5-((4-chloro-6-((8-hydroxy-3,6-disulfo-7-((2-sulfo-4-((2(sulfooxy)ethyl)sulfonyl)phenyl)diazenyl)naphthalen-1-yl)amino)-1,3,5-triazin-2-yl)amino)-4-hydroxy-3-((Z)-(3-sulfophenyl)diazenyl)naphthalene-2,7-disulfonic acid

Endpoint summary

Description of key information

Short term toxicity to aquatic invertebrates

On the basis of the experimental studies of the structurally and functionally similar read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, the 48 hr EC50 value can be expected to be >100 mg/l.

Toxicity to aquatic algae and cyanobacteria

On the basis of the experimental studies of the structurally and functionally similar read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, the 48 hr ErC50 value can be expected to be in the range 135.323 to 152.76 mg/l, respectively

Additional information

Short term toxicity to aquatic invertebrates

Data available of the structurally and functionally similar read across chemicals has been reviewed to determine the effect of the test chemical on aquatic invertebrates. The studies are as mentioned below:

 

An acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical. The test was performed following the principles of the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test).The study was performed under static conditions using Daphnia magna (Water flea) as a test organism. Test organism was taken from Daphtoxkit F™ magna, a commercial kit that provides daphnids in the form of dormant eggs (ephippia) that need to be incubate 3 days before the use to obtain neonates used for the test. Neonates less than 24 h old (25 for each condition) were fed with lyophilized spirulina as dietary supplement for 2 h prior to the test and then moved to the exposure chamber. Test chemical solutions were prepared by directly dissolving the test chemical in specific test medium (ISO 6341 Standard Freshwater). Test chemical concentration used for the study was 100 mg/l. Thus, a limit test was performed using 100 mg/l test chemical concentration. Selected bottled water was used only in the daphnid assays and satisfied the acceptability criteria for holding and dilution water described by the OECD 202 guideline. ISO 6341 Standard Freshwater was used as a test medium for the study. Media were aerated for at least 1 h prior to use in order to ensure that the dissolved oxygen concentration has reached saturation. Test daphnids (25 test organisms) were exposed with the test chemical at 20±1°C for a period of 48 hrs. No illumination of light was provided to the test vessel during the study. No significant changes in pH were recorded in the test solutions. Untreated ISO formulation freshwater was used as negative control. Potassium dichromate (0.32 – 3.2 mg/L) was used as a reference substance during the study. 48 h LC50 were derived by non-linear regression analysis. All statistical analyses were done using Prism5 (GraphPad Software, Inc.). One-way ANOVA was used for statistical comparisons with Bonferroni's, Dunnett's and Tukey's post hoc tests for multiple comparisons. Significance was set at p<0.05. REGTOX macro Excel™ for dose-response was used to obtain EC50 values by a non-linear regression analysis with Hill's model. Dissolved oxygen concentration was measured with HI9142 Portable Dissolved Oxygen Meter (Hanna Instruments srl) in all test chambers at the end of the test and was higher than 3 mg/l, according to the OECD 202 guidelines. On the basis of the toxic effect of the test chemical on mobility of the test organism Daphnia magna, the 48 hr median lethal concentration (LC50) value was determined to be >100 mg/l (nominal conc.). Thus, test chemical was consideredas non-toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be ‘not classified’ as per the CLP classification criteria.

 

Another short term toxicity to aquatic invertebrate study was conducted for 48 hrs for assessing the effect of test chemical. The test was performed following the principles of the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test).The study was performed under static conditions using Daphnia magna (Water flea) as a test organism. Test was conducted using the same test procedure and test conditions as mentioned in above study. On the basis of the toxic effect of the test chemical on mobility of the test organism Daphnia magna, the 48 hr median lethal concentration (LC50) value was determined to be >100 mg/l (nominal conc.). Thus, test chemical was considered as non-toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be ‘not classified’ as per the CLP classification criteria.

 

On the basis of the experimental studies of the structurally and functionally similar read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, the 48 hr EC50 value can be expected to be >100 mg/l.

Toxicity to aquatic algae and cyanobacteria

Data available of the structurally and functionally similar read across chemicals has been reviewed to determine the effect of the test chemical on aquatic algae. The studies are as mentioned below:

 

Toxicity to aquatic algae study was carried out for 72 hrs for assessing the effect of test chemical on Raphidocelis subcapitata (green algae). Algae were routinely cultured at 25±1 °C with light in Cyanobacteria BG-11 Freshwater Solution. The culture was split twice a week, adding fresh medium. Test chemical solution was prepared directly by dissolving the formulation powder in specific test medium for algae (ISO 8692 medium). Limit test was performed using a nominal test chemical conc. of 100 mg/l. Test organism were exposed to the test chemical (2 ml) in sterile 24-well plates for 72 hrs under static conditions. Algal stock solution containing 1010000 cells/mL was added (20 μL) to each well to obtain a starting algal density of 10000 cells/mL. Untreated ISO formulation freshwater and Potassium dichromate (0.10 to 1.8 mg/l) were used as a negative and positive control. All experiments was performed in triplicates. Plates were incubated at 25±1 °C for 72 hr on an orbital shaker (90 rpm) in a thermostatic chamber. Light was provided by a 2 W LED Unit for each plate. Algal cell density was measured at the beginning and end of treatment with a TC20™ Automated Cell Counter (Bio-Rad Laboratories, Inc), selecting a gate of 4–14 μm or by visual scoring with a Burker's chamber. Results were expressed as effect on biomass (EbC50) and on growth rate (ErC50). All statistical analyses were done using Prism5 (GraphPad Software, Inc.). One-way ANOVA was used for statistical comparisons with Bonferroni's, Dunnett's and Tukey's post hoc tests for multiple comparisons. Significance was set at p<0.05. REGTOX macro Excel™ for dose-response was used to obtain EC50 values by a non-linear regression analysis with Hill's model. On basis of effect of the test chemical on growth rate of the test organism Raphidocelis subcapitata, the 72 hr ErC50 value was determined to be 135.323 mg/l (95% C. I. – 116.974 to 179.365 mg/l) & on the basis of biomass, the 72 hr EbC50 value was determined to be 45.365 mg/l, respectively. Thus, based on the ErC50 value (135.323mg/l) alongwith growth rate effect, test chemical was considered as non-toxic to aq. algae and hence, considered to be ‘not classified’ as per CLP classification criteria.

 

Another toxicity to aquatic algae study was carried out for 72 hrs for assessing the effect of test chemical on Raphidocelis subcapitata (green algae). Algae were routinely cultured at 25±1 °C with light in Cyanobacteria BG-11 Freshwater Solution. The culture was split twice a week, adding fresh medium. Test chemical solution was prepared directly by dissolving the formulation powder in specific test medium for algae (ISO 8692 medium). Limit test was performed using a nominal test chemical conc. of 100 mg/l. Test was conducted using the same test procedure and test conditions as mentioned in above study. On basis of effect of the test chemical on growth rate of the test organism Raphidocelis subcapitata, the 72 hr ErC50 value was determined to be 152.76 mg/l (95% C. I. – 134.406 to 179.274 mg/l) & on the basis of biomass, the 72 hr EbC50 value was determined to be 25.904 mg/l (95% C. I. – 22.233 to 32.632 mg/l), respectively. Thus, based on the ErC50 value (152.76 mg/l) alongwith growth rate effect, test chemical was considered as non-toxic to aq. algae and hence, considered to be ‘not classified’ as per CLP classification criteria.

 

On the basis of the experimental studies of the structurally and functionally similar read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, the 48 hr ErC50 value can be expected to be in the range 135.323 to 152.76 mg/l, respectively. Thus, based on the ErC50 value alongwith growth rate effect, test chemical was considered as non-toxic to aq. algae and hence, considered to be ‘not classified’ as per CLP classification criteria.

On the basis of the available information of aquatic toxicity studies, it can be concluded that the test chemical was considered as non-toxic to aquatic organisms at environmental relevant concentrations andhence, considered to be ‘not classified’ as per the CLP classification criteria.