2,7-Naphthalenedisulfonic acid, 5-(acetylamino)-3-[2-[5-[[4-chloro-6-[[3-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-2-sulfophenyl]diazenyl]-4-hydroxy-, sodium salt (1:4)

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:

From January 16, 2019 to July 27, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test organisms (species):

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

Details on test organisms:

- Source: Procured from ATCC, USA and culture are maintained at the department of Ecotoxicology, ROSS Lifescience.
- Justification of the Test System: Green algae, Pseudokirchneriella subcapitata suggested by OECD guildline for testing growth inhibition.
- Age of culture: 2-4 days old preculture

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

Range finding study: 22.7~23.4°C
Definitive study: 21.9~23.9°C

pH:

Range finding study: 6.21~7.41
Definitive study: 6.43~7.80

Details on test conditions:

Photo period: Continuous light
Light intensity: Range finding study: 5696~5934 lux
Definitive study: 6238~6516 lux
Test container: Petri dish (to transfer light to algal cells because of colored test item)
Volume of the medium: 20mL/replicate
Test Chamber: Shaker incubator with 100 rpm

Duration:

72 h

Dose descriptor:

EC50

Remarks:

ErC50

Effect conc.:

6.13 mg/L

Duration:

72 h

Dose descriptor:

EC50

Remarks:

EyC50

Effect conc.:

0.352 mg/L

Details on results:

1. Range finding study:
The results of range finding study revealed that the growth rate and yield inhibition of algae were ranged from 15.42 to 115.06% and 52.10 to 100.48%, respectively for the concentrations tested.

2. Definitive study:
2.1 Validity of the study:
The test met all the validity criteria, such as biomass in control (84.98 times against 16 times), mean coefficient of variation for section by section specific growth rates in the control (6.15 to 9.59% against 35% at 0 to 72 hr) and the coefficient of variation of average specific growth rate in control replicates (09.95% against 7%)(Table 1).

2.2 Physicochemical Parameter:
The exposure medium pH value was 6.43 to 6.50 at test start and 7.58 to 7.80 at end of the test for control and treatment (Table 2).

2.3 Growth rate and yield:
The average specific growth rate in control was recorded 1.48 and in test item concentrations ranged from 0.24 to 1.47.
The growth yield inhibition in was ranged test item concentration was recorded on ranged from 3.47% to 98.67% over control.
Similarly, the growth rate inhibition in test item concentration was recorded in ranged from 0.77% to 83.71% over control (Table 3).

2.4 Effective concentration for growth rate and yield inhibition:
The effective concentrations for 10%, 20% and 50% growth rate inhibition were of -1.63, 1.04 and 6.13 mg a.i/L respectively based on mean measured concentration. Similarly, the effective concentrations for 10%, 20% and 50% for yield inhibition were of 0.041, 0.085 and 0.352 mg a.i/L, respectively based on mean measured concentration (Table 4).

3. Reference study (Potassium dichromate)
A reference study was conducted to validate the test conditions using potassium dichromate. The EC50 for growth inhibition (0.85 mg/L), NOEC (0.1 mg/L) and LOEC (0.2 mg/L) of the reference item against Pseudokirchneriella subcapitata and EC50 value of 0.27 mg/L for yield inhibition at 72 hours after treatment.

4. Chemistry Analysis:
The results indicated that the test item was not stable under the test conditions up to 48 hours. The recovery of test item concentration in the test medium was 97.79, 75.90, 65.64 and 63.77% for 0, 24, 48 and 72 hours of analysis, respectively. The data on dose verification indicated that recovery was ranged from 77.21 to 95.46%, 71.01 to 112.24%, 56.63 to 91.15 and 42.74 to 83.29% at 0, 24, 48 and 72 hours for all the concentrations analyzed.

Table 1. Validity criteria for the study with CR SR94 - Definitive study

Control	Average specific growth rate* section by section
	Replicates	0~24 hrs	24~48 hrs	48~72 hours	0~72 hours
	1	2.02	1.23	1.20	1.48
	2	1.85	1.45	1.20	1.50
	3	1.97	1.36	1.06	1.46
	4	1.72	1.60	1.09	1.47
	5	1.91	1.29	1.28	1.49
	6	2.02	1.30	1.11	1.48
	Average	1.91	1.37	1.16	1.48
	SD	0.12	0.13	0.08	0.01
	CV%	6.15	9.59	7.06	0.95
Average Initial cell count (0 hr)	6296 cells/mL
Average Initial cell count 72 hrs	535000 cells/mL
Biomass	84.98 time

Table 2. pH of test solution treated with CR SR94 - Definitive study

T. No.	Conc. (mg/L)	pH at start (0hr)						Mean	pH at end (72hrs)						Mean
		R1	R2	R3	R4	R5	R6		R1	R2	R3	R4	R5	R6
1	Control	6.49	6.44	6.49	6.49	6.46	6.48	6.48	7.61	7.68	7.64	7.69	7.80	7.79	7.70
2	0.06	6.48	6.47	6.46	-	-	-	6.47	7.70	7.77	7.72	-	-	-	7.73
3	0.18	6.47	6.48	6.47	-	-	-	6.47	7.75	7.61	7.72	-	-	-	7.69
4	0.54	6.50	6.47	6.47	-	-	-	6.48	7.69	7.68	7.58	-	-	-	7.65
5	1.6	6.48	6.49	6.43	-	-	-	6.47	7.70	7.78	7.79	-	-	-	7.76
6	4.9	6.48	6.47	6.49	-	-	-	6.48	7.64	7.69	7.79	-	-	-	7.71
7	14.6	6.47	6.48	6.46	-	-	-	6.47	7.74	7.68	7.66	-	-	-	7.69

Table 3. Growth rate and yield of Pseudokirchneriella subcapitata treated with CR SR94 - Definitive study

T. No.	Conc. (mg/L)	Average specific growth rate	Growth rate inhibition (%) (%Ir)	Yield inhibition (%) (%Iy)
1	Control	1.48	-	-
2	0.06 (0.050)	1.47^n.s.	0.77	3.47
3	0.18 (0.154)	1.28*	13.65	46.02
4	0.54 (0.501)	1.18*	20.52	60.37
5	1.6 (0.966)	1.12*	24.57	67.15
6	4.9 (4.001)	0.7*	52.63	91.42
7	14.6 (13.328)	0.24*	83.71	98.67

*Statistically significant from control (Dunnett Multiple comparison test, a=0.05); n.s. - Not significant from control; Figure in parentheses are mean measured.

Table 4. Effective concentrations for Growth rate and Yield inhibition of Pseudokirchneriella subcapitata treated with CR SR94

Growth rate inhibition (%Ir)	Effective concentration (mg active/L)	ErC10	E r C 20	E r C50
		-1.63	1.04	6.13
	95% Fiducial limits (mg active/L)	-11.70 ~ -1.45	-4.94 ~ 3.98	3.25 ~ 13.58
	Regression equation	Y = -1.01 + 0.17X
	NOEC (mg active/L)	0.05
	LOEC (mg active/L)	0.154
Yield inhibition (% Iy)	Effective concentration (mg active/L)	EyC 10	E y C20	E y C 50
		0.041	0.085	0.352
	95% Fiducial limits (mg active/L)	0.005 ~ 0.104	0.017 ~ 0.184	0.157 ~ 0.686
	Regression equation	Y = 0.622 + 1.37 log X

Validity criteria fulfilled:

yes

Conclusions:

It is concluded from the study the EC50 of CR SR94 with respect to growth rate and yield inhibition of Pseudokirchneriella subcapitata was estimated as 6.13mg active/L and 0.352 mg active/L, respectively based on mean measured concentration. The NOEC and LOEC with respect to growth rate inhibition was estimated as 0.050 mg active and 0.154 mg active/L, respectively based on mean measured concentration.

Executive summary:

In order to assess the growth inhibition effect of CR SR94 on algae (green alga Pseudokirchneriella subcapitata), a study was performed as per OECD guideline No. 201 by the department of Ecotoxicology, ROSS Life Science Pvt. Ltd., Bhosari, India during January - July 2019.

 

Method validation was done with CR SR94 in TG201 medium. Stability of the test item concentration in TG 201 medium was performed and found to be unstable, the recovery were 97.79, 75.90, 65.64 and 63.77 for 0, 24, 48 and 72 hrs respectively. Verification of the test item concentrations was done during definitive study and found to be the range of 42.74 to 112.24%.

 

A range finding study was conducted with the concentrations viz., 1, 3.2, 10, 32 and 100 mg/L of CR SR94 with two replicates. Control (without test item) was maintained for comparison with four replicates. Observation for the cell counts was made at 24, 48 and 72 hours after treatment. The growth rate and yield inhibition of CR SR94 was ranged from 15.42 to 115.06% and 52.10 to 100.46%, respectively.

 

Based on the range finding results, definitive study was conducted with the test item concentration viz., 0.06, 0.18, 0.54, 1.6, 4.9 and 14.6 mg/L selected with a geometric factor of 3. Three replicates for treatment and 6 replicates for control. The results of the study indicated that all the validity criteria specified by the guideline were met. As the test item is not stable up to 72 hours in the test medium, the results are expressed based on geometric mean measured concentrations. The growth and yield inhibition of the test item, CR SR94 was ranged from 0.77 to 83.71% and 3.47 to 98.67%, respectively. The summary of results is tabulated below:

Summary of results:

Growth inhibition (%Ir)	Result Parameters	Concentration (mg active/L)
	ErC50	6.13
	95% Fiducial limits	3.25 to 13.58
	ErC10	-1.63
	ErC20	1.04
	NOEC	0.050
	LOEC	0.154
Yield inhibition (% Iy)	EyC50	0.352
	95% Fiducial limits	0.157 to 0.686
	EyC10	0.041
	EyC20	0.085

*Based on geometric mean measured concentrations.

Description of key information

Respectively,the E_rC₅₀and the E_yC₅₀of CR SR94 was 6.13mg active/L and 0.352 mg active/L for Pseudokirchneriella subcapitata (OECD TG201).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.05 mg/L

EC10 or NOEC for marine water algae:

0.154 mg/L