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

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 March 09, 2018 to May 17, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar 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 (laboratory, culture collection): National Chiayi University Freshwater Bioresource Center
- Acclimation period: 2-4 days

Water media type:

freshwater

Total exposure duration:

72 h

Nominal and measured concentrations:

Nominal concentrations: 4.28, 9.41, 20.70, 45.54 and 100.20 mg/L

Details on test conditions:

- Test vessel: 125 mL glass flask
- Test volume: 60 mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Light intensity and quality: 4440-8880 lux

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

ErC50

Effect conc.:

ca. 13.51 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: average specific growth rate

Duration:

72 h

Dose descriptor:

EC50

Remarks:

EyC50

Effect conc.:

ca. 3.56 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Table 1. The cell concentration for the definitive test

Group (mg/L)		Cell concentration (×10^4cells/mL)
		0 hour	24 ± 2 hours	48± 2 hours	72 ± 2 hours
Control group^a	0.00	1.00 ± 0.00	3.41 ± 0.17	8.29 ± 0.32	17.49 ± 0.69
Test group A^a	100.20	1.00 ± 0.00	1.62 ± 0.06	1.65 ± 0.06	2.09 ± 0.25
	45.54	1.00 ± 0.00	1.59 ± 0.05	1.75 ± 0.05	2.20 ± 0.02
	20.70	1.00 ± 0.00	1.83 ± 0.04	2.66 ± 0.31	6.31 ± 0.66
	9.41	1.00 ± 0.00	1.88 ± 0.05	5.00 ± 1.05	10.58 ± 1.03
	4.28	1.00 ± 0.00	2.02 ± 0.15	7.02 ± 0.16	9.61 ± 1.06
Test group B^b	100.20	1.00	1.87	3.65	15.6
	45.54	1.00	1.96	4.85	11.19
	20.70	1.00	2.17	5.27	15.28
	9.41	1.00	2.09	4.84	17.59
	4.28	1.00	2.00	7.76	17.91

^a All of control group and test group A conducted three replicates, and the data showed as mean ± standard deviation.

^b Test group B conducted one replicate.

 

Table 2. The average specific growth rate for the definitive test

Group (mg/L)		Average specific growth rate (d^-1)
		0 ~ 24 ± 2 hour	24 ± 2 ~ 48 ± 2 hours	48 ± 2 ~ 72 ± 2 hours	0 ~ 72 ± 2 hours
Control group^a	0.00	1.22 ± 0.05 (4.15)	0.89 ± 0.09 (9.98)	0.75 ± 0.08 (10.40)	0.95 ± 0.01 (1.39)
Test group A^a	100.20	0.48 ± 0.03 (7.17)	0.02 ± 0.01 (45.26)	0.23 ± 0.09 (41.52)	0.24 ± 0.04* (17.08)
	45.54	0.47 ± 0.03 (6.88)	0.09 ± 0.02 (18.41)	0.23 ± 0.02 (10.72)	0.26 ± 0.00* (0.88)
	20.70	0.60 ± 0.02 (3.27)	0.37 ± 0.10 (27.32)	0.87 ± 0.04 (4.80)	0.61 ± 0.03* (5.69)
	9.41	0.63 ± 0.02 (3.82)	0.97 ± 0.19 (19.50)	0.76 ± 0.27 (35.59)	0.79 ± 0.03* (4.03)
	4.28	0.70 ± 0.07 (10.65)	1.25 ± 0.05 (4.11)	0.31 ± 0.09 (30.35)	0.75 ± 0.04* (4.77)
Test group B^a	100.20	0.63	0.67	1.45	0.92
	45.54	0.67	0.91	0.84	0.81
	20.70	0.77	0.89	1.06	0.91
	9.41	0.74	0.84	1.29	0.96
	4.28	0.69	1.36	0.84	0.96

^a All of control group and test group A conducted three replicates, and the data showed as mean ± standard deviation (Coefficient of variation [%]).

^b Test group B conducted one replicate.

* SigmaStat 3.5 statistical software was used by One way ANOVA-Dunnett analysis, the results showed a significant difference between mean of the average specific growth rate of the test group A and the control group within 0~72± 2 hours (ρ< 0.05).

 

Table 3. The percent inhibition in average specific growth rate and percent inhibition of yield for the definitive test

Group (mg/L)		Geometric mean conc. (mg/L)	Logarithm conc. (mg/L)	Percent inhibition in average specific growth rate^c(%)	Percent inhibition of yield^d(%)
Test group A^a	100.20	55.55	1.74	70.74	92.06
				73.41	93.09
				79.26	95.09
	45.54	21.53	1.33	72.13	92.60
				72.44	92.72
				72.60	92.78
	20.70	10.06	1.00	36.01	68.23
				31.99	63.62
				39.29	71.62
	9.41	4.08	0.61	20.09	46.40
				13.89	34.82
				19.04	44.58
	4.28	2.28	0.36	16.77	40.46
				22.63	50.59
				23.80	52.41
Test group B^b	100.20	55.55	1.74	3.99	11.48
	45.54	21.53	1.33	15.60	38.22
	20.70	10.06	1.00	4.71	13.42
	9.41	4.08	0.61	-0.21	-0.59
	4.28	2.28	0.36	-0.84	-2.53

^a Test group A conducted three replicates.

^b Test group B conducted one replicate.

^c Percent inhibition in average specific growth rate (I_r) only within 0~72 ± 2 hours need to be calculated.

^d Percent inhibition of yield (I_y) only within 0~72 ± 2 hours need to be calculated.

 

Table 4. EC_X and 95% confidence interval for the definitive test

Parameter	ErC10^a	ErC20	ErC50	EyC10^b	EyC20	EyC50
ECX(mg/L)	1.88	3.08	13.51	0.38	0.66	3.56
95% confidence interval-lower	1.38	2.24	9.66	0.28	0.48	2.54
95% confidence interval-upper	2.58	4.26	19.08	0.52	0.91	5.04

^a E_rC_x was calculated by percent inhibition in average specific growth rate.

^b E_yC_x was calculated by percent inhibition of yield.

 

Table 5. The pH value for the definitive test

Group (mg/L)		pH
		0 hours	72 ± 2 hours
Control group^a	0.00	7.57 ± 0.12	7.58 ± 0.09
Test group A^a	100.20	7.50 ± 0.03	7.37 ± 0.03
	45.54	7.41 ± 0.03	7.48 ± 0.06
	20.70	7.60 ± 0.07	7.62 ± 0.12
	9.41	7.43 ± 0.02	7.60 ± 0.07
	4.28	7.57 ± 0.11	7.56 ± 0.09
Test group B^a	100.20	7.45	7.53
	45.54	7.31	7.36
	20.70	7.31	7.31
	9.41	7.40	7.41
	4.28	7.40	7.43

^a All of control group and test group A conducted three replicates, and the data showed as mean ± standard deviation (Coefficient of variation [%]).

^b Test group B conducted one replicate.

Validity criteria fulfilled:

yes

Conclusions:

According to OECD 201 test method, the ErC50 and the EyC50 of CR SB37 was 13.51 mg/L and 3.56 mg/L respectively.

Executive summary:

This test using the procedures outlined in the SuperLab Study Plan for M62-170900015001EN which is based on the SOP for the OECD 201 (SOPE-512) and referred to OECD 201 (OECD, 2011). The results of this test for CR SB37 show that test validity criteria was met. In the alga growth inhibition test (definitive test), the test article concentrations applied were 4.28, 9.41, 20.70, 45.54 and 100.20 mg/L. The alga growth would partially inhibited after CR SB37 shading of light, but less than toxicity of CR SB37. Therefore, the inhibition of algal growth was due to the toxicity of CR SB37. Under the test conditions, the E_rC₅₀and the E_yC₅₀of CR SB37 was 13.51 mg/L and 3.56 mg/L respectively.

Description of key information

Respectively, the E_rC₅₀ and the E_yC₅₀ of CR SB37 was 13.51 mg/L and 3.56 mg/L for Pseudokirchneriella subcapitata (OECD TG201).

Key value for chemical safety assessment

EC50 for freshwater algae:

13.51 mg/L

Additional information

The biomass in the control group have increased exponentially by a factor of at least 16 within the 72-hour test period. The specific growth rate in the control group was more than 0.92 d^-1. The mean coefficient of variation for specific growth rates (0 ~ 24 ± 2 hour, 24 ± 2 ~ 48 ± 2 hours, 48 ± 2 ~ 72 ± 2 hours and 0 ~ 72 ± 2 hours) in the control group were not exceed 35%. The coefficient of variation of average specific growth rates during the whole test period in replicate control groups were not exceed 7%. The variation in the pH values in the beginning and end of the tests was less than 1.5 units.Under the test conditions, the E_rC₅₀ and the E_yC₅₀ of CR SB37 was 13.51 mg/L and 3.56 mg/L respectively.