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)

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

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 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

yes

Vehicle:

yes

Remarks:

Deionized water, acetonitrile, ammonium formate, reconstitute water.

Details on test solutions:

Deionized water: Prepared from the Arium® Pro ultrapure water system
Acetonitrile: J. T. Baker; liquid chromatography level; 99.9% purity
Ammonium formate: SIGMA, 99.0% purity
Reconstitute water: Method of preparation was shown in Table 1. Expiration date: No longer than six months.

Test organisms (species):

Daphnia magna

Details on test organisms:

- Source: National Chiayi University Freshwater Biological Resource Center

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

19-21°C

Nominal and measured concentrations:

In limit test: nominal concentration was 100 mg/L

Details on test conditions:

- Test volume: 30 mL
- Aeration: None
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Photoperiod: 16-hr light: 8-hr dark

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

Calibration curve: The analytical results of the test solution of each concentration at 0.10, 1.00, 10.00, 50.00 and 100.00 µg/mL showed that the linear equation of the calibration curve was y = 48.877383x - 0.626373, while R = 1.000.

LOQ(limit of quantification): Added the test article into deionized water and made its final concentration was 0.10 µg/mL. The results showed that the signal-to-noise ratio (S/N) ranged between 21.058 ~ 28.903.

Recovery rate: The test article was added into deionized water and made its final concentration was 0.10 and 100.00 µg/mL, respectively. The results showed that the average recovery rates were 111.60%, 101.13% at 0.10 and 100.00 µg/mL, respectively.

Stability test: The average recovery rate were respectively 100.50% and 101.22% at refrigeration. After stored at room temperature (RT, 25 ± 2℃) for 1 day. The average recovery rate were respectively 109.00% and 101.47%.

Quantitative analysis: The results indicated that the concentrations of samples from the test group of the day 2 were 149.58%~154.05% higher than the initial level.

Range-finding test: The results showed that the number of immobilized daphnids in control and test groups after the end of the test was 0, and thus the percentages immobilized was 0.00%.

Limit test:

1. The results showed that, at the end of the test, the number of immobilised daphnids in 100.00 mg/L nominal concentration group was 0 (Table 1), percentages immobilised was 0.00%, and EC50 was thus greater than 100.00mg/L nominal concentrations (Table 2).

2. The analytical results of test article concentration (Table 3) showed that test article was undetected in concrol group at 0 and 48 hours of the test. In test group, the test article concentration determined was at the range of 108.055~108.695 µg/mL at 0 hour of the test and was at the range of 166.059~166.883 µg/mL at 48 hours.

Table 1. The numbers of immobilized daphnids for the limit test

Group (mg/L)		Time (hours)	Numbers of immobilised activity^a	Number of discoloration	Numbers of trapping at surface of water
Control1	0.00	24	0	0	0
Control2	0.00	24	0	0	0
Control3	0.00	24	0	0	0
Control4	0.00	24	0	0	0
Test1	100.00	24	0	0	0
Test2	100.00	24	0	0	0
Test3	100.00	24	0	0	0
Test4	100.00	24	0	0	0
Control1	0.00	48	0	0	0
Control2	0.00	48	0	0	0
Control3	0.00	48	0	0	0
Control4	0.00	48	0	0	0
Test1	100.00	48	0	0	0
Test2	100.00	48	0	0	0
Test3	100.00	48	0	0	0
Test4	100.00	48	0	0	0

^aDeath of daphnids was also viewed as one of immobilisation responses.

Table 2. The percentages immobilized and EC50 for the limit test

Group (mg/L)		Percentages immobilised (%) 24 hours 48 hours		EC50(mg/L)
Control1	0.00	0.00	0.00	-
Control2	0.00	0.00	0.00	-
Control3	0.00	0.00	0.00	-
Control4	0.00	0.00	0.00	-
Test1	100.00	0.00	0.00	>100.00
Test2	100.00	0.00	0.00	>100.00
Test3	100.00	0.00	0.00	>100.00
Test4	100.00	0.00	0.00	>100.00

Table 3: Analytical results of concentrations in test solution of limit test

Sampling time	Sampling date	Analysis date	Nominal concentration (µg/mL)	Sample (test iquid)  concentration (µg/mL)
0 hour	2019/3/26	2019/3/26	0.000	0.000
			0.000	0.000
			0.000	0.000
			0.000	0.000
			100.000	108.332
			100.000	108.695
			100.000	108.462
			100.000	108.055
48 hours	2019/3/28	2019/3/28	0.000	0.000
			0.000	0.000
			0.000	0.000
			0.000	0.000
			100.000	166.883
			100.000	162.585
			100.000	166.216
			100.000	164.059

Table 4. Temperature, dissolved oxygen and pH value of the limit test

Group (mg/L)		Time	Item	Mean±SD
Control	0.00	0 hour	Temperature (℃)	21.0
			Dissolved oxygen (mg/L)	7.94±0.02
			pH value	7.19 ±0.01
		48 hours	Temperature (℃)	21.0
			Dissolved oxygen (mg/L)	7.96±0.08
			pH value	7.32±0.03
Test	100.00	0 hour	Temperature (℃)	21.0
			Dissolved oxygen (mg/L)	8.06±0.03
			pH value	7.27±0.01
		48 hours	Temperature (℃)	21.0
			Dissolved oxygen (mg/L)	8.05±0.09
			pH value	7.48±0.04

^aTemperature referred to the air temperature in the ambient environment measured when testing

Validity criteria fulfilled:

yes

Conclusions:

According to OECD 202 test method, the 48-hr EC50 of CR SB33 was >100 mg/L.

Executive summary:

This test using the procedures outlined in the SuperLab Study Plan for MZ6-181200027 which is based on the SOP for the OECD 202 (SOPE-505) and OECD 202 (OECD, 2004). The results of this OECD 202 test for CR SB33 show that all validity criteria were met. Under the conditions of this test, the 48-hr EC50 of CR SB33 was >100 mg/L.

Description of key information

The 48-hr EC50 of CR SB33 was >100 mg/L for Daphnia magna (OECD TG202).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

In both range-finding and limit test, not more than 10 % of the Daphnia were immobilised in the control. All test validity criteria were met. The pH of the test solutions was within the ranges outlined in the OECD 202 test guideline. Under the conditions of this test, the 48-hr EC50 of CR SB33 was >100 mg/L.