Tetrabutylammonium bromide

Endpoint summary

Description of key information

Stability:

Hydrolysis:

The hydrolysis half-life value of the test chemical can be expected to be ≥ 1 yr at pH 4, 7 and 9 & at a temperature of 20°C or 5 to 63 days, respectively. Thus, based on this half-life value, it can be concluded that the test chemical undergoes slow to negligible hydrolysis in water.

Biodegradation:

Biodegradation in water

42-days Closed Bottle test following the OECD guideline 301 D was performed to determine the ready biodegradability of the test chemical. The study was performed at a temperature of 20°C. The test system included control, test item and reference item and toxicity control. the test was conducted using activated sludge, . The sampling site for collection of the activated sludge was selected ensuring that no known history of its contamination with the test item within the previous four years considering the history of possible agricultural, industrial or domestic inputs. The sampling depth was 1-2 feet from the aeration tank. The temperature of the activated sludge was measured (38º C) at the site of collection. Oxygen concentration of the activated sludge sample was 2.9 mg/L. The sample was transported to the test facility within 3 hours from collection and kept it aerobic during transport. This was pre-conditioned at the test temperature. 1 mL supernatant was diluted to 100 mL with mineral medium and from this solution 0.125 mL was added to 125 mL test bottles. This gave the bacterial count as 10e4 to 10e6 CFU/L. .The concentration of test and reference item (Sodium Benzoate) chosen for both the study was 4 mg/L. OECD mineral medium was used for the study. ThOD (Theoretical oxygen demand) of test and reference item was determined by calculation. % degradation was calculated using the values of BOD and ThOD for test item and reference item. The oxygen consumed by the test systems was corrected for oxygen consumption occurring in the blank test systems. The BOD Values (mgO2/mg) and percent biodegradation results for each test system are reported in tabes in additional information. The BOD42 value of test chemical was observed to be 1.03 mgO2/mg. ThOD was determined by calculation as 2.41 mgO2/mg. % degradation was calculated using the values of BOD and ThOD for test item and was determined to be  42.7 % at 20 ± 1°C. The % degradation of procedure control (reference item) was also calculated using BOD & ThOD and was determined to be 89.82 %. Degradation of Sodium Benzoate exceeds 64.67 % on 7 days & 88.62 % on 14th day. The activity of the inoculum is thus verified and the test can be considered as valid. The toxicity control was > 25% after 14 days of exposure. Based on the results, the test chemical, under the test conditions, was considered to be inherently biodegradable in nature.

Biodegradation in water: simulation testing on ultimate degradation in surface water

Aerobic mineralisation of test chemical in water was studies as per the principles of the OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test) (Adopted 13th April 2004) under aerobic conditions. The surface water was collected from Kaveri River, Sangama, Ramnagar District, Karnataka State, India in a thoroughly cleansed container. The sampling site for collection of the surface water was selected ensuring that no known history of its contamination with the test item or its structural analogues within the previous four years considering the history of possible agricultural, industrial or domestic inputs. The pH and temperature of the water was measured at the site of collection and the depth of sampling and the appearance of the water sample. (e.g. color and turbidity) was also noted. Oxygen concentration of the surface layer was measured in order to demonstrate aerobic conditions. Depth of sampling was 1-2 feet and surface water was clear with no turbidity. The test water was stored at 4 to 6°C with continuous aeration prior use for a period not more than 4 weeks. Temperature (°C) at time of collection was 21.8°C, pH of temperature was 6.83, Oxygen concentration (mg/l) of 4.8 mg/l,  Dissolved organic carbon (%) of 3.9 mg/l, colony count consists of 4500 CFU/ml, Total organic carbon (TOC) of 3.8 mg/l, Nitrate (NO3- ) of 4.5 mg/l, Nitrite (NO2- ) of 0.62 mg/l, P of <0.1 mg/l, Orthophosphates (PO43-) of 0.19 mg/l, Total ammonia tot (NH4+ ) of <0.3 mg/l and BOD of <2.0 mg/l, respectively. Prior to use of surface water, the coarse particles were removed by filtration through a 100 μm mesh sieve. Test chemical conc. used in the study was 10 μg/L as low dose and 100 μg/L as high dose, respectively. Study was performed in duplicates in a 250 ml conical flasks which was covered with cotton plugs under continuous darkness. Test conditions involve a temperature of 12±2°C, pH of  6.83. Test vessel was kept in an incubator shaker at 12 ± 2°C in dark. Aerobic condition was maintained in the test system by continuous shaking. Agitation was provided to facilitate oxygen transfer from the headspace to the liquid so that aerobic conditions were adequately maintained. Additional to test vessels, 1 blank test vessel containing only the test water for all sampling intervals was included, 1 blank test vessel containing only the sterile test water was also treated at 10 µg/L (0.01 µg/mL) and 100 µg/L (0.1 µg/mL) conc. and duplicate test vessels with reference (aniline) (conc. 10 μg/l i.e. 0.01 mg/l) was also kept in the study. The concentration of test chemical residues in samples collected at different pre-determined interval zero-time (immediately after treatment day 0), day 1, day 3 day 7, day 14, day 28, day 45 and day 60 were diluted suitably with acetonitrile and at each sampling occasion, duplicate aliquots from each test concentration were subjected to analysis by a validated LC-MS/MS method. Simutaneously, samples were removed at regular intervals, measured pH and oxygen concentration. After that the samples were diluted at 1:1, v/v ratio with methanol to prevent further degradation prior to LC-MS/MS analysis. Shaking was continued at 12 ± 2°C in dark for using in other sampling intervals. The surface water samples were analyzed for the residues of test item by liquid chromatography with positive-ion electrospray ionization (ESI) tandem mass spectrometry using the mass ion transition m/z 244.1 -> 143.2 for primary quantification and the mass ion transition m/z 244.1 -> 101.1 for qualitative confirmation. High performance liquid chromatograph (Exion HPLC) equipped with a mass spectrometer (TQ 5500) was used with a column of  Phenomenex Luna, C18 (2), 4.6mm×150mm i.d., 3.0µm, column oven temperature of 40°C, mobile phase consists of Solvent A : 5 mM ammonium formate in Milli-Q® water and Solvent B : Acetonitrile in a ratio of 15 : 85, v/v, flow rate of 0.6 mL/min with splitter, respectively. Detection method involve the use of MS. Linearity range was evaluated to be in the range of 0.00026-0.02064 µg/ml, respectively. During method validation, acceptable recoveries were generated for the samples fortified at LOQ and 10 LOQ level. The % RSD (precision) was ≤20% at each fortification level. Recovery data from these samples demonstrated that test chemical was stable during analysis. The recoveries of all the samples analyzed were in the range of 70-110% with %RSD ≤ 20%. Analysis of the Day 0 samples at 10 μg/L and 100 μg/L test concentrations demonstrated quantitative recovery of test chemical. The average amount of test chemical present was 107.5% and 2.8% & 96.9% and 3.0% at Day 0 and Day 60, respectively following application of test chemical to test water at 10 μg/L (low dose) and 100μg/L (high dose). The average amount of test chemical present was 105.1% and 61.6% & 108.0% and 59.1% at Day 0 and Day 60, respectively following application of test chemical to sterile test water at 10 μg/L (low dose) and 100μg/L (high dose). The DT50 value was determined to be 10.2 d and 10.4 d at test chemical conc. of 10 μg/l and 100 μg/l at 12°C, respectively. 90% of test chemical in natural surface water was determined after 33.7 d and 34.5 d at test chemical conc. of 10 μg/l and 100 μg/l, respectively. Based on the these results, test chemical was degraded in surface water and sterile surface water. Hence, test chemical was considered to be not persistent in water.

Biodegradation in water: sediment simulation testing

In accordance with Annex IX column 2 of REACH regulation, test for this endpoint is scientifically not necessary and does not need to be conducted, since the substance is readily biodegradable i.e. not persistent based on the experimental result of surface water simulation biodegradation study.

 

Biodegradation in soil

The half-life period of test chemical in soil was estimated using Level III Fugacity Model by EPI Suite version 4.1 estimation database (2018). If released into the environment, 81% of the chemical will partition into soil according to the Mackay fugacity model level III. The half-life period of test chemical in soil is estimated to be 17.33 days (416 hrs). Based on half-life value, it is concluded that the test chemical is not persistent in the soil environment and the exposure risk to soil dwelling animals is moderate to low.

Bioaccumulation:

Bioaccumulation study in fish was conducted for determining the bioconcentration factor of test chemical. The study was performed in accordance with the OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test and EPA OPPTS 850.1730 (Fish Bioconcentration Test), respectively. Danio rerio (Zebra fish) (male) of total length 2.5 to 3.2 cm obtained from commercial fish farm, Manimangalam was used as a test fishes. Fish used in the tests were free from observable diseases and abnormalities. Test fishes were fed daily throughout the study period. Fishes were fed with an appropriate diet of known lipid and total protein content in an amount sufficient to keep them in a healthy condition and to maintain body weight. Fish species identification was done externally at Fisheries College and Research Institute (FCRI), Ponneri, Tiruvallur District, Tamil Nadu. Acclimatization of the stock population of test fishes were done for 2 weeks. Test conditions were same as the test. Feeding to test fishes were done in the same manner as that done during the study. Preliminary study was carried out. A limit test was conducted at 100 mg/l alongwith control for 96 hrs. 7 fishes were exposed to 100 mg/l for a period of 96 hrs. An equal control was also maintained. No mortality or toxicity signs were observed during the study period upto 96 hrs. The highest conc. (1 mg/l) of the test chemical was selected base on 1% of its acute LC50 (100 mg/l). The second conc. (0.1 mg/l) was selected from the one above by factor of 10. Thus, exposure conc. were 0.1 and 1.0 mg/l, respectibely. The blended water was used in the study. It was prepared by mixing the reverse osmosis water and well water in the ratio of 3:1. This water was used during the acclomatization, preliminary experiment, uptake and depuration phase. The pH, hardness, total solids, total organic carbon (TOC), anions, cations, heavy metals and the presence of pesticides were analyzed.Glass aquaria (glass tank) of 30 lit capacity was used as a test vessel. Test chemical conc. used for the study were 0.1 and 1.0 mg/l. Total 72 fishes/vessel for each test group were exposed with the test chemical. Study was performed under semi-static conditions and test medium was renewed thrice in a week and durng the renewal. fish from each group was transferred to a new medium in which test conc. are exposed in a test chamber. Biomass loading rate during the study was 0.1 to 1.0 g/l, respectively. One control was run in addition to the test series. A control group of fish was held under experimental conditions except for the absence of the test chemical. The leftout food and faeces were siphoned daily from the test chambers shortly after feeding (30 mins to 1 hr). Test chambers were kept clean as possible throughout the test to keep the conc. of organic matter as low as possible to avoid the presence of organic carbon that may limit the bioavailability of the test chemical. The blended water was used in the study. It was prepared by mixing the reverse osmosis water and well water in the ratio of 3:1. This water was used during the acclomatization, preliminary experiment, uptake and depuration phase. The pH, hardness, total solids, total organic carbon (TOC), anions, cations, heavy metals and the presence of pesticides were analyzed. Test conditions involve a pH of 7.39 to 7.97, hardness of 139 to 140 mg/l as CaCO3, temperature of 23.4 to 25°C, dissolved oxygen of 84.3 to 102.7%, TOC of < 5 mg/l, respectively. Uptake phase was carried out for 28 d and depuration phase for 14 d, respectively. Photoperiod during the uptake phase was 12: 12 light:dark conditions controlled by an automatic timer and depuration phase was 6:6 light:dark conditions, respectively. All experiments were performed in triplicates.The leftout food and faeces were siphoned daily from the test chambers shortly after feeding (30 mins to 1 hr). Test chambers were kept clean as possible throughout the test to keep the conc. of organic matter as low as possible to avoid the presence of organic carbon that may limit the bioavailability of the test chemical. After removal of lipid content from the sample, the aqueous layer was transferred into 50 mL centrifuge tube and the volume was adjusted to 10 mL using acetonitrile. The sample was homogenized for 2 minutes using tissumizer. The sample solution was centrifuged at 3000 rpm and decanted, A 2 mL of sample solution was filtered through 0.2 um PTFE and analysed for active content. The final solution was filtered and analyzed by LC-MS/MS conditions. HPLC system consists of Agilent 1290 Infinity UHPLC, Agilent Zorbax Eclipse XDB (2.1 mm X 75 mm, 3.5 μm), column temperature of 40°C, injection volume of 10 μl, flow rate of 1.0 ml/min, auto dampler temp. of 10°C. Gradient conditions involve the use of 0.1% acetic acid in 90/10 (MilliQ water/ Methanol) and 0.1%  acetic acid in acetonitrile, respectively. Total run time was 9.0 mins with a retention time of 1.5 mins. MS conditions involves the detector of Agilent 6490 LC-MS/MS System, coupled with Mass Hunter instrument control and data processing software, AJS-ESI interface, Positive polarity, Gas temperature of 260°C, Gas flow of 16 L/min, Nebulizer gas flow of 50 Psi, Sheath gas temperature of 400°C, Sheath gas flow of 11 L/min, Capillary voltage of 3500V, CAV of 4 V and Dwell time of 200 sec., respectively. During the uptake and depuration phase, the fish samples were extracted immediately and analyzed for active content. The uptake phase rate constants (k1), depuration phase rate constant (k2) was calculated by plotting the Ln (comc. of fish, mg/kg) Vs occasions (days). During the acclimatization period, there was no mortaliy observed and fish was found to be healthy. In preliminary test: there was no mortalirty or toxicity signs in test fishes. During the study, the water temperature variation was less than + 2°C which ranged from 23.4 to 25.0°C, the concentration of dissolved oxygen had not been fallen below 60% saturation and the range was 84.3 to 102.7%, the concentration of the test item (0.1 & 1.0 mg/L) in the chambers was maintained within + 20% of the mean of the measured values during the uptake phase, the concentration of the test item (0.1 & 1.0 mg/L) was below its limit of solubility in water, taking into account the effect that the test water may have on effective solubility, no mortality and other adverse effect was found in both control and treated fish during the uptake and depuration phases. No significant differences in average growth between the test and the control groups of sampled fish (Hence there was no indication of toxic effect of the test chemical, thereby fulfilling the validity criteria and hence, study was considered to be valid.The Bio-concentration factor (BCF) was determined as 0,8394 and 0.4624 for 0.1 mg/L and 1.0 mg/L concentrations respectively in the uptake phase. Further, based on the overall rate constants of the uptake and depuration phases, the kinetic Bio-concentration factor (BCFk) was determined to be -0.1261 and -0.3114 for the concentrations 0.1 mg/L and 1.0 mg/L respectively. From the growth rate analysis, the growth corrected kinetic bio-concentration factor (BCFkg) was determined to be -0.1240 and -0.3173 for the concentrations 0.1 mg/L and 1.0 mg/L respectively. Based on the lipid content analysis, the lipid normalized kinetic bio-concentration factor (BCFkl) was determined to be -0.1852 and -0.5097 for the concentrations 0.1 mg/L and 1.0 mg/L respectively in the uptake phase. Since, the BCF value did not exceeded the threshold limit of 2000, thus, test chemical was considered to be non-bioaccumulative,

Transport and distribution:

Adsorption/Desorption:

The Adsorption Coefficient of test chemical was determined as per the HPLC method (OECD Guideline-121). The Log Koc value was determined to be 1.280 ± 0.003 at 25°C.This log Koc value indicates that the test chemical has a negligible sorption to soil and sediment therefore has rapid migration potential to ground water.

Additional information

Stability:

Hydrolysis:

Data available for the test chemicals has been reviewed to determine the half-life of the test chemical. The studies are as mentioned below:

 

The half-life of the test chemical was determined at different pH range. The study was performed at pH of 4, 7 and 9, & at a temperature of 20°C, respectively. The half-life period of test chemical was determined to be ≥ 1 yr at pH 4, 7 and 9, respectively & at a temperature of 20°C and thus test chemical was reported to be hydrolytically stable. On the basis of this, test chemical is considered to be not hydrolysable.

                               

In another study, the half-life of the test chemical was determined. The half-life value of test chemical was determined to be ranges from 5 to 63 days, respectively. Thus, based on the value, test chemical is considered to undergo slow to negligible hydrolysis in water.

 

For the test chemical, the hydrolysis half-life value was determined. The hydrolysis of the quaternary nitrogen to a tertiary amine and an alcohol is thermodynamically very unfavorable with a delta G estimated from the delta H of reaction greater than +400 kJ mole. This is predicted to be a very endothermic reaction and should occur only under conditions of very high temperature. Thus, it can be concluded that the test chemical is stable in water and has a hydrolysis half-life of > 1 yr. On the basis of this, test chemical is considered to be not hydrolysable.

 

On the basis of the available experimental studies, the hydrolysis half-life value of the test chemical can be expected to be ≥ 1 yr at pH 4, 7 and 9 & at a temperature of 20°C or 5 to 63 days, respectively. Thus, based on this half-life value, it can be concluded that the test chemical undergoes slow to negligible hydrolysis in water.

Biodegradation:

Biodegradation in water:

Experimental studies have been reviewed biodegradation in water endpoint and their results are summarized below.

42-days Closed Bottle test following the OECD guideline 301 D was performed to determine the ready biodegradability of the test chemical. The study was performed at a temperature of 20°C. The test system included control, test item and reference item and toxicity control. the test was conducted using activated sludge, . The sampling site for collection of the activated sludge was selected ensuring that no known history of its contamination with the test item within the previous four years considering the history of possible agricultural, industrial or domestic inputs. The sampling depth was 1-2 feet from the aeration tank. The temperature of the activated sludge was measured (38º C) at the site of collection. Oxygen concentration of the activated sludge sample was 2.9 mg/L. The sample was transported to the test facility within 3 hours from collection and kept it aerobic during transport. This was pre-conditioned at the test temperature. 1 mL supernatant was diluted to 100 mL with mineral medium and from this solution 0.125 mL was added to 125 mL test bottles. This gave the bacterial count as 10e4 to 10e6 CFU/L. .The concentration of test and reference item (Sodium Benzoate) chosen for both the study was 4 mg/L. OECD mineral medium was used for the study. ThOD (Theoretical oxygen demand) of test and reference item was determined by calculation. % degradation was calculated using the values of BOD and ThOD for test item and reference item. The oxygen consumed by the test systems was corrected for oxygen consumption occurring in the blank test systems. The BOD Values (mgO2/mg) and percent biodegradation results for each test system are reported in tabes in additional information. The BOD42 value of test chemical was observed to be 1.03 mgO2/mg. ThOD was determined by calculation as 2.41 mgO2/mg. % degradation was calculated using the values of BOD and ThOD for test item and was determined to be  42.7 % at 20 ± 1°C. The % degradation of procedure control (reference item) was also calculated using BOD & ThOD and was determined to be 89.82 %. Degradation of Sodium Benzoate exceeds 64.67 % on 7 days & 88.62 % on 14th day. The activity of the inoculum is thus verified and the test can be considered as valid. The toxicity control was > 25% after 14 days of exposure. Based on the results, the test chemical, under the test conditions, was considered to be inherently biodegradable in nature.

Next study was reviewed from authoritative database (J check, 2018) in this Biodegradation experiment was conducted for 28 days for evaluating the percentage biodegradability of test chemical in this experiment activated sludge was used as a test inoculums. The initial test substance conc. used in the study was 7.2 mg/l. The percentage degradation of test substance was determined to be 0% by O2 consumption, DOC removal and HPLC parameter in 28 days. Thus, based on percentage degradation, test chemical is considered to be not readily biodegradable in nature.

 

By considering the result of first study which was performed according to OECD guideline and of Klimisch rating 1 the test chemical is considered to be ultimate inherently biodegradable in nature.

Biodegradation in water: simulation testing on ultimate degradation in surface water

Aerobic mineralisation of test chemical in water was studies as per the principles of the OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test) (Adopted 13th April 2004) under aerobic conditions. The surface water was collected from Kaveri River, Sangama, Ramnagar District, Karnataka State, India in a thoroughly cleansed container. The sampling site for collection of the surface water was selected ensuring that no known history of its contamination with the test item or its structural analogues within the previous four years considering the history of possible agricultural, industrial or domestic inputs. The pH and temperature of the water was measured at the site of collection and the depth of sampling and the appearance of the water sample. (e.g. color and turbidity) was also noted. Oxygen concentration of the surface layer was measured in order to demonstrate aerobic conditions. Depth of sampling was 1-2 feet and surface water was clear with no turbidity. The test water was stored at 4 to 6°C with continuous aeration prior use for a period not more than 4 weeks. Temperature (°C) at time of collection was 21.8°C, pH of temperature was 6.83, Oxygen concentration (mg/l) of 4.8 mg/l,  Dissolved organic carbon (%) of 3.9 mg/l, colony count consists of 4500 CFU/ml, Total organic carbon (TOC) of 3.8 mg/l, Nitrate (NO3- ) of 4.5 mg/l, Nitrite (NO2- ) of 0.62 mg/l, P of <0.1 mg/l, Orthophosphates (PO43-) of 0.19 mg/l, Total ammonia tot (NH4+ ) of <0.3 mg/l and BOD of <2.0 mg/l, respectively. Prior to use of surface water, the coarse particles were removed by filtration through a 100 μm mesh sieve. Test chemical conc. used in the study was 10 μg/L as low dose and 100 μg/L as high dose, respectively. Study was performed in duplicates in a 250 ml conical flasks which was covered with cotton plugs under continuous darkness. Test conditions involve a temperature of 12±2°C, pH of  6.83. Test vessel was kept in an incubator shaker at 12 ± 2°C in dark. Aerobic condition was maintained in the test system by continuous shaking. Agitation was provided to facilitate oxygen transfer from the headspace to the liquid so that aerobic conditions were adequately maintained. Additional to test vessels, 1 blank test vessel containing only the test water for all sampling intervals was included, 1 blank test vessel containing only the sterile test water was also treated at 10 µg/L (0.01 µg/mL) and 100 µg/L (0.1 µg/mL) conc. and duplicate test vessels with reference (aniline) (conc. 10 μg/l i.e. 0.01 mg/l) was also kept in the study. The concentration of test chemical residues in samples collected at different pre-determined interval zero-time (immediately after treatment day 0), day 1, day 3 day 7, day 14, day 28, day 45 and day 60 were diluted suitably with acetonitrile and at each sampling occasion, duplicate aliquots from each test concentration were subjected to analysis by a validated LC-MS/MS method. Simutaneously, samples were removed at regular intervals, measured pH and oxygen concentration. After that the samples were diluted at 1:1, v/v ratio with methanol to prevent further degradation prior to LC-MS/MS analysis. Shaking was continued at 12 ± 2°C in dark for using in other sampling intervals. The surface water samples were analyzed for the residues of test item by liquid chromatography with positive-ion electrospray ionization (ESI) tandem mass spectrometry using the mass ion transition m/z 244.1 -> 143.2 for primary quantification and the mass ion transition m/z 244.1 -> 101.1 for qualitative confirmation. High performance liquid chromatograph (Exion HPLC) equipped with a mass spectrometer (TQ 5500) was used with a column of  Phenomenex Luna, C18 (2), 4.6mm×150mm i.d., 3.0µm, column oven temperature of 40°C, mobile phase consists of Solvent A : 5 mM ammonium formate in Milli-Q® water and Solvent B : Acetonitrile in a ratio of 15 : 85, v/v, flow rate of 0.6 mL/min with splitter, respectively. Detection method involve the use of MS. Linearity range was evaluated to be in the range of 0.00026-0.02064 µg/ml, respectively. During method validation, acceptable recoveries were generated for the samples fortified at LOQ and 10 LOQ level. The % RSD (precision) was ≤20% at each fortification level. Recovery data from these samples demonstrated that test chemical was stable during analysis. The recoveries of all the samples analyzed were in the range of 70-110% with %RSD ≤ 20%. Analysis of the Day 0 samples at 10 μg/L and 100 μg/L test concentrations demonstrated quantitative recovery of test chemical. The average amount of test chemical present was 107.5% and 2.8% & 96.9% and 3.0% at Day 0 and Day 60, respectively following application of test chemical to test water at 10 μg/L (low dose) and 100μg/L (high dose). The average amount of test chemical present was 105.1% and 61.6% & 108.0% and 59.1% at Day 0 and Day 60, respectively following application of test chemical to sterile test water at 10 μg/L (low dose) and 100μg/L (high dose). The DT50 value was determined to be 10.2 d and 10.4 d at test chemical conc. of 10 μg/l and 100 μg/l at 12°C, respectively. 90% of test chemical in natural surface water was determined after 33.7 d and 34.5 d at test chemical conc. of 10 μg/l and 100 μg/l, respectively. Based on the these results, test chemical was degraded in surface water and sterile surface water. Hence, test chemical was considered to be not persistent in water.

Biodegradation in water: sediment simulation testing

In accordance with Annex IX column 2 of REACH regulation, test for this endpoint is scientifically not necessary and does not need to be conducted, since the substance is readily biodegradable i.e. not persistent based on the experimental result of surface water simulation biodegradation study.

 

Biodegradation in soil

The half-life period of test substance in soil was estimated using Level III Fugacity Model by EPI Suite version 4.1 estimation database (2018). If released into the environment, 81% of the chemical will partition into soil according to the Mackay fugacity model level III. The half-life period of test substance in soil is estimated to be 17.33 days (416 hrs). Based on this half-life value of test substance, it is concluded that the chemical is not persistent in the soil environment and the exposure risk to soil dwelling animals is moderate to low.

 

Bioaccumulation:

Bioaccumulation study in fish was conducted for determining the bioconcentration factor of test chemical. The study was performed in accordance with the OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test and EPA OPPTS 850.1730 (Fish Bioconcentration Test), respectively. Danio rerio (Zebra fish) (male) of total length 2.5 to 3.2 cm obtained from commercial fish farm, Manimangalam was used as a test fishes. Fish used in the tests were free from observable diseases and abnormalities. Test fishes were fed daily throughout the study period. Fishes were fed with an appropriate diet of known lipid and total protein content in an amount sufficient to keep them in a healthy condition and to maintain body weight. Fish species identification was done externally at Fisheries College and Research Institute (FCRI), Ponneri, Tiruvallur District, Tamil Nadu. Acclimatization of the stock population of test fishes were done for 2 weeks. Test conditions were same as the test. Feeding to test fishes were done in the same manner as that done during the study. Preliminary study was carried out. A limit test was conducted at 100 mg/l alongwith control for 96 hrs. 7 fishes were exposed to 100 mg/l for a period of 96 hrs. An equal control was also maintained. No mortality or toxicity signs were observed during the study period upto 96 hrs. The highest conc. (1 mg/l) of the test chemical was selected base on 1% of its acute LC50 (100 mg/l). The second conc. (0.1 mg/l) was selected from the one above by factor of 10. Thus, exposure conc. were 0.1 and 1.0 mg/l, respectibely. The blended water was used in the study. It was prepared by mixing the reverse osmosis water and well water in the ratio of 3:1. This water was used during the acclomatization, preliminary experiment, uptake and depuration phase. The pH, hardness, total solids, total organic carbon (TOC), anions, cations, heavy metals and the presence of pesticides were analyzed.Glass aquaria (glass tank) of 30 lit capacity was used as a test vessel. Test chemical conc. used for the study were 0.1 and 1.0 mg/l. Total 72 fishes/vessel for each test group were exposed with the test chemical. Study was performed under semi-static conditions and test medium was renewed thrice in a week and durng the renewal. fish from each group was transferred to a new medium in which test conc. are exposed in a test chamber. Biomass loading rate during the study was 0.1 to 1.0 g/l, respectively. One control was run in addition to the test series. A control group of fish was held under experimental conditions except for the absence of the test chemical. The leftout food and faeces were siphoned daily from the test chambers shortly after feeding (30 mins to 1 hr). Test chambers were kept clean as possible throughout the test to keep the conc. of organic matter as low as possible to avoid the presence of organic carbon that may limit the bioavailability of the test chemical. The blended water was used in the study. It was prepared by mixing the reverse osmosis water and well water in the ratio of 3:1. This water was used during the acclomatization, preliminary experiment, uptake and depuration phase. The pH, hardness, total solids, total organic carbon (TOC), anions, cations, heavy metals and the presence of pesticides were analyzed. Test conditions involve a pH of 7.39 to 7.97, hardness of 139 to 140 mg/l as CaCO3, temperature of 23.4 to 25°C, dissolved oxygen of 84.3 to 102.7%, TOC of < 5 mg/l, respectively. Uptake phase was carried out for 28 d and depuration phase for 14 d, respectively. Photoperiod during the uptake phase was 12: 12 light:dark conditions controlled by an automatic timer and depuration phase was 6:6 light:dark conditions, respectively. All experiments were performed in triplicates.The leftout food and faeces were siphoned daily from the test chambers shortly after feeding (30 mins to 1 hr). Test chambers were kept clean as possible throughout the test to keep the conc. of organic matter as low as possible to avoid the presence of organic carbon that may limit the bioavailability of the test chemical. After removal of lipid content from the sample, the aqueous layer was transferred into 50 mL centrifuge tube and the volume was adjusted to 10 mL using acetonitrile. The sample was homogenized for 2 minutes using tissumizer. The sample solution was centrifuged at 3000 rpm and decanted, A 2 mL of sample solution was filtered through 0.2 um PTFE and analysed for active content. The final solution was filtered and analyzed by LC-MS/MS conditions. HPLC system consists of Agilent 1290 Infinity UHPLC, Agilent Zorbax Eclipse XDB (2.1 mm X 75 mm, 3.5 μm), column temperature of 40°C, injection volume of 10 μl, flow rate of 1.0 ml/min, auto dampler temp. of 10°C. Gradient conditions involve the use of 0.1% acetic acid in 90/10 (MilliQ water/ Methanol) and 0.1%  acetic acid in acetonitrile, respectively. Total run time was 9.0 mins with a retention time of 1.5 mins. MS conditions involves the detector of Agilent 6490 LC-MS/MS System, coupled with Mass Hunter instrument control and data processing software, AJS-ESI interface, Positive polarity, Gas temperature of 260°C, Gas flow of 16 L/min, Nebulizer gas flow of 50 Psi, Sheath gas temperature of 400°C, Sheath gas flow of 11 L/min, Capillary voltage of 3500V, CAV of 4 V and Dwell time of 200 sec., respectively. During the uptake and depuration phase, the fish samples were extracted immediately and analyzed for active content. The uptake phase rate constants (k1), depuration phase rate constant (k2) was calculated by plotting the Ln (comc. of fish, mg/kg) Vs occasions (days). During the acclimatization period, there was no mortaliy observed and fish was found to be healthy. In preliminary test: there was no mortalirty or toxicity signs in test fishes. During the study, the water temperature variation was less than + 2°C which ranged from 23.4 to 25.0°C, the concentration of dissolved oxygen had not been fallen below 60% saturation and the range was 84.3 to 102.7%, the concentration of the test item (0.1 & 1.0 mg/L) in the chambers was maintained within + 20% of the mean of the measured values during the uptake phase, the concentration of the test item (0.1 & 1.0 mg/L) was below its limit of solubility in water, taking into account the effect that the test water may have on effective solubility, no mortality and other adverse effect was found in both control and treated fish during the uptake and depuration phases. No significant differences in average growth between the test and the control groups of sampled fish (Hence there was no indication of toxic effect of the test chemical, thereby fulfilling the validity criteria and hence, study was considered to be valid.The Bio-concentration factor (BCF) was determined as 0,8394 and 0.4624 for 0.1 mg/L and 1.0 mg/L concentrations respectively in the uptake phase. Further, based on the overall rate constants of the uptake and depuration phases, the kinetic Bio-concentration factor (BCFk) was determined to be -0.1261 and -0.3114 for the concentrations 0.1 mg/L and 1.0 mg/L respectively. From the growth rate analysis, the growth corrected kinetic bio-concentration factor (BCFkg) was determined to be -0.1240 and -0.3173 for the concentrations 0.1 mg/L and 1.0 mg/L respectively. Based on the lipid content analysis, the lipid normalized kinetic bio-concentration factor (BCFkl) was determined to be -0.1852 and -0.5097 for the concentrations 0.1 mg/L and 1.0 mg/L respectively in the uptake phase. Since, the BCF value did not exceeded the threshold limit of 2000, thus, test chemical was considered to be non-bioaccumulative,

Transport and distribution:

Adsorption/Desorption:

The adsorption coefficient Koc in soil and in sewage sludge of test chemical was determined by the Reverse Phase High Performance Liquid Chromatographic method according to OECD Guideline No. 121 for testing of Chemicals. The solutions of the test substance and reference substances were prepared in appropriate solvents. A test item solution was prepared by accurately weighing 4 mg of test item and diluted with Acetonitrile up to 10 ml. Thus, the test solution concentration was 400 mg/l. The pH of test substance was 6.7 .Each of the reference substance and test substance were analysed by HPLC at 210 nm. After equilibration of the HPLC system, Urea was injected first, the reference substances were injected in duplicate, followed by the test chemical solution in duplicate. Reference substances were injected again after test sample, no change in retention time of reference substances was observed. Retention time tR were measured, averaged and the decimal logarithms of the capacity factors k were calculated. The graph was plotted between log Koc versus log k(Annex - 2).The linear regression parameter of the relationship log Koc vs log k were also calculated from the data obtained with calibration samples and therewith, log Koc of the test substance was determined from its measured capacity factor. The reference substances were chosen according to estimated Koc range of the test substance and generalized calibration graph was prepared or chosen according to functionalyl similarity with the test substance and calibration graph prepared. The reference substances wereAcetanilide, 4-chloroaniline, 4-methylaniline(p-Tolouidine), N-methylaniline, p-toluamide, Aniline, 2,5-Dichloroaniline, 4-nitrophenol, 2 - nitrophenol, 2-nitrobenzamide, 3-nitrobenzamide, Nitrobenzene, 4-Nitrobenzamide, 1-naphthylamine, 1-naphthol, Direct Red 81, Benzoic acid methylester, Carbendazim, Benzoic acid phenylester, Xylene, Ethylbenzene, Toluene, Naphthalene, 1,2,3-trichlorobenzene, Pentachlorophenol, Phenol, N,N-dimethylbenzamide, 3,5-dinitrobenzamide, N-methylbenzamide, Benzamide, phenanthrene, DDT having Koc value ranging from 1.25 to 5.63

The Log Koc value of test substance test chemical was determined to be 1.280 ± 0.003 at 25°C. This log Koc value indicates that the test chemical has a negligible sorption to soil and sediment therefore has rapid migration potential to ground water.