2-[[(4-methoxyphenyl)methylhydrazono]methyl]-1,3,3-trimethyl-3H-indolium acetate

Endpoint summary

Description of key information

Short term toxicity to fish:

Study was conducted to access the effect of test chemical 2-[[(4-methoxyphenyl)methylhydrazono]methyl]-1,3,3-trimethyl-3H-indolium acetate on the growth of fish Danio rerio. Test conducted according to OECD Guideline 203 (Fish, Acute Toxicity Test).

The test substance was soluble in water. Therefore, the stock solution was prepared by dissolving 1 g of the test substance in 1 liters of potable water (passed through reverse osmosis system) with continuous 1 hour stirring for achieving test concentrations of 6.25 mg/L,12.5 mg/L,25 mg/L,50 mg/L,100 mg/L,respectively.

Bowl aquaria containing 2 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes. A static procedure was used for the study and it was conducted in compliance with the OECD guideline 203. After 96 hours of exposure to test item to various nominal test concentrations, LC50 was determine to be 25 mg/l . Based on the LC50, it can be consider that the chemical was toxic and can be consider to be classified as aquatic chronic 3 as per the CLP classification criteria.

Short term toxicity to aquatic invertebrate:

Daphnia sp., Acute Immobilization Test according to OECD Guideline 203 was conducted for disodium butanedioate. The test substance was soluble in water. Therefore, the stock solution was prepared by dissolving 100 mg of the test substance in 100 ml of ADaM’s media. Achieving test concentrations of 0.125 mg/L,0.25 mg/L,0.5 mg/L,1 mg/L, and 2 mg/L, respectively

The nominal concentration selected for the experiment were 0.125 mg/L,0.25 mg/L,0.5 mg/L,1 mg/L, and 2 mg/L

and test Daphnia magna were exposed to these concentration for 48 hours. The median lethal concentration (EC50) for 2-[[(4-methoxyphenyl)methylhydrazono]methyl]-1,3,3-trimethyl-3H-indolium acetate on Daphnia magna in a 48 hours study on the basis of immobilization effect was found to be 2 mg/l.

Thus, on the basis of this EC50 value and according to CLP criteria for aquatic classification of the substance, it is concluded that the substance, 2-[[(4-methoxyphenyl)methylhydrazono]methyl]-1,3,3-trimethyl-3H-indolium acetate does not exhibit short term toxicity to Daphnia.

EC50 (48 hours) Experimental is 2 mg/l.After 48 hours of exposure to test item 2-[[(4-methoxyphenyl)methylhydrazono]methyl]-1,3,3-trimethyl-3H-indolium acetate to various nominal test concentrations, EC50 was determine to be 2 mg/l . Based on the EC50, it can be consider that the chemical was toxic and can be consider to be classified as aquatic chronic 2 as per the CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria:

The study was designed to assess the toxic effects of the test compound 2-[[(4-methoxyphenyl)methylhydrazono]methyl]-1,3,3-trimethyl-3H-indolium acetate on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).

Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left.

The test solution was prepared in aseptic condition. The test item 2-[[(4- methoxyphenyl)methylhydrazono]methyl]-1,3,3-trimethyl-3H-indolium acetate was prepared by adding 0.278 mg of test item in 250 ml of BBM to get the final concentration of 1.112 mg/L. This

stock solution was kept for stirring for 01 minutes to obtain a homogenous solution for the experiment. The test concentrations were chosen according to the available data of the test item. The concentrations chosen were set up to the water solubility limit. 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 1 X 104cells/ml. Care was taken to have a homogeneous solution for the experiment.

For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined.

Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations the cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate.

As per OECD 201, the biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72 hr test period. This corresponds to a specific growth rate of 0.92 per day. Thus, the observed specific growth rate in the control cultures during the experiment was 0.358 per day. Secondly the mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 & 2-3, for 72 hr tests) in the control cultures must not exceed 35%. Thus, the observed mean coefficient of variation in the control cultures during the experiment was 33.42%. Thirdly the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%. Thus, the observed coefficient of variation of average specific growth rates during the experiment in control cultures was 8.26%. Hence, the test is considered valid as per OECD guideline, 201

After 72 hours of exposure to test item 2-[[(4-methoxyphenyl)methylhydrazono]methyl]-1,3,3-trimethyl-3H-indolium acetate to various nominal test concentrations, EC50 was determine to be 1.079 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was toxic and can be consider to classified as aquatic chronic 2 as per the CLP classification criteria.

Toxicity to microorganism:

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the short term toxicity of fish of the test chemical 2-[[(4-methoxyphenyl)methylhydrazono]methyl]-1,3,3-trimethyl-3H-indolium acetate .The studies are as mentioned below:

A screening method based on the measurement of the respiration rate of activated sludge for assessing the possible inhibitory effect of dyestuffs on aerobic waste-water bacteria. The test principle involves measuring the respiration rate of an activated sludge and comparing it with the respiration rate of the same activated sludge under identical conditions, but in the presence of the chemical under test. The test was carried out in activated sludge respiration rate apparatus with constant 20 ± 2°C and pH about 7-8. The test concentration used was 100 mg/l. OECD recommended synthetic sewage was used as feed, while activated sludge was obtained from a sewage works treating predominantly domestic sewage or from a sewage works treating predominantly industrial waste water. The respiration rate of an activated sludge and the respiration rate of activated sludge with test chemical were noted down. In order to calculate the inhibitory effect of a particular chemical at 100 mg/l test concentration its respiration rate is expressed as a percentage of the mean of the two control respiration rates. Thus, IC50 value (concentration for 50% inhibition of respiration rate) for the test chemical on activated sludge (aerobic bacteria) is determined to be10 - 100 mg/1 after 3 hrs of exposure.

The test chemical 2-[[(4-methoxyphenyl)methylhydrazono]methyl]-1,3,3-trimethyl-3H-indolium acetate is  likely to be toxic to microorganism  in the concentration  range of 10-100 mg/l