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Short term toxicity to fish

Short term toxicity to fish was conducted for 96 hrs for assessing the effect of test chemical. The test was performed in accordance to OECD guideline No. 203 “Fish Acute Toxicity Test” (Study report, 2015). Zebra fish (Danio rerio) of average weight 0.272 g and average length of 2.5 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 7.4 mg/l, pH 7.3, water temperature 24°C and under a photoperiod of 12:12 hr light: dark conditions, respectively. The limit test (100 mg/l) was conducted for this substance. Thus, on the basis of this limit test, definitive test was performed at different test chemical nominal concentrations, i.e., at 0, 0.1, 0.2, 0.4, 0.8 and 1.6 mg/l (nominal concentrations), 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 23°C, pH of 6.9, hardness of water 46.25 mg of CaCO3 and under a photoperiod of 12:12 hr light: dark conditions, respectively. Aeration in test vessels was provided 1 day before the start of the experiment. Test fishes were moving slowly as compared to control. On the basis of effect of test chemical on mortality of the test organism, the LC0 and median lethal concentration (LC50 (96 h)) value was determined to be 1.6 mg/l and > 1.6 mg/l, respectively.

Short term toxicity to aquatic invertebrates

The short-term toxicity of the test chemical to aquatic invertebrates was predicted using EPI Suite ECOSAR version 1.11. On the basis of effect of test chemical observed in a static system on the mobility of the test organism during the 48 hr exposure duration, the lethal effect concentration (LC50) for the test chemical was estimated to be 1.636 mg/l. Thus, based on the LC50 value, test chemical can be considered as toxic to aquatic invertebrates and hence, considered to be classified in 'aquatic chronic category 2' as per the 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 Chlorella vulgaris (Study report, 2015). The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. Bold’s Basal Medium (BBM) composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test substance was prepared by dissolving 12.5 mg of test substance in 250 ml of BBM to get the final concentration of 50 mg/L. 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 10000 cells/ml. Green algae were exposed to nominal concentration of test chemical ( 0, 1.5625, 3.125, 6.25, 12.5, 25 and 50 mg/l) in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 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. Control containing medium without test chemical was used for the study. The cultures were counted and observed daily with the help of an automated cell counter to verify a normal and healthy appearance of the algae cells and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test chemical). All the cells appeared healthy, round and green throughout the study duration in the control while changes were observed at the higher concentration which includes, decrease in cell count and substance was both adsorbed and absorbed on the cells. On the basis of growth rate of the test organism Chlorella vulgaris, the 72 hrs median effect concentration (ErC50) value calculated from equation and graphically through probit analysis was determined to be 14.05 and 12.59 mg/l. On the basis of the EC50 value, chemical was considered to be 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 structurally similar read across chemical and applying the weight of evidence approach, the EC50 value of the test chemical on test organism can be expected to be in the range of 0.53 to > 39.9 mg/l.

Additional information

Short term toxicity to fish

Short term toxicity to fish was conducted for 96 hrs for assessing the effect of test chemical. The test was performed in accordance to OECD guideline No. 203 “Fish Acute Toxicity Test” (Study report, 2015). Zebra fish (Danio rerio) of average weight 0.272 g and average length of 2.5 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 7.4 mg/l, pH 7.3, water temperature 24°C and under a photoperiod of 12:12 hr light: dark conditions, respectively. The limit test (100 mg/l) was conducted for this substance. Thus, on the basis of this limit test, definitive test was performed at different test chemical nominal concentrations, i.e., at 0, 0.1, 0.2, 0.4, 0.8 and 1.6 mg/l (nominal concentrations), 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 23°C, pH of 6.9, hardness of water 46.25 mg of CaCO3 and under a photoperiod of 12:12 hr light: dark conditions, respectively. Aeration in test vessels was provided 1 day before the start of the experiment. Test fishes were moving slowly as compared to control. On the basis of effect of test chemical on mortality of the test organism, the LC0 and median lethal concentration (LC50 (96 h)) value was determined to be 1.6 mg/l and > 1.6 mg/l, respectively.

Short term toxicity to aquatic invertebrates

The short-term toxicity of the test chemical to aquatic invertebrates was predicted using EPI Suite ECOSAR version 1.11. On the basis of effect of test chemical observed in a static system on the mobility of the test organism during the 48 hr exposure duration, the lethal effect concentration (LC50) for the test chemical was estimated to be 1.636 mg/l. Thus, based on the LC50 value, test chemical can be considered as toxic to aquatic invertebrates and hence, considered to be classified in 'aquatic chronic category 2' as per the 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 Chlorella vulgaris (Study report, 2015). The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. Bold’s Basal Medium (BBM) composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test substance was prepared by dissolving 12.5 mg of test substance in 250 ml of BBM to get the final concentration of 50 mg/L. 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 10000 cells/ml. Green algae were exposed to nominal concentration of test chemical ( 0, 1.5625, 3.125, 6.25, 12.5, 25 and 50 mg/l) in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 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. Control containing medium without test chemical was used for the study. The cultures were counted and observed daily with the help of an automated cell counter to verify a normal and healthy appearance of the algae cells and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test chemical). All the cells appeared healthy, round and green throughout the study duration in the control while changes were observed at the higher concentration which includes, decrease in cell count and substance was both adsorbed and absorbed on the cells. On the basis of growth rate of the test organism Chlorella vulgaris, the 72 hrs median effect concentration (ErC50) value calculated from equation and graphically through probit analysis was determined to be 14.05 and 12.59 mg/l. On the basis of the EC50 value, chemical was considered to be 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 for its structurally similar read across chemicals has been reviewed to determine the toxic effect of the test chemical on microorganisms. The studies are as mentioned below:

 

Toxicity to microorganisms study was carried out for 30 mins. The study was performed in accordance with the ISO 11348-2 guideline. Bacteria V. fischeri (NRRL B-1117) was supplied as freeze-dried reagent, BioFix Lumi, from Macherey-Nagel, Du¨ ren, Germany. Test chemcal was dissolved in distilled water; osmolality adjusted to 2 % NaCl. Test chemical concentration used for the study was <=39.9 mg/L. Light production from luminescent bacteria was measured with a luminometer equipped with a constant temperature water bath (15°C). On the basis of the effect of test chemical on light production of the test organism V. fischeri, the 30 mins EC50 value was determined to be > 39.9 mg/l.

 

Another toxicity to micro-organism study was carried out using Bacillus subtilis as test organism. On the basis on effect of test chemical on growth rate of the test organism, the EC50 value was determined to be 0.53 mg/l.

 

On the basis of the experimental studies of the structurally similar read across chemical and applying the weight of evidence approach, the EC50 value of the test chemical on test organism can be expected to be in the range of 0.53 to > 39.9 mg/l.

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