Pyridinium, 1-[2-[[4-[[3-[2-[6-amino-1-hydroxy-3-sulfo-5-[2-[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-2-naphthalenyl]diazenyl]-4-sulfophenyl]amino]-4-oxobutyl]sulfonyl]ethyl]-3-carboxy-, inner salt, 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 March 14, 2019 to July 15, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, 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

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

20.5-24.5°C

pH:

At the 0 hour of the test, the average pH value of the control and the test group were 7.70±0.01 and 7.85±0.02, respectively; and the end(72 hours), the average pH value of the control group and the test group were 8.12±0.02 and 8.23±0.03 respectively.

Nominal and measured concentrations:

0.16, 0.80, 4.00, 20.00 and 100.00 mg/L

Details on test conditions:

- Test vessel: 125 mL glass flask
- Test volume: 60 mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- Light intensity and quality: 4640-5800 lux
- Shaking rate: Maintained at 100 rpm by water bath shaker

Duration:

72 h

Dose descriptor:

EC50

Remarks:

ErC50

Effect conc.:

> 100 mg/L

Duration:

72 h

Dose descriptor:

EC50

Remarks:

EyC50

Effect conc.:

> 100 mg/L

Details on results:

1. Range-finding test: The results showed that there were no abnormalities in the growth of algae cells in the test group of 0.16, 0.80, 4.00, 20.00 and 100.00 mg/L, and inhibition in average specific growth rate was less than 5%, so the limit test was conducted.
2. Limit test:
2.1 The results of the algae cell concentration (Table 1) and the algae growth curve during the test showed that the average cell concentration of the control group at the end of the test was 98.33 x 10^4 cells/mL, and the average cell concentration of the test group was 118.33 x 10^4 cells/mL. After microscopic observation, there was no abnormality in the growth of algae cells in each test group.
2.2 The specific growth rate of each group during the test period (Table) showed that the average specific growth rate of the control group was 1.53 d^(-1) from 0 to 72 hours, and the average specific growth rate of the test group was 1.59 d^(-1). The difference in specific growth rate between control and test groups was not statistically significant (P>0.05), and, thus, the NOEC was 100.00 mg/L nominal concentration.
2.3 The results of the percent inhibition in average specific growth rate and the percent inhibition percent inhibition of yield of each test groups (Table 3) showed the percent inhibition in average specific growth rate of test group was range between -6.14% ~ -2.50% and the percent inhibition of yield was range between -32.53% ~ -11.99%.
2.4 ECx were shown in Table 4, ErC10, ErC20 and ErC50 were > 100.00mg/L nominal concentration; EyC10, EyC20 and EyC50 were > 100.00mg/L nominal concentration.
2.5 At the 0 hour of the test, the average pH value of the control group and the test group were 7.70±0..01 and 7.85±0.02, respectively (Table 5); and at the end (72 hours), the average pH value of the control group and the test group were 8.12±0.02 and 8.23±0.03 respectively.
2.6 The test article concentration analysis results (Table 6) showed that the concentration from 0 to 72 hours was between 107.89~172.71 mg/L.

Table 1. The algal biomass for the limit test

Group (mg/L)^a		Average specific growth rate (d^-1) (Coefficient of variation [%]) 0 hour           24 hours          48 hours          72 hours
Control group	0.00	1.00±0.00	2.47±0.30	22.67±1.51	98.33±7.53
Test group	100.00	1.00±0.00	3.27±0.69	27.67±5.20	118.33± 7.53

^aResult are presented as mean±standard deviation. N=6. Raw data is shown in Appendix6.

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

Group (mg/L)^a		Algal biomass (x10^4cell/mL) 0 hour           24 hours          48 hours          72 hours
Control group	0.00	0.90±0.12 (13.94)	2.22±0.17 (7.48)	1.47±0.08 (5.63)	1.53±0.03 (1.66)
Test group	100.00	1.17±0.21 (17.82)	2.14±0.34 (15.81)	1.47±0.20 (13.93)	1.59±0.02 (1.33)

^aResult are presented as mean±standard deviation. N=6. Raw data is shown in Appendix7.

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

Group (mg/L)	Logarithm conc. (mg/L)	Percent inhibition in average specific growth rate^a(%)	Percent inhibition of yield^b(%)
100.0	2.00	-6.14 -4.40 -2.50 -4.40 -4.40 -2.50	-32.53 -22.26 -11.99 -22.26 -22.26 -11.99

^aPercent inhibition in average specific growth rate (I_r) during the entire period of the test (0~72 hours).

^bPercent inhibition of yield (I_y) during the entire period of the test (0~72 hours).

Table 4. ECx for the limit test

Parameter	ErC10^a   ErC20     ErC50    EyC10^b     EyC20     EyC50   (mg/L)
ECx	>100.00	>100.00	>100.00	>100.00	>100.00	>100.00

^aErCx was calculated by percent inhibition in average specific growth rate

^bErCx was calculated by percent inhibition of yield.

Table 5. pH value for the limit test

Group (mg/L)^a		pH value 0 hour 72 hours
Contol group	0.00	7.70±0.01	8.12±0.02
Test group	100.00	7.85±0.02	8.23±0.03

^aResult are presented as mean±standard deviation. N=6. Raw data is shown in Appendix8.

Table 6. Test article concentration analysis result of limit test

Sampling time	Sampling date	Analysis date	Nominal concentration (µg/mL)	Sample (test liquid) concentration  (µg/mL)
0 hour	2019/03/26	2019/03/26	0.000	0.000
			0.000	0.000
			0.000	0.000
			0.000	0.000
			0.000	0.000
			0.000	0.000
			100.000	107.890
			100.000	108.802
			100.000	108.778
			100.000	109.323
			100.000	108.972
			100.000	111.382
72 hour	2019/03/29	2019/03/29	0.000	0.000
			0.000	0.000
			0.000	0.000
			0.000	0.000
			0.000	0.000
			0.000	0.000
			100.000	168.521
			100.000	164.672
			100.000	170.797
			100.000	169.337
			100.000	172.811
			100.000	171.288

Validity criteria fulfilled:

yes

Conclusions:

According to OECD 201 test method, the ErC50 and the EyC50 of CR SB33 were both >100 mg/L.

Executive summary:

This test using the procedures outlined in the SuperLab Study Plan for MZ6-181200028 which is based on the SOP for the OECD 201 (SOPE-504) and OECD 201 (OECD, 2011). The results of this OECD 201 test for CR SB33 show that test validity criteria was met. In the limit test, the test article concentrations applied was 100.00 mg/L. Under these test conditions, the ErC50 and the EyC50 of CR SB33 were both >100 mg/L.

Description of key information

The E_rC₅₀ and the E_yC₅₀ of CR SB33 were both >100 mg/L for Pseudokirchneriella subcapitata (OECD TG201).

Key value for chemical safety assessment

EC50 for freshwater algae:

100 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 (Day 0-1, Day 1-2, Day 2-3, and Day 0-3) 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 SB33 were both >100 mg/L.