2,2,2-trichloro-1-phenylethyl acetate

Endpoint summary

Description of key information

Short term toxicity to fish:

This study was conducted as per OECD 203 (2019) to assess the acute toxicity effects of test chemical on zebrafish (Danio rerio) following exposure up to 96 h under static condition. Juvenile fish of same age and normal in appearance were used in this (originate from same source and population). The average length and weight (10 fish) were observed 1.4 cm and 0.042g, respectively. Fish were fed (commercial fish food) daily during acclimatization. Photoperiod and light intensity were maintained such as16 h light- 8 h dark, 698 to 745 Lux during experiment. Hardness of water was measured once during acclimatization and found to be 165 mg CaCO3/L, temperature, pH and dissolve oxygen were maintained between 21.9-22.5 °C, 7.0 to 7.8, 5.61-9.9 mg/L, respectively, throughout the test. Fish were acclimatized for 7 days prior dosing. Natural water was used as dilution control and acetone in natural water (100µL/1000 mL conc) was used as vehicle control and the same concentration was used for formulation of test chemical. The study was initiated with a range finding test by using following concentrations of 0 (control), 0 (vehicle control), 2.80, 5.04, 9.07, 16.33 and 29.39 mg/L. During range finding test, no mortality or abnormality was found in control and test conc. groups. Hence, a main study (limit test) was conducted using 0 (control), 0 (vehicle control), 30 mg/L concentrations. No mortality or abnormality was found in control and test groups. 7 fish were used/concentration during range finding and main study. HPLC method was used for method validation and active ingredient analysis along with stability of the test item in the test medium. Test item was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during main study 0 h and 96 h avg. recovery for 1, 30 mg/L conc were 95.01 to 96.19% and 94.52 to 96.16%, respectively., were found in acceptable range. The test is valid as all the validity criteria are fulfilled: No mortality in control or vehicle control were found throughout the 96-h test duration; Dissolve oxygen conc. was maintained above 60% in all test vessels throughout the test; The recovery active ingredient content was found between 80-120% up to 96h. The 96 h LC50 of test chemical to zebrafish (Danio rerio) was determined to be >30 mg/L. Since, the test chemical is readily biodegradable in water, test chemical was considered as non-toxic to aquatic fishes and hence, considered 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 days post hatch for assessing the effect of test chemical on survival rate and hatching success. Study was performed following the principles of the OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test) under semi-static conditions. The stock solution of test chemical was prepared by dissolving 2000 mg in 2000 mL of test media to get the final concentration of 1000 mg/L followed by analytical determination. The final solubility obtained after analytical determination was 13.25 mg/L which was later on used to prepare the remaining test solutions by diluting the above stock solution. The received samples were stirred and done filtrations for the preparation of stock solution. This was later analyzed under UV-Vis spectrophotometer. Internal calibration was done and LOD, LOQ was determined to be 0.654164 and 1.982315, respectively. In spent samples of the test substance concentrations, the determined concentrations of test item were between 72 and 129.6% of the concentrations, respectively. Therefore, the concentrations of test item were stable during 96 h under test conditions. 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.  Which were collected from breeding groups, mixed, and randomly selected for 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. The fertilised eggs were collected and eggs were rinsed with reconstituted water after collection from spawning traps. Post hatch feeding was given to test fishes. Which was as follows, Commercial dry food was provided for 30 days. From day 15 onwards live food (artemia) was provided along with dry food. Dry flake food was given thrice a day and Brine shrimp Artemia was given to test fishes once daily. Surplus food and feces were removed where required, to avoid accumulation of waste. Reconstituted water was used as a test medium. Test fishes (total 20embryos/vessel) were exposed to different test chemical conc. (i.e., 0 (Control), 0 (Control), 0.625, 1.25, 2.5, 5 and 10 mg/l) in a 2 lit glass aquaria. 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: 16 light: dark conditions, Light intensity ranges from 500 to 1000 lux, temperature at the beginning of the test: 26.5°C, pH of 6.8 to 7.4 and dissolved oxygen of 84.72 to 99.25% air saturation value, respectively. Data were analyzed using 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 7.2 & 7.1 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. 20% mortality was observed in control after 30 days whereas mortality at the end of exposure to test chemical at concentrations of 0.625, 1.25, 2.5, 5 and 10 mg/l was 7.81, 14.06, 37.5, 53.13 and 59.38%, respectively. At 96 hr, all the embryos were hatched in control. Hatching success in the control and in the tested concentration of 0.625, 1.25, 2.5, 5 and 10 mg/ L was 91.25%, 90%, 87.5%, 86.25%, 81.25% and 75%, respectively. Larval survival until day 30 post-hatch in the control group was 87.67% thereby exceeding and satisfying the validity criteria for post-hatch survival (>75%) whereas in the group treated with test chemical at concentrations of 0.625, 1.25, 2.5, 5 and 10 mg/l was 81.94%, 78.57%, 57.97%, 46.15% and 43.33%,respectively.All surviving juveniles were healthy in the control. For fish total lengths, determined on Day 30 post-hatch were 11.28, 9.33, 8.78, 8.41, 7.87 and 7.72 mm for test item at concentrations control, 0.625, 1.25, 2.5, 5 and 10mg/L, respectively.In fresh samples of the test substance concentrations, the determined concentrations of test chemical were 89.92 to 120% for 0.0625 mg/L, 89.8 to 104.8% for 1.25 mg/L, 100 to 115.6% for 2.5 mg/l, 95.4% to 111.8% for 5 mg/l and 88.9 to 111.8% for 10 mg/L, respectively whereas in old samples of the test substance concentrations, the determined concentrations of test chemical were 72 to 129.6% for 0.0625 mg/L, 89.6 to 126.4% for 1.25 mg/L, 94 to 170% for 2.5 mg/l, 83.6% to 136.2% for 5 mg/l and 88.2 to 129.8% for 10 mg/L, respectively. The results confirm that the test substance concentrations were within the acceptability range. All validity criteria were satisfied during the test, therefore the test was considered to be valid. Based on the effect on mortality of test fishes, the 30 d NOEC, LOEC and LC50 value was determined to be 1.25, 2.5 and 5.39±0.499 mg/l (95% C. I. = 4.34 to 6.70 mg/l), respectively. On the basis of the effect on survival larvae of test fishes, the 30 d NOEC, LOEC and EC50 value was determined to be 1.25, 2.5 and 6.93± 0.51 mg/l mg/l, respectively. Where, based on hatching success, the 30 d NOEC, LOEC and EC10 value was determined to be 2.5, 5 and 4.42±0.55 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.

 

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 48 hours 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. Natural water was used as control, acetone with natural water was used as vehicle control and the same was used for test item formulation and test medium (15 µL acetone/150 mL natural water concentration was used). 25 mL glass beakers having a solution volume of 20 mL were used in the test. A range finding test (2 replicates/concentration having 5 daphnids/replicate) with the test concentrations of 0 (control), 0 (vehicle control), 1.5, 3, 6, 12, 24 mg/L was done prior to main study. No immobilization or abnormality was found in control groups and 1.5 mg/l, 3 mg/l, conc., but 20%, 30% and 100% in 6, 12, 24 mg/L concentrations were found, respectively. Hence, a main study (definitive test) (using a spacing factor of 1.65) was conducted using 0 (control), 0 (vehicle control), 3.23, 5.33, 8.79, 14.51 and 23.94 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 3.23 mg/L but 10%, 25%, 35% and 95% mortality were observed in the test concentrations of 5.33, 8.79, 14.51 and 23.94 mg/L, respectively. HPLC method was used for method validation and active ingredient analysis along with stability of the test item in the test medium. Test item was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during main study 0 h and 48 h avg. recovery 3.23, 8.79, 23.94 mg/L conc were 93.29% to 95.64%, 95.06% to 96.12% and 94.70% to 95.88% respectively, were found in acceptable range. 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 of 7.4 to 7.7 (at the begining of the test) and 7.0 to 7.5 (at the end of the test), temperature (20-21 °C), dissolve oxygen of 7.8 to 8.3 mg/l (at the begining of the test) and 7.2-7.9 mg/L (at the end of the test), hardness (165 mg CaCO3/L), photoperiod (16 h light- 8 h dark) and light intensity (1330 to 1340 Lux) was maintained in acceptable range throughout the test. Feed was not provided during the test. The 48-h EC50 of test chemical to daphnid,Daphnia magnaare was determined to be 13.47 mg/L with a 95% confidence interval of 12.33 to 14.61 mg/l, respectively. The 48-h EC50 of 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. 48h EC50 value with 95% confidence limits (upper limit, lower limit) were calculated by probit analysis using NCSS Software 2019, version 19.0.3. Since, the test chemical is readily biodegradable in water, test chemical was considered as non-toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be not classified as per the 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 Adams 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 Selenestrum capricornutum and it was done thrice a week. The test chemical will be prepared by dissolving 100 mg of test chemical in 100 mL of Adams 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 0.1, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 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 not been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on measured concentration. Test chemical conc. used in the defiinite study were 0, 0.1562, 0.3125, 0.625, 1.25 and 5 mg/l. 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. Adams medium was used as a test medium. Test conditions involve a temperature range of 18.4-21.5°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 and LOEC value was determined to be 0.625 and 1.25 mg/l, respectively. Thus, based on the NOEC value, test chemical was considered as toxic to aquatic invertebrates and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria:

This study was conducted as per OECD 201 (2011) to assess the acute toxicity effects of test chemical on the growth of green alga Pseudokirchneriella subcapitata(ATCC)following exposure of alga up to 72 h under static condition. Test system was initially procured from “American Type Culture Collection”, Chromachemie Laboratory Private Limited, later test system was sub-cultured (using OECD growth medium, OECD 201) in the test facility. 250 mL sterilized conical flasks covered with cap were used in the study. The test item was found to be soluble in acetone. Hence, acetone (concentration <0.1mL/L) with OECD growth medium was used as test medium. An inoculum culture in the test medium was prepared 3-4 days prior to test and culture conditions were maintained same as the test conditions. A range finding study (3 replicates/ concentration, 6 replicates for control group)with the test concentrations of1.5, 3, 6, 12, 24 mg/L were tested along with a control (0), vehicle control (0) group, prior to the main study. The inhibition in growth rate (0%, 0%, 0.27%, 28.39%, 39.15%, 89.02%, 94.64%) and yield (0%, 0%, 1.25%, 70.43%, 81.65%, 99.08%, 99.60%) were observed at 72 h in the test concentrations of 0, 0, 1.5, 3, 6, 12, 24 mg/L. Based on the results of range finding test following concentrations of 0 (control), 0 (vehicle control), 0.6, 1.3, 2.9, 6.4 and 14.1 mg/L were selected for the main study(3 replicates/ concentration, 6 replicates for control groups)as significant changes (inhibition algal growth rate) were observed in treatment groups during 72h test period. The inhibition in growth rate (0%, 0%, 0.46%, 2.91%, 33.92%, 45.76%, 88.98%) and yield (0%, 0%, 1.94%, 11.57%, 76.72%, 86.35%, 99.06%) were observed during 72h test period in the test concentrations of 0 (control), 0 (vehicle control), 0.6, 1.3, 2.9, 6.4 and 14.1 mg/L. A reference standard (Potassium dichromate) was tested to ensure the authenticity of the test in the lab. And the inhibition of growth rate (ErC50-72h) and yield (EyC50-72h) were found to be0.620 mg/L & 0.334 mg/L, respectively; and were found to be in acceptable range. The initial cell density was 10000 cells/mL.The algal cells in the control and vehicle control increased by 65.62, 63.3 times (>16 times) of the initial cell count during the 72-h exposure period, respectively. The mean coefficient of variation for section-by-section growth rate for the control cultures over the test period (0-72-h) was 25.84% (<35%). The coefficient of variation of average specific growth rate was 0.6% (<7%). Hence, fulfilling the all the validity criteria of the test. HPLC method was used for method validation and active ingredient analysis along with stability of the test item in the test medium. Test item was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during main study 0 hr and 72 hr avg. recovery for 0.6, 2.9 and 14.1 mg/L conc were 93.44% to 94.61%, 95.65% to 96.19%, 95.40% to 95.45%, respectively, were found in acceptable range. Continuous light having and average light intensity of 6748 to 6814 Lux, pH of 7.5 -7.8, temperature 21.2 -23.5°C along with a shaking speed of 110 RPM were provided and maintained for the test system throughout the test. 72h EC50 (growth rate and yield) values with 95% confidence limits were calculated by probit analysis. NOEC and LOEC of growth rate and yield were calculated by one way ANOVA (Kruskal-Wallis-Comparison Z-Value Test) using NCSS Software 2019, version 19.0.3. The cells were counted using Haemocytometer under illumination of the microscope at 24, 48, and 72 h after inoculation. 72-h EC50 of test item to alga on growth rate and yield are 5.47, 2.41 mg/L, respectively. 72-h LOEC of test item to alga on growth rate and yield is 6.4 mg/L. 72-h NOEC of test item to alga on growth rate and yield is 2.9 mg/L. Since, the test chemical is readily biodegradable in water, test chemical was considered as non-toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be not classified as per the CLP classification criteria.

Toxicity to microorganisms:

Study provide a means to predict the ‘minimum’ toxicity that a phenol willexert in the Microtox test. Toxicity data noted as ‘pT’ are listed as the negative logarithm of the millimolar concentration required to elicit a 50% reduction in light emission in the stated time (i.e. 5-, 15, or 30-min) and pT 30 was predicted to be 1.09.

Additional information

Short term toxicity to fish:

This study was conducted as per OECD 203 (2019) to assess the acute toxicity effects of test chemical on zebrafish (Danio rerio) following exposure up to 96 h under static condition. Juvenile fish of same age and normal in appearance were used in this (originate from same source and population). The average length and weight (10 fish) were observed 1.4 cm and 0.042g, respectively. Fish were fed (commercial fish food) daily during acclimatization. Photoperiod and light intensity were maintained such as16 h light- 8 h dark, 698 to 745 Lux during experiment. Hardness of water was measured once during acclimatization and found to be 165 mg CaCO3/L, temperature, pH and dissolve oxygen were maintained between 21.9-22.5 °C, 7.0 to 7.8, 5.61-9.9 mg/L, respectively, throughout the test. Fish were acclimatized for 7 days prior dosing. Natural water was used as dilution control and acetone in natural water (100µL/1000 mL conc) was used as vehicle control and the same concentration was used for formulation of test chemical. The study was initiated with a range finding test by using following concentrations of 0 (control), 0 (vehicle control), 2.80, 5.04, 9.07, 16.33 and 29.39 mg/L. During range finding test, no mortality or abnormality was found in control and test conc. groups. Hence, a main study (limit test) was conducted using 0 (control), 0 (vehicle control), 30 mg/L concentrations. No mortality or abnormality was found in control and test groups. 7 fish were used/concentration during range finding and main study. HPLC method was used for method validation and active ingredient analysis along with stability of the test item in the test medium. Test item was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during main study 0 h and 96 h avg. recovery for 1, 30 mg/L conc were 95.01 to 96.19% and 94.52 to 96.16%, respectively., were found in acceptable range. The test is valid as all the validity criteria are fulfilled: No mortality in control or vehicle control were found throughout the 96-h test duration; Dissolve oxygen conc. was maintained above 60% in all test vessels throughout the test; The recovery active ingredient content was found between 80-120% up to 96h. The 96 h LC50 of test chemical to zebrafish (Danio rerio) was determined to be >30 mg/L. Since, the test chemical is readily biodegradable in water, test chemical was considered as non-toxic to aquatic fishes and hence, considered 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 days post hatch for assessing the effect of test chemical on survival rate and hatching success. Study was performed following the principles of the OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test) under semi-static conditions. The stock solution of test chemical was prepared by dissolving 2000 mg in 2000 mL of test media to get the final concentration of 1000 mg/L followed by analytical determination. The final solubility obtained after analytical determination was 13.25 mg/L which was later on used to prepare the remaining test solutions by diluting the above stock solution. The received samples were stirred and done filtrations for the preparation of stock solution. This was later analyzed under UV-Vis spectrophotometer. Internal calibration was done and LOD, LOQ was determined to be 0.654164 and 1.982315, respectively. In spent samples of the test substance concentrations, the determined concentrations of test item were between 72 and 129.6% of the concentrations, respectively. Therefore, the concentrations of test item were stable during 96 h under test conditions. 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.  Which were collected from breeding groups, mixed, and randomly selected for 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. The fertilised eggs were collected and eggs were rinsed with reconstituted water after collection from spawning traps. Post hatch feeding was given to test fishes. Which was as follows, Commercial dry food was provided for 30 days. From day 15 onwards live food (artemia) was provided along with dry food. Dry flake food was given thrice a day and Brine shrimp Artemia was given to test fishes once daily. Surplus food and feces were removed where required, to avoid accumulation of waste. Reconstituted water was used as a test medium. Test fishes (total 20embryos/vessel) were exposed to different test chemical conc. (i.e., 0 (Control), 0 (Control), 0.625, 1.25, 2.5, 5 and 10 mg/l) in a 2 lit glass aquaria. 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: 16 light: dark conditions, Light intensity ranges from 500 to 1000 lux, temperature at the beginning of the test: 26.5°C, pH of 6.8 to 7.4 and dissolved oxygen of 84.72 to 99.25% air saturation value, respectively. Data were analyzed using 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 7.2 & 7.1 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. 20% mortality was observed in control after 30 days whereas mortality at the end of exposure to test chemical at concentrations of 0.625, 1.25, 2.5, 5 and 10 mg/l was 7.81, 14.06, 37.5, 53.13 and 59.38%, respectively. At 96 hr, all the embryos were hatched in control. Hatching success in the control and in the tested concentration of 0.625, 1.25, 2.5, 5 and 10 mg/ L was 91.25%, 90%, 87.5%, 86.25%, 81.25% and 75%, respectively. Larval survival until day 30 post-hatch in the control group was 87.67% thereby exceeding and satisfying the validity criteria for post-hatch survival (>75%) whereas in the group treated with test chemical at concentrations of 0.625, 1.25, 2.5, 5 and 10 mg/l was 81.94%, 78.57%, 57.97%, 46.15% and 43.33%,respectively.All surviving juveniles were healthy in the control. For fish total lengths, determined on Day 30 post-hatch were 11.28, 9.33, 8.78, 8.41, 7.87 and 7.72 mm for test item at concentrations control, 0.625, 1.25, 2.5, 5 and 10mg/L, respectively.In fresh samples of the test substance concentrations, the determined concentrations of test chemical were 89.92 to 120% for 0.0625 mg/L, 89.8 to 104.8% for 1.25 mg/L, 100 to 115.6% for 2.5 mg/l, 95.4% to 111.8% for 5 mg/l and 88.9 to 111.8% for 10 mg/L, respectively whereas in old samples of the test substance concentrations, the determined concentrations of test chemical were 72 to 129.6% for 0.0625 mg/L, 89.6 to 126.4% for 1.25 mg/L, 94 to 170% for 2.5 mg/l, 83.6% to 136.2% for 5 mg/l and 88.2 to 129.8% for 10 mg/L, respectively. The results confirm that the test substance concentrations were within the acceptability range. All validity criteria were satisfied during the test, therefore the test was considered to be valid. Based on the effect on mortality of test fishes, the 30 d NOEC, LOEC and LC50 value was determined to be 1.25, 2.5 and 5.39±0.499 mg/l (95% C. I. = 4.34 to 6.70 mg/l), respectively. On the basis of the effect on survival larvae of test fishes, the 30 d NOEC, LOEC and EC50 value was determined to be 1.25, 2.5 and 6.93± 0.51 mg/l mg/l, respectively. Where, based on hatching success, the 30 d NOEC, LOEC and EC10 value was determined to be 2.5, 5 and 4.42±0.55 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.

 

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 48 hours 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. Natural water was used as control, acetone with natural water was used as vehicle control and the same was used for test item formulation and test medium (15 µL acetone/150 mL natural water concentration was used). 25 mL glass beakers having a solution volume of 20 mL were used in the test. A range finding test (2 replicates/concentration having 5 daphnids/replicate) with the test concentrations of 0 (control), 0 (vehicle control), 1.5, 3, 6, 12, 24 mg/L was done prior to main study. No immobilization or abnormality was found in control groups and 1.5 mg/l, 3 mg/l, conc., but 20%, 30% and 100% in 6, 12, 24 mg/L concentrations were found, respectively. Hence, a main study (definitive test) (using a spacing factor of 1.65) was conducted using 0 (control), 0 (vehicle control), 3.23, 5.33, 8.79, 14.51 and 23.94 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 3.23 mg/L but 10%, 25%, 35% and 95% mortality were observed in the test concentrations of 5.33, 8.79, 14.51 and 23.94 mg/L, respectively. HPLC method was used for method validation and active ingredient analysis along with stability of the test item in the test medium. Test item was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during main study 0 h and 48 h avg. recovery 3.23, 8.79, 23.94 mg/L conc were 93.29% to 95.64%, 95.06% to 96.12% and 94.70% to 95.88% respectively, were found in acceptable range. 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 of 7.4 to 7.7 (at the begining of the test) and 7.0 to 7.5 (at the end of the test), temperature (20-21 °C), dissolve oxygen of 7.8 to 8.3 mg/l (at the begining of the test) and 7.2-7.9 mg/L (at the end of the test), hardness (165 mg CaCO3/L), photoperiod (16 h light- 8 h dark) and light intensity (1330 to 1340 Lux) was maintained in acceptable range throughout the test. Feed was not provided during the test. The 48-h EC50 of test chemical to daphnid,Daphnia magnaare was determined to be 13.47 mg/L with a 95% confidence interval of 12.33 to 14.61 mg/l, respectively. The 48-h EC50 of 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. 48h EC50 value with 95% confidence limits (upper limit, lower limit) were calculated by probit analysis using NCSS Software 2019, version 19.0.3. Since, the test chemical is readily biodegradable in water, test chemical was considered as non-toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be not classified as per the 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 Adams 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 Selenestrum capricornutum and it was done thrice a week. The test chemical will be prepared by dissolving 100 mg of test chemical in 100 mL of Adams 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 0.1, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 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 not been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on measured concentration. Test chemical conc. used in the defiinite study were 0, 0.1562, 0.3125, 0.625, 1.25 and 5 mg/l. 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. Adams medium was used as a test medium. Test conditions involve a temperature range of 18.4-21.5°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 and LOEC value was determined to be 0.625 and 1.25 mg/l, respectively. Thus, based on the NOEC value, test chemical was considered as toxic to aquatic invertebrates and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

 

Toxicity to aquatic algae and cyanobacteria:

This study was conducted as per OECD 201 (2011) to assess the acute toxicity effects of test chemical on the growth of green alga Pseudokirchneriella subcapitata(ATCC)following exposure of alga up to 72 h under static condition. Test system was initially procured from “American Type Culture Collection”, Chromachemie Laboratory Private Limited, later test system was sub-cultured (using OECD growth medium, OECD 201) in the test facility. 250 mL sterilized conical flasks covered with cap were used in the study. The test item was found to be soluble in acetone. Hence, acetone (concentration <0.1mL/L) with OECD growth medium was used as test medium. An inoculum culture in the test medium was prepared 3-4 days prior to test and culture conditions were maintained same as the test conditions. A range finding study (3 replicates/ concentration, 6 replicates for control group)with the test concentrations of1.5, 3, 6, 12, 24 mg/L were tested along with a control (0), vehicle control (0) group, prior to the main study. The inhibition in growth rate (0%, 0%, 0.27%, 28.39%, 39.15%, 89.02%, 94.64%) and yield (0%, 0%, 1.25%, 70.43%, 81.65%, 99.08%, 99.60%) were observed at 72 h in the test concentrations of 0, 0, 1.5, 3, 6, 12, 24 mg/L. Based on the results of range finding test following concentrations of 0 (control), 0 (vehicle control), 0.6, 1.3, 2.9, 6.4 and 14.1 mg/L were selected for the main study(3 replicates/ concentration, 6 replicates for control groups)as significant changes (inhibition algal growth rate) were observed in treatment groups during 72h test period. The inhibition in growth rate (0%, 0%, 0.46%, 2.91%, 33.92%, 45.76%, 88.98%) and yield (0%, 0%, 1.94%, 11.57%, 76.72%, 86.35%, 99.06%) were observed during 72h test period in the test concentrations of 0 (control), 0 (vehicle control), 0.6, 1.3, 2.9, 6.4 and 14.1 mg/L. A reference standard (Potassium dichromate) was tested to ensure the authenticity of the test in the lab. And the inhibition of growth rate (ErC50-72h) and yield (EyC50-72h) were found to be0.620 mg/L & 0.334 mg/L, respectively; and were found to be in acceptable range. The initial cell density was 10000 cells/mL.The algal cells in the control and vehicle control increased by 65.62, 63.3 times (>16 times) of the initial cell count during the 72-h exposure period, respectively. The mean coefficient of variation for section-by-section growth rate for the control cultures over the test period (0-72-h) was 25.84% (<35%). The coefficient of variation of average specific growth rate was 0.6% (<7%). Hence, fulfilling the all the validity criteria of the test. HPLC method was used for method validation and active ingredient analysis along with stability of the test item in the test medium. Test item was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during main study 0 hr and 72 hr avg. recovery for 0.6, 2.9 and 14.1 mg/L conc were 93.44% to 94.61%, 95.65% to 96.19%, 95.40% to 95.45%, respectively, were found in acceptable range. Continuous light having and average light intensity of 6748 to 6814 Lux, pH of 7.5 -7.8, temperature 21.2 -23.5°C along with a shaking speed of 110 RPM were provided and maintained for the test system throughout the test. 72h EC50 (growth rate and yield) values with 95% confidence limits were calculated by probit analysis. NOEC and LOEC of growth rate and yield were calculated by one way ANOVA (Kruskal-Wallis-Comparison Z-Value Test) using NCSS Software 2019, version 19.0.3. The cells were counted using Haemocytometer under illumination of the microscope at 24, 48, and 72 h after inoculation. 72-h EC50 of test item to alga on growth rate and yield are 5.47, 2.41 mg/L, respectively. 72-h LOEC of test item to alga on growth rate and yield is 6.4 mg/L. 72-h NOEC of test item to alga on growth rate and yield is 2.9 mg/L. Since, the test chemical is readily biodegradable in water, test chemical was considered as non-toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be not classified as per the CLP classification criteria.

 

Toxicity to microorganisms:

First study provides a means to predict the ‘minimum’ toxicity that a phenol willexert in the Microtox test. Toxicity data noted as ‘pT’ are listed as the negative logarithm of the millimolar concentration required to elicit a 50% reduction in light emission in the stated time (i.e. 5-, 15, or 30-min) and pT 30 was predicted to be 1.09.

First study was supported by second from peer reviewed journal, provide a means to predict the ‘minimum’ toxicity that a phenol willexert in the Microtox test.Toxicity data noted as ‘pT’ are listed as the negative logarithm of the millimolar concentration required to elicit a 50% reduction in light emission in the stated time (i.e. 5-, 15, or 30-min) and pT 30, pT15 and pT5 was predicted to be 1.52, 1.59 and 1.66 respectively.

Thus based on the above all results and studies conducted on the fish, invertebrates and algae, it was observed that the chemical is readily biodegradable in water and thus not classified in case of short term toxicity study, but while considering long term toxicity results chemical consider to be toxic and classified in aquatic chronic category 3. Thus on the basis of long term effects observation on aquatic lifes, chemical can be consider to be toxic and classified in aquatic chronic category 3 as per the CLP classification criteria.