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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 fish (Experimental study report, 2018). The test was performed in accordance with the OECD guideline No. 203 “Fish Acute Toxicity Test”. Zebra fish (Danio rerio) of average weight 0.0792 g and average length of 1.88 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 7.1, water temperature 24°C and under a photoperiod of 16:8 hr light: dark conditions, respectively. Test chemical concentrations were not verified analytically. Nominal concentrations of test chemical used for the study were 0, 6.25,12.5, 25, 50 and 100mg/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 6.9, hardness of water 200 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 vessel. On the basis of effect of test chemical on mortality of the test organism, the median lethal concentration (LC50 (96 h)) and LC100 value was determined to be > 6.25 and 12.5 mg/l. Thus, test chemical was considered as toxic to aquatic fishes and hence, considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.

Long term toxicity to fish

Based on the prediction done using ECOSAR version 1.11, the long term toxicity on fish was predicted for test chemical (2018). On the basis of effects observed in a flow through freshwater system on test organism, the NOEC value for the substance was estimated to be 4.5 mg/l for fish for 28 days of exposure duration. Thus, it can be concluded that the test chemical can be considered as non-toxic to fish at environmentally relevant concentrations

Short term toxicity to aquatic invertebrates

An acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on aquatic invertebrates (Experimental study report, 2018). 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 100 mg/l was prepared by dissolving test chemical in reconstituted water. The test solutions of required concentrations were prepared by mixing the stock solution of the test sample in reconstituted water. Test chemical concentrations were not verified analytically. Nominal test chemical concentrations used for the study were 0, 10, 20, 40 and 80 mg/l, respectively. Study was performed using total 5 organisms per vessel/replicates in a static 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. The 24 hr EC50 value of the reference substance was determined to be 0.73 mg/l. 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 and the dissolved oxygen concentration at the end of the test was evaluated to be ≥ 3 mg/l (i.e, reported as > 7 mg/l) both in the control and test vessels. Thus, the validity criterion of the test has been fulfilled. 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 30.1 mg/l (95 % C. I. - 23.9 to 37.9 mg/l). Thus, test chemical is considered as toxic to aquatic invertebrates at environmental related concentrations and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

Long term toxicity to aquatic invertebrates

Chronic toxicity to aquatic invertebrate study was conducted for 21 days for assessing the effect of test chemical on aquatic invertebrates (Experimental study report, 2019). The study was performed following the OECD Guideline 211 (Daphnia magna Reproduction Test) under semi-static conditions. The solution of test chemical was prepared by dissolving 100 mg of test chemical in 100 ml of Adams medium to get the final concentration of 1000 mg/L. Test chemical concentrations were verified analytically by UV-Vis Spectrophotometer and the final solubility value obtained after analytical detection was 296.79 mg/l. The remaining test solutions were prepared by dilution from the stock solution. Thus, test chemical concentrations used for the study were 0 (control), 0.5, 1, 2, 4 and 8 mg/L, respectively. Daphnia magna (water flea) of length 0.37 cm was used as a test organism. A population of parthenogenetic females of synchronized age structure has been maintained for more than 2 years in the test facility under constant temperature conditions (18 to 22 °C) at a 16 : 8 hour light-dark photoperiod (illumination: < 1000 lux). The culture media (Adams medium') was partly renewed once a week. The Daphnia were exclusively fed with unicellular green algae (Selenestrum capricornutum) thrice a week. Total 50 test organisms were exposed to five test chemical concentrations (n = 10) in 100 ml glass beaker. In addition to this, control test vessel was also setup during the study. Test vessels were then placed at temperature of 18 – 22°C, hardness 200 mg/l as CaCO3, pH 7.8, dissolved oxygen 6.3 to 8.3 mg/l and under a photoperiod of 16 hour light and 8 hour dark with 1000 – 1500 lux light intensity. Reproduction rate and the mobility behaviour / mortality rate of parent Daphnia was assessed at least three times a week. The test concentrations were measured and was observed be maintained within 80-120% of nominal concentration, thus, effect concentrations (EC) would be evaluated as the nominal concentrations. The mortality in the control groups did not exceeded 20% percent. Thus, the validity criterion of the test has been fulfilled. On the basis of the effect of test chemical on the reproduction of the test organism Daphnia magna, the 21 days EC50 value was determined to be 0.5581 mg/l (nominal concentration). Thus, test chemical was 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 (Experimental study report, 2019). 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. Sterile, unicellular, suspension cultures of algae Pseudokirchneriella subcapitata of length 8 – 14 μm and width 2 - 3 μm was used as a test organism. 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 300 mg of test chemical in 300 ml of OECD medium to get the final concentration of 1000 mg/L. Test chemical concentrations were verified analytically by UV-VIS spectrophometer. Green algae were exposed to six different nominal concentrations of test chemical (0, 5 mg/L, 10 mg/L, 20 mg/L, 40 mg/L, 80 mg/L and 160 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 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. 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). Apart from this, the cell count of each test vessel was also noted with the help of a microscope. Cell densities were recorded in section by section growth rate at 24 hr intervals, which was calculated as specific growth rate. The 72 hr ErC10 (based on growth rate [r]), was calculated or read from the dose/concentration/percentage response curve. Potassium dichromate (K2Cr2O7) was used as a reference substance for the study. Since the concentration of the test chemical being tested has been satisfactorily maintained within ±20% of the nominal concentration throughout the test, all concentrations will be reported as nominal concentration. As per the OECD guideline No. 201 – Alga growth inhibition test, the biomass in the control cultures have increase exponentially by a factor of at least 17 within the 72 hr test period, the mean coefficient of variation by section specific growth rate in the control cultures not exceeded 35% and the coefficient of variation of average specific growth rate during the whole test period in replicate control cultures was not exceeded 10%, thus, fulfilling the validity of the criteria. The 72 hr EC50 value of reference substance was determined to be 0.809 mg/l. On the basis of growth rate of the test organism Pseudokirchneriella subcapitata, the 72 hrs effect concentration (EC10) value was determined to be 0.45 mg/l (nominal concentration) and median effect concentration (ErC50) was determined to be ≥ 160 (nominal concentration) & ≥ 184.46 mg/l (initial measured concentration). On the basis of the EC50 value, chemical was considered to be non-toxic to aquatic algae at environmental relevant concentrations.

Toxicity to microorganisms

Toxicity to microorganism study was performed using Tetrahymena pyriformis as a test organism (T. W. Schultz et. al., 1999 and authoritative database, 2012). The study was carried out under static conditions. Stock solutions of test chemical were prepared in dimethyl sulfoxide. Exact test chemical concentrations used for the study was not known, but each test replicate consists of six to eight different concentrations. Range finding study was performed in duplicates prior to the initiation of study. Flasks were used as a test vessel. To each test flask were added test organism and a solution of test chemical. Two control vessels, one control with no test chemical but inoculated with T. pyriformis, and the other, a blank control, which has neither test chemical nor test organism were run simultaneously during the study. All test concentrations experiments were performed in duplicates. Test conditions allow for 8-9 cell cycles in control cultures. Only replicates with control-absorbency values > 0.6 but < 0.75 were used for the analysis. Population density of test organism was measured spectrophotometrically at 540 nm. The 50% growth inhibitory concentrations, IGC50, were determined by Probit Analysis of Statistical Analysis System (SAS) software. On the basis of the effect on growth inhibition of the test organism Tetrahymena pyriformis, the 40 hr IGC50 value was determined to be 204.64 mg/l.

Additional information

Short term toxicity to fish

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

 

In an experimental study from study report (2018), an acute toxicity test was conducted for 96 hrs for assessing the effect of test chemical on fish. The test was performed in accordance with the OECD guideline No. 203 “Fish Acute Toxicity Test”. Zebra fish (Danio rerio) of average weight 0.0792 g and average length of 1.88 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 7.1, water temperature 24°C and under a photoperiod of 16:8 hr light: dark conditions, respectively. Test chemical concentrations were not verified analytically. Nominal concentrations of test chemical used for the study were 0, 6.25, 12.5, 25, 50 and 100mg/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 6.9, hardness of water 200 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 vessel. On the basis of effect of test chemical on mortality of the test organism, the median lethal concentration (LC50 (96 h)) and LC100 value was determined to be > 6.25 and 12.5 mg/l.

 

Another short term toxicity to fish study was conducted for 96 hrs for assessing the effect of test chemical (J-CHECK, 2019). The study was performed in accordance to the OECD Guideline 203 (Fish, Acute Toxicity Test) in a static system. On the basis of the effect on mortality of the test organism, the 96 hr LC50 was determined to be 8.8 mg/l (nominal concentration).

 

On the basis of the above results, it can be concluded that the test chemical can be considered as toxic to fish and considered to be classified in ‘aquatic chronic category 2’ as per CLP classification criteria.

Long term toxicity to fish

Based on the prediction done using ECOSAR version 1.11, the long term toxicity on fish was predicted for test chemical (2018). On the basis of effects observed in a flow through freshwater system on test organism, the NOEC value for the substance was estimated to be 4.5 mg/l for fish for 28 days of exposure duration. Thus, it can be concluded that the test chemical can be considered as non-toxic to fish at environmentally relevant concentrations

Short term toxicity to aquatic invertebrates

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

 

In an experimental study from study report (2018), an acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on aquatic invertebrates. 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 100 mg/l was prepared by dissolving test chemical in reconstituted water. The test solutions of required concentrations were prepared by mixing the stock solution of the test sample in reconstituted water. Test chemical concentrations were not verified analytically. Nominal test chemical concentrations used for the study were 0, 10, 20, 40 and 80 mg/l, respectively. Study was performed using total 5 organisms per vessel/replicates in a static 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. The 24 hr EC50 value of the reference substance was determined to be 0.73 mg/l. 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 and the dissolved oxygen concentration at the end of the test was evaluated to be ≥ 3 mg/l (i.e, reported as > 7 mg/l) both in the control and test vessels. Thus, the validity criterion of the test has been fulfilled. 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 30.1 mg/l (95 % C. I. - 23.9 to 37.9 mg/l).

 

In a supporting weight of evidence study, short term toxicity to aq. invertebrate study was conducted for 48 hrs for assessing the effect of test chemical (authoritative database, 2019 and secondary source, 2019). This test was performed using Daphnia magna (water flea) of < 24 hr old under static conditions. No analytical monitoring was performed of exposed test chemical concentrations. On the basis of effect on mobility of the test organism Daphnia magna, the 48 hr EC50 was determined to be 17 mg/l (nominal concentration).

 

For the test chemical from peer reviewed journal (A.M. Api et. al., 2019), short term toxicity to aquatic invertebrate study was conducted for 48 hrs for assessing the effect of test chemical. The study was performed following the American Society of Testing and Materials (ASTM) method in a static system. Daphnia magna was used as a test organism. On the basis of effect on mobility of the test organism Daphnia magna, the 48 hr EC50 was determined to be 30 mg/l (nominal concentration).

 

On the basis of the above results, it can be concluded that the test chemical can be considered as toxic to aquatic invertebrates at environmental related concentrations and considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

Long term toxicity to aquatic invertebrates

Chronic toxicity to aquatic invertebrate study was conducted for 21 days for assessing the effect of test chemical on aquatic invertebrates (Experimental study report, 2019). The study was performed following the OECD Guideline 211 (Daphnia magna Reproduction Test) under semi-static conditions. The solution of test chemical was prepared by dissolving 100 mg of test chemical in 100 ml of Adams medium to get the final concentration of 1000 mg/L. Test chemical concentrations were verified analytically by UV-Vis Spectrophotometer and the final solubility value obtained after analytical detection was 296.79 mg/l. The remaining test solutions were prepared by dilution from the stock solution. Thus, test chemical concentrations used for the study were 0 (control), 0.5, 1, 2, 4 and 8 mg/L, respectively. Daphnia magna (water flea) of length 0.37 cm was used as a test organism. A population of parthenogenetic females of synchronized age structure has been maintained for more than 2 years in the test facility under constant temperature conditions (18 to 22 °C) at a 16 : 8 hour light-dark photoperiod (illumination: < 1000 lux). The culture media (Adams medium') was partly renewed once a week. The Daphnia were exclusively fed with unicellular green algae (Selenestrum capricornutum) thrice a week. Total 50 test organisms were exposed to five test chemical concentrations (n = 10) in 100 ml glass beaker. In addition to this, control test vessel was also setup during the study. Test vessels were then placed at temperature of 18 – 22°C, hardness 200 mg/l as CaCO3, pH 7.8, dissolved oxygen 6.3 to 8.3 mg/l and under a photoperiod of 16 hour light and 8 hour dark with 1000 – 1500 lux light intensity. Reproduction rate and the mobility behaviour / mortality rate of parent Daphnia was assessed at least three times a week. The test concentrations were measured and was observed be maintained within 80-120% of nominal concentration, thus, effect concentrations (EC) would be evaluated as the nominal concentrations. The mortality in the control groups did not exceeded 20% percent. Thus, the validity criterion of the test has been fulfilled. On the basis of the effect of test chemical on the reproduction of the test organism Daphnia magna, the 21 days EC50 value was determined to be 0.5581 mg/l (nominal concentration). Thus, test chemical was 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

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

 

In an experimental study from study report (2019), a freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae. 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. Sterile, unicellular, suspension cultures of algae Pseudokirchneriella subcapitata of length 8 – 14 μm and width 2 - 3 μm was used as a test organism. 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 300 mg of test chemical in 300 ml of OECD medium to get the final concentration of 1000 mg/L. Test chemical concentrations were verified analytically by UV-VIS spectrophometer. Green algae were exposed to six different nominal concentrations of test chemical (0, 5 mg/L, 10 mg/L, 20 mg/L, 40 mg/L, 80 mg/L and 160 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 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. 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). Apart from this, the cell count of each test vessel was also noted with the help of a microscope. Cell densities were recorded in section by section growth rate at 24 hr intervals, which was calculated as specific growth rate. The 72 hr ErC10 (based on growth rate [r]), was calculated or read from the dose/concentration/percentage response curve. Potassium dichromate (K2Cr2O7) was used as a reference substance for the study. Since the concentration of the test chemical being tested has been satisfactorily maintained within ±20% of the nominal concentration throughout the test, all concentrations will be reported as nominal concentration. As per the OECD guideline No. 201 – Alga growth inhibition test, the biomass in the control cultures have increase exponentially by a factor of at least 17 within the 72 hr test period, the mean coefficient of variation by section specific growth rate in the control cultures not exceeded 35% and the coefficient of variation of average specific growth rate during the whole test period in replicate control cultures was not exceeded 10%, thus, fulfilling the validity of the criteria. The 72 hr EC50 value of reference substance was determined to be 0.809 mg/l. On the basis of growth rate of the test organism Pseudokirchneriella subcapitata, the 72 hrs effect concentration (EC10) value was determined to be 0.45 mg/l (nominal concentration) and median effect concentration (ErC50) was determined to be ≥ 160 (nominal concentration) & ≥ 184.46 mg/l (initial measured concentration). On the basis of the EC50 value, chemical was considered to be non-toxic to aquatic algae at environmental relevant concentrations.

 

Another toxicity to aquatic algae study was conducted for 72 hrs for assessing the effect of test chemical on green algae (Experimental study report. 2018). The test was performed in accordance to OECD Guideline 201 (Alga, Growth Inhibition Test). Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) of strain 86.81 SAG obtained from Institute of botany of the ASCR with an initial biomass conc. 5000 cells /ml was used as a test organism. The stock solution 100 mg/l was prepared by dissolving test chemical in OECD growth medium. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. Test chemical concentrations were not verified analytically. Nominal test chemical conc. used for the study were 0, 2.5, 5.0, 10, 20, 40 and 80 mg/l, respectively. Study was performed in a static system for 72 hrs. Desmodesmus subspicatus were exposed to test chemical in 50 ml glass vessel in a volume of 15 ml of liquid solution containing both the chemical and media. Control solution vessel containing OECD medium without the test chemical was also setup during the study. The beakers were placed in a room at a temperature of 23±2°C with a continuous light intensity of 6000-8000 lx, respectively. Alongwith the test chemical, one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. The 24 hr EC50 value of the reference substance was determined to be 0.77 mg/l. Cell counting was carried out using microscope with counting chamber Cyrus I or electronic particle counter. ErC50 was calculated using non-linear regression by the software Prism 4.0. In the control test vessel containing OECD growth medium without test chemical, the coefficient of variation of average growth rate in replicates during the whole test period was 4.5% and the specific growth rate in the control was 1.77 per day, indicating that the biomass in the control cultures have increased exponentially by a factor of more than 16 within the 72 hr exposure duration. Thus, the validity criterion of the test has been fulfilled. On the basis of the effect of test chemical on the growth rate of the test organism Desmodesmus subspicatus, the 72 hr median effect concentration (ErC50) value was determined to be 20.5 mg/l (95% CL: 10.7 to 39.3 mg/l). Thus, based on the EC50 value, test chemical can be considered to be toxic to aquatic algae and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

 

For the test chemical, an acute toxicity test was conducted for 96 hrs for assessing the effect of test chemical on green algae. The test was performed in accordance with the Scenedesmus cell proliferation inhibition test, DIN 38412 part 9.Test chemical concentrations were not verified analytically. Test chemical conc. used for the study were 0 (control), 7.8, 15.6, 31.3, 62.5, 125, 250 and 500 mg/l (nominal conc.). Study was performed using Scenedesmus subspicatus as a test organism in a static system in quadruplicates (for test conc.). Initial cell concentration used for the study was 10000 cells/ml. Test organisms were exposed to test chemical in 250 mL Erlenmeyer flask. The test vessels were placed at a temperature of 19.85°C under continuous illumination with a light intensity of 6.2 miS/cm. Cell growth was measured fluorometrically in all flasks at 24, 48, 72 and 96 hours of incubation period. On the basis of effect on growth rate of the test organism Scenedesmus subspicatus, the 96 hr EC20, ErC50 and EC90 was determined to be 20, 79 and > 500 mg/l (nominal conc.), respectively. Thus, based on this, 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. 

 

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

Toxicity to microorganisms

Toxicity to microorganism study was performed using Tetrahymena pyriformis as a test organism (T. W. Schultz et. al., 1999 and authoritative database, 2012). The study was carried out under static conditions. Stock solutions of test chemical were prepared in dimethyl sulfoxide. Exact test chemical concentrations used for the study was not known, but each test replicate consists of six to eight different concentrations. Range finding study was performed in duplicates prior to the initiation of study. Flasks were used as a test vessel. To each test flask were added test organism and a solution of test chemical. Two control vessels, one control with no test chemical but inoculated with T. pyriformis, and the other, a blank control, which has neither test chemical nor test organism were run simultaneously during the study. All test concentrations experiments were performed in duplicates. Test conditions allow for 8-9 cell cycles in control cultures. Only replicates with control-absorbency values > 0.6 but < 0.75 were used for the analysis. Population density of test organism was measured spectrophotometrically at 540 nm. The 50% growth inhibitory concentrations, IGC50, were determined by Probit Analysis of Statistical Analysis System (SAS) software. On the basis of the effect on growth inhibition of the test organism Tetrahymena pyriformis, the 40 hr IGC50 value was determined to be 204.64 mg/l.

On the basis of the available information of aquatic toxicity studies, it can be concluded that the test chemical can be considered as toxicto aquatic organisms at environmental relevant concentrations and considered to be classified in 'aquatic chronic category 2' as per CLP classification criteria.