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)

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 January 22, 2019 to August 1, 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

Details on test solutions:

Name Make Batch/Lot Number Exp. date
pH Buffer 7 Fluka 10610 Aug 2021
pH Buffer 4 Fluka SZBG0140V November 2019
pH Buffer 9 Fluka SZBG1180V November 2019
Hardness test kit Hanna K0432/16 April 2021
Chlorine kit Chlorine test kit Prerana Laboratories R1102

Test organisms (species):

Daphnia magna

Details on test organisms:

Source: Daphnia culture, RLS. (Culture originally came from Daphtox kit F magna, Microbiotests, Belgium)
Age of Daphnids: <24 hrs old
Progeny: second progeny
Justification of the Test System: The Daphnia species, Daphnia magna is recommended by the test guideline, OECD 202.

Test type:

semi-static

Water media type:

other: chlorine free water

Limit test:

yes

Total exposure duration:

48 h

Hardness:

180 mg/L

Test temperature:

Range finding study: 19.8~21.0°C
Definitive study: 19.3~20.4°C

pH:

Range finding study: 6.2~6.4
Definitive study: 6.4~6.5

Nominal and measured concentrations:

In limit test: nominal concentration was 100 mg/L

Details on test conditions:

- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 4
- Photoperiod: 16-hr light: 8-hr dark
- Light intensity: Range finding study: 1236~1294 Lux
Definitive study: 1216.2~1254 Lux
- DO of exposure medium: Range finding study: 6.6~8.2mg/L
Definitive study: 6.9~8 mg/L
- Test container: 250mL Glass Beakers
- Volume of medium: 100mL/replicate for range finding study,
100mL/replicate for Definitive study

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:

1. Range finding study:
No daphnia immobility was observed in the concentrations tested and control at 48 hours after treatment. There were no toxic symptoms were observed at all the concentrations tested and control. The physicochemical parameters observed during Range finding study are presented in Table 3.

2. Definitive study:
2.1 Validity of the study:
The study met all the validity criteria, such as control mortality (0% against<=10%), Dissolved oxygen content of exposure medium (was more than 6.9 mg/L against >3mg/L (Table 4 and 5).
2.2 Daphnia immobilization and Toxic symptoms:
There was no Daphnia immobility and toxic sign were observed in the concentration tested at 48 hours after treatment.
The untreated control also recorded no immobilization of Daphnia throughout the study.
The EC50, NOEC and LOEC of CR SR94 was estimated as >100mg/L, at 48 hours after treatment.
2.3 Physicochemical Parameter:
The exposure medium pH value was in the range of 6.4 to 6.5 for fresh medium and 6.4 to 6.5 for 24 hrs renewed medium, respectively. The mean dissolved oxygen concentration at the end of 24 hrs in the treatment and control were in the range of 6.9 to 8.0mg/L. The exposure temperature value was in the range of 19.4 to 20.3℃ for fresh medium and 19.3 to 20.4℃ for 24 hours renewed medium, respectively (Table 4). The hardness and chlorine of exposure medium were observed as 180 and 0 mg/L respectively.

3. Reference study (Potassium dichromate)
A reference study was conducted to validate the test conditions using potassium dichromate during October 2018. The EC50 0.67 mg/L NOEC 0.1 mg/L and LOEC 0.2 mg/L was recorded against Daphnia magna at 48 hrs after.

4. Chemistry analysis:
The results of method validation using chloride free water medium, Stability analysis was done 0 and 24 hrs. The results of stability study indicated that the test item recovery was observed as 85.71 and 72.71% for 0 and 24 hrs analysis respectively. The results of test concentration verification indicated that the mean recovery of test item active was 93.10 & 89.29% for 0 and 24 hrs and 94.59 & 91.22% for 0 renewal and 24 hrs of exposure, respectively.

Table 3. Physico-chemical Parameters of medium - Range finding study 

T No.	Concentrations (mg/L)	0 hrs (Fresh medium)			24 hrs (Old medium)			24 hrs (Fresh medium)			48 hrs (Old medium)
		pH	DO (mg/L)	Temp (℃)	pH	DO (mg/L)	Temp (℃)	pH	DO (mg/L)	Temp (℃)	pH	DO (mg/L)	Temp (℃)
T1	Control	6.2	7.3	20.1	6.2	7.3	20.0	6.2	6.9	20.1	6.3	7.8	21.0
T2	0.1	6.2	7.4	20.0	6.3	7.2	19.9	6.2	6.9	20.0	6.3	7.7	21.0
T3	0.5	6.2	7.3	20.0	6.3	7.2	19.8	6.3	6.7	20.1	6.4	7.8	21.0
T4	1	6.2	8.2	20.0	6.3	7.3	20.2	6.2	6.6	20.3	6.4	7.9	21.0
T5	5	6.2	8.2	20.0	6.3	7.2	20.1	6.2	6.6	20.0	6.4	7.8	21.0
T6	10	6.2	8.1	20.0	6.3	7.4	20.1	6.2	6.8	20.0	6.4	7.8	21.0
T7	50	6.2	8.1	20.0	6.3	7.5	20.0	6.2	6.8	20.5	6.3	7.8	21.0
T8	100	6.2	8.0	20.0	6.3	7.6	20.0	6.2	6.9	20.6	6.3	7.9	21.0

Table 4. Physico-chemical Parameters of medium - Definitive study

T No.	Concentrations (mg/L)	0 hrs (Fresh medium)			24 hrs (Old medium)			24 hrs (Fresh medium)			48 hrs (Old medium)
		pH	DO (mg/L)	Temp (℃)	pH	DO (mg/L)	Temp (℃)	pH	DO (mg/L)	Temp (℃)	pH	DO (mg/L)	Temp (℃)
T1	Control	6.4	7.4	20.3	6.4	7.1	19.4	6.4	8	20.0	6.4	6.9	20.4
T2	100	6.5	7.6	20.3	6.5	7.2	19.3	6.4	7.8	19.4	6.5	6.9	20.1

Table 5. Daphnia magna Immobilization - Definitive Study

Treatment No.	Conc. (mg/L)	*Immobility (24hrs)				Mean (%)		*Cumulative immobility (48 hrs)				Mean (%)
		R1	R2	R3	R4			R1	R2	R3	R4
T1	Control	0	0	0	0	0	0	0	0	0	0	0
T2	100	0	0	0	0	0	0	0	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

According to OECD 202 test method, the 48-hr EC50 of CR SR94 was >100 mg/L. The NOEC and LOEC of CR SR94 was estimated as >100 mg/L.

Executive summary:

In order to determine acute immobilization effect of CR SR94 on Daphnia magna, a study was performed as per OECD guideline No. 202 by the department of Ecotoxicology, ROSS Life Science Pvt. Ltd., Bhosari, India during January - July 2019. The study was conducted with 5 Daphnids (<24 hrs old) per replicate and control (without test item) was maintained for comparison, Test item related effects, if any in Daphnia and sub lethal effects were observed at 24 and 48 hrs after exposure.

 

The stability of test item was performed in chlorine free water medium and the test item recovery was observed as 85.71 and 72.71% for 0 and 24 hrs analysis, respectively. A range finding semi static study was conducted with concentrations viz., 0.1, 0.5, 1.0, 5.0, 10.0, 50.0 and 100.0 mg/L with two replicates. The results of range finding study revealed that no immobility was observed up to the maximum concentration of 100 mg/L on Daphnia magna at 48 hours after treatment.

 

Based on the range finding results, definitive study was conducted as Limit test at the concentration of 100mg/L of test item with 4 replicates. The test medium for the test item concentration and control were renewed once after 24 hours. The results of the study indicated that all the validity criteria specified by the guideline were met. No immobilisation on Daphnia magna was observed in the limit test concentration and control. No toxic symptoms were observed during the test. The analysis of concentration verification revealed that the test item recovery in the exposure medium were 93.10, 89.29, 94.59 and 91.22% for 0, 24(fresh), 0 and 24 hrs (renewal) of exposure, respectively. As the analysed concentration recovery was within the range of 80-120%, the result is expressed as nominal concentration. The summary of results is tabulated below:

Result Parameters (at 48 hours)	Nominal Concentrations (mg/L)
EC50	>100
Fiducial limits (with 95% confidence)	NA
NOEC (immobility)	>100
LOEC (immobility)	>100

Description of key information

The 48-hr EC50 of CR SR94 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

Based on the range finding results, definitive study was conducted as Limit test at the concentration of 100mg/L of test item with 4 replicates. The test medium for the test item concentration and control were renewed once after 24 hours. The results of the study indicated that all the validity criteria specified by the guideline were met. No immobilisation on Daphnia magna was observed in the limit test concentration and control. No toxic symptoms were observed during the test. 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 SR94 was >100 mg/L.