Reaction products of diazotised sodium 2-amino-5-{[2-(sulfonatooxy)ethyl]sulfonyl}benzenesulfonate coupled with sodium 2-(4-aminobenzene-1-sulfonyl)ethyl sulfate, subsequently coupled with diazotised sodium 2-(4-aminobenzene-1-sulfonyl)ethyl sulfate

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Experimental test result performed using standard OECD test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 100 mg/l
- Sampling method: The test solution was prepared by dissolving 400 mg of test chemical in 4000 ml of RO water to get final concentration of 100 mg/l. The same stock solution was used as test concentration.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
-Method: The test solution was prepared by dissolving 400 mg of test chemical in 4000 ml of RO water to get final concentration of 100 mg/l. The same stock solution was used as test concentration.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish
- Source: Anu Pet Shop Nagpur
- Length at study initiation (length definition, mean, range and SD): 1.9 cm
- Weight at study initiation (mean and range, SD): 0.15 g


ACCLIMATION
- Acclimation period: 7 days
- Feeding frequency during acclimation: Daily once
- Type and amount of food: Food was provided during acclimatization.
- Health during acclimation (any mortality observed): No
- Aeration: Continuous

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

100 mg of CaCO3

Test temperature:

21 to 25 °C

pH:

7.7

Dissolved oxygen:

86.83 %

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria having 7 lit capacity
- Type (delete if not applicable): open
- Total volume of solution: 4 L
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): Replicate
- No. of vessels per control (replicates): Replicate
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: 7 fishes/ 4 L of test solution

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: RO water

OTHER TEST CONDITIONS
- Photoperiod: 16 hr light and 8 hr darkness
- Light Intensity : 821.25 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality
VEHICLE CONTROL PERFORMED: Not applicable
RANGE-FINDING STUDY
Based on the available data on the test chemical range finding test was not conducted directly main study was intaited to draw the conclusion on test substance toxicity.

Reference substance (positive control):

not required

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Other details not known

Details on results:

- Other abnormalities: No abnormal symptoms in control and test vessel
- Mortality of control: No mortality
- Other adverse effects control: No adverse effects

Sublethal observations / clinical signs:

Table 1 : Result on Mortality and Visible Symptoms 

Sr. No	Test concentrations           (mg/ L)	3 hr	6 hr	24 hr	48 hr	72 hr	96 hr	Cumulative Mortality	Percent Inhibition
1	control	-	-	-	-	-	-	-	-
2	100	-	-	-	-	-	-	-	-

Table 2 : Result on Mortality and Visible Symptoms 

Sr. No	Test concentrations (mg/ L)	3 hr	6 hr	24 hr	48 hr	72 hr	96 hr
1	control	NS 7	NS 7	NS 7	NS 7	NS 7	NS 7
2	100	NS 7	NS 7	NS 7	NS 7	NS 7	NS 7
Any other symptoms		-	-	-	-	-	-

 

EXPLANATION OF SYMPTOMS:

 The number behind symbol for symptom = number of affected fish

A:  apathy,   NR : Narcotic-like state,  C:   Swimming  near  water  surface,  M:   Slow  movements   compared  to   control,  NS:  No abnormal symptoms, SC: Schooling effect , L : Loss of equilibrium,  SM:  Shimming  movement, H 1 : Hyperactivity, H 2 : Hypoactivity B : Bending behavior, F : Fin rot ,IS:  Invert  swimming, SB: Swimming behavior, RF: Respiratory Function, P: Pigmentation, E: Exophthalmos

 

Table 3: Result on pH

Test Concentrations (mg/L)		p H
control	7.9	7.5	7.5	7.7
100	7.8	7.9	7.8	8.1

Table 4: Result on temperature

Test concentrations (mg /L)	Temperature °C
	24 Hr	48 Hr	72 Hr	96 Hr
control	23.3	23.1	23.5	22.4
100	23.3	23.3	23.5	22.4

 

Table 5: Result on Dissolved oxygen

Test Concentrations ( mg/L)	Dissolved Oxygen
	24 Hr	48 Hr	72 Hr	96 Hr
control	80.7	75.0	68.0	70
100	74.7	69.4	64.6	86.7

Validity criteria fulfilled:

yes

Remarks:

No mortality was found in the control at end of test. The DO concentration remained above 60% of air- saturation value throughout, exposure period (reported as 86.83%).Test concentrations were measured, found to remain at 80-120% of nomina concentration.

Conclusions:

Based on mortality effect of test chemical on test organism Danio rerio (Zebra fish) the 96 hr LC50 value was determined to be > 100 mg/l.

Executive summary:

The study was conducted in accordance with OECD Guideline 203, Fish, Acute Toxicity Test ( 2019 ). A study was performed to assess the Acute toxicity of test chemical, to Danio rerio (Zebra fish) under static conditions. Source of test organisms was Anu Pet Shop Nagpur. Length at study initiation 1.9 cm (avg).  Weight at study initiation was 0.15 g (avg). Test organism was acclimatized for a period of 7 days. Feeding frequency during acclimation once in a day.  Food was provided during acclimatization. No mortality observed was observed during acclimatization. Continuous aeration was also provided during acclimatization.  The test solution was prepared by dissolving 400 mg of test chemical in 4000 ml of RO water to get final concentration of 100 mg/l. The same stock solution was used as test concentration. The main study (i.e. limit test) was conducted using 0 (control) and 100 mg/L test concentration. Test vessel was taken as Glass aquaria having 7 lit capacity. Type of test vessel was of open type. Total volume of solution was 4 L. Number of organisms per vessel was taken as  seven. Number of vessel per concentration was taken in replicate. The number of vessels per control was taken in the replicate. The biomass loading rate was of 7 fishes/ 4 L of test solution. The analytical determinations were performed by UV- VIS spectrophotometer. Analytical assessments were performed for selected test concentrations at 0 Hr and 96 Hr. Environmental parameters such as pH (7.7), temperature (21 to 25 °C), dissolved oxygen (86.83 %), hardness (100 mg of CaCO3), photoperiod (16 h light- 8 h dark) with light intensity of 821.25 lux was maintained in acceptable range throughout the test. Thus, fulfilling the validity criteria includes, no mortality was found in the control by the end of the test. The dissolved oxygen concentration remained above 60 % of the air- saturation value throughout, exposure period. And the test concentrations were measured and found to remain at 120-80 % of nominal concentration. Based on mortality effect of test chemical on test organism Danio rerio (Zebra fish) the 96 hr LC50 value was determined to be > 100 mg/l. As per value of LC50 the  test chemical was  considered to be ‘not classified' as per the CLP classification criteria Thus, considered as non-toxic to fishes. 

 

Description of key information

Based on mortality effect of test chemical on test organism Danio rerio (Zebra fish) the 96 hr LC50 value was determined to be > 100 mg/l.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

> 100 mg/L

Additional information

Various experimental studies of test chemical and its read across chemical were reviewed for short term toxicity to fishes endpoint which are summarised as below:

 

In an experimental study from study report (2022) for the target chemical, the study was conducted in accordance with OECD Guideline 203, Fish, Acute Toxicity Test (2019). A study was performed to assess the Acute toxicity of test chemical, to Danio rerio (Zebra fish) under static conditions. Source of test organisms was Anu Pet Shop Nagpur. Length at study initiation 1.9 cm (avg).  Weight at study initiation was 0.15 g (avg). Test organism was acclimatized for a period of 7 days. Feeding frequency during acclimation once in a day.  Food was provided during acclimatization. No mortality observed was observed during acclimatization. Continuous aeration was also provided during acclimatization.  The test solution was prepared by dissolving 400 mg of test chemical in 4000 ml of RO water to get final concentration of 100 mg/l. The same stock solution was used as test concentration. The main study (i.e. limit test) was conducted using 0 (control) and 100 mg/L test concentration. Test vessel was taken as Glass aquaria having 7 lit capacity. Type of test vessel was of open type. Total volume of solution was 4 L. Number of organisms per vessel was taken as  seven. Number of vessel per concentration was taken in replicate. The number of vessels per control was taken in the replicate. The biomass loading rate was of 7 fishes/ 4 L of test solution. The analytical determinations were performed by UV- VIS spectrophotometer. Analytical assessments were performed for selected test concentrations at 0 Hr and 96 Hr. Environmental parameters such as pH (7.7), temperature (21 to 25 °C), dissolved oxygen (86.83 %), hardness (100 mg of CaCO3), photoperiod (16 h light- 8 h dark) with light intensity of 821.25 lux was maintained in acceptable range throughout the test. Thus, fulfilling the validity criteria includes, no mortality was found in the control by the end of the test. The dissolved oxygen concentration remained above 60 % of the air- saturation value throughout, exposure period. And the test concentrations were measured and found to remain at 120-80 % of nominal concentration. Based on mortality effect of test chemical on test organism Danio rerio (Zebra fish) the 96 hr LC50 value was determined to be > 100 mg/l. As per value of LC50 the  test chemical was  considered to be ‘not classified' as per the CLP classification criteria Thus, considered as non-toxic to fishes. 

Another acute study to fish has been carried for assessing the toxic effect of test chemical. The study was conducted by following the OECD Guideline 203 (Fish, Acute Toxicity Test) in a static system for a period of 96 days. Brachydanio rerio (Zebra fish) was used as a test organism.Test chemical concentrations were verified analytically. On the basis of the effect of the test chemical on mortality of test organism Brachydanio rerio (Zebra fish), the 96 hr LC0 and LC50 value was determined to be 500 and >500 mg/l (nominal conc.), respectively. Thus on the basis of these values, test chemical was considered as non-toxic to aquatic fish and hence, considered to be ‘’not classified’’ as per the CLP classification criteria.

 

Also, another acute toxicity study was conducted for 96 hrs for assessing the effect of test chemical on fish. The test was performed following the OECD guideline No. 203 “Fish Acute Toxicity Test”. Zebra fish (Danio rerio) of average weight 0.066 g and average length of 1.7 cm was used as a test organism for the study. Test fishes were kept in a static tank in tap water passed through reverse osmosis system, under natural conditions along with proper feed and aeration. During the housing period, test fishes were fed once daily with standard brand fed. The test conditions during the housing of the test organisms were oxygen content of 7.0 mg/l, pH 7.4, water temperature 24°C and under a photoperiod of 16:8 hr light: dark conditions, respectively. Test concentrations selected for the study was were 0, 6.25, 12.5, 25, 50 and 100 mg/L, respectively. Total 8 fishes were exposed to test chemical in a 5 lit bowl aquaria containing 4 liters of potable water. The test vessels were placed in a room at a temperature of 24°C, pH 7.2, hardness of water 150.5 mg of CaCO3 and under a photoperiod of 16:8 hr light: dark conditions, respectively. Aeration in test vessels was provided 1 day before the start of the experiment. No mortalities were observed in the control and test vessel. On the basis of effect of test chemical on mortality of the test organism, the median lethal concentration (LC50 (96 h)) value was determined to be > 100 mg/l. Thus, test chemical was considered as non-toxic to aquatic fishes and hence, considered to be not classified as per the CLP classification criteria.

On the basis of the above-mentioned information, the test chemical was considered to be 'not classified' as per the CLP classification criteria. Thus, test chemical was considered as non-toxic to fishes.