3-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexan-1-ol

Endpoint summary

Description of key information

Short term toxicity to fish

1: Based on the mortality of freshwater fish Brachydanio rerio by the chemical exposure for 96 hrs, the LC50 was determine to be 17.6 mg/l and the LC0 and LC100 was 11.3 mg/l and 26.2 mg/l.

2: Based on the mortality of fishes Pimephales promelas after the exposure of chemical for 96 hrs, the LC50 was determine to be 18.9 mg/l.

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

 

Long term toxicity to fish

The Early-Life Stage Fish Toxicity Test was performed using Danio rerio (zebrafish). The study was performed in compliance with OECD 210 Guideline for the testing of chemicals; Fish, Early-life Stage Toxicity Test, (adopted 26 July, 2013). Solubility of the test item was performed by weighing 25.7 mg of test item in a glass vial and dissolved in 25 µL of acetone and transferring into the 250 ml volumetric flask, and made up to the mark using natural water for solubility check. The stability of the test item in natural water determined by analyzing the test concentrations of 0.01 and 100 mg/L at 0 hour, 24 hour, 48 hour, 72 hour and 96 hour showed that the test item concentration remained 80% to 120% (100.55% to 101.51 % for 0.01 mg/L and 98.66% to 99.94% for 100 mg/L).Range finding test was conducted with test concentrations at 0 (Control), 0 (Solvent Control), 5, 10, 25, 50 and 100 mg/ L. Each concentration contained two replicate and each replicates were exposed 20 fertilized eggs. Test item was formulated in acetone + natural water (not exceeding 100 µL /L). Hatching (percent) observed in control and solvent control groups were 92.5% and 85.0% whereas, 82.5% and 2.5% in the tested concentration of 5 and 10 mg/L for a period of 96 hours. Non-detachment of the tail observed in the control (3), solvent control (6) and in the tested concentration of 5 mg/ L (7) for a period of 96 hours. Coagulation observed was 39 in the tested concentration of 10 mg/ L, wheras Coagulation observed (40) in the tested concentration of 25, 50 and 100 mg/ L for a period of 96 hours. Based on the results of range finding test, the definitive was conducted with 8 test concentrations at 0 (Control), 0 (Solvent Control), 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/L, in a geometric series with a spacing factor of 2.5 by static renewal method. Test item was formulated in acetone+ natural water (not exceeding lO0µL /L). At the start of the test, each test concentration contained four replicate and each replicates were randomly distributed 20 fertilized eggs. Hatching success in the control and solvent control groups were 98.75% and 95.00% whereas, 93.75%, 90.00%, 88.75%, 87.50%, 71.25%, 63.75%, 57.50% and 47.50%, in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. Post-hatch larval survival in the control and solvent control groups were 91.139% and 92.105% whereas,  89.333%, 88.889%, 85.915%, 84.286%, 78.947%, 76.471 %, 69.565% and 60.526%, in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. Larval mortality (Corrected mortality) was 4.29%, 8.57%, 12.86%, 15.71 %, 35.71 %, 44.29%,54.29% and  67.14% in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. During the definitive test, no clinical sign exhibited in control group, solvent control group and in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391 and 0.977 mg/ L, whereas loss of buoyancy control (2+2 larvae) behaviour was observed in the tested concentration of 2.441 and 6.104 mg/ L. Abnormal behaviour like hypoactivity (2 larvae) observed in in the tested concentration of 2.441 mg/ L for a period of 30 after post hatch. Total mean weight in the control and solvent control groups were 0.034 and 0.032 g whereas, 0.028 g, 0.027 g, 0.025 g, 0.023 g, 0.022 g, 0.021 g, 0.018 g and 0.015 g in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. Total mean lengths in the control and solvent control groups were 11 mm whereas, 10.93 mm, 10.90 mm, 10.64 mm, 10.57 mm, 10.52 mm, 10.03 mm, 9.80 mm and 9.42 mm in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. During the definitive test period, all the beakers were incubated in the room under test conditions. The pH of the control and solvent control at the test start 7.3 and 7.2 and end of the test was 7.1 and 7.2 and therefore did not vary more than 1.5 units during the study. The pH of  all the tested concentrations was 7.3 to 7.9 at the beginning of the test and 7.3 to 7.6 at test termination. The temperature at the beginning was 26.0°C to 26.5°C and at test termination was 26.1 °C to 26.5°C. The Dissolved oxygen of the concentrations ranged from 74.5 to 91.5% ASV. The mean intensity of light ranged from 698 to 735 Lux. The test item content obtained in during the dose concentration analysis New sample solution range was 99.94 % to 101.22 % for 0.01 mg/L, 99.27 % to 100.84 % for 0.391 mg/Land 99.98 % to 101.22 % for 6.104 mg/L. For old sample solution range was 99.76 % to 101.56 % for 0.01 mg/L, 99.52 % to 101.01 % for 0.391 mg/Land 99.05 % to 101.04 % for 6.104 mg/L. Based on the mortality data, LC5o /EC,o value (Based on the larvae mortality, Fertility reduction (%) and Surviving larval reduction) was determined statistically by probit analysis using NCSS software 2022, v22.0.2. the 95% confidence limits were calculated by using the formula: LC,oIEC,o±1.96xstandard error. Based on the mortality data, NOEC and LOEC value were determined statistically by Fisher's Exact test (p value > 0.05) using NCSS software 2022, v22.0.2. Based on the hatching success data, NOEC and LOEC value were determined statistically by Kruskal-Wallis Multiple. Comparison Z-Value Test (z value > 1.96) using NCSS software 2022, v22.0.2. Based on the hatching success data, NOEC and LOEC value were determined statistically by Dunnett's Two Sided Multiple-Comparison Test (Alpha value > 0.05) using NCSS software 2022, v22.0.2. The LC50 of the test chemical in Dania rerio larvae mortality (30 days post hatch) was found to be 1.640 mg/L with 95% confidence limits between 1.062 mg/L and 2.218 mg/L. EC50 value for hatching reduction was found to be 5.037 mg/L with 95% confidence limits between 2.544 mg/L and 7.531 mg/L. EC50 value for surviving larval reduction was found to be > 6.104 mg/L. The NOEC and LOEC for larvae mortality was found to be 0.063 mg/L and 0.156 mg/L, respectively. On the basis of survival effect, the NOEC and LOEC were found to be 0.156 mg/L and 0.391 mg/L, respectively. Based on the NOEC value (i.e., 0.156 mg/l) (based on larave survival effect), the test chemical was considered to be classified in ''aquatic chronic category 2'' as per CLP classification criteria.

 

Short term toxicity to aquatic invertebrates

An acute immobilisation test was used to test how a range of concentrations of test chemical exerts different degrees of toxic effects on the swimming capability of Daphnia magna under otherwise identical test conditions. The test was performed in close resemblance to OECD guideline 202. The testing aim was to determine a EC50 after 48 hours of exposure to D. magna. The stock solution used for the exposure assessment was prepared by dissolving colourless dense liquid in acetone. Daphnids were exposed to chemical in 50 ml beakers in a volume of 25 ml of liquid solution containing both the chemical and media as specified in OECD 202. The beakers were placed in a temperature controlled room at 20±1 degrees Celsius. The D. magna (age ≤24) used for the test. The animals were exposed to stock solution prepared by adding dense colourless liiquid in acetone. There were 5 Daphnia per test vessels and 4 replicates per concentration. The positive control/reference substance used in the teste showed an expected result and gave a EC50 that corresponded to previous exposures with this chemical in D. magna. Immobilisation effects in D. mangaby chemical exposure was evident after 48 hours in exposure concentrations above 1 mg/L (i.e. EC20). The EC50 was defined as a concentration that immobilizes 50% of the exposed D. magna. The % of immobilization in D. magna after 48 hours of exposure to test chemical were used in a nonlinear regression by Graphpad Software Prism 4.0 (San Diego, US). Based on the immobility of daphnia magna by the chemical exposure for 48 hrs, the EC50 was determine to be 2.59 mg/l (95% C.I: 1.81-3.7 mg/L). Thus, based on this value, test chemical can be considered as toxic to aquatic organisms and thus can be classified as aquatic chronic category 2 as per the CLP classification criteria.

 

Long term toxicity to aquatic invertebrates

The test substance was used to evaluate its adverse effects to Daphnia magna. The test was conducted in accordance with the OECD 211 guidleines, for the period of 21 days. The test solutions was prepared in the ADAMS medium, the test concentration chosen for the exposure was 0.25 - 4 mg/L, with the geometric series of 2. the test was performed in semi static conditions, wherein the test chemical was renewed every alternate day, of the exposure period. The EC10 was calculated to be 0.148±0.85 mg/L. Based on the Effective concentration it can be classified into chronic 2 category of CLP criteria.

 

Toxicity to aquatic algae and cyanobacteria

The study was conducted to assess the effects of the test chemical on the growth of green Algae, Pseudokirchneriella subcapitata. This study was performed in compliance with OECD Guideline for Testing of chemicals OECD NO. 201, Adopted by the Council on 23 March 2006 and Council Regulation (EC) No. 440/2008, Annex Part C.3 'Freshwater Algae and Cyanobacteria, Growth Inhibition Test. Solubility of the test item was performed by weighing 103.0 mg of the test chemical in a glass vial and dissolving it in 10 µL of acetone and transferred it into the 100 ml volumetric flask and made up to the mark using natural water for solubility check. The stability of the test chemical in natural water determined by analyzing the test concentrations of 1 and 100 mg/L at O hour, 24 hours, 48 hours and 72 hours showed that the test chemical concentration remained 80% to 120% (98.27 % to 100.75 % for 1 mg/Land 98.87 % to 99.51 % for 100 mg/L) with respect to initial measured concentration and hence the dose verification for definitive test was performed at the beginning and at the termination of the test. The test concentrations were prepared in OECD TG 201 medium by dilution of stock and transferred 100 ml of each test solution into 250 ml Erlenmeyer flasks. Three replicates per test item concentration and six replicates for the controls were used for both range finding test and definitive test. The controls and treatment flasks were inoculated with Algae, Pseudokirchneriella subcapitata (pre-cultured for 3 days) and tested at an initial cell density of 10000 cells per ml, incubated under test condition for 72 hours The study was performed by a static method. The cells were counted using an Improved Neubaur's Haemocytometer under illumination of the microscope at 24, 48 and 72 hour after inoculation. A range finding test was conducted at test chemical concentrations of 5, 10, 25, 50 and 100 mg/L along with control groups without test chemicals. No significant changes in the appearance of algae cells were observed in controls and in the test concentrations during 72 hours test period. At the end of 72-hour observation, 1.95 %, 3.36 %, 5.40 %, 27.50 %, and 85.34 % in terms of the percent inhibition of growth rate and 7.27 %, 12.18 %, 18.86 %, 66.01 % and 98.23 % % in terms of the percent inhibition of yield was observed in the test concentrations of 5, 10, 25, 50 and 100 mg/L respectively. Based on the results of the range finding test, the definitive test was conducted at test chemical concentrations of 2.70, 6.48, 15.55, 37.32 and 89.58 mg/L in a geometric series with a factor of 2.4 along with control groups without test chemical. No significant changes in the appearance of algae cells were observed in controls and in the test concentrations during 72 hours test period. At the end of 72-hour observation, 1.69 %, 9.32 %, 18.74 %, 35.20 % and 84.73 % in terms of the percent inhibition of growth rate and 6.58 %, 31.44 %, 53.37 %, 76.60 % and 98.38 % % in terms of the percent inhibition of yield was observed in the test concentrations of 2.70, 6.48, 15.55, 37.32 and 89.58 mg/L respectively. During the Definitive test period, all the flasks were incubated in the room under test condition. The pH of the solvent control at the test start was 8.0 and at the termination of the test was 8.1 and therefore did not vary more than 1.5 units during the study. The pH of all the tested concentrations was 7.7 to 7.8 at the beginning of the test and 7.5 to 7.6 at test termination. The room temperature (Average) was between 21.3°C and 22.8°C. The temperature of the control and test concentration at the beginning was between 22.3°C and 22.8C and at test termination was between 22.5°C and 23.0°C. The average intensity of light ranged from 6716 to 6734 Lux and the % CV ranged from 0.29 % to 1.50 %, which proved that the light intensity is maintained within ±15% from the average light intensity. The test chemical available in the test medium (Acetone + natural water) was determined by a validated GC method. The test chemical concentration of test chemical in the test medium at the initiation (0 hour) was between 98.26 % and 99.04 % and the end of test (96 hours) was between 98.55 % and 99.92 % for 2.7 and 89.58 mg/L of the nominal test concentrations. The concentration of the test chemical has been satisfactorily maintained within 80 to 120% of the nominal concentration during the exposure period. Based on the results of this study, the ErCso (percent inhibition of growth rate) value for 72 hours of the test chemical was found to be 39.76 mg/L with 95% confidence limits between 32.88 mg/L and 46.64 mg/L. The EyCs (percent inhibition of yield) value for 72 hours of the test chemical was found to be 13.32 mg/L with 95% confidence limits between 11.30 mg/L and 15.34 mg/L. The LOEC and NOEC (inhibition of growth rate and yield) value for 72 hours was 15.55 mg/L and 6.48 mg/L. The results observed in this study meets all the validity criteria as per OECD 201 test guideline. Based on the ErC50 value, it can be concluded that the substance is likely to be hazardous to aquatic algae and can be considered to be classified as aquatic chronic category 3 as per the CLP classification criteria.

 

Toxicity to micro-organisms

The test chemical is likely to be toxic to microorganism  atleast in the concentartion range of 1 to 8 mg/l.

Additional information

Short term toxicity to fish:

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

 

Aim of this study was to determine the nature of chemical on the mortality of freshwater fish Brachydanio rerio for total exposure of 96 hrs. Chemical was analytically monetarized by GC. Test conducted under static system. After incubation period of 96 hrs, slow and inactive swimming behaviour and loss of equilibrium (uncontrolled movements) were also observed. Based on the mortality of freshwater fish Brachydanio rerio by the chemical exposure for 96 hrs, the LC50 was determine to be 17.6 mg/l and the LC0 and LC100 was 11.3 mg/l and 26.2 mg/l. Based on the LC50, chemical was consider as toxic and can be consider to to be classfied as aquatic chronic 3 category as per the CLP classification criteria.

 

Similar study was also conducted on other fish. Aim of this study was to determine the nature of chemical on the mortality of fish Pimephales promelas for total exposure of 96 hrs. Study was conducted comparable to standard method and in accordance with general accepted scientific standards. Nominal 5 concentrations in the range of 4.39 to 24.6 mg/l were used and the sample was analytically monetarized by GC method. 0.079 g 30 days old Pimephales promelas (Fathead Minnow) were used. Test conducted under the flow- through system. Observations of fish behaviour and body morphology at regular intervals. Affected fish lost schooling behavior, were hypoactive and underreactive to external stimuli. They had increased respiration were darkly colored and lost equilibrium prior to death. Based on the mortality of fishes Pimephales promelas after the exposure of chemical for 96 hrs, the LC50 was determine to be 18.9 mg/l. based on the LC50, chemical was consider as toxic and can be consider to not classified as aquatic chronic 3 category as per the CLP classification criteria.

 

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

 

Long term toxicity to fish:

The Early-Life Stage Fish Toxicity Test was performed using Danio rerio (zebrafish). The study was performed in compliance with OECD 210 Guideline for the testing of chemicals; Fish, Early-life Stage Toxicity Test, (adopted 26 July, 2013). Solubility of the test item was performed by weighing 25.7 mg of test item in a glass vial and dissolved in 25 µL of acetone and transferring into the 250 ml volumetric flask, and made up to the mark using natural water for solubility check. The stability of the test item in natural water determined by analyzing the test concentrations of 0.01 and 100 mg/L at 0 hour, 24 hour, 48 hour, 72 hour and 96 hour showed that the test item concentration remained 80% to 120% (100.55% to 101.51 % for 0.01 mg/L and 98.66% to 99.94% for 100 mg/L).Range finding test was conducted with test concentrations at 0 (Control), 0 (Solvent Control), 5, 10, 25, 50 and 100 mg/ L. Each concentration contained two replicate and each replicates were exposed 20 fertilized eggs. Test item was formulated in acetone + natural water (not exceeding 100 µL /L). Hatching (percent) observed in control and solvent control groups were 92.5% and 85.0% whereas, 82.5% and 2.5% in the tested concentration of 5 and 10 mg/L for a period of 96 hours. Non-detachment of the tail observed in the control (3), solvent control (6) and in the tested concentration of 5 mg/ L (7) for a period of 96 hours. Coagulation observed was 39 in the tested concentration of 10 mg/ L, wheras Coagulation observed (40) in the tested concentration of 25, 50 and 100 mg/ L for a period of 96 hours. Based on the results of range finding test, the definitive was conducted with 8 test concentrations at 0 (Control), 0 (Solvent Control), 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/L, in a geometric series with a spacing factor of 2.5 by static renewal method. Test item was formulated in acetone+ natural water (not exceeding lO0µL /L). At the start of the test, each test concentration contained four replicate and each replicates were randomly distributed 20 fertilized eggs. Hatching success in the control and solvent control groups were 98.75% and 95.00% whereas, 93.75%, 90.00%, 88.75%, 87.50%, 71.25%, 63.75%, 57.50% and 47.50%, in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. Post-hatch larval survival in the control and solvent control groups were 91.139% and 92.105% whereas,  89.333%, 88.889%, 85.915%, 84.286%, 78.947%, 76.471 %, 69.565% and 60.526%, in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. Larval mortality (Corrected mortality) was 4.29%, 8.57%, 12.86%, 15.71 %, 35.71 %, 44.29%,54.29% and  67.14% in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. During the definitive test, no clinical sign exhibited in control group, solvent control group and in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391 and 0.977 mg/ L, whereas loss of buoyancy control (2+2 larvae) behaviour was observed in the tested concentration of 2.441 and 6.104 mg/ L. Abnormal behaviour like hypoactivity (2 larvae) observed in in the tested concentration of 2.441 mg/ L for a period of 30 after post hatch. Total mean weight in the control and solvent control groups were 0.034 and 0.032 g whereas, 0.028 g, 0.027 g, 0.025 g, 0.023 g, 0.022 g, 0.021 g, 0.018 g and 0.015 g in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. Total mean lengths in the control and solvent control groups were 11 mm whereas, 10.93 mm, 10.90 mm, 10.64 mm, 10.57 mm, 10.52 mm, 10.03 mm, 9.80 mm and 9.42 mm in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. During the definitive test period, all the beakers were incubated in the room under test conditions. The pH of the control and solvent control at the test start 7.3 and 7.2 and end of the test was 7.1 and 7.2 and therefore did not vary more than 1.5 units during the study. The pH of  all the tested concentrations was 7.3 to 7.9 at the beginning of the test and 7.3 to 7.6 at test termination. The temperature at the beginning was 26.0°C to 26.5°C and at test termination was 26.1 °C to 26.5°C. The Dissolved oxygen of the concentrations ranged from 74.5 to 91.5% ASV. The mean intensity of light ranged from 698 to 735 Lux. The test item content obtained in during the dose concentration analysis New sample solution range was 99.94 % to 101.22 % for 0.01 mg/L, 99.27 % to 100.84 % for 0.391 mg/Land 99.98 % to 101.22 % for 6.104 mg/L. For old sample solution range was 99.76 % to 101.56 % for 0.01 mg/L, 99.52 % to 101.01 % for 0.391 mg/Land 99.05 % to 101.04 % for 6.104 mg/L. Based on the mortality data, LC5o /EC,o value (Based on the larvae mortality, Fertility reduction (%) and Surviving larval reduction) was determined statistically by probit analysis using NCSS software 2022, v22.0.2. the 95% confidence limits were calculated by using the formula: LC,oIEC,o±1.96xstandard error. Based on the mortality data, NOEC and LOEC value were determined statistically by Fisher's Exact test (p value > 0.05) using NCSS software 2022, v22.0.2. Based on the hatching success data, NOEC and LOEC value were determined statistically by Kruskal-Wallis Multiple. Comparison Z-Value Test (z value > 1.96) using NCSS software 2022, v22.0.2. Based on the hatching success data, NOEC and LOEC value were determined statistically by Dunnett's Two Sided Multiple-Comparison Test (Alpha value > 0.05) using NCSS software 2022, v22.0.2. The LC50 of the test chemical in Dania rerio larvae mortality (30 days post hatch) was found to be 1.640 mg/L with 95% confidence limits between 1.062 mg/L and 2.218 mg/L. EC50 value for hatching reduction was found to be 5.037 mg/L with 95% confidence limits between 2.544 mg/L and 7.531 mg/L. EC50 value for surviving larval reduction was found to be > 6.104 mg/L. The NOEC and LOEC for larvae mortality was found to be 0.063 mg/L and 0.156 mg/L, respectively. On the basis of survival effect, the NOEC and LOEC were found to be 0.156 mg/L and 0.391 mg/L, respectively. Based on the NOEC value (i.e., 0.156 mg/l) (based on larave survival effect), the test chemical was considered to be classified in ''aquatic chronic category 2'' as per CLP classification criteria.

 

Short term toxicity to aquatic invertebrate:

An acute immobilisation test was used to test how a range of concentrations of test chemical exerts different degrees of toxic effects on the swimming capability of Daphnia magna under otherwise identical test conditions. The test was performed in close resemblance to OECD guideline 202. The testing aim was to determine a EC50 after 48 hours of exposure to D. magna. The stock solution used for the exposure assessment was prepared by dissolving colourless dense liquid in acetone. Daphnids were exposed to chemical in 50 ml beakers in a volume of 25 ml of liquid solution containing both the chemical and media as specified in OECD 202. The beakers were placed in a temperature controlled room at 20±1 degrees Celsius. The D. magna (age ≤24) used for the test. The animals were exposed to stock solution prepared by adding dense colourless liquid in acetone. There were 5 Daphnia per test vessels and 4 replicates per concentration. The positive control/reference substance used in the teste showed an expected result and gave a EC50 that corresponded to previous exposures with this chemical in D. magna. Immobilisation effects in D. manga by chemical exposure was evident after 48 hours in exposure concentrations above 1 mg/L (i.e. EC20). The EC50 was defined as a concentration that immobilizes 50% of the exposed D. magna. The % of immobilization in D. magna after 48 hours of exposure to test chemical were used in a nonlinear regression by Graphpad Software Prism 4.0 (San Diego, US). Based on the immobility of daphnia magna by the chemical exposure for 48 hrs, the EC50 was determine to be 2.59 mg/l (95% C.I: 1.81-3.7 mg/L). Thus, based on this value, test chemical can be considered as toxic to aquatic organisms and thus can be classified as aquatic chronic category 2 as per the CLP classification criteria.

 

Long term toxicity to aquatic invertebrate:

The test substance was used to evaluate its adverse effects to Daphnia magna. The test was conducted in accordance with the OECD 211 guidleines, for the period of 21 days. The test solutions was prepared in the ADAMS medium, the test concentration chosen for the exposure was 0.25 - 4 mg/L, with the geometric series of 2. the test was performed in semi static conditions, wherein the test chemical was renewed every alternate day, of the exposure period. The EC10 was calculated to be 0.148±0.85 mg/L. Based on the Effective concentration it can be classified into chronic 2 category of CLP criteria.

 

 

Toxicity to algae and cyanobacteria:

The study was conducted to assess the effects of the test chemical on the growth of green Algae, Pseudokirchneriella subcapitata. This study was performed in compliance with OECD Guideline for Testing of chemicals OECD NO. 201, Adopted by the Council on 23 March 2006 and Council Regulation (EC) No. 440/2008, Annex Part C.3 'Freshwater Algae and Cyanobacteria, Growth Inhibition Test. Solubility of the test item was performed by weighing 103.0 mg of the test chemical in a glass vial and dissolving it in 10 µL of acetone and transferred it into the 100 ml volumetric flask and made up to the mark using natural water for solubility check. The stability of the test chemical in natural water determined by analyzing the test concentrations of 1 and 100 mg/L at O hour, 24 hours, 48 hours and 72 hours showed that the test chemical concentration remained 80% to 120% (98.27 % to 100.75 % for 1 mg/Land 98.87 % to 99.51 % for 100 mg/L) with respect to initial measured concentration and hence the dose verification for definitive test was performed at the beginning and at the termination of the test. The test concentrations were prepared in OECD TG 201 medium by dilution of stock and transferred 100 ml of each test solution into 250 ml Erlenmeyer flasks. Three replicates per test item concentration and six replicates for the controls were used for both range finding test and definitive test. The controls and treatment flasks were inoculated with Algae, Pseudokirchneriella subcapitata (pre-cultured for 3 days) and tested at an initial cell density of 10000 cells per ml, incubated under test condition for 72 hours The study was performed by a static method. The cells were counted using an Improved Neubaur's Haemocytometer under illumination of the microscope at 24, 48 and 72 hour after inoculation. A range finding test was conducted at test chemical concentrations of 5, 10, 25, 50 and 100 mg/L along with control groups without test chemicals. No significant changes in the appearance of algae cells were observed in controls and in the test concentrations during 72 hours test period. At the end of 72-hour observation, 1.95 %, 3.36 %, 5.40 %, 27.50 %, and 85.34 % in terms of the percent inhibition of growth rate and 7.27 %, 12.18 %, 18.86 %, 66.01 % and 98.23 % % in terms of the percent inhibition of yield was observed in the test concentrations of 5, 10, 25, 50 and 100 mg/L respectively. Based on the results of the range finding test, the definitive test was conducted at test chemical concentrations of 2.70, 6.48, 15.55, 37.32 and 89.58 mg/L in a geometric series with a factor of 2.4 along with control groups without test chemical. No significant changes in the appearance of algae cells were observed in controls and in the test concentrations during 72 hours test period. At the end of 72-hour observation, 1.69 %, 9.32 %, 18.74 %, 35.20 % and 84.73 % in terms of the percent inhibition of growth rate and 6.58 %, 31.44 %, 53.37 %, 76.60 % and 98.38 % % in terms of the percent inhibition of yield was observed in the test concentrations of 2.70, 6.48, 15.55, 37.32 and 89.58 mg/L respectively. During the Definitive test period, all the flasks were incubated in the room under test condition. The pH of the solvent control at the test start was 8.0 and at the termination of the test was 8.1 and therefore did not vary more than 1.5 units during the study. The pH of all the tested concentrations was 7.7 to 7.8 at the beginning of the test and 7.5 to 7.6 at test termination. The room temperature (Average) was between 21.3°C and 22.8°C. The temperature of the control and test concentration at the beginning was between 22.3°C and 22.8C and at test termination was between 22.5°C and 23.0°C. The average intensity of light ranged from 6716 to 6734 Lux and the % CV ranged from 0.29 % to 1.50 %, which proved that the light intensity is maintained within ±15% from the average light intensity. The test chemical available in the test medium (Acetone + natural water) was determined by a validated GC method. The test chemical concentration of test chemical in the test medium at the initiation (0 hour) was between 98.26 % and 99.04 % and the end of test (96 hours) was between 98.55 % and 99.92 % for 2.7 and 89.58 mg/L of the nominal test concentrations. The concentration of the test chemical has been satisfactorily maintained within 80 to 120% of the nominal concentration during the exposure period. Based on the results of this study, the ErCso (percent inhibition of growth rate) value for 72 hours of the test chemical was found to be 39.76 mg/L with 95% confidence limits between 32.88 mg/L and 46.64 mg/L. The EyCs (percent inhibition of yield) value for 72 hours of the test chemical was found to be 13.32 mg/L with 95% confidence limits between 11.30 mg/L and 15.34 mg/L. The LOEC and NOEC (inhibition of growth rate and yield) value for 72 hours was 15.55 mg/L and 6.48 mg/L. The results observed in this study meets all the validity criteria as per OECD 201 test guideline. Based on the ErC50 value, it can be concluded that the substance is likely to be hazardous to aquatic algae and can be considered to be classified as aquatic chronic category 3 as per the CLP classification criteria.

 

Toxicity to microorganisms:

Data available for the test chemical and read across chemicals selected on the basis of structural and functional similarity has been reviewed to determine its effect on microorganism. The studies are as mentioned below:

 

In the first study toxicity of test material was evaluated for microorganisms by disc diffusion method and the minimum inhibition concentration was evaluated using broth dilution method. The organisms used for the test were S. aureus, B. cereus , E. coli , P. aeruginosa. The turbidity of microorganisms was adjusted to 0.5. The inoculum were 1×1081×106CFU/ml for bacteria and fungi respectively. Inoculate was swabbed on Muller Hinton Agar. Sterile blank discs impregnated with 3, 5, 10 μl of oil in 10 μl of dimethyl sulfoxide (DMSO) were used and put on cultured plates. Disc containing DMSO and antibiotics were used as control. The minimum inhibition concentration of test material on bacteria S. aureus, B. cereus , E. coli , P. aeruginosa was observed to be > 8 mg/l.

 

Above study was supported by the second from secondary source. Toxicity of test material was evaluated for microorganisms by disc diffusion method and the minimum inhibition concentration was evaluated using broth dilution method. The organisms used for the test were S. aureus, B. cereus , E. coli , P. aeruginosa. The turbidity of microorganisms was adjusted to 0.5. The inoculum were 1×1081×106CFU/ml for bacteria and fungi respectively. Inoculate was swabbed on Muller Hinton Agar. Sterile blank discs impregnated with 3, 5, 10 μl of oil in 10 μl of dimethyl sulfoxide (DMSO) were used and put on cultured plates. Disc containing DMSO and antibiotics were used as control. The minimum inhibition concentration of test material on bacteria S. aureus, B. cereus, E. coli , P. aeruginosa was observed to be 1 mg/l.

 

The test chemical is likely to be toxic to microorganism atleast in the concentration range of 1 to 8 mg/l.

 

Thus based on the effects on the aquatic lifes, chemical consider to be toxic and classified as aquatic chronic 2 as per the CLP classification criteria.