Trisodium hydrogen [2-[[α-[[3-[[4-chloro-6-[ethyl[3-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-2-hydroxy-5-sulphophenyl]azo]benzyl]azo]-4-sulphobenzoato(6-)]cuprate(4-)

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

Justification for type of information:

Experimental test result performed according to the OECD test guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

The analytical determinations were performed by UV-VIS spectrophotometer. The pre-treated stock solution was then diluted with media in order to get the required test solutions ranging from concentrations 1.25, 2.5, 5, 25, 50, 75, 100, 125, 135, 150 mg/L. The absorbance of resulting solution was measured using UV-VIS spectrophotometer against corresponding blank at lambda max (λmax). Standard curve was plotted against concentration verses absorbance and the maximum solubility was determined from the below standard curve. Analytical assessments were performed for selected test concentration at 0 hour and 96 hours. The concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration.

Vehicle:

no

Details on test solutions:

The test chemical was prepared by dissolving 1500 mg of test chemical in 1500 ml of RO water to get the final concentration of 1000 mg/L which was then analytically determined. The remaining test solutions were prepared by dilution from the above stock solution. Test chemical concentrations used for the study were 0, 11.25, 20.25, 36.45, 65.61, 118.09 mg/l, respectively.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra Fish
- Length at study initiation (length definition, mean, range and SD): average 0.21 cm
- 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 hour light and 8 hour darkness
- Diet : Standard brand feed, once daily
- Acclimatization: seven days
- Feeding frequency: Once in a day

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

21-25°C

pH:

Control:
at 0 hour: 7.8
at 96 hour: 7.6

Dissolved oxygen:

Control:
at 0 hour: 7.9 mg/l
at 96 hour: 5.9 mg/l

Nominal and measured concentrations:

Test chemical concentrations used for the study were 0, 11.25, 20.25, 36.45, 65.61, 118.09 mg/l, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel: Plastic aquaria
- Material, size, headspace, fill volume: 7 liters of plastic aquaria filled with 4000 ml 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: 16 hours light and 8-hour darkness

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 118.09 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Sublethal observations / clinical signs:

Table: Assessment of test concentrations

Sr. no.	Concentrations (mg/l)	Wavelength (nm)	Absorbance	Temperature (°C)
1	blank	523	0.00	25
2	1.25	523	0.01	25
3	2.50	523	0.03	25
4	5.00	523	0.05	25
5	25	523	0.03	25
6	50	523	0.69	25
7	75	523	1.01	25
8	100	523	1.37	25
9	125	523	1.68	25
10	135	523	1.78	25
11	150	523	1.99	25

 

The absorbance and concentrations were recorded at 523 nm.

Table: Concentration after analytical Determination

Sr. No	Concentrations (mg/L)	Absorbance (mean) (0 hour)	Analytical Concentrations (0 hour)	Absorbance (mean) (96 hour)	Analytical Concentrations (96 hour)
1	blank	0.00	0.00	0.00	0.00
2	200 mg/l	0.56	189.3	0.58	195.42
3	36.45	0.50	37.51	0.51	38.70
4	65.61	0.84	62.86	0.94	70.22
5	118.09	1.57	117.5	1.62	121.35

 

Table: 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	11.25	-	-	-	-	-	-	-	-
3	20.25	-	-	-	-	-	-	-	-
4	36.45	-	-	-	-	-	-	-	-
5	65.61	-	-	-	-	-	-	-	-
6	118.09	-	-	-	-	-	-	-	-

 

Sr. No	Test concentrations(mg/L)	3 hr	6 hr	24 hr	48 hr	72 hr	96 hr
1	Control	NS8SC8	NS8SC8	NS8SC8	NS8SC8	NS8SC8	NS8SC8
2	11.25	NS8SC8	NS8SC8	NS8SC8	NS8SC8	NS8SC8	NS8SC8
3	20.25	NS8SC8	NS8SC8	NS8SC8	NS8SC8	NS8SC8	NS8SC8
4	36.45	NS8SC8	NS8SC8	NS8SC8	NS8SC8	NS8SC8	NS8SC8
5	65.61	NS8SC8	NS8SC8	NS8SC8	NS8SC8	NS8SC8	NS8SC8
6	118.09	NS8SC8	NS8SC8	NS8SC8	NS8SC8	NS8SC8	NS8SC8

 

 

Table: pH, Temperature and dissolve oxygen

Test Concentration (mg/L)	pH
	24 Hours	48 Hours	72 Hours	96 Hours
Control	7.7	7.2	7.6	7.6
11.25	7.3	7.4	7.2	7.4
20.25	7.3	7.2	7.1	7.2
36.45	7.1	7.0	7.1	7.1
65.61	6.9	7.2	7.2	7.1
118.09	7.2	7.1	7.0	7.0

 

Test Concentration (mg/L)	Temperature °C
	24 Hours	48 Hours	72 Hours	96 Hours
Control	21.3	20.2	21.3	21.1
11.25	21.6	20.9	21.3	21.6
20.25	21.6	20.4	21.6	21.6
36.45	21.6	20.4	21.6	21.6
65.61	21.6	20.1	21.6	21.6
118.09	21.6	20.1	21.5	21.6

 

Test Concentration (mg/L)	Dissolved Oxygen
	24 Hours	48 Hours	72 Hours	96 Hours
Control	6.9	5.2	5.1	5.9
11.25	4.5	5.6	4.5	5.0
20.25	6.0	5.4	4.3	4.0
36.45	4.5	4.1	4.4	4.0
65.61	4.1	4.3	3.7	3.8
118.09	5.0	5.9	5.1	3.4

Validity criteria fulfilled:

yes

Conclusions:

Based on nominal concentrations, the median lethal concentration [LC50 (96 h)] for test chemical on Danio rerio (Zebra Fish) was determined to be > 118.09 mg/L.

Executive summary:

An acute toxicity test was conducted for 96 hrs for assessing the effect of test chemical on Zebra fish (Danio rerio). The test was performed in accordance to OECD guideline No. 203 “Fish Acute Toxicity Test”. Zebra fish (Danio rerio) of average length of 0.21 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 6.9 mg/l, pH 7.4, water temperature 20.7°C and under a photoperiod of 16:8 hr light: dark conditions, respectively. The test chemical was prepared by dissolving 1500 mg of test chemical in 1500 ml of RO water to get the final concentration of 1000 mg/L which was then analytically determined by UV-VIS spectrophotometer. The remaining test solutions were prepared by dilution from the above stock solution. Test chemical concentrations used for the study were 0, 11.25, 20.25, 36.45, 65.61, 118.09 mg/l, respectively. Total 8 fishes were exposed to test chemical in a 7 lit bowl aquaria containing 4000 ml of potable water. The test vessels were placed in a room at a temperature of 21 -25°C, pH of control at 0 and 96 hour was 7.8 & 76. and DO of control at 0 and 96 hour was 7.9 & 5.9 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. The dissolved oxygen concentration remained above 60% of the air saturation value throughout the exposure period. Thus, fulfilling the validity criterion. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of effect of test chemical on mortality of the test organism, the median lethal concentration [LC50 (96 h)] for test chemical on Danio rerio (Zebra Fish) was determined to be > 118.09 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.

Description of key information

An acute toxicity test was conducted for 96 hrs for assessing the effect of test chemical on Zebra fish (Danio rerio) (Experimental study report, 2019). The test was performed in accordance to OECD guideline No. 203 “Fish Acute Toxicity Test”. Zebra fish (Danio rerio) of average length of 0.21 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 6.9 mg/l, pH 7.4, water temperature 20.7°C and under a photoperiod of 16:8 hr light: dark conditions, respectively. The test chemical was prepared by dissolving 1500 mg of test chemical in 1500 ml of RO water to get the final concentration of 1000 mg/L which was then analytically determined by UV-VIS spectrophotometer. The remaining test solutions were prepared by dilution from the above stock solution. Test chemical concentrations used for the study were 0, 11.25, 20.25, 36.45, 65.61, 118.09 mg/l, respectively. Total 8 fishes were exposed to test chemical in a 7 lit bowl aquaria containing 4000 ml of potable water. The test vessels were placed in a room at a temperature of 21 -25°C, pH of control at 0 and 96 hour was 7.8 & 76. and DO of control at 0 and 96 hour was 7.9 & 5.9 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. The dissolved oxygen concentration remained above 60% of the air saturation value throughout the exposure period. Thus, fulfilling the validity criterion. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of effect of test chemical on mortality of the test organism, the median lethal concentration [LC50 (96 h)] for test chemical on Danio rerio (Zebra Fish) was determined to be > 118.09 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.

Additional information

Experimental study of the test chemical and various supporting weight of evidence studies for its structurally and functionally similar read across chemical were reviewed for short term toxicity to fish end point which are summarized as below:

 

In an experimental study from study report (2019),an acute toxicity test was conducted for 96 hrs for assessing the effect of test chemical on Zebra fish (Danio rerio). The test was performed in accordance to OECD guideline No. 203 “Fish Acute Toxicity Test”. Zebra fish (Danio rerio) of average length of 0.21 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 6.9 mg/l, pH 7.4, water temperature 20.7°C and under a photoperiod of 16:8 hr light: dark conditions, respectively. The test chemical was prepared by dissolving 1500 mg of test chemical in 1500 ml of RO water to get the final concentration of 1000 mg/L which was then analytically determined by UV-VIS spectrophotometer. The remaining test solutions were prepared by dilution from the above stock solution. Test chemical concentrations used for the study were 0, 11.25, 20.25, 36.45, 65.61, 118.09 mg/l, respectively. Total 8 fishes were exposed to test chemical in a 7 lit bowl aquaria containing 4000 ml of potable water. The test vessels were placed in a room at a temperature of 21 -25°C, pH of control at 0 and 96 hour was 7.8 & 76. and DO of control at 0 and 96 hour was 7.9 & 5.9 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. The dissolved oxygen concentration remained above 60% of the air saturation value throughout the exposure period. Thus, fulfilling the validity criterion. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of effect of test chemical on mortality of the test organism, the median lethal concentration [LC50 (96 h)] for test chemical on Danio rerio (Zebra Fish) was determined to be > 118.09 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.

 

Another acute toxicity study was conducted for 96 hrs for assessing the effect of test chemical on fish (Experimental study report, 2018). 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.

 

For the test chemical from peer reviewed journal and secondary sources, short term fish toxicity was conducted for 96 hrs for assessing the effect of test chemical. The test was performed in accordance with procedures described in the Fish-Pesticide Acute Toxicity Test Method prepared by the Animal Biology Section of the Pesticides Regulation Division of the USDA. Salmo gairdneri (rainbow trout) obtained from a commercial fish hatchery in New Jersey of average weight 0.9 g and average length of 42 mm was used as a test organism for the study. During the acclimation period (10 days), mortality was < 1% and the fish were judged to be in excellent physical condition. Fish were conditioned to test water for at least 24 hours prior to testing. Total 4 test chemical concentrations were taken. Exact test chemical conc. was not known. Test solutions were prepared by adding appropriate amounts of test chemical (mixed in 500 ml of test water) to vessels containing 14.5 liters of test water. The test chemical appeared to be in solution at all concentrations tested. Study was performed using fishes in a static system at 13 ± 0.5°C temperature and 7.1 pH. Total 40 fishes (10 fish/conc.) were exposed to test chemical in a gallon glass vessel for 96 hr. At the end of the 96 hour test period, 1-liter water samples were taken from each vessel and analyzed for concentration of test chemical. Test water consisted of 15 liters of deionized water (at least 1 million ohms resistivity) that was reconstituted by adding 3 mg KCl, 30 mg CaSO4, 30 mg MgSO4, alkalinity 35 mg/l and 48 mg NaHCO3 per liter. Fish were identified according to concentration tested and length of survival and analyzed for bioaccumulation. On the basis of effect on mortality of the test organism Salmo gairdneri (rainbow trout), the 96 hr LC50 was determined to be 750 mg/l. Thus, based on this value, test chemical was considered as non-toxic to fish and hence, considered to be ‘not classified’ as per the CLP classification criteria. 

 

On the basis of the above results, it can be concluded that the test chemical was considered as non-toxic to fishes and hence, considered to be ‘not classified’ as per the CLP classification criteria.