3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethylanilinium chloride

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:

key study

Study period:

22-03-2018 - 27-03-2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experimental test result performed according to the guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Principles of method if other than guideline:

Study was conducted to access the effect of test chemical 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethylanilinium chloride(CAS No.68391-31- 1) on the mortality of fish Danio rerio. Test conducted according to OECD Guideline 203 (Fish, Acute Toxicity Test).

GLP compliance:

no

Analytical monitoring:

no

Remarks:

According to test (If substance is stable and if not analytical done by spectrophotometer)

Vehicle:

no

Details on test solutions:

The test substance was highly soluble in water; In house solubility was found to be 4.745 g/L. Therefore the stock solution prepared as 400mg/4L, with the concentration of 100mg/L, and was kept for 48 hr. stirring. After this filter the stock, give it for analytical detection and then stock taken for experiment.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name:Zebra Fish
- Strain:Danio rerio
- Source: Unique Aqua Fish Corner
- Length at study initiation (length definition, mean, range and SD): average 1.9 cm
- Weight at study initiation (mean and range, SD): average 0.699 g
-Housing:The fishes were kept in a static tank in tap water passed through reverse osmosis system, under natural conditions along with proper feed and aeration.
-Duration of housing: 15 days
-Photoperiod: 16 hours light and 8 hours dark
-Diet : Standard brand feed, once daily
-Acclimatization: seven days
-feeding frequency: Once in a day

Test type:

static

Water media type:

freshwater

Total exposure duration:

96 h

Hardness:

158.4 mg of CaCO3

Test temperature:

22.6 °C

pH:

6.78-

Dissolved oxygen:

8.0 mg/l

Nominal and measured concentrations:

100mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Plastic aquaria
- Material, size, headspace, fill volume: 5 liters of plastic aquaria filled with 2 liter of water
- Aeration: Aeration in test vessels was provided 1 day before the start of experiment
- No. of organisms per vessel: 8 fishes

OTHER TEST CONDITIONS
- Photoperiod: -----------------

Reference substance (positive control):

not specified

Key result

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: No mortality was observed

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: No mortality was observed

Details on results:

No mortality was observed

Sublethal observations / clinical signs:

(In each test, result varies always check)

8.1. Mortality and symptoms:

Nominal Concentration (mg/l)	Number of fish		Mortality (cumulated) after
			3h		6h		24h		48h		72h		96h
			V1	V2	V1	V2	V1	V2	V1	V2	V1	V2	V1	V2
Control	4	4	-	-	-	-	-	-	-	-	-	-	-	-
100	4	4	-	-	-	-	-	-	-	-	-	-	-	-

Visible Symptoms:

Nominal Concentration (mg/l)

Number of fish

symptom (cumulated) after

3h

6h

24h

48h

72h

96h

V1

V2

V1

V2

V1

V2

V1

V2

V1

V2

V1

V2

Control

4

4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

100

4

4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

Explanation of symptoms:

A: apathy

N: Narcotic-like state

C: Swimming near water surface

M: Slow movements compared to control

NS: No abnormal symptoms

S: Surfacing behaviour

L: Loss of equilibrium

SM: Shimming movement

H1: Hyperactivity

H2: Hypo activity

B: Bending behavior

F: Fin rot

IS: Invert Swimming

SB: Swimming behaviour

RF: Respiratory Function

P: Pigmentation

pH and oxygen content:

Nominal Concentration (mg/l)	Number of fish		pH readings after
			24 h		48 h		72 h		96 h
			V1	V2	V1	V2	V1	V2	V1	V2
Control	4	4	7.20	7.22	8.33	8.30	8.37	8.38	8.35	8.39
100	4	4	7.30	7.35	7.57	7.58	7.58	7.58	7.54	7.54

 

 

Nominal Concentration (mg/l)	Number of fish		Oxygen content (mg/L) after
			24 h		48 h		72 h		96 h
			V1	V2	V1	V2	V1	V2	V1	V2
Control	4	4	8.6	8.6	8.1	8.1	8.1	8.1	8.2	8.2
100	4	4	8.5	8.5	8.1	8.1	8.0	8.0	8.1	8.1

8.3. Test temperature

Nominal Concentration (mg/l)	Number of fish		Temperature (°C) after
			24 h		48 h		72 h		96 h
			V1	V2	V1	V2	V1	V2	V1	V2
Control	4	4	22.1	22.1	23.9	23.9	23.8	23.8	21.3	21.3
100	4	4	21.9	21.9	23.9	23.9	23.9	23.9	21.6	21.6

 

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

Based on nominal concentrations, experimental median lethal Concentrations [LC-50 (96 h)] for 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethylanilinium chloride(CAS No.68391-31- 1) on Zebra Fish Danio rerio was determine to be >100 mg/L..

Executive summary:

Study was conducted to access the effect of test chemical 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1Hpyrazol-4-yl)azo]-N,N,N-trimethylanilinium chloride(CAS No.68391-31-1) on the growth of fish Danio rerio. Test conducted according to OECD Guideline 203 (Fish, Acute Toxicity Test).

The test substance was highly soluble in water; In house solubility was found to be 4.745 g/L. Therefore the stock solution prepared as 400mg/4L, with the concentration of 100mg/L, and was kept for 48 hr. stirring. After this filter the stock, give it for analytical detection and then stock taken for experiment and Zebra Fish Danio rerio were exposed to these concentration for 96 hours.

Bowl aquaria containing 2 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes. A static procedure was used for the study and it was conducted in compliance with the OECD guideline 203. After 96 hours of exposure of test chemical at limit concentration of 100mg/L, no mortality was observed. Therefore the LC50was determined to be 100mg/L. Based on the LC50, the test chemical 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1Hpyrazol-4-yl)azo]-N,N,N-trimethylanilinium chloride(CAS No.68391-31-1) can be considered as non-toxic and can be concluded as not classified as per the CLP classification criteria.

Description of key information

After 72 hours of exposure to test item 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4- yl)azo]-N,N,N-trimethylanilinium chloride (CAS No. 68391-31-1) to various nominal test concentrations, EC50 was determine to be 109.09 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4- yl)azo]-N,N,N-trimethylanilinium chloride (CAS No. 68391-31-1)can be considered as non-toxic to algae and thus can be consider as not classified as per the CLP classification criteria.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

109.09 mg/L

Additional information

The study was designed to assess the toxic effects of the test compound 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4- yl)azo]-N,N,N-trimethylanilinium chloride (CAS No. 68391-31-1) on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).

 

The test solution was prepared in aseptic condition. The test item 3-[(4,5-dihydro-3-methyl-5-oxo- 1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethylanilinium chloride was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200 mg/L. This stock solution was kept for stirring for 24 hours to obtain a homogenous solution for the experiment. The test concentrations were chosen according to the available data of the test item. The concentrations chosen were set up to the water solubility limit. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 104cells/ml. Care was taken to have a homogeneous solution for the experiment.

 

 

For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined.

 

 

Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations the cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate.

 

As per OECD 201, the he biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72 hr test period. This corresponds to a specific growth rate of 0.92 per day. Thus, the observed specific growth rate in the control cultures during the experiment was 0.602 per day. The mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 & 2- 3, for 72 hr tests) in the control cultures must not exceed 35%. Thus, the observed mean coefficient of variation in the control cultures during the experiment was 10.506 %. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%. Thus, the observed coefficient of variation of average specific growth rates during the experiment in control cultures was 2.363 %. Hence, the test is considered valid as per OECD guideline, 201.

 

After 72 hours of exposure to test item 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4- yl)azo]-N,N,N-trimethylanilinium chloride (CAS No. 68391-31-1) to various nominal test concentrations, EC50 was determine to be 109.09 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4- yl)azo]-N,N,N-trimethylanilinium chloride (CAS No. 68391-31-1)can be considered as non-toxic to algae and thus can be consider as not classified as per the CLP classification criteria.