Registration Dossier

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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Ecotoxicological information

Endpoint summary

Administrative data

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 1.951 cm and 0.047g, respectively. Fish were fed (commercial fish food) daily (except one day prior to the exposure)during acclimatization. Photoperiod and light intensity were maintained such as16 h light- 8 h dark, 775-836 Lux during experiment. Hardness of water was measured once during acclimatization and found to be 170 mg CaCO3/L, temperature, pH and dissolve oxygen were maintained between 22-23 °C,6.5 to 7.1, 6.4 to 7.0 mg/L, respectively, throughout the test. Fish were acclimatized for 7 days prior dosing. Natural water was used as dilution control and the same was used for formulation of test chemical. The study was initiated with a range finding test by using following concentrations of 0 (control), 6.25, 12.5, 25, 50, 100 mg/L. During range finding test,no mortality or abnormality was found in control group as well as in all the tested groups. Hence, a limit test was conducted at 100 mg/L test concentration, along with a control group (0). No mortality or abnormality was found in control group and 100 mg/L concentration. 7 fish were used/concentration during range finding and limit test. HPLC method was used for method validation and active ingredient analysis along with stability of the test chenical in the test medium. Test chemical was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during limit test 0 h and 96 h avg. recovery for 100 (100.04) mg/L conc were 96.21% & 96.10%, 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 chenical to zebrafish (Danio rerio) was determined to be >100 mg/L. 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.

 

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 50 mg in 500 mL of test media to get the final concentration of 100 mg/L followed by analytical determination. The final solubility obtained after analytical determination was 126.62 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 1.4599 and 4.4238, respectively. In spent samples of the test substance concentrations, the determined concentrations of test item were between 87.2 and 130.6% 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 20 embryos/vessel) were exposed to different test chemical conc. (i.e., 0 (Control), 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 16:8 light: dark conditions, Light intensity ranges from 500 to 1000 lux, temperature at the beginning of the test: 26.5°C, pH of 7.0 to 7.3 and dissolved oxygen of 88.35 to 99.61% air saturation value, respectively. Data were analyzed using appropriate statistical methods i.e., student t-test to calculate the EC50, LOEC and NOEC. The pH of the control at the test start and end was 7.1 and therefore did not vary more than 1.5 units during the study. 89.74% survival was observed at embryo stage in the control. 10.26% mortality was observed in control after 30 days whereas mortality at the end of exposure to test chemical at concentrations of 5 and 10 mg/l was 10.39% and 10.53%, respectively. At 96 hr, all the embryos were hatched in control. Hatching success in the control and in the tested concentration of 5 and 10 mg/ L was 97.5%, 96.25% and 95%, respectively. Larval survival until day 30 post-hatch in the control group was 89.74% thereby exceeding and satisfying the validity criteria for post-hatch survival (>75%) whereas in the group treated with test chemical at concentrations of 5 and 10 mg/l was 89.61% and 89.47%, respectively. All surviving juveniles were healthy in the control. For fish total lengths, determined on Day 30 post-hatch were 11, 7.52, 7.66 mm for test item at concentrations control, 5 and 10mg/L, respectively. In fresh samples of the test substance concentrations, the determined concentrations of test chemical were 89.4 to 117.6% for 5 mg/L and 87.2 to 125.2% for 10 mg/L, respectively whereas in old samples of the test substance concentrations, the determined concentrations of test chemical were 94 to 130.6% for 5 mg/l and 89.7 to 135.5% 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, survival larvae and hatching success of test fishes, the 30 d NOEC, LOEC, EC10, LC50, EC50 value was determined to be >10 mg/l. 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 invertebrates:

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 test daphnids were acclimatized 48 hours prior to the test chemical exposure. After exposure on day 0, daphnids were observed for immobilization at 24 and 48 h. M7 medium was used as control, and the same was used for test chemical formulation . 25 mL glass beakers having a solution volume of 20 mL were used in the test. . Main study (i.e. limit test) was conducted using 0 (control), 100 mg/L concentrations. 4 replicates/concentration having 5 daphnids/replicate was used for the main study. UV-Visible spectrophotometer method was used for 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. Feed was not provided during the test. The active ingredient content results were considered acceptable (80-120%) as during main study 0 h and after 48 hours of exposure, which 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 (7.2 -7.5), temperature (21-22°C), dissolve oxygen (7.1 - 8.6 mg/L), hardness (>140 mg CaCO3/L), photoperiod (16 h light- 8 h dark) was maintained in acceptable range throughout the test. Normal behavioural response and no immobilization (0% mortality) were observed up to 48 h followed by control groups and in the test concentrations of 100 mg/L. DO was reported to be ≥ 3 mg/l in the control and test vessel. Thus, fulfilling the validity criteria. The 48-h EC50 of test chemical to daphnid, Daphnia magna are >100 mg/l. The 48-h EC50 of reference substance (Potassium dichromate) to daphnid, Daphnia magna (found to be in acceptable range) is 0.65 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 chemical. Thus, 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 invertebrates:

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 linearity range selected for concentrations analysis and stock analysis was 0.3254, 0.6108, 6.8485, 1.1290, 1.4311, 1.6983 and 1.945 mg/L. The absorbance of resulting solution was measured using UV-VIS spectrophotometer against corresponding blank at lambda max (λmax). Standard curve was plotted against concentration verses absorbance and the maximum solubility was determined from the below standard curve. Analytical assessments were performed for selected test concentration at 1st, 2nd and 3rd week. The concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. Test chemical conc. used in the defiinite study were 0 and 10 mg/l. Thus, limit test was performed at test chemical conc. of 10 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 has 10 replicates and each replicate has 1 dapnids same number were taken for control goups. The evaluation of the NOEC was determined based on the number of offspring’s produced per living parent Daphnia. Defined concentrations of the test chemical led to a certain percentage reduction of the parthenogenetic reproduction rate at the end of the 21 day study period. The living offspring was counted daily along with the renewal of the test medium. However, test vessels were inspected daily for the occurrence of juveniles and marked accordingly. On the basis of the effect on reproduction of the test daphnids, the 21 d NOEC value was determined to be 10 mg/l. Thus, based on the NOEC value, test chemical was considered as non-toxic to aquatic invertebrates and hence, considered to be 'not classified' as per the CLP classification criteria.

 

Toxicity to algae and cyanobacteria:

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test solution was prepared by dissolving 200 mg of test chemical in 200 ml of OECD media, and allowed for stirring for 96 hours, which was then filtered and the final stock solution obtained was 1000 mg/L, verified analytically by UV-Vis Spectrophotometer. To have a better growth and visibility of cells, the initial cell count of the culture was kept 10,000 cells/mL. Further, exposure concentrations of 0 and 100 mg/l was from the stock test concentrations. Thus, limit test was performed. Test vessel were placed in orbital shaking incubator for 72 hrs at a temperature of 21 to 24 ± 2°C and with a continuous uniform illumination of 7000 -8000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. All control and test vessel conc. were performed in six replicates. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16, the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% (i.e., reported to be 2.63%) and the mean coefficient of variation of specific growth rate was not exceeded 35% (i.e., reported to be 13.75%), respectively 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 growth rate of the test organism, the 72 hr median effect concentration (ErC50) value was determined to be >100 mg/l (nominal conc.).Thus, test chemical was considered as non-toxic to aquatic algae at environmental relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.

 

Toxicity to microorganism:

1st study: Based on the growth rate inhibition of Pseudomonas putida by the test chemical exposure for 18 hrs, the EC10 was determine at 2050 mg/l.

2nd study: Based on the effect observed on Mycobacterium smegmatis by the test chemical, the EC0 was determine at 9707 mg/l.

Thus based on the above effects, chemical toxicity ranges from 2050 mg/l to 9707 mg/l.

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 1.951 cm and 0.047g, respectively. Fish were fed (commercial fish food) daily (except one day prior to the exposure)during acclimatization. Photoperiod and light intensity were maintained such as16 h light- 8 h dark, 775-836 Lux during experiment. Hardness of water was measured once during acclimatization and found to be 170 mg CaCO3/L, temperature, pH and dissolve oxygen were maintained between 22-23 °C,6.5 to 7.1, 6.4 to 7.0 mg/L, respectively, throughout the test. Fish were acclimatized for 7 days prior dosing. Natural water was used as dilution control and the same was used for formulation of test chemical. The study was initiated with a range finding test by using following concentrations of 0 (control), 6.25, 12.5, 25, 50, 100 mg/L. During range finding test,no mortality or abnormality was found in control group as well as in all the tested groups. Hence, a limit test was conducted at 100 mg/L test concentration, along with a control group (0). No mortality or abnormality was found in control group and 100 mg/L concentration. 7 fish were used/concentration during range finding and limit test. HPLC method was used for method validation and active ingredient analysis along with stability of the test chenical in the test medium. Test chemical was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during limit test 0 h and 96 h avg. recovery for 100 (100.04) mg/L conc were 96.21% & 96.10%, 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 chenical to zebrafish (Danio rerio) was determined to be >100 mg/L. 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.

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 50 mg in 500 mL of test media to get the final concentration of 100 mg/L followed by analytical determination. The final solubility obtained after analytical determination was 126.62 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 1.4599 and 4.4238, respectively. In spent samples of the test substance concentrations, the determined concentrations of test item were between 87.2 and 130.6% 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 20 embryos/vessel) were exposed to different test chemical conc. (i.e., 0 (Control), 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 16:8 light: dark conditions, Light intensity ranges from 500 to 1000 lux, temperature at the beginning of the test: 26.5°C, pH of 7.0 to 7.3 and dissolved oxygen of 88.35 to 99.61% air saturation value, respectively. Data were analyzed using appropriate statistical methods i.e., student t-test to calculate the EC50, LOEC and NOEC. The pH of the control at the test start and end was 7.1 and therefore did not vary more than 1.5 units during the study. 89.74% survival was observed at embryo stage in the control. 10.26% mortality was observed in control after 30 days whereas mortality at the end of exposure to test chemical at concentrations of 5 and 10 mg/l was 10.39% and 10.53%, respectively. At 96 hr, all the embryos were hatched in control. Hatching success in the control and in the tested concentration of 5 and 10 mg/ L was 97.5%, 96.25% and 95%, respectively. Larval survival until day 30 post-hatch in the control group was 89.74% thereby exceeding and satisfying the validity criteria for post-hatch survival (>75%) whereas in the group treated with test chemical at concentrations of 5 and 10 mg/l was 89.61% and 89.47%, respectively. All surviving juveniles were healthy in the control. For fish total lengths, determined on Day 30 post-hatch were 11, 7.52, 7.66 mm for test item at concentrations control, 5 and 10mg/L, respectively. In fresh samples of the test substance concentrations, the determined concentrations of test chemical were 89.4 to 117.6% for 5 mg/L and 87.2 to 125.2% for 10 mg/L, respectively whereas in old samples of the test substance concentrations, the determined concentrations of test chemical were 94 to 130.6% for 5 mg/l and 89.7 to 135.5% 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, survival larvae and hatching success of test fishes, the 30 d NOEC, LOEC, EC10, LC50, EC50 value was determined to be >10 mg/l. 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 invertebrates:

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 test daphnids were acclimatized 48 hours prior to the test chemical exposure. After exposure on day 0, daphnids were observed for immobilization at 24 and 48 h. M7 medium was used as control, and the same was used for test chemical formulation . 25 mL glass beakers having a solution volume of 20 mL were used in the test. . Main study (i.e. limit test) was conducted using 0 (control), 100 mg/L concentrations. 4 replicates/concentration having 5 daphnids/replicate was used for the main study. UV-Visible spectrophotometer method was used for 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. Feed was not provided during the test. The active ingredient content results were considered acceptable (80-120%) as during main study 0 h and after 48 hours of exposure, which 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 (7.2 -7.5), temperature (21-22°C), dissolve oxygen (7.1 - 8.6 mg/L), hardness (>140 mg CaCO3/L), photoperiod (16 h light- 8 h dark) was maintained in acceptable range throughout the test. Normal behavioural response and no immobilization (0% mortality) were observed up to 48 h followed by control groups and in the test concentrations of 100 mg/L. DO was reported to be ≥ 3 mg/l in the control and test vessel. Thus, fulfilling the validity criteria. The 48-h EC50 of test chemical to daphnid, Daphnia magna are >100 mg/l. The 48-h EC50 of reference substance (Potassium dichromate) to daphnid, Daphnia magna (found to be in acceptable range) is 0.65 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 chemical. Thus, 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 invertebrates:

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 linearity range selected for concentrations analysis and stock analysis was 0.3254, 0.6108, 6.8485, 1.1290, 1.4311, 1.6983 and 1.945 mg/L. The absorbance of resulting solution was measured using UV-VIS spectrophotometer against corresponding blank at lambda max (λmax). Standard curve was plotted against concentration verses absorbance and the maximum solubility was determined from the below standard curve. Analytical assessments were performed for selected test concentration at 1st, 2nd and 3rd week. The concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. Test chemical conc. used in the defiinite study were 0 and 10 mg/l. Thus, limit test was performed at test chemical conc. of 10 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 has 10 replicates and each replicate has 1 dapnids same number were taken for control goups. The evaluation of the NOEC was determined based on the number of offspring’s produced per living parent Daphnia. Defined concentrations of the test chemical led to a certain percentage reduction of the parthenogenetic reproduction rate at the end of the 21 day study period. The living offspring was counted daily along with the renewal of the test medium. However, test vessels were inspected daily for the occurrence of juveniles and marked accordingly. On the basis of the effect on reproduction of the test daphnids, the 21 d NOEC value was determined to be 10 mg/l. Thus, based on the NOEC value, test chemical was considered as non-toxic to aquatic invertebrates and hence, considered to be 'not classified' as per the CLP classification criteria.

 

Toxicity to algae and cyanobacteria:

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test solution was prepared by dissolving 200 mg of test chemical in 200 ml of OECD media, and allowed for stirring for 96 hours, which was then filtered and the final stock solution obtained was 1000 mg/L, verified analytically by UV-Vis Spectrophotometer. To have a better growth and visibility of cells, the initial cell count of the culture was kept 10,000 cells/mL. Further, exposure concentrations of 0 and 100 mg/l was from the stock test concentrations. Thus, limit test was performed. Test vessel were placed in orbital shaking incubator for 72 hrs at a temperature of 21 to 24 ± 2°C and with a continuous uniform illumination of 7000 -8000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. All control and test vessel conc. were performed in six replicates. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16, the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% (i.e., reported to be 2.63%) and the mean coefficient of variation of specific growth rate was not exceeded 35% (i.e., reported to be 13.75%), respectively 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 growth rate of the test organism, the 72 hr median effect concentration (ErC50) value was determined to be >100 mg/l (nominal conc.).Thus, test chemical was considered as non-toxic to aquatic algae at environmental relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.

Toxicity to microorganism:

Based on the experimental studies for the test chemical and structurally and functionally similar read across chemicals, studies have been were reviewed and mention as below:

 

Objective of this study was to determine the effect of test chemical on the growth of microorganism. Test conducted for 18 hrs. Based on the growth rate inhibition of Pseudomonas putida by the test chemical exposure for 18 hrs, the EC10 was determine at 2050 mg/l.

Above first study was supported by the second study from handbook. Aim of this study was to determine the effect of test chemical on the growth of microorganism Mycobacterium smegmatis. Based on the effect observed on Mycobacterium smegmatis by the test chemical, the EC0 was determine at 9707 mg/l.

Thus based on the above effects, chemical toxicity ranges from 2050 mg/l to 9707 mg/l.

Hence on the basis of effects observation on fish, invertebrates and algae, chemical consider to be nontoxic and not classified as per the CLP classification criteria.