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Short- term toxicity to aquatic invertebrate:

Data available for the structurally similar read across chemicals and predicted study has been reviewed to determine the short term toxicity of aquatic invertebrate of the test chemical tetrasodium 2-[[4-[[4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino] -5-sulphonato-1-naphthyl]azo] -7-sulphonato-1-naphthyl]azo] benzene-1,4-disulphonate (70161 -16 -9).The studies are as mentioned below:

1.Based on the prediction done by EPI suite, ECOSAR version 1.1, on the basis of similarity of structure to chemicals for which the aquatic toxicity has been previously measured by structure-activity relationships (SARs) program, the LC 50 value for short term toxicity to aquatic invertebrates was predicted.

On the basis of this program, the LC 50 value for short term toxicity to aquatic invertebrates was predicted to be 42.8 mg/l for CAS nametetrasodium 2-[[4-[[4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulphonato-1-naphthyl]azo]-7-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonatein 48 hrs.

Based on this value it can be concluded that the substance is considered to be toxic to aquatic environment and can be classified in aquatic acute 3 category as per the criteria mentioned in CLP regulation.

2.Test material was used to evaluate short term toxicity on 24 h old neonates Daphnia magna as per OECD guideline 202. Untreated ISO formulation freshwater and potassium dichromate (0.32 – 3.2 mg/L) were used as negative and positive controls respectively. The effect concentration (LC50) with Daphnia magna at 48 h was observed to be 58.22 mg/l.

3.Short term toxicity study t oDaphnia similiswas carried out for 48 hrs. Neonates of less than 24 h old isolated from Daphnia culture were used for the study. Five test organisms were placed in each screw cap tubes, filled with 10 mL solution, and then incubated at 21±0.3°C in the dark. After 48 h of exposure, the number of immobile organisms was recorded. Jspear software was used to calculate the median 50% immobilization concentration (EC50). Based on immobilization of test organism by test chemical , the EC50 value was determine to be 87 mg/l along with confidence interval (p – 0.05) of 76-99 mg/l.

Thus, based on the above summarised studies, and it’s structurally similar read across substance, it can be concluded that effect concetration value is in the range of 42 - 87 mg/L. Thus, comparing this value with the criteria of CLP regulation, tetrasodium 2-[[4-[[4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulphonato-1-naphthyl]azo]-7-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate can be classified as aquatic acute 3 for short term toxicity for aquatic invertebrate .

Toxicity to aquatic algae and cyanobacteria:

Data available for the structurally similar read across chemicals and predicted study has been reviewed to determine the toxicity of aquatic algae and cyanobacteria of the test chemical tetrasodium 2-[[4-[[4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino] -5-sulphonato-1-naphthyl]azo] -7-sulphonato-1-naphthyl]azo] benzene-1,4-disulphonate (70161 -16 -9).The studies are as mentioned below:

1.The short-term toxicity of the test substancetetrasodium 2-[[4-[[4-[(4-amino-6-chloro-1,3,5-triazin-2-yl) amino]-5-sulphonato-1-naphthyl]azo]-7- sulphonato -1-naphthyl]azo]benzene-1,4-disulphonate (CAS no.70161 -16 -9) to green algae is predicted using EPI Suite ECOSAR version 1.10. On the basis of effects observed in a static freshwater system during a 96 hr exposure, the effect concentration (EC50) for the substance is estimated to be 49.0 mg/L. Based on this value, it can be concluded that the test chemical  can be considered as toxicto green algae at environmentally relevant concentrations and can be considered to be classified in aquatic acute category 3 as per the CLP classification criteria.

 

2.The effect of test item was studied on the growth of fresh water green algaChlorella vulgaris.The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 0.3 mg/L, 0.96 mg/L, 3.072 mg/L, 9.830 mg/L, 31.457 mg/L and 100.663 mg/L. The test concentrations were prepared using stock solution of the test item using mineral media. The green alga was exposed to the test concentration for a period of 72 hours to observe average specific growth rate and % growth inhibition under the effect of the test item. EC50 calculated graphically through probit analysis was observed to be 24.342 mg/L.

3.The 72 h algal growth inhibition test with Raphidocelis subcapitata was done according to OECD 201. Algae were routinely cultured at 25±1 °C with light in Cyanobacteria BG-11 Freshwater Solution. Algae were exposed to the treatment in sterile 24-well plates with 2 mL of test material.

 The effect concentration (EC50) for the test material against Raphidocelis subcapitata was observed to be 67.2 mg/l.

Thus, based on the above summarised studies, and it’s structurally similar read across substance, it can be concluded that effect concetration value is in the range of 49 - 67 mg/L. Thus, comparing this value with the criteria of CLP regulation, tetrasodium 2-[[4-[[4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulphonato-1-naphthyl]azo]-7-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate can be classified as aquatic acute 3 for toxicity for aquatic algae and cyanobacteria.

Additional information

Short- term toxicity to aquatic invertebrate:

Data available for the structurally similar read across chemicals and predicted study has been reviewed to determine the short term toxicity of aquatic invertebrate of the test chemical tetrasodium 2-[[4-[[4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino] -5-sulphonato-1-naphthyl]azo] -7-sulphonato-1-naphthyl]azo] benzene-1,4-disulphonate (70161 -16 -9).The studies are as mentioned below:

1.Based on the prediction done by EPI suite, ECOSAR version 1.1, on the basis of similarity of structure to chemicals for which the aquatic toxicity has been previously measured by structure-activity relationships (SARs) program, the LC 50 value for short term toxicity to aquatic invertebrates was predicted.

On the basis of this program, the LC 50 value for short term toxicity to aquatic invertebrates was predicted to be 42.8 mg/l for CAS nametetrasodium 2-[[4-[[4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulphonato-1-naphthyl]azo]-7-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonatein 48 hrs.

Based on this value it can be concluded that the substance is considered to be toxic to aquatic environment and can be classified in aquatic acute 3 category as per the criteria mentioned in CLP regulation.

2.Test material was used to evaluate short term toxicity on 24 h old neonates Daphnia magna as per OECD guideline 202. Untreated ISO formulation freshwater and potassium dichromate (0.32 – 3.2 mg/L) were used as negative and positive controls respectively. The effect concentration (LC50) with Daphnia magna at 48 h was observed to be 58.22 mg/l.

3.Short term toxicity study t oDaphnia similiswas carried out for 48 hrs. Neonates of less than 24 h old isolated from Daphnia culture were used for the study. Five test organisms were placed in each screw cap tubes, filled with 10 mL solution, and then incubated at 21±0.3°C in the dark. After 48 h of exposure, the number of immobile organisms was recorded. Jspear software was used to calculate the median 50% immobilization concentration (EC50). Based on immobilization of test organism by test chemical , the EC50 value was determine to be 87 mg/l along with confidence interval (p – 0.05) of 76-99 mg/l.

Thus, based on the above summarised studies, and it’s structurally similar read across substance, it can be concluded that effect concetration value is in the range of 42 - 87 mg/L. Thus, comparing this value with the criteria of CLP regulation, tetrasodium 2-[[4-[[4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulphonato-1-naphthyl]azo]-7-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate can be classified as aquatic acute 3 for short term toxicity for aquatic invertebrate .

Toxicity to aquatic algae and cyanobacteria:

Data available for the structurally similar read across chemicals and predicted study has been reviewed to determine the toxicity of aquatic algae and cyanobacteria of the test chemical tetrasodium 2-[[4-[[4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino] -5-sulphonato-1-naphthyl]azo] -7-sulphonato-1-naphthyl]azo] benzene-1,4-disulphonate (70161 -16 -9).The studies are as mentioned below:

1.The short-term toxicity of the test substancetetrasodium 2-[[4-[[4-[(4-amino-6-chloro-1,3,5-triazin-2-yl) amino]-5-sulphonato-1-naphthyl]azo]-7- sulphonato -1-naphthyl]azo]benzene-1,4-disulphonate (CAS no.70161 -16 -9) to green algae is predicted using EPI Suite ECOSAR version 1.10. On the basis of effects observed in a static freshwater system during a 96 hr exposure, the effect concentration (EC50) for the substance is estimated to be 49.0 mg/L. Based on this value, it can be concluded that the test chemical  can be considered as toxicto green algae at environmentally relevant concentrations and can be considered to be classified in aquatic acute category 3 as per the CLP classification criteria.

 

2.The effect of test item was studied on the growth of fresh water green algaChlorella vulgaris.The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 0.3 mg/L, 0.96 mg/L, 3.072 mg/L, 9.830 mg/L, 31.457 mg/L and 100.663 mg/L. The test concentrations were prepared using stock solution of the test item using mineral media. The green alga was exposed to the test concentration for a period of 72 hours to observe average specific growth rate and % growth inhibition under the effect of the test item. EC50 calculated graphically through probit analysis was observed to be 24.342 mg/L.

3.The 72 h algal growth inhibition test with Raphidocelis subcapitata was done according to OECD 201. Algae were routinely cultured at 25±1 °C with light in Cyanobacteria BG-11 Freshwater Solution. Algae were exposed to the treatment in sterile 24-well plates with 2 mL of test material.

 The effect concentration (EC50) for the test material against Raphidocelis subcapitata was observed to be 67.2 mg/l.

Thus, based on the above summarised studies, and it’s structurally similar read across substance, it can be concluded that effect concetration value is in the range of 49 - 67 mg/L. Thus, comparing this value with the criteria of CLP regulation, tetrasodium 2-[[4-[[4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulphonato-1-naphthyl]azo]-7-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate can be classified as aquatic acute 3 for toxicity for aquatic algae and cyanobacteria.