Phenethyl phenylacetate

Endpoint summary

Description of key information

Short term toxicity to fish:

Principle of this study was to determine the effect of test chemical on the mortality rate of fishes. Test conducted in accordance with OECD Guideline 203 (Fish, Acute Toxicity Test). Zebra fish (Danio rerio) was used as a test organism. The stock solution was prepared by dissolving 15.56 mg of the test substance in 1 liters of potable water (passed through reverse osmosis system) and kept in sonicator for the sonication process of 30 min. From this stock solution required test concentration were prepared for achieving the test concentrations of 0.8 mg/L, 1.68 mg/L, 3.52 mg/L, 7.40 mg/L & 15.56 mg/L, respectively and Zebra fish (Danio rerio) were exposed to these concentration for 96 hours. Test performed under the static system. Effects on the mortality rate of fishes was calculated and were observed in the interval of 24, 48, 72 and 96 hours. The fishes were moving slowly as compared to control. No mortalities were observed in the control aquaria. Based on nominal concentrations, experimental median lethal Concentrations [LC-50 (96 h)] of test material on Zebra fish (Danio rerio) was determined to be 11.47 mg/l calculated through geometric mean based on LC0 and LC100 value. Thus based on the LC50 value, it can be concluded that the test chemical was toxic to fish and can be classified in "aquatic chronic 3"category under CLP regulation.

 

Long-term toxicity to fish: On the basis of the experimental studies of the structurally similar read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, the 28 days NOEC value of test chemical was determined to be > 0.154 to < 0.92 mg/l and LOEC value was determined to be > 0.435 to < 1.92 mg/l.

 

 

Short term toxicity to aquatic invertebrates:

An acute immobilisation test was conducted for 48 hrs for assessing the short term toxicity of test chemical to aquatic invertebrate. Study was performed according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). Based on the effects on the immobilisation of Daphnia magna after the chemical exposure for 48 hours, it was observed that the EC50 value ranges from 16.6 to 63.86 mg/l. Thus based on the overall studies from various sources, it was concluded that the test chemical was toxic and classified in aquatic chronic category 3 as per the CLP classification criteria.

 

Long-term toxicity to aquatic invertebrates: On the basis of the experimental studies of the structurally similar read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, daphnia magna the 21 days NOEC, LOEC and EC50 value was determined to be > 0.17 to < 0.519 mg/l, 1.18 mg/l and > 1.18 to < 1.2 mg/l respectively.

 

Toxicity to algae:

The study was designed to access the toxic effects of the test chemical on the green alga. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). Chlorella vulgaris was used as test organism. The test substance was prepared by adding 6.06 mg of test substance in 390 ml of BBM to get the final concentration of 15.56 mg/L. The sock solution was ultrasonically agitated for 30 minutes to obtain a homogenous solution for the experiment. The remaining test solutions were prepared by dilution from the above prepared stock solution under aseptic conditions. Six test concentrations were 2 mg/l, 3 mg/l, 4.5 mg/l, 6.75 mg/l, 10.13 mg/l and 15.19 mg/l was used and the study conducted under the static system. 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. The test is considered to be valid as met all the standard parameters. After 72 hours of exposure to test chemical to various nominal test concentrations with algae Chlorella vulgaris, the EC50 value based on the growth rate inhibition of algae was determined to be 13.89 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 be classified aq aquatic chronic 3 as per the CLP classification criteria.

 

Toxicity to microorganisms:

WoE 2: For the test chemical, based on the effect on reduction in light output of the test organism Photobacterium phosphoreum, the EC50 value during 5, 15 and 30 min exposure period was determined to be 3.273, 3.859 and 4.52 mg/l, respectively.

 

WoE 3: Based on growth rate inhibition of test organism tetrahymena thermophila, the EC50 value after 24 and 28 hrs exposure duration was observed to be 4.3 and 5.7 mg/l, respectively and LOEC value ewas determine to be 0.48 mg/l.

 

WoE 4: Based on decrease in bacterial light production by the test organism Photobacterium leiognathi SB strain, the EC50 value after 15 and 30 exposure duration with the test chemical, EC50 was observed to be 1.3 and 1.6 mg/l, respectively and LOEC value was observed to be 0.25 mg/l.

 

Thus based on the above studies, chemical toxicity were ranges from 1.3 mg/l to 16.819 mg/l.

Additional information

Short term toxicity to fish:

Data available for the test chemical and structurally and functionally similar read across chemicals has been reviewed to determine the toxicity of the test chemical on fishes. The studies are as mentioned below:

 

Principle of this study was to determine the effect of test chemical on the mortality rate of fishes. Test conducted in accordance with OECD Guideline 203 (Fish, Acute Toxicity Test). Zebra fish (Danio rerio) was used as a test organism. The stock solution was prepared by dissolving 15.56 mg of the test substance in 1 liters of potable water (passed through reverse osmosis system) and kept in sonicator for the sonication process of 30 min. From this stock solution required test concentration were prepared for achieving the test concentrations of 0.8 mg/L, 1.68 mg/L, 3.52 mg/L, 7.40 mg/L & 15.56 mg/L, respectively and Zebra fish (Danio rerio) were exposed to these concentration for 96 hours. Test performed under the static system. Effects on the mortality rate of fishes was calculated and were observed in the interval of 24, 48, 72 and 96 hours. The fishes were moving slowly as compared to control. No mortalities were observed in the control aquaria. Based on nominal concentrations, experimental median lethal Concentrations [LC-50 (96 h)] of test material on Zebra fish (Danio rerio) was determined to be 11.47 mg/l calculated through geometric mean based on LC0 and LC100 value. Thus based on the LC50 value, it can be concluded that the test chemical was toxic to fish and can be classified in "aquatic chronic 3"category under CLP regulation.

 

 

Above study further supported by the second study from experimental sources. This study was designed to access the toxic effects of the test compound on the fishes. Test conducted in accordance with OECD Guideline 203 (Fish, Acute Toxicity Test). Zebra fish (Danio rerio) was used as a test organism. The stock solution was prepared by dissolving 1ml of the test substance in 1 liter of potable water (passed through reverse osmosis system) with 24 hrs of continuous stirring. From this stock solution, further test concentration was prepared for achieving test concentrations of 6.25 mg/L, 12.5 mg/L, 25 mg/L, 50 mg/L & 100 mg/L, respectively. Aquaria containing 4 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes each. A static procedure was used for the study. Effects on the mortality rate of fishes was calculated and were observed in the interval of 24, 48, 72 and 96 hours. The median lethal concentration (LC50) value of test chemical on Danio rerio in a 96 hours study on the basis of mortality effect was determine to be >12.5 mg/L. As 100 % mortality was observed at the concentration of 25 mg/l, thus on that basis, chemical consider to be toxic and classified in aquatic chronic category 3 as per the CLP classification criteria.

 

Similar an acute toxicity test was conducted for 96 hrs for assessing the effect of test chemical on Zebra fish (Danio rerio). The test was performed in accordance to OECD guideline No. 203 “Fish Acute Toxicity Test”. Zebra fish (Danio rerio) of average length of 2.7 cm and average weight of 0.19 g 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 8.8 mg/l, pH 7.5, water temperature 22°C and under a photoperiod of 16:8 hr light: dark conditions, respectively. The test chemical was prepared by dissolving 5 gm of test chemical in 5 lit of potable water (passed through reverse osmosis system). After stirring, the stock solution was filtered and analytically detected & the concentration was found to be 236.34 mg/l. The remaining test solutions were prepared by dilution from the stock solution. Test chemical concentrations were analytically determined by UV-VIS spectrophotometer. Test chemical concentrations used for the study were 0, 6.25, 12.5, 50 and 100 mg/l, respectively. Total 7 fishes were exposed to test chemical in a 4 lit Polypropylene (PP) fish tank containing 4000 ml of potable water. The test vessels were placed in a room at a temperature of 21 -22°C, pH of control at 0 and 96 hr was 7.8 & 7.9 and DO of control at 0 and 96 hr was 7.9 & 4.8 mg/l 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. Mortality in the control was 0%. The dissolved oxygen concentration remained above 60% of the air saturation value throughout the exposure period. Thus, fulfilling the validity criterion. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of effect of test chemical on mortality of the test organism, the 96 hr median lethal concentration [LC50 (96 h)] for test chemical on Danio rerio (Zebra Fish) was determined to be in the range of > 25 to < 50 mg/L. Thus, test chemical was considered as toxic to aquatic fishes and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

 

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

 

Long-term toxicity to fish: 

 

Data available of the structurally similar read across chemicals have been reviewed to determine the effect of the test chemical on fishes. The studies are as mentioned below:

 

The first study of long-term toxicity to fish was carried out according to OECD 210 guideline. Oryzias latipes (Japanese rice fish) was taken as test organism. The duration of test was 42 days, exposed from fertilized egg, until after 30 days from the day when hatching rate became more than 70 % in control. Post-hatch feeding includes BSN type of food (*: brine shrimp hatching larva just after hatching for larval and juvenile fish). Full feeding was given to test fishes, frequency of feeding was 1- 2 times per day. N,N-Dimethylformamide, HCO-60 was taken as vehicle, N,N-Dimethylformamide was taken as ca. 100 microL/L, HCO-60 was taken as ca. 2.0 mg/L. Test concentrations were taken as 0.020 mg/L,  0.058 mg/L, 0.17 mg/L, 0.48 mg/L, 1.4 mg/L in geometric ratio of 2.9. The renewal rate of the test solution was 19 times/day, 48 L/vessel/day. The liquid volume in test vessel was 2.5 L. Number of fertilized eggs/embryos per vessel was 60 per concentration, 20 per vessel. The number of vessels per concentration replicates was taken as three. Adjustment of pH was not adjusted. Photoperiod was given as 16 hours of light per 8 hours darkness. Light intensity was given as less than 1000 lux. Test temperature at egg and embryo stages was 24 ±1 °C and at  larval -juvenile fish stages was 23 ± 2 °C. Dissolved oxygen was maintained at more than 60% of the saturated concentration. Analytical monitoring was done by HPLC. On the basis of survival rate effect of test chemical on test organisms Oryzias latipes (Japanese rice fish) the 42 days NOEC and LOEC value of test chemical was determined to be 0.154 mg/l and 0.435 mg/l respectively. Based of the value of NOEC the test chemical was classified as Chronic category 2 as per CLP criteria. 

The second study of long-term toxicity to fish was carried out. Oryzias latipes (Japanese rice fish) was taken as test organism. The duration of test was 28 days. On the basis of growth effect of test chemical on test organisms Oryzias latipes (Japanese rice fish) the 28 days NOEC and LOEC value of test chemical was determined to be 0.92 mg/l and 1.92 mg/l respectively. Based of the value of NOEC the test chemical was classified as 'Chronic category 2' as per CLP criteria. 

 

On the basis of the experimental studies of the structurally similar read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, the 28 days NOEC value of test chemical was determined to be > 0.154 to < 0.92 mg/l and LOEC value was determined to be > 0.435 to < 1.92 mg/l. Based of the value of NOEC the test chemical was classified as 'Chronic category 2' as per CLP criteria. 

 

 

Short term toxicity to aquatic invertebrates:

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the toxicity of the test chemical on aquatic invertebrates Daphnia magna. The studies are as mentioned below:

 

Aim of this key weight of evidence study was to assess the short term toxicity of test chemical to aquatic invertebrate. Study was performed according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). Daphnia magna was used a test organism. The animals used for the test was Daphnia magna, which should be less than 24 h old and should not be of first brood progeny. The stock solution 100 g/L was prepared in acetone. Test solutions of required concentration as were prepared by mixing the stock solution of the test sample with reconstituted test water. 0, 0, 5, 10, 20, 40 and 80 mg/L concentrations were used in the study. Effects on immobilisation were observed for 48 hours and conducted under the static system. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. The median effective concentration (EC50) value of the test substance on Daphnia magna was determined to be 60.7 mg/L on the basis of immobilisation effects in a 48 hour study. Based on the EC50 value, substance consider likely to be hazardous to aquatic invertebrate Daphnia magna and can be classified in aquatic chronic 3 category as per the CLP classification criteria.

 

Above study further supported by the third weight of evidence study from experimental report. An acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on Daphnia magna. The test was performed in accordance to OECD guideline No. 202“Daphnia sp., Acute Immobilization Test”. The saturated test solution was prepared by dissolving 500mg of test chemical in 500ml of M7 media, and allowed for stirring for 96 hours, which was then filtered and the final saturated stock solution obtained was 162.82 mg/L, verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 6.25, 13.12, 27.55, 57.85 and 121.48 mg/l, respectively was from the saturated test concentration. Study was performed using 10 daphnids in a static system. Total 10 Daphnids/conc. were exposed to test chemical in 25 ml beakers in a volume of 20 ml of liquid solution containing both the chemical and media. The beakers were placed in a room at a temperature of 20°C, hardness of water > 140 mg of CaCO3 and under a photoperiod of 16:8 hr light: dark conditions with light intensity 1000 – 1500 Lux, respectively. One control vessel was also run simultaneously during the study. The animals in control and test chemical concentrations were exposed for a period of 48 hour. Potassium dichromate was used as a reference substance for the study. The 24 hr EC50 value of reference substance was determined to be 0.831 mg/l. No Immobility were found in the control test animals and the dissolved oxygen concentration at the end of the test in the control and test vessel was ≥ 3 mg/l, thus validity criterion of the study has been fulfilled. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of effect of test chemical on mobility of the test organism, the median effect concentration (EC50 (48 h)) value was determined to be 63.86 mg/L. Thus, based on the EC50 value, chemical was considered as toxic to aquatic invertebrates and hence, considered to be classified in 'aquatic chronic category 3' as per CLP classification criteria.

 

Similar an acute immobilisation test was conducted for 48 hrs for assessing the short term toxicity of test chemical to aquatic invertebrate. Study was performed according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). Daphnia magna was used a test organism. The stock solution 100 mg/l was prepared in reconstituted water. 0, 2.5, 5, 10, 20, 40 mg/l nominal concentrations were used in the study. Effects on immobilisation were observed for 48 hours and conducted under the static system. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Based on the immobilisation of Daphnia magna due to the exposure of test chemical for 48 hours, the EC50 value was determine to be 16.6 mg/l. EC50 value indicates that the substance is likely to be hazardous to aquatic invertebrates and can be classified in aquatic chronic 3 category as per the CLP criteria.

 

Thus based on the overall studies from various sources, it was concluded that the test chemical was toxic and classified in aquatic chronic category 3 as per the CLP classification criteria.

 

Long-term toxicity to aquatic invertebrates: 

 

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

 

The first study of long term toxicity to aquatic invertebrates was carried out according to OECD 211 guideline. The test organisms was taken as daphnia magna. The test was performed in semi-static conditions. The duration of study was 21 days. Dimethylformamide was used as a vehicle with a concentration of 100 microL/L. Test concentrations were taken as (nominal values) control, vehicle control of 0.040, 0.097, 0.240, 0.580, 1.40 mg/L in the geometric ratio: 2.4. The adjustable highest concentration of test solution was 1.40 mg/l. Test vessel was of closed, covered water surface with teflon sheet. Test solution volume was taken as 80 mL/vessel. Total amount of test solution was renewed every day. Number of organisms per vessel was taken as 10 per concentration, one test organism per vessel was taken for study. The number of vessels per concentration (replicates) was taken as ten. Photoperiod was given for 16 hours light with 8 hours darkness. Light intensity provided was less than 800 lux. Test conditions includes the temperature of 20 ±1 °C. Analytical monitoring was done by HPLC. On the basis of reproduction inhibition effect of test chemical on daphnia magna the 21 days NOEC, LOEC and EC50 value of test chemical was determined to be 0.519 mg/l, 1.18 mg/l and > 1.18 mg/L respectively. On the basis of the value of NOEC the test chemical was classified as 'Chronic category 2' as per CLP criteria.

 

The second study of long-term toxicity to aquatic invertebrates was carried out according to OECD 211 guideline. The test organisms was taken as daphnia magna. The test was performed in semi-static conditions. the duration of study was 21 days. On the basis of reproduction inhibition effect of test chemical on daphnia magna the 21 days EC50 and NOEC value of test chemical was determined to be 1.2 mg/l and 0.71 mg/l respectively. On the basis of the value of NOEC the test chemical was classified as Chronic category 2 as per CLP criteria.  

 

On the basis of the experimental studies of the structurally similar read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, daphnia magna the 21 days NOEC, LOEC and EC50 value was determined to be > 0.17 to < 0.519 mg/l, 1.18 mg/l and > 1.18 to < 1.2 mg/l respectively. Based on the value of NOEC the test chemical was classified as 'Chronic category 2' as per CLP criteria.  

 

Toxicity to algae:

Various experimental data of the test chemical and supporting weight of evidence studies for its structurally and functionally similar read across chemical were reviewed for the toxicity of test chemical on algae end point which are summarized as below:

 

The study was designed to access the toxic effects of the test chemical on the green alga. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). Chlorella vulgaris was used as test organism. The test substance was prepared by adding 6.06 mg of test substance in 390 ml of BBM to get the final concentration of 15.56 mg/L. The sock solution was ultrasonically agitated for 30 minutes to obtain a homogenous solution for the experiment. The remaining test solutions were prepared by dilution from the above prepared stock solution under aseptic conditions. Six test concentrations were 2 mg/l, 3 mg/l, 4.5 mg/l, 6.75 mg/l, 10.13 mg/l and 15.19 mg/l was used and the study conducted under the static system. 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. The test is considered to be valid as met all the standard parameters. After 72 hours of exposure to test chemical to various nominal test concentrations with algae Chlorella vulgaris, the EC50 value based on the growth rate inhibition of algae was determined to be 13.89 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 be classified aq aquatic chronic 3 as per the CLP classification criteria.

 

Similar freshwater algal growth inhibition test was carried out on green algae. Test conducted in accordance with OECD Guideline 201 (Alga, Growth Inhibition Test). Desmodesmus subspicatus was used as test organism. The stock solution 100 mg/L was prepared by dissolving colourless liquid in acetone. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. 0, 6.25, 12.5, 25, 50 and 100 mg/L, respectively concentrations were used in the study. The test was performed under static conditions at a temp. of 23±2°C. Initial cell density of test organism used was 5x10e3 cells/ml. Determination of cell counting involve the use of microscope with counting chamber Cyrus I or electronic particle counter. ErC50 was calculated using non-linear regression by the software Prism 4.0. Effects on the growth rate of algae was calculated in the interval of 24, 48 and 72 hours. The median effective concentration (ErC50) for the test substance on Desmodesmus subspicatus was determined to be 34.5 mg/L on the basis of effects on growth rate in a 72 hour study. Thus based on this value, test chemical can be considered as toxic to aquatic organisms and thus can be classified in aquatic chronic category 3 as per the CLP criteria.

 

Above studies further supported by the third weight of evidence study from experimental study report. Principle of this study was to evaluate the nature of test chemical when comes in contact with the test organism. Test was conducted according to the OECD guideline 201. Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) was used as test organism. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. 0, 10, 16, 26, 41, 66 mg/l, respectively concentration were used. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Effect on the growth of algae was determined after an exposure period of 72 hrs. The median effective concentration (ErC50) of the test substance on algae was determined to be 23.6 mg/L with 95% CI of 28.9 mg/l to 33.7 mg/l on the basis of growth rate inhibition effects in a 72 hour study. Based on the ErC50 value, the substance chemical was consider likely to be hazardous to aquatic algae and can be consider to be classified in aquatic chronic 3 category.

 

Thus based on the overall studies from various sources, it was concluded that the test chemical was toxic and classified in aquatic chronic category 3 as per the CLP classification criteria.

 

Toxicity to microorganisms:

Various studies available for the test chemical and structurally and functionally similar read across chemicals have been reviewed to determine the toxic nature of test chemical on the growth and other activity of microorganisms. The studies are as mentioned below:

 

Toxicity study from handbook, micro-organism study was carried out for assessing the effect of the test chemical. Study was performed using Photobacterium phosphoreum, strain NRRL-B-11177 (also referred to as Vibrio fischerii, strain NRRL-B-11177) as a test organism at a temperature of 15°C and pH range 5 to 9. Recommended reference substance that can be used for the study were Phenol and Sodium pentachlorophenate, respectively. When the test bacterium was exposed to the test chemical, reduction in light output was observed. Thus, based on this effect, the EC50 value during 5, 15 and 30 min exposure period was determined to be 3.273, 3.859 and 4.52 mg/l, respectively.

 

 

Above study was supported by the third study from peer reviewed journal. Toxicity to micro-organisms study was conducted on Tetrahymena thermophile for 28 hrs. Protoxkit bioassay test was performed for the toxicity study. All stock solutions were prepared in methanol and serially diluted in distilled water to obtain the target concentrations. The methanol concentration in the exposure solutions, including controls, was 0.01% (v/v) in distillate water in the tested solutions. Potassium dichromate (K2Cr2O7) was used as control substance. The test organism was exposed to the test substance with exposure duration 28 hrs. Inhibition in bacterial growth was measured after 24 and 28 hrs of exposure to tested solutions. Based on growth rate inhibition of test organism Tetrahymena thermophila, the EC50 value after 24 and 28 hrs exposure duration was observed to be 4.3 and 5.7 mg/l, respectively and LOEC value ewas determine to be 0.48 mg/l.

 

 

Similar toxicity to micro-organisms study was conducted on Photobacterium leiognathi SB strain for 30 mins. Toxscreen bioassay test was performed for the toxicity study. All stock solutions were prepared in methanol and serially diluted in distilled water to obtain the target concentrations. The methanol concentration in the exposure solutions, including controls, was 0.01% (v/v) in distillate water in the tested solutions. 2-4-dichlorophenol (DCP) was used as control solution.   The test bacteria was exposed to the test substance with exposure duration of 15 and 30 mins. Bacterial luminescence inhibition was measured after 15 and 30 mins of exposure to tested solutions.   Based on decrease in bacterial light production by the test organism Photobacterium leiognathi SB strain, the EC50 value after 15 and 30 exposure duration with the test chemical, EC50 was observed to be 1.3 and 1.6 mg/l, respectively and LOEC value was observed to be 0.25 mg/l.

 

 

 

However, based on the above all studies it is the test chemical is considered to be classified in the 'aquatic chronic 2' category as per the CLP classification criteria.