Tetrabutylammonium bromide

Endpoint summary

Description of key information

Short term toxicity to fish:

Aim of this study was to evaluate the effect of test chemical on the mortality rate of Zebra 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 one 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. Potable water (passed through reverse osmosis system) was used. Fishes were exposed to these concentrations for 96 hours. The Stability of Test substance was determined as per the Spectrophotometric method and found to be stable for 4 days at 25°C. Plastic aquaria having the water capacity of 5 liters filled with 2 liters of water and were loaded with 8 fishes. A static procedure was used for the study and it was conducted in compliance with the OECD guideline 203 thus consider to be valid. Also fulfills all the necessary criteria. The mortality in the control was found to be 0 per cent at the end of the test. Constant conditions were maintained as far as possible throughout the test and, static procedures used. The dissolved oxygen concentration was found to be 79.76 per cent of the air saturation value throughout the test. After 96 hours of exposure to test item, LC50 was determine to be > 100 mg/l. As no effect (LC0) were observed at the limit concentration of 100 mg/l. Based on the LC50, it can be consider that the chemical was nontoxic and can be consider to be not classified as per the CLP classification criteria.

Long term toxicity to fish:

Chronic toxicity study to aquatic fishes was carried for 30 dayspost hatchfor assessing the effect of test chemicalon survival rate and hatchingsuccess. Study was performed following the principles of the OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test) under semi-static conditions. The test chemical was dissolved in the dilution water and the selected test chemical concentrations were prepared by dilution of a stock solution. Solubility of the test item was performed by weighed 25.5 mg of the test item in a 250 ml volumetric flask dissolved and made up to the mark.  The stability test was conducted toanalysethe chemical stability for the duration of 96 hours(at regular intervals24 hours).At 0.01 mg/L and 100 mg/Lat 0 hour, 24 hour, 48 hour, 72 hour and 96 hour showed that the test item concentration remained 80% to 120% (98.37% to 99.09% for 0.01 mg/L and 98.29% to 99.03% for 100 mg/L) with respect to initial measured concentrationthus semi static conditions were opted for main study. Danio rerio (Zebra fish) of both sex of ratio (2 male: 1female)were used for spawning.Spawning traps were placed in the breeding tank for the collection of eggs.  Whichwere collected from breeding groups,mixed,and randomly selectedfor the exposure. To prevent predation of eggs by adult zebra fish, the spawn traps were covered with inert wire mesh of size (2.5±0.5 mm). The spawn traps with the collected eggs were carefully removed. Thefertilizedeggs were collected by usingpasteurpipette on next day morning (on the day of exposure) and eggs were rinsed with natural water after collection from spawning traps. Fertilized eggs were placed into respective test vessels by using transparent plastic pipette for hatching. Post hatch feeding wasgivento test fishes.Which was as follows,Commercial dry food was provided for 14 days. From day 15 onwards live food (artemia) was provided along with dry food. Dry feed was given twice a day and Brine shrimp Artemia was given to test fishes once daily. Surplus food andfeceswere removed where required, to avoid accumulation of waste.dilutionwater was used as atest medium.Range finding test was conducted with test concentrations at 0 (Control), 5, 10, 25, 50 and 100 mg/. Each concentration contained two replicate and eachreplicatewere exposed 20 fertilized eggs. Test item was formulated inDilutionwater. Hatching (percent) observed in control group was 100% whereas, 97.5%, 92.5%, 90.0%, 87.5% and 85.0% in the tested concentration of 5, 10, 25, 50 and 100 mg/L for a period of 96 hours. Allfertilisedeggs hatched in the control group whereas non-detachment of the tail observed in the tested concentration of 5, 10, 25, 50 and  100 mg/ L for a period of 96 hours. Based on the mortality of embryos and hatching success concentrations for the main study was determined.The concentrations ranging from0 (Control), 0.0164, 0.041, 0.102, 0.256, 0.64, 1.6, 4 and 10 mg/lwith geometric factor 2.5 was selected formianstudy.Test fishes (total 20embryos/vessel) were exposed to different test chemical conc. (i.e., 0 (Control), 0.0164, 0.041, 0.102, 0.256, 0.64, 1.6, 4 and 10 mg/l) in a 2 lit glass beaker. No aeration was provided during the study. Renewal rate of test solution was 96 hrs. Biomass loading rate contains 80 eggs/conc. All control and test experiments were performed in 4 replicates. Test conditions involve a photoperiod of 12: 12 light: dark conditions, Light intensity ranges from 694 to 768 lux, temperature at the beginning of the test: 26.5°C to 27.0°C and at the end of the test: 26.2°C to 26.5°C, pH at the beginning of the test: 7.5 to 8.0 and at the end of the test: 7.5 to 8.2 and dissolved oxygen of 69.8 to 94.3% air saturation value, respectively. Data wereanalyzedusing appropriate statistical methods to calculate the EC50, LOEC and NOEC. The 95% confidence Limits were calculated. The pH of the control at the test start and end was 7.5 and therefore did not vary more than 1.5 units during the study. No mortality/100% survival was observed at embryo stage in the control; 7.5% mortality was observed in control after 30 days whereas mortality at the end of exposure to test chemical at concentrations of 0.0164, 0.041, 0.102, 0.256, 0.64,1.6, 4 and 10 mg/l was 4.05%, 2.70%, 2.70%, 4.05%, 5.41%, 6.76%, 10.81% and 10.81%, respectively. At 96hr, all the embryos were hatched in control. Hatching success in the control and in the tested concentration of 0.0164, 0.041, 0.102, 0.256, 0.64 and 1.6 mg/ L was 100%, whereas 96.3% and 90.0 %, in the tested concentration of 4 and 10 mg/l. Larval survival until day 30 post-hatch in the control group was 92.5% thereby exceeding and satisfying the validity criteria for post-hatch survival (>75%).whereas in the group treated with test chemical at concentrations of 0.0164, 0.041, 0.102, 0.256, 0.64, 1.6, 4 and 10 mg/l was 88.8, 90, 90, 88.8, 87.5, 86.3, 81.8 and 80.6%, respectively. All fishes were healthy in the control. For fish total lengths, the NOEC and LOEC determined on Day 30 post-hatch were 0.102 and 0.256 mg/L, respectively. At concentration of 1.6 mg/L in two hatching out of which one has tail deformities and other was hypoactive, at concentration 4 mg/L one hatchling was hypoactive and at concentration of 10 mg/L one hatchling had oedema and other has loss of buoyancy control. Length and weightweremeasured after 30 days post hatch, in control reported to be 11.20 mm and 0.032 g. Samples wereanalytically determined at pre and post renewal. The received test sample concentrations (0.0164 mg/L, 0.64 mg/L and 10 mg/L on 0-hour fresh samples, 96-Hour spent and fresh samples and 48-hour spent samples) were mixed well with uniformity. An aliquot of required volume of sample solutions was transfer to a clean round bottom flask (RB) and dry on a 60°C heating condition. After dry eluate of sample solutions made up to the final volume using acetonitrile and analyzed under high performance liquid chromatography (HPLC).In fresh samples of the test substance concentrations, the determined concentrations of test chemicalwere93.19 % to 95.79 % for 0.0164 mg/L, 91.37 % to 94.48 % for 0.640 mg/L and 93.60 % to 95.34 % for 10 mg/L, respectively. The results confirm that the test substance concentrations were within the acceptability range. In spent samples of the test substance concentrations, the determined concentrations of test chemical was 93.94 % to 96.03 % for 0.0164 mg/L, 92.67 % to 94.55 % for 0.640 mg/L and 94.05 % to 95.67 % for 10 mg/L, respectively. All validity criteria were satisfied during the test, therefore the test was considered to be valid. Based on the results and effect on mortality of test fishes, the 30 d NOEC and LC50 value was determined to be ≥ 10 mg/l and >10 mg/l, respectively. On the basis of the effect on hatching success and survival larvae of test fishes,the 30 d NOEC, LOEC and EC50 value was determined to be 1.6, 4 and >10 mg/l, respectively. Thus, test chemical was considered as non-toxic to aquatic fishes at environmental relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.Thus based on the above results chemical cannot be classified as per CLP classification criteria. 

Short term toxicity to aquatic invertebrate:

This study was designed (as per OECD 202, adopted in 2004) to assess the acute toxicity of test chemical following exposure of daphnids up to 48h by static method.The brood daphnids were acclimatized > 2weeks prior to the test item exposure. Less than 24 h old daphnids were collected from the acclimatized gravid females and exposed to the test item. After exposure on day 0, daphnids were observed for immobilization at 24 and 48 h. M7 medium was used as control, and the same was used for test item formulation and test medium . 25 mL glass beakers having a solution volume of 20 mL were used in the test. A main study (using a spacing factor of 2) was conducted using 0 (control), 0 (vehicle control), 25, 50, 100, 200, and 400 mg/L concentrations. 4 replicates/concentration having 5 daphnids/replicate was used for the main study. Normal behavioural response and no immobilization (0% mortality) were observed up to 48 h followed by control groups, and 5%, 15%, 65%, 80, and 90% mortality were observed in the test concentrations of 25, 50, 100, 200, and 400 mg/L, respectively. The analytial monitoring was done, and the test chemicals were maintained within the acceptable range i.e., 80 -120% of the nominal concentrations. Hence the results were based on nominal concentration, since the deviation in the initial measured concentration didn’t exceed 20%. Environmental parameters such as pH (7.2-7.8), temperature (20-21 °C), dissolve oxygen (6.8-7.2 mg/L), hardness (160 mg CaCO₃/L), photoperiod (16 h light- 8 h dark) and light intensity (1330-1352 Lux) was maintained in acceptable range throughout the test. Feed was not provided during the test. The 48-h EC₅₀of test chemical to daphnid,Daphnia magnaare 98.1mg/L. The 48-h EC₅₀of reference item (Potassium dichromate) to daphnid,Daphnia magna(found to be in acceptable range) is 0.690 mg/L. Hence, the results of the test with reference item establish the acceptability of the test system response, test procedures followed and results obtained with test item. Thus, based on the EC50 value, test chemical can considered as toxic and hazardous to aquatic invertebrates. Thus, chemical can be classified into aquatic chronic category 3 as per CLP classification criteria

Long term toxicity to aquatic invertebrate:

A chronic study was conducted for assesing the effect of test chemical. The study was performed in accordance with the principles of the OECD Guideline 211 (Daphnia magna Reproduction Test). Parent daphina were acclimitised in the similar conditions as maintined on the test. From this, gravid parents were isolated and miantined in the M7 medium offsprings were collected, and these nenonates were used in the study. The test organims was obatined from MicroBio tests Kleimoer 15B-9030 MARIAKERKE(GENT)BELGIUM. The Neonates whose age was less than <24 hours were selected. Test daphnids were fed with living cells of C. vulgaris and it was done thrice a week. The test chemical will be prepared by dissolving 1000 mg of test chemical in 1000 mL of M7 media with 48 hours stirring to get the final concentration of 1000 mg/L. This stock solution was filtered by using whatman filter paper no. 42, which was then analytically determined. The final solubility value obtained in media was used to prepare the remaining test concentrations from the above stock solution. The analytical determinations were performed by UV-VIS spectrophotometer. The pre-treated stock solution was then diluted with media in order to get the required test solutions. the leniarity range selected for concentrations analysis and stock analysis was 5.0, 10, 20, 30, 40, and 50 mg/L. The absorbance of resulting solution was measured using UV-VIS spectrophotometer against corresponding blank at lambda max (λmax). Standard curve was plotted against concentration verses absorbance and the maximum solubility was determined from the below standard curve. Analytical assessments were performed for selected test concentration at 1st, 2nd and 3rd week. 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. Test chemical conc. used in the defiinite study was 10 mg/l. Thus, limit test was performed at 10 mg/l conc. Test daphnids were exposed to test chemical conc. in a glass beaker for an exposure period of 21 days. Vessels were not aerated during the study. M7 medium was used as a test medium. Test conditions involve a temperature range of 20.1 -21.5°C, 16:8 light:dark conditions and light intensity not exceeding 1000-1500 lux, respectively. Each test concentrations has10 replicates and each replicate has 1 dapnids same number were taken for control goups. The evaluation of the NOEC was determined based on the number of offspring’s produced per living parent Daphnia. Defined concentrations of the test chemical led to a certain percentage reduction of the parthenogenetic reproduction rate at the end of the 21 day study period. The living offspring was counted daily along with the renewal of the test medium. However, test vessels were inspected daily for the occurrence of juveniles and marked accordingly. On the basis of the effect on reproduction of the test daphnids, the 21 d NOEC value was determined to be 10 mg/l. Thus based on the outcomes chemical could not be classified as per CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria:

Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). The test substance was prepared by adding 50 mg of test substance in 100 ml of BBM to get the final concentration of 500 mg/L. The remaining test solutions were prepared by dilution from the above stock solution. The test solution was prepared in aseptic condition. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 10000 cells/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. After 72 hours of exposure to test item to various nominal test concentrations, EC50 was determine to be 204.70 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was nontoxic and can be consider to be not classified as per the CLP classification criteria.

Toxicity to aquatic plants other than algae:

Tests were conducted in 15-ml Petri dishes over 96 h. In petri dish approximately two fronds of L. minor (a single colony) with roots cut to less than 1 mm was added. Then the root length was measured and the number of new fronds were counted at the end of the experiment. For each Petri dish, the combined root length was divided by the number of fronds to calculate the mean root length per frond, which were used as metric of root growth. Root growth was normalized to frond number since roots must grow from fronds and because preliminary tests showed that this parameter was sensitive to concentrations of chemicals below the concentration that would cause effects on frond number. The cultures were maintained and tests was conducted in continuous light from four 122- cm, wide-spectrum General Electric plant and aquarium bulbs suspended 20 cm above the fronds. Lemna minor strain number 492 from the University of Toronto Culture Collection. Prior to tests, cultures were maintained for one month in growth medium. No sexual reproduction was observed in the cultures. The concentration (and 95% confidence interval) at which a 50% reduction in growth (the EC50) occurred was calculated for both frond growth (measured as number of new fronds) and root length per frond using the sigmoidal dose–response (variable slope) equation in the ligand-binding response macro for Sigmaplot 7.0. After the exposure of test chemical for 96 hrs, EC50 was observed to be at 51.4 mg/l. Hence, it is said to have low toxicity for aquatic plants in aquatic chronic category 3.

Toxicity to microorganisms:

Toxicity was determine on the bacteria vibrio fischeri. Toxicity value of test chemical to luminescent bacterium Vibrio fischeri NRRL B-11177 observed was EC50 (15 minutes) was observed to be 1.862 mg/L. The toxic effect depicted by test chemical was much less toxic than the pyridinium and imidazolium compounds tested.

Additional information

Summarized result for the toxicity of test chemical on the growth and mortality of aquatic life’s including fish, invertebrates, algae and microorganism were studied and are as follows:

 

Short term toxicity to fish:

Based on the various experimental data for the target chemical study have been reviewed to determine the toxic nature of test chemical on the mortality of fish. The studies are as mentioned below:  

Aim of this study was to evaluate the effect of test chemical test chemical on the mortality rate of Zebra 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 one 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. Potable water (passed through reverse osmosis system) was used. Fishes were exposed to these concentrations for 96 hours. The Stability of Test substance was determined as per the Spectrophotometric method and found to be stable for 4 days at 25°C. Plastic aquaria having the water capacity of 5 liters filled with 2 liters of water and were loaded with 8 fishes. A static procedure was used for the study and it was conducted in compliance with the OECD guideline 203 thus consider to be valid. Also fulfills all the necessary criteria. The mortality in the control was found to be 0 per cent at the end of the test. Constant conditions were maintained as far as possible throughout the test and, static procedures used. The dissolved oxygen concentration was found to be 79.76 per cent of the air saturation value throughout the test. After 96 hours of exposure to test item, LC50 was determine to be > 100 mg/l. As no effect (LC0) were observed at the limit concentration of 100 mg/l. Based on the LC50, it can be consider that the chemical was nontoxic and can be consider to be not classified as per the CLP classification criteria.

 

First study was supported by the second experimental study for target chemical. Study was conducted to access the effect of test chemical on the mortality rate of Guppy Fish (Poecilia reticulata). Test conducted according to OECD Guideline 203 (Fish, Acute Toxicity Test). The test solution was prepared by dissolving 1 g of the test substance in 10 liters deionized water with continuous stirring for achieving the test concentration of100 mg/L, respectively. Deionized water was used. A limit test performed at 100 mg/l and Guppy Fish (Poecilia reticulata) were exposed to these concentration for 96 hours. Glass bowl aquaria 12” each were filled with 10 liters of water and 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 nominal concentration 100 mg/l, LC50 was determine to be > 100 mg/l. As no effect (LC0) were observed at the limit concentration of 100 mg/l. Based on the LC50, it can be consider that the chemical was nontoxic and can be consider to be not classified as per the CLP classification criteria.

Based on the above studies, it was conclude that the chemical was nontoxic and can be consider to be not classified as per the CLP classification criteria.

 

Long term toxicity to fish:

Chronic toxicity study to aquatic fishes was carried for 30 dayspost hatchfor assessing the effect of test chemicalon survival rate and hatchingsuccess. Study was performed following the principles of the OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test) under semi-static conditions. The test chemical was dissolved in the dilution water and the selected test chemical concentrations were prepared by dilution of a stock solution. Solubility of the test item was performed by weighed 25.5 mg of the test item in a 250 ml volumetric flask dissolved and made up to the mark.  The stability test was conducted toanalysethe chemical stability for the duration of 96 hours(at regular intervals24 hours).At 0.01 mg/L and 100 mg/Lat 0 hour, 24 hour, 48 hour, 72 hour and 96 hour showed that the test item concentration remained 80% to 120% (98.37% to 99.09% for 0.01 mg/L and 98.29% to 99.03% for 100 mg/L) with respect to initial measured concentrationthus semi static conditions were opted for main study. Danio rerio (Zebra fish) of both sex of ratio (2 male: 1female)were used for spawning.Spawning traps were placed in the breeding tank for the collection of eggs.  Whichwere collected from breeding groups,mixed,and randomly selectedfor the exposure. To prevent predation of eggs by adult zebra fish, the spawn traps were covered with inert wire mesh of size (2.5±0.5 mm). The spawn traps with the collected eggs were carefully removed. Thefertilizedeggs were collected by usingpasteurpipette on next day morning (on the day of exposure) and eggs were rinsed with natural water after collection from spawning traps. Fertilized eggs were placed into respective test vessels by using transparent plastic pipette for hatching. Post hatch feeding wasgivento test fishes.Which was as follows,Commercial dry food was provided for 14 days. From day 15 onwards live food (artemia) was provided along with dry food. Dry feed was given twice a day and Brine shrimp Artemia was given to test fishes once daily. Surplus food andfeceswere removed where required, to avoid accumulation of waste.dilutionwater was used as atest medium.Range finding test was conducted with test concentrations at 0 (Control), 5, 10, 25, 50 and 100 mg/. Each concentration contained two replicate and eachreplicatewere exposed 20 fertilized eggs. Test item was formulated inDilutionwater. Hatching (percent) observed in control group was 100% whereas, 97.5%, 92.5%, 90.0%, 87.5% and 85.0% in the tested concentration of 5, 10, 25, 50 and 100 mg/L for a period of 96 hours. Allfertilisedeggs hatched in the control group whereas non-detachment of the tail observed in the tested concentration of 5, 10, 25, 50 and  100 mg/ L for a period of 96 hours. Based on the mortality of embryos and hatching success concentrations for the main study was determined.The concentrations ranging from0 (Control), 0.0164, 0.041, 0.102, 0.256, 0.64, 1.6, 4 and 10 mg/lwith geometric factor 2.5 was selected formianstudy.Test fishes (total 20embryos/vessel) were exposed to different test chemical conc. (i.e., 0 (Control), 0.0164, 0.041, 0.102, 0.256, 0.64, 1.6, 4 and 10 mg/l) in a 2 lit glass beaker. No aeration was provided during the study. Renewal rate of test solution was 96 hrs. Biomass loading rate contains 80 eggs/conc. All control and test experiments were performed in 4 replicates. Test conditions involve a photoperiod of 12: 12 light: dark conditions, Light intensity ranges from 694 to 768 lux, temperature at the beginning of the test: 26.5°C to 27.0°C and at the end of the test: 26.2°C to 26.5°C, pH at the beginning of the test: 7.5 to 8.0 and at the end of the test: 7.5 to 8.2 and dissolved oxygen of 69.8 to 94.3% air saturation value, respectively. Data wereanalyzedusing appropriate statistical methods to calculate the EC50, LOEC and NOEC. The 95% confidence Limits were calculated. The pH of the control at the test start and end was 7.5 and therefore did not vary more than 1.5 units during the study. No mortality/100% survival was observed at embryo stage in the control; 7.5% mortality was observed in control after 30 days whereas mortality at the end of exposure to test chemical at concentrations of 0.0164, 0.041, 0.102, 0.256, 0.64,1.6, 4 and 10 mg/l was 4.05%, 2.70%, 2.70%, 4.05%, 5.41%, 6.76%, 10.81% and 10.81%, respectively. At 96hr, all the embryos were hatched in control. Hatching success in the control and in the tested concentration of 0.0164, 0.041, 0.102, 0.256, 0.64 and 1.6 mg/ L was 100%, whereas 96.3% and 90.0 %, in the tested concentration of 4 and 10 mg/l. Larval survival until day 30 post-hatch in the control group was 92.5% thereby exceeding and satisfying the validity criteria for post-hatch survival (>75%).whereas in the group treated with test chemical at concentrations of 0.0164, 0.041, 0.102, 0.256, 0.64, 1.6, 4 and 10 mg/l was 88.8, 90, 90, 88.8, 87.5, 86.3, 81.8 and 80.6%, respectively. All fishes were healthy in the control. For fish total lengths, the NOEC and LOEC determined on Day 30 post-hatch were 0.102 and 0.256 mg/L, respectively. At concentration of 1.6 mg/L in two hatching out of which one has tail deformities and other was hypoactive, at concentration 4 mg/L one hatchling was hypoactive and at concentration of 10 mg/L one hatchling had oedema and other has loss of buoyancy control. Length and weightweremeasured after 30 days post hatch, in control reported to be 11.20 mm and 0.032 g. Samples wereanalytically determined at pre and post renewal. The received test sample concentrations (0.0164 mg/L, 0.64 mg/L and 10 mg/L on 0-hour fresh samples, 96-Hour spent and fresh samples and 48-hour spent samples) were mixed well with uniformity. An aliquot of required volume of sample solutions was transfer to a clean round bottom flask (RB) and dry on a 60°C heating condition. After dry eluate of sample solutions made up to the final volume using acetonitrile and analyzed under high performance liquid chromatography (HPLC).In fresh samples of the test substance concentrations, the determined concentrations of test chemicalwere93.19 % to 95.79 % for 0.0164 mg/L, 91.37 % to 94.48 % for 0.640 mg/L and 93.60 % to 95.34 % for 10 mg/L, respectively. The results confirm that the test substance concentrations were within the acceptability range. In spent samples of the test substance concentrations, the determined concentrations of test chemical was 93.94 % to 96.03 % for 0.0164 mg/L, 92.67 % to 94.55 % for 0.640 mg/L and 94.05 % to 95.67 % for 10 mg/L, respectively. All validity criteria were satisfied during the test, therefore the test was considered to be valid. Based on the results and effect on mortality of test fishes, the 30 d NOEC and LC50 value was determined to be ≥ 10 mg/l and >10 mg/l, respectively. On the basis of the effect on hatching success and survival larvae of test fishes,the 30 d NOEC, LOEC and EC50 value was determined to be 1.6, 4 and >10 mg/l, respectively. Thus, test chemical was considered as non-toxic to aquatic fishes at environmental relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.Thus based on the above results chemical cannot be classified as per CLP classification criteria. 

 

Short term toxicity to aquatic invertebrate:

Based on the various experimental data for the target chemical study have been reviewed to determine the nature of test chemical on the mobility and growth of aquatic invertebrate. The studies are as mentioned below:  

 

Acute Immobilization study of test chemical on Daphnia magna in a Semi-Static System for 48 hrs. Test performed according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). Chemical was analytically monitored. The Stability of Test substance was determined as per the Spectrophotometric method and found to be stable for 2 days at 22°C. The test substance was soluble in Adams media. Therefore, the test solution was prepared by dissolving 100 mg of the test substance in 100 ml of ADaM’s media. Achieving test concentrations of 3.125 mg/L,6.25 mg/L,12.5 mg/L,25 mg/L, and 50 mg/L, respectively and test Daphnia magna were exposed to these concentration for 48 hours. Test consider to be valid as conducted accordance to the OECD guideline. Firstly in the control, 0 percent of the daphnids found to be immobilized. Secondly the dissolved oxygen concentration at the end of the test found to be 6.6 mg/l in control and 6.4 in test vessels. Based on nominal concentrations, experimental median effective Concentrations [EC-50 (48 h)] for test chemical on test daphnia was observed to be 50 mg/l. Based on the immobilization of test animal daphnia magna after 48 hrs of exposure, chemical consider to be toxic and can be consider to be classified as aquatic chronic 3 as per the CLP classification criteria.

 

First study was supported by the second experimental from peer reviewed journal. Aim of this study was to evaluate the effect of test chemical on the mortality rate of aquatic arthropod Echinogammarus tibaldii. Organisms of the species E. tibaldii Pink & Stock (Crustacea, Amphipoda) were collected near the spring of the river Vera (L'Aquila, Italy). The animals were screened from mayor detritus, transported to the laboratory under continuous aeration and held for acclimation in cool, aerated water in 20-L aquaria. Mature adult male individuals obtained from precopula pairs and kept for about 3 d in cool "reconstituted water'". Received artificial oxygenation and were fed on dry poplar leaves previously soaked in spring water in order to enrich them with fungi and bacteria. The definitive tests were carried out in duplicate by adding 20 animals (Mature adult male) in each I-L glass jar containing 250 mL of each precooled and aerated solution. During the test, carried out in static conditions and without oxygenation, the animals were not fed. After the exposure of test chemical for 24 hrs with aquatic arthropod Echinogammarus tibaldii, the LC50 was determine to > 2000 mg/l. Based on the LC50 value, chemical consider to be nontoxic and not classified as per the CLP classification criteria.

 

Thus based on the experimental study which is according to the guideline, it was concluded that the test chemical was toxic and the can be consider to be classified as aquatic chronic 3 category as per the CLP classification criteria.

 

Long term toxicity to aquatic invertebrate:

Various long term studies available for the test chemical and structually and functionally similar read across were reviewed to determine the toxic nature of test chemical on the growth and mobility of aquatic invertebrates. The studies are as mentioned below:

 

A chronic study was conducted for assesing the effect of test chemical. The study was performed in accordance with the principles of the OECD Guideline 211 (Daphnia magna Reproduction Test). Parent daphina were acclimitised in the similar conditions as maintined on the test. From this, gravid parents were isolated and miantined in the M7 medium offsprings were collected, and these nenonates were used in the study. The test organims was obatined from MicroBio tests Kleimoer 15B-9030 MARIAKERKE(GENT)BELGIUM. The Neonates whose age was less than <24 hours were selected. Test daphnids were fed with living cells of C. vulgaris and it was done thrice a week. The test chemical will be prepared by dissolving 1000 mg of test chemical in 1000 mL of M7 media with 48 hours stirring to get the final concentration of 1000 mg/L. This stock solution was filtered by using whatman filter paper no. 42, which was then analytically determined. The final solubility value obtained in media was used to prepare the remaining test concentrations from the above stock solution. The analytical determinations were performed by UV-VIS spectrophotometer. The pre-treated stock solution was then diluted with media in order to get the required test solutions. the leniarity range selected for concentrations analysis and stock analysis was 5.0, 10, 20, 30, 40, and 50 mg/L. The absorbance of resulting solution was measured using UV-VIS spectrophotometer against corresponding blank at lambda max (λmax). Standard curve was plotted against concentration verses absorbance and the maximum solubility was determined from the below standard curve. Analytical assessments were performed for selected test concentration at 1st, 2nd and 3rd week. 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. Test chemical conc. used in the defiinite study was 10 mg/l. Thus, limit test was performed at 10 mg/l conc. Test daphnids were exposed to test chemical conc. in a glass beaker for an exposure period of 21 days. Vessels were not aerated during the study. M7 medium was used as a test medium. Test conditions involve a temperature range of 20.1 -21.5°C, 16:8 light:dark conditions and light intensity not exceeding 1000-1500 lux, respectively. Each test concentrations has10 replicates and each replicate has 1 dapnids same number were taken for control goups. The evaluation of the NOEC was determined based on the number of offspring’s produced per living parent Daphnia. Defined concentrations of the test chemical led to a certain percentage reduction of the parthenogenetic reproduction rate at the end of the 21 day study period. The living offspring was counted daily along with the renewal of the test medium. However, test vessels were inspected daily for the occurrence of juveniles and marked accordingly. On the basis of the effect on reproduction of the test daphnids, the 21 d NOEC value was determined to be 10 mg/l. Thus based on the outcomes chemical could not be classified as per CLP classification criteria.

In the first study (from authoritative database) toxicity was measured on the basis of immobilisation. Aim of this study was to determine the effect of test chemical on the reproductive inhibition and immobilisation of daphnia magna. Test conducted in accordance with the OECD Guideline 202 (Daphnia sp., Acute Immobilisation Test and Reproduction Test). Test conducted under the semi-static system for 21 days. Nominal concentration including control, vehicle control (2.94 mg/L), 0.35, 0.60, 1.02, 1.73, 2.94 mg/L (geometric ratio: 1.7) were used. Test vessel filled with 400 ml of solution and 10 daphnia per vessel were studied. After the exposure of 21 days effect were measured. Based on the effect observed on the daphnia magna after the exposure of 21 days with the test chemical, No effect were observed (NOEC) at 0.33 mg/l and EC50 was observed at 0.99 mg/l. Based on the EC50, chemical consider to be toxic and classified as aquatic chronic 2 category. But the test chemical was readily biodegradable in water and thus on that basis chemical classified in aquatic chronic 3 category.

 

First study was supported by the second study from authoritative database. Study was conducted to access the effect of test chemical on the growth of aquatic invertebrates by providing the exposure rate of 21days. Test conducted in accordance with the OECD Guideline 211 (Daphnia magna Reproduction Test). Study was conducted to access the effect of test chemical on the growth of aquatic invertebrates by providing the exposure rate of 21days. Test conducted in accordance with the OECD Guideline 211 (Daphnia magna Reproduction Test). Based on the readily biodegradability of test chemical and NOEC, chemical consider to be toxic and classified as aquatic chronic 3 as per the CLP classification criteria.

 

Thus based on the overall studies, it was concluded that the test chemical was toxic and the can be consider to be classified as aquatic chronic 3 category as per the CLP classification criteria.

 

Toxicity to aquatic algae and cyanobacteria:

Based on the various experimental data for the target chemical study have been reviewed to determine the nature of test chemical on the growth of aquatic algae and cyanobacteria. The studies are as mentioned below:  

The study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). The test substance was prepared by adding 50 mg of test substance in 100 ml of BBM to get the final concentration of 500 mg/L. The remaining test solutions were prepared by dilution from the above stock solution. The test solution was prepared in aseptic condition. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 10000 cells/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. After 72 hours of exposure to test chemical to various nominal test concentrations, EC50 was determine to be 204.70 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was nontoxic and can be consider to be not classified as per the CLP classification criteria.

 

First study was supported by the second experimental study for target chemical from peer reviewed journal. Freshwater algal growth inhibition test was carried out for 96 hr on Pseudokirchneriella subcapitata with the test chemical according to OECD Guideline 201 (Alga, Growth Inhibition Test) and EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II), respectively. The study was based on the effects of the test compound on Pseudokirchneriella subcapitata in a flow through fresh water system. The green microalga used as the model algal strain in this study, Selenastrum capricornutum ATCC-22662, was obtained from the National Institute Environmental Research, Korea. The stock alga was cultivated in 250 ml Erlenmeyer flasks, containing 200 ml sterilized nitrate-enriched BBM medium prepared in triple distilled water, to avoid nitrogen limitation during the high-density culture. Experiments were performed in 250 ml Erlenmeyer flasks, containing 55ml of sterilized culture medium, inoculated with 5ml samples of 7-day cultured algae. Solutions of ionic liquids and organic solvents were subsequently added to the test flasks. The flasks then were placed on a shaker incubator at 170 rpm and 25 deg C, with 24 h light supplied via warm-white fluorescent tubes, with an average illumination of 3075 mEm2 s1. At each determined exposure date, the optical density of the algal biomass was estimated at 438nm using a spectrophotometer (UV mini-1240, Shimadzu, Kyoto, Japan) and average specific growth rate determined. The dry cell weight, corresponding to the optical density, was determined from the linear relationship; dry cell weight (g/l) = 0.1329 optical density. Based upon the effect on the growth rate of the test organism Pseudokirchneriella subcapitata, the 48, 72 and 96hr EC50 value was determined to be 300.84, 1542.94 and 721.689 mg/L, respectively. Thus, based on this value, it can be concluded that the test chemical can be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP criteria.

 

Thus based on the above studies, it was conclude that the test chemical was nontoxic to the growth of algae and cyanobacteria and can be consider to be not classified as per the CLP classification criteria.

 

 

 

Toxicity to aquatic plants other than algae:

Based on the various experimental data for the target chemical study have been reviewed to determine the nature of test chemical on the growth of aquatic plants. The studies are as mentioned below:  

 

In the first experimental study from peer reviewed journal toxicity were measured. Tests were conducted in 15-ml Petri dishes over 96 h. In petri dish approximately two fronds of L. minor (a single colony) with roots cut to less than 1 mm was added. Then the root length was measured and the number of new fronds were counted at the end of the experiment. For each Petri dish, the combined root length was divided by the number of fronds to calculate the mean root length per frond, which were used as metric of root growth. Root growth was normalized to frond number since roots must grow from fronds and because preliminary tests showed that this parameter was sensitive to concentrations of chemicals below the concentration that would cause effects on frond number. The cultures were maintained and tests was conducted in continuous light from four 122- cm, wide-spectrum General Electric plant and aquarium bulbs suspended 20 cm above the fronds. Lemna minor strain number 492 from the University of Toronto Culture Collection. Prior to tests, cultures were maintained for one month in growth medium. No sexual reproduction was observed in the cultures. The concentration (and 95% confidence interval) at which a 50% reduction in growth (the EC50) occurred was calculated for both frond growth (measured as number of new fronds) and root length per frond using the sigmoidal dose–response (variable slope) equation in the ligand-binding response macro for Sigmaplot 7.0. After the exposure of test chemical for 96 hrs, EC50 was observed to be at 51.4 mg/l. Hence, it is said to have low toxicity for aquatic plants in aquatic chronic category 3.

 

Above study was supported by the second study from peer reviewed journal 2008. Toxicity value of tetrabutyl ammonium bromide in terms of root length per frond to Lemna minor is EC50: 32.66 mg/L. Hence, the substance is said to have low toxicity to aquatic plants in aquatic chronic category 3.

 

Thus based on the above studies, chemical consider to be toxic and classified as aquatic chronic 3 as per the CLP classification criteria.

 

Toxicity to microorganisms:

Summarized result for the toxicity of test chemical on the mortality rate of microorganisms.

In the first study from peer reviewed journal toxicity was determine on the bacteria vibrio fischeri. Toxicity value of test chemical to luminescent bacterium Vibrio fischeri NRRL B-11177 observed was EC50 (15 minutes) was observed to be 1.862 mg/L. The toxic effect depicted by test chemical was much less toxic than the pyridinium and imidazolium compounds tested.

 

Above study was supported by the second study from peer reviewed journal. The anti-fungal activity of test chemical was evaluated against two wood decay fungi. 21 day MIC (minimum inhibition concentration) was determine to be >4.2 micro mol/cm2. Tetrabutyl ammonium bromide did not inhibit fungi (Perenniporia tephropora) growth under bioassay conditions.

From the above studies, some supports the nontoxic nature of test chemical where when consider long term studies chemical consider to be toxic. Thus based on the effect observed on fish, aquatic invertebrate and algae, test chemical consider to be toxic and can be consider to be classified as aquatic chronic 3 as per the CLP classification criteria.