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-)

Endpoint summary

Description of key information

Short term toxicity to fish

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.

Short term toxicity to aquatic invertebrates

On the basis of the experimental studies of the read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism Daphnia magna, the 48hr EC50 value can be expected to be > 100 mg/l. Thus, test chemical was considered as non-toxic and hence, considered to be 'not classified' as per CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata (Experimental study report, 2020). The test was performed in accordance to the principles of the OECD guideline no. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test solution was prepared by dissolving 200 mg of test chemical in 200 mL of OECD medium to get the final concentration of 1000 mg/L, which was then analytically determined by UV-VIS spectrophotometer. Green algae were exposed to nominal concentration of test chemical (0, 6.25, 12.5, 25, 50 and 100 mg/l) (factor 2) in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 21 to 24 ± 2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16, the mean coefficient of variation of for section-by-section specific growth rate in the control cultures was not exceeded 35% (i.e., reported as 5.84%) and the mean coefficient of variation of average specific growth rates in the replicate control culture was not exceeded 7% in test with Pseudokirchneriella subcapitata (i.e., reported as 0.34%), respectively. Thus, all validity criteria of the test guideline were fulfilled. 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 growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be 77.75 mg/l (calculated from equation through probit analysis). On the basis of this value, chemical was considered as toxic to aquatic algae and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

Toxicity to microorganisms

On the basis of the experimental studies of the read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism activated sludge, the 3 hr EC50 value can be expected to be > 100 mg/l.

Additional information

Short term toxicity to fish

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.

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 on Daphnia magna. The test was performed in accordance to OECD guideline No. 202“Daphnia sp.,Acute Immobilization Test”. The test solution was prepared by dissolving 50 mg of the test chemical in 100 ml of ADaM’s media achieving a final test concentrations of 500 mg/l. Test chemical concentrations used for the study were 0, 7.5, 15, 30, 60, 120 mg/l, respectively. Test concentrations were verified analytically by UV-Vis Spectrophotometer. Study was performed using 10 daphnids in a static system. Total 10 Daphnids/conc. were exposed to test chemical in 25 ml beakers in a volume of 20 ml of liquid solution containing both the chemical and media. The beakers were placed in a room at a temperature of 18 -22°C, hardness of water 140 mg of CaCO3 and under a photoperiod of 16:8 hr light: dark conditions with light intensity 1000 – 1500 Lux, respectively. One control vessel was also run simultaneously during the study. The animals were exposed to medium (i.e.a beaker containing only medium) and the test chemical concentrations for a period of 48 hour. Potassium dichromate was used as a reference substance for the study. The 48 hr EC50 value of reference substance was determined to be 0.831 mg/l. No Immobility were found in the control test animals and the dissolved oxygen concentration at the end of the test in the control and test vessel was ≥ 3 mg/l, thus validity criterion of the study has been fulfilled. 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 mobility of the test organism, the median effect concentration (EC50 (48 h)) value was determined to be > 120 mg/L. Thus, based on the EC50 value, chemical was considered as non-toxic to aquatic invertebrates and hence, considered to be 'not classified' as per 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 in accordance to OECD guideline No. 202 “Daphnia sp.,Acute Immobilization Test”. Daphnia magna was used as a test organism for the study. The stock solution 150 mg/l was prepared by dissolving test chemical in reconstituted water. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with reconstituted test water. Nominal test chemical concentrations used for the study were 0, 30, 45, 67.5, 100 and 150 mg/l, respectively. Study was performed using 5 organisms per vessel/replicates in a static fresh water system. Daphnids were exposed to test chemical in 50 ml glass vessel in a volume of 25 ml of liquid solution containing both the chemical and media. Control solution vessel containing reconstituted water without the test chemical was also setup during the study. The beakers were placed in a room at a temperature of 20±1°C. With the test substance one positive control Potassium dichromate (K2Cr2O7) was run simultaneously. EC50 was calculated using non linear regression by the software Prism 4. In the control vessel containing reconstituted water without the test chemical, no daphnids were immobilized at the end of the test. On the basis of the mobility of the test organism Daphnia magna due to the exposure of test chemical, the 48hr median effect concentration (EC50) value was determined to be 103.8 mg/l (95 % CI. = 77.2 to 139.6 mg/l). Thus, test chemical was considered as non-toxic to aquatic invertebrates at environmental related concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.

 

On the basis of the experimental studies of the read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism Daphnia magna, the 48hr EC50 value can be expected to be > 100 mg/l. Thus, test chemical was considered as non-toxic and hence, considered to be 'not classified' as per CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria

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

 

In an experimental study from study report (2020), a freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to the principles of the OECD guideline no. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test solution was prepared by dissolving 200 mg of test chemical in 200 mL of OECD medium to get the final concentration of 1000 mg/L, which was then analytically determined by UV-VIS spectrophotometer. Green algae were exposed to nominal concentration of test chemical (0, 6.25, 12.5, 25, 50 and 100 mg/l) (factor 2) in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 21 to 24 ± 2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16, the mean coefficient of variation of for section-by-section specific growth rate in the control cultures was not exceeded 35% (i.e., reported as 5.84%) and the mean coefficient of variation of average specific growth rates in the replicate control culture was not exceeded 7% in test with Pseudokirchneriella subcapitata (i.e., reported as 0.34%), respectively. Thus, all validity criteria of the test guideline were fulfilled. 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 growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be 77.75 mg/l (calculated from equation through probit analysis).

 

Another algal growth inhibition test was conducted for 96 hrs for assessing the growth inhibition potential of test chemical on green algae (from handbook, 2008 and secondary sources). This test was performed by following the OECD 201 guideline (Alga, Growth Inhibition Test) under static condition for 96 hrs. Desmodesmus subspicatus (former scientific name: Scenedesmus subspicatus) was used as the test organism. The algae was cultured in a nutrient solution prepared according to OECD Guideline 201. Initial cell density of the culture used was 10,000 cells/ml of nutrient solution. The cells were taken from a pre-culture, which was set up 3 days prior to the test. On the basis of preliminary study, series of sequential dilutions of test chemical are prepared in a test medium. 400 mg test material was suspended up to 100 ml with test medium. Nominal test chemical concentrations used for the study were 0, 3.125, 6.25, 12.5, 25, 50, 100 and 200 mg/l, respectively. Due to precipitation, test chemical concentrations were not verified analytically. Green algae were exposed to various nominal concentration of test chemical in 50 ml Erlenmeyer flask containing 30 ml of algal suspension were set up per each concentration of test chemical. These flasks were stoppered with cotton wool plugs. Test vessel were placed in shaking incubator for 96 hrs at a room at a temperature of 21°C, pH 7.5 with a continuous uniform illumination of 800 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. Control (containing only the OECD medium) was also included in the test. Samples (2 to 5 ml) of algae were taken after 24, 48, 72 and 96 hours of incubation and the cell count was noted with the help of a microscope. The pH of one solution per concentration (including the control) was also measured at 96 hours. As per the OECD guideline No. 201 – Alga growth inhibition test, the drift in the pH did not increase by > 1.5 units after 96 hrs as compared to 0 hrs in the control vessel. Algal growth inhibition was determined from the growth curves (biomass integral). The EC50 values with confidence limits were estimated by logit analysis whereas the NOEC and LOEC values were determined statistically using the Dunnett’s test. On the basis of effect on biomass of the test organism Desmodesmus subspicatus, the 96 hrs NOEC, LOEC, EbC50 value was determined to be 25 mg/l, 50 mg/l and 41.1 mg/l, respectively.

 

On the basis of the above results, it can be concluded that the test chemical was considered as toxic to aquatic algae and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

Toxicity to microorganisms

Data available of the read across chemicals has been reviewed to determine the effect of the test chemical on toxicity to microorganisms. The studies are as mentioned below:

 

Toxicity to micro-organism study was carried out for 3 hours. The study was performed following the OECD Guideline 209 "Activated Sludge, Respiration Inhibition Test" at 21.1°C temperature. Test organism activated sludge (domestic) was obtained from laboratory sewage treatment apparatus (Husman). Test chemical concentration used for the study were 1, 3.2, 10, 32 and 100 mg/l. The test concentrations were not verified analytically. 3,5 -dichlorophenol was used as a reference substance. Oxygen-consumption was measured with an electrode system. The IC50 value of the reference substance 3,5 -dichlorophenol was determined to be within 10 -25 mgO2/l. No significant inhibition of the respiration rate was observed at any concentration. Thus, on the basis of effect on respiration rate of the test organism activated sludge, the EC0 and EC50 value was determined to be 100 and > 100 mg/l.

 

For the test chemical, toxicity to micro-organism study was carried out for 3 hours. The study was performed following the OECD Guideline 209 "Activated Sludge, Respiration Inhibition Test". Activated sludge (domestic) was used as a test organism. On the basis of effect on respiration rate of the test organism, the EC50 value was determined to be > 100 mg/l.

 

On the basis of the experimental studies of the read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism activated sludge, the 3 hr EC50 value can be expected to be > 100 mg/l.

On the basis of the available information of aquatic toxicity studies, it can be concluded that the test chemical was considered as toxicto aquatic organisms at environmental relevant concentrations and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.