.2103	10.3890	0.05	0.00			22500	0.00
	Average		10000	27917	9.2103	10.2325	0.04	0.00			17917	0.00
	SD		0	2923	0.00	0.10	0.00
	% C.V		0.00	10.47	0.00	1.01	10.07
Solventcontrol	T1R1		10000	25000	9.2103	10.1266	0.04	0.00			15000	0.00
	T1R2		10000	22500	9.2103	10.0213	0.03	0.00			12500	0.00
	T1R3		10000	22500	9.2103	10.0213	0.03	0.00			12500	0.00
	T1R4		10000	25000	9.2103	10.1266	0.04	0.00			15000	0.00
	T1R5		10000	27500	9.2103	10.2219	0.04	0.00			17500	0.00
	T1R6		10000	22500	9.2103	10.0213	0.03	0.00			12500	0.00
	Average		10000	24167	9.2103	10.0898	0.04	0.00			14167	0.00
	SD		0	2041	0.00	0.08	0.00
	% C.V		0.00	8.45	0.00	0.82	9.41
0.8	T2R1		10000	20000	9.2103	9.9035	0.03	21.19		10000		29.41
	T2R2		10000	17500	9.2103	9.7700	0.02	36.37		7500		47.06
	T2R3		10000	22500	9.2103	10.0213	0.03	7.80		12500		11.76
	Average		10000	20000	9.2103	9.8982	0.03	21.78		10000		29.41
	SD		0	2500	0.00	0.13	0.01
	% C.V		0.00	12.50	0.00	1.27	18.28
1.6	T3R1		10000	17500	9.2103	9.7700	0.02	36.37		7500		47.06
	T3R2		10000	20000	9.2103	9.9035	0.03	21.19		10000		29.41
	T3R3		10000	17500	9.2103	9.7700	0.02	36.37		7500		47.06
	Average		10000	18333	9.2103	9.8145	0.03	31.31		8333		41.18
	SD		0	1443	0.00	0.08	0.00
	% C.V		0.00	7.87	0.00	0.79	12.76
3.2	T4R1		10000	12500	9.2103	9.4335	0.01	74.63		2500		82.35
	T4R2		10000	15000	9.2103	9.6158	0.02	53.90		5000		64.71
	T4R3		10000	12500	9.2103	9.4335	0.01	74.63		2500		82.35
	Average		10000	13333	9.2103	9.4943	0.01	67.72		3333		76.47
	SD		0	1443	0.00	0.11	0.00
	% C.V		0.00	10.83	0.00	1.11	37.08
6.4	T5R1		10000	12500	9.2103	9.4335	0.01	74.63		2500		82.35
	T5R2		10000	10000	9.2103	9.2103	0.00	100.00		0		100.00
	T5R3		10000	12500	9.2103	9.4335	0.01	74.63		2500		82.35
	Average		10000	11667	9.2103	9.3591	0.01	83.09		1667		88.24
	SD		0	1443	0.00	0.13	0.01
	% C.V		0.00	12.37	0.00	1.38	86.60
12.8	T6R1		10000	10000	9.2103	9.2103	0.00	100.00		0		100.00
	T6R2		10000	10000	9.2103	9.2103	0.00	100.00		0		100.00
	T6R3		10000	10000	9.2103	9.2103	0.00	100.00		0		100.00
	Average		10000	10000	9.2103	9.2103	0.00	100.00		0		100.00
	SD		0	0	0.00	0.00	0.00
	% C.V		0.00	0.00	0.00	0.00	0.00
A - 0 hour cell count					B = 24/48/72 hours cell count
Caluclation: Growth rate(µ)=(ln(X24/48/72 hours) - ln(X0/24/48 hours))/ number of days, %Ir=(µcontrol - µTreatment)/µcontrol*100
Yield(Y)= X24/48/72 hours - X0/24/48 hours), %Iy=(Ycontrol - YTreatment)/Ycontrol*100
S.D=Standard deviation		%C.V=Standard deviation/Average×100							R=Replication

Percentage Inhibition of Growth Rate and Yield – 48 hours (Definitive test)

Treatments (mg/L)	R. No	A	B	ln A	ln B	(ln B - ln A)/Ocassion	Inhibition growth rate (%Ir)	Yield B - A		Inhibition yield (%Iy)
Control	CR1	10000	142500	9.2103	11.8671	0.06	0.00	132500		0.00
	CR2	10000	127500	9.2103	11.7559	0.05	0.00	117500		0.00
	CR3	10000	140000	9.2103	11.8494	0.05	0.00	130000		0.00
	CR4	10000	137500	9.2103	11.8314	0.05	0.00	127500		0.00
	CR5	10000	165000	9.2103	12.0137	0.06	0.00	155000		0.00
	CR6	10000	137500	9.2103	11.8314	0.05	0.00	127500		0.00
	Average	10000	141667	9.2103	11.8581	0.06	0.00	131667		0.00
	SD	0	12517	0.00	0.09	0.00
	% C.V	0.00	8.84	0.00	0.72	3.22
Solvent control	T1R1	10000	132500	9.2103	11.7943	0.05	0.00	122500		0.00
	T1R2	10000	120000	9.2103	11.6952	0.05	0.00	110000		0.00
	T1R3	10000	145000	9.2103	11.8845	0.06	0.00	135000		0.00
	T1R4	10000	135000	9.2103	11.8130	0.05	0.00	125000		0.00
	T1R5	10000	135000	9.2103	11.8130	0.05	0.00	125000		0.00
	T1R6	10000	157500	9.2103	11.9672	0.06	0.00	147500		0.00
	Average	10000	137500	9.2103	11.8279	0.05	0.00	127500		0.00
	SD	0	12649	0.00	0.09	0.00
	% C.V	0.00	9.20	0.00	0.77	3.49
0.8	T2R1	10000	120000	9.2103	11.6952	0.05	5.07		110000	13.73
	T2R2	10000	107500	9.2103	11.5852	0.05	9.27		97500	23.53
	T2R3	10000	135000	9.2103	11.8130	0.05	0.57		125000	1.96
	Average	10000	120833	9.2103	11.6978	0.05	4.97		110833	13.07
	SD	0	13769	0.00	0.11	0.00
	% C.V	0.00	11.39	0.00	0.97	4.58
1.6	T3R1	10000	77500	9.2103	11.2580	0.04	21.77		67500	47.06
	T3R2	10000	70000	9.2103	11.1563	0.04	25.66		60000	52.94
	T3R3	10000	80000	9.2103	11.2898	0.04	20.56		70000	45.10
	Average	10000	75833	9.2103	11.2347	0.04	22.66		65833	48.37
	SD	0	5204	0.00	0.07	0.00
	% C.V	0.00	6.86	0.00	0.62	3.45
3.2	T4R1	10000	57500	9.2103	10.9595	0.04	33.17		47500	62.75
	T4R2	10000	47500	9.2103	10.7685	0.03	40.47		37500	70.59
	T4R3	10000	42500	9.2103	10.6573	0.03	44.72		32500	74.51
	Average	10000	49167	9.2103	10.7951	0.03	39.46		39167	69.28
	SD	0	7638	0.00	0.15	0.00
	% C.V	0.00	15.53	0.00	1.42	9.65
6.4	T5R1	10000	22500	9.2103	10.0213	0.02	69.02		12500	90.20
	T5R2	10000	22500	9.2103	10.0213	0.02	69.02		12500	90.20
	T5R3	10000	17500	9.2103	9.7700	0.01	78.62		7500	94.12
	Average	10000	20833	9.2103	9.9375	0.02	72.22		10833	91.50
	SD	0	2887	0.00	0.15	0.00
	% C.V	0.00	13.86	0.00	1.46	19.95
12.8	T6R1	10000	12500	9.2103	9.4335	0.00	91.48		2500	98.04
	T6R2	10000	12500	9.2103	9.4335	0.00	91.48		2500	98.04
	T6R3	10000	10000	9.2103	9.2103	0.00	100.00		0	100.00
	Average	10000	11667	9.2103	9.3591	0.00	94.32		1667	98.69
	SD	0	1443	0.00	0.13	0.00
	% C.V	0.00	12.37	0.00	1.38	86.60
A - 0 hour cell count				B = 24/48/72 hours cell count
Caluclation: Growth rate(µ)=(ln(X24/48/72 hours) - ln(X0/24/48 hours))/ number of days, %Ir=(µcontrol - µTreatment)/µcontrol*100
Yield(Y)= X24/48/72 hours - X0/24/48 hours), %Iy=(Ycontrol - YTreatment)/Ycontrol*100
S.D=Standard deviation		%C.V=Standard deviation/Average×100					R=Replication

EC₅₀Value of test item

Response variable based on % inhibition (0-72 h)		Effective concentration (mg/L)	95% confidence limits
			Lower limit (mg/L)	Upper Limit (mg/L)
Growth rate(mg/L)	ErC50	6.19	5.43	6.95
	ErC20	3.03	2.60	3.46
	ErC10	2.09	0.20	2.44
Yield(mg/L)	EyC50	2.73	2.42	3.04
	EyC20	1.34	1.12	1.56
	EyC10	0.93	0.75	1.11
Growth rate (mg/L)	LOEC	3.2
	NOEC	1.6
Yield (mg/L)	LOEC	3.2
	NOEC	1.6

Key:LC₅₀- Median Lethal Concentration, LOEC – Lowest Observed Effect Concentration,

Validity criteria fulfilled:

yes

Remarks:

The control and vehicle control increased by 64.25, 62.58 times (>16 times) of the initial cell count. The mean CoV for growth rate for the control cultures was 32.8%. The coefficient of variation of average specific growth rate was 0.6%.

Conclusions:

The effect of test chemical on fresh water algae was studied as per OECD 201 test guidelines, the 72-h EC50 of test item to alga on growth rate and yield are 6.19, 2.73 mg/L, respectively. 72-h LOEC of test item to alga on growth rate and yield is 3.2 mg/L. 72-h NOEC of test item to alga on growth rate and yield is 1.6 mg/L.

Executive summary:

This study was conducted as per OECD 201 (2011) to assess the acute toxicity effects of test chemical on the growth of green alga Pseudokirchneriella subcapitata(ATCC)following exposure of alga up to 72 h under static condition. Test system was initially procured from “American Type Culture Collection”, Chromachemie Laboratory Private Limited, later test system was sub-cultured (using OECD growth medium, OECD 201) in the test facility. 250 mL sterilized conical flasks covered with cap were used in the study. The test item was found to be soluble in acetone. Hence, acetone (concentration <0.1mL/L) with OECD growth medium was used as test medium. An inoculum culture in the test medium was prepared 3-4 days prior to test and culture conditions were maintained same as the test conditions. A range finding study (3 replicates/ concentration, 6 replicates for control group)with the test concentrations of1.5, 3, 6, 12, 24 mg/L were tested along with a control (0), vehicle control (0) group, prior to the main study. The inhibition in growth rate (0%, 0%, 3.04%, 22.18%, 31.09%, 87.92%, 100%) and yield (0%, 0%, 11.69%, 60.46%, 73.02%, 98.85%, 100%) were observed at 72 h in the test concentrations of 0, 0, 1.5, 3, 6, 12, 24 mg/L. Based on the results of range finding test following concentrations of 0 (control), 0 (vehicle control), 0.8, 1.6, 3.2, 6.4, 12.8 mg/L were selected for the main study(3 replicates/ concentration, 6 replicates for control groups)as significant changes (inhibition algal growth rate) were observed in treatment groups during 72h test period. The inhibition in growth rate (0%, 0%, 1.78%, 6.68%, 24.76%, 33.68%, 90.82%) and yield (0%, 0%, 7.17%, 24.49%, 65.09%, 76.32%, 99.19%) were observed during 72h test period in the test concentrations of 0 (control), 0 (vehicle control), 0.8, 1.6, 3.2, 6.4, 12.8 mg/L. A reference standard (Potassium dichromate) was tested to ensure the authenticity of the test in the lab. And the inhibition of growth rate (ErC50-72h) and yield (EyC50-72h) were found to be 0.620 mg/L & 0.334 mg/L, respectively; and were found to be in acceptable range. The initial cell density was 10000 cells/mL.The algal cells in the control and vehicle control increased by 64.25, 62.58 times (>16 times) of the initial cell count during the 72-h exposure period, respectively. The mean coefficient of variation for section-by-section growth rate for the control cultures over the test period (0-72-h) was 32.8% (<35%). The coefficient of variation of average specific growth rate was 0.6% (<7%). Hence, fulfilling the all the validity criteria of the test. HPLC method was used for method validation and active ingredient analysis along with stability of the test item in the test medium. Test item was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during main study 0 hr and 72 hr avg. recovery for 0.8, 3.2, 12.8 mg/L conc were 94.34%, 94.44%, 95.84% & 94.11%, 94.59%, 96.11%, respectively, were found in acceptable range. Continuous light having and average light intensity of 6766-6794 Lux, pH of 7.6-8.1, temperature 21.7-23.9 °C along with a shaking speed of 110 RPM were provided and maintained for the test system throughout the test. 72h EC50 (growth rate and yield) values with 95% confidence limits were calculated by probit analysis. NOEC and LOEC of growth rate and yield were calculated by one way ANOVA (Kruskal-Wallis-Comparison Z-Value Test) using NCSS Software 2019, version 19.0.3. The cells were counted using Haemocytometer under illumination of the microscope at 24, 48, and 72 h after inoculation. 72-h EC50 of test item to alga on growth rate and yield are 6.19, 2.73 mg/L, respectively. 72-h LOEC of test item to alga on growth rate and yield is 3.2 mg/L. 72-h NOEC of test item to alga on growth rate and yield is 1.6 mg/L. Thus based on the outcomes test chemical was classified into aquatic chronic category 2 as CLP classification criteria.

Description of key information

This study was conducted as per OECD 201 (2011) to assess the acute toxicity effects of test chemical on the growth of green alga Pseudokirchneriella subcapitata(ATCC)following exposure of alga up to 72 h under static condition. Test system was initially procured from “American Type Culture Collection”, Chromachemie Laboratory Private Limited, later test system was sub-cultured (using OECD growth medium, OECD 201) in the test facility. 250 mL sterilized conical flasks covered with cap were used in the study. The test item was found to be soluble in acetone. Hence, acetone (concentration <0.1mL/L) with OECD growth medium was used as test medium. An inoculum culture in the test medium was prepared 3-4 days prior to test and culture conditions were maintained same as the test conditions. A range finding study (3 replicates/ concentration, 6 replicates for control group)with the test concentrations of1.5, 3, 6, 12, 24 mg/L were tested along with a control (0), vehicle control (0) group, prior to the main study. The inhibition in growth rate (0%, 0%, 3.04%, 22.18%, 31.09%, 87.92%, 100%) and yield (0%, 0%, 11.69%, 60.46%, 73.02%, 98.85%, 100%) were observed at 72 h in the test concentrations of 0, 0, 1.5, 3, 6, 12, 24 mg/L. Based on the results of range finding test following concentrations of 0 (control), 0 (vehicle control), 0.8, 1.6, 3.2, 6.4, 12.8 mg/L were selected for the main study(3 replicates/ concentration, 6 replicates for control groups)as significant changes (inhibition algal growth rate) were observed in treatment groups during 72h test period. The inhibition in growth rate (0%, 0%, 1.78%, 6.68%, 24.76%, 33.68%, 90.82%) and yield (0%, 0%, 7.17%, 24.49%, 65.09%, 76.32%, 99.19%) were observed during 72h test period in the test concentrations of 0 (control), 0 (vehicle control), 0.8, 1.6, 3.2, 6.4, 12.8 mg/L. A reference standard (Potassium dichromate) was tested to ensure the authenticity of the test in the lab. And the inhibition of growth rate (ErC50-72h) and yield (EyC50-72h) were found to be 0.620 mg/L & 0.334 mg/L, respectively; and were found to be in acceptable range. The initial cell density was 10000 cells/mL.The algal cells in the control and vehicle control increased by 64.25, 62.58 times (>16 times) of the initial cell count during the 72-h exposure period, respectively. The mean coefficient of variation for section-by-section growth rate for the control cultures over the test period (0-72-h) was 32.8% (<35%). The coefficient of variation of average specific growth rate was 0.6% (<7%). Hence, fulfilling the all the validity criteria of the test. HPLC method was used for method validation and active ingredient analysis along with stability of the test item in the test medium. Test item was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during main study 0 hr and 72 hr avg. recovery for 0.8, 3.2, 12.8 mg/L conc were 94.34%, 94.44%, 95.84% & 94.11%, 94.59%, 96.11%, respectively, were found in acceptable range. Continuous light having and average light intensity of 6766-6794 Lux, pH of 7.6-8.1, temperature 21.7-23.9 °C along with a shaking speed of 110 RPM were provided and maintained for the test system throughout the test. 72h EC50 (growth rate and yield) values with 95% confidence limits were calculated by probit analysis. NOEC and LOEC of growth rate and yield were calculated by one way ANOVA (Kruskal-Wallis-Comparison Z-Value Test) using NCSS Software 2019, version 19.0.3. The cells were counted using Haemocytometer under illumination of the microscope at 24, 48, and 72 h after inoculation. 72-h EC50 of test item to alga on growth rate and yield are 6.19, 2.73 mg/L, respectively. 72-h LOEC of test item to alga on growth rate and yield is 3.2 mg/L. 72-h NOEC of test item to alga on growth rate and yield is 1.6 mg/L. Thus based on the outcomes test chemical was classified into aquatic chronic category 2 as CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:

6.19 mg/L

Additional information

This study was conducted as per OECD 201 (2011) to assess the acute toxicity effects of test chemical on the growth of green alga Pseudokirchneriella subcapitata(ATCC)following exposure of alga up to 72 h under static condition. Test system was initially procured from “American Type Culture Collection”, Chromachemie Laboratory Private Limited, later test system was sub-cultured (using OECD growth medium, OECD 201) in the test facility. 250 mL sterilized conical flasks covered with cap were used in the study. The test item was found to be soluble in acetone. Hence, acetone (concentration <0.1mL/L) with OECD growth medium was used as test medium. An inoculum culture in the test medium was prepared 3-4 days prior to test and culture conditions were maintained same as the test conditions. A range finding study (3 replicates/ concentration, 6 replicates for control group)with the test concentrations of1.5, 3, 6, 12, 24 mg/L were tested along with a control (0), vehicle control (0) group, prior to the main study. The inhibition in growth rate (0%, 0%, 3.04%, 22.18%, 31.09%, 87.92%, 100%) and yield (0%, 0%, 11.69%, 60.46%, 73.02%, 98.85%, 100%) were observed at 72 h in the test concentrations of 0, 0, 1.5, 3, 6, 12, 24 mg/L. Based on the results of range finding test following concentrations of 0 (control), 0 (vehicle control), 0.8, 1.6, 3.2, 6.4, 12.8 mg/L were selected for the main study(3 replicates/ concentration, 6 replicates for control groups)as significant changes (inhibition algal growth rate) were observed in treatment groups during 72h test period. The inhibition in growth rate (0%, 0%, 1.78%, 6.68%, 24.76%, 33.68%, 90.82%) and yield (0%, 0%, 7.17%, 24.49%, 65.09%, 76.32%, 99.19%) were observed during 72h test period in the test concentrations of 0 (control), 0 (vehicle control), 0.8, 1.6, 3.2, 6.4, 12.8 mg/L. A reference standard (Potassium dichromate) was tested to ensure the authenticity of the test in the lab. And the inhibition of growth rate (ErC50-72h) and yield (EyC50-72h) were found to be 0.620 mg/L & 0.334 mg/L, respectively; and were found to be in acceptable range. The initial cell density was 10000 cells/mL.The algal cells in the control and vehicle control increased by 64.25, 62.58 times (>16 times) of the initial cell count during the 72-h exposure period, respectively. The mean coefficient of variation for section-by-section growth rate for the control cultures over the test period (0-72-h) was 32.8% (<35%). The coefficient of variation of average specific growth rate was 0.6% (<7%). Hence, fulfilling the all the validity criteria of the test. HPLC method was used for method validation and active ingredient analysis along with stability of the test item in the test medium. Test item was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during main study 0 hr and 72 hr avg. recovery for 0.8, 3.2, 12.8 mg/L conc were 94.34%, 94.44%, 95.84% & 94.11%, 94.59%, 96.11%, respectively, were found in acceptable range. Continuous light having and average light intensity of 6766-6794 Lux, pH of 7.6-8.1, temperature 21.7-23.9 °C along with a shaking speed of 110 RPM were provided and maintained for the test system throughout the test. 72h EC50 (growth rate and yield) values with 95% confidence limits were calculated by probit analysis. NOEC and LOEC of growth rate and yield were calculated by one way ANOVA (Kruskal-Wallis-Comparison Z-Value Test) using NCSS Software 2019, version 19.0.3. The cells were counted using Haemocytometer under illumination of the microscope at 24, 48, and 72 h after inoculation. 72-h EC50 of test item to alga on growth rate and yield are 6.19, 2.73 mg/L, respectively. 72-h LOEC of test item to alga on growth rate and yield is 3.2 mg/L. 72-h NOEC of test item to alga on growth rate and yield is 1.6 mg/L. Thus based on the outcomes test chemical was classified into aquatic chronic category 2 as CLP classification criteria.