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Description of key information

Hydrolysis

The half-life of the test chemical was estimatedusing the Bond SAR method (secondary source, 2017).The half-life value of test chemical in river and lake water was estimated to be 8.655 hrs and 168.9 hrs (7 days), respectively. Thus based on the value, test chemical is considered to undergoe moderate to slow hydrolysis in water.

Biodegradation in water

28-days Closed Bottle test following the OECD guideline 301 D to determine the ready biodegradability of the test chemical (Experimental study report, 2018). The study was performed at a temperature of 20°C. The test system included control, test item and reference item. Polyseed were used for this study. 1 polyseed capsule were added in 500 ml D.I water and then stirred for 1 hour for proper mixing and functioning of inoculum. This gave the bacterial count as 10E7 to 10E8 CFU/ml. At the regular interval microbial plating was also performed on agar to confirm the vitality and CFU count of microorganism. The concentration of test and reference item (Sodium Benzoate) chosen for both the study was 4 mg/L, while that of inoculum was 32 ml/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 % degradation of procedure control (reference item) was also calculated using BOD & ThOD and was determined to be 76.5%. Degradation of Sodium Benzoate exceeds 39.15% on 7 days & 70.48% on 14th day. The activity of the inoculum was thus verified and the test can be considered as valid.The BOD28 value of test chemical was observed to be 1.25 mgO2/mg. ThOD was calculated as 2.94 mgO2/mg. Accordingly, the % degradation of the test item after 28 days of incubation at 20 ± 1°C according to Closed Bottle test was determined to be 42.51%. Based on the results, the test item, under the test conditions, was considered to be ultimate inherently biodegradable in nature.

Biodegradation in water & sediment

Estimation Programs Interface (2018) prediction model was run to predict the half-life in water and sediment for the test chemical. If released in to the environment, 12.5% of the chemical will partition into water according to the Mackay fugacity model level III and the half-life period of test chemical in water is estimated to be 37.5 days (900 hrs). The half-life (37.5 days estimated by EPI suite) indicates that the chemical is not persistent in water and the exposure risk to aquatic animals is moderate to low whereas the half-life period of test chemical in sediment is estimated to be 337.5 days (8100 hrs). However, as the percentage release of test chemical into the sediment is less than 1% (i.e, reported as 0.758%), indicates that test chemical is not persistent in sediment.

 

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, 86.7% 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 75 days (1800 hrs). Based on this half-life value of test chemical, 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.

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 (Experimental study report, 2018). The solutions of the test substance and reference substances were prepared in appropriate solvents. A test item solution was prepared by accurately weighing 5 microliter of test item and diluted with acetonitrile up to 10 ml. Thus, the test solution concentration was 465.5 mg/l. The pH of test substance was 4.7. 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 The reference substances were Acetanilide, 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-naphtol, 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, Nmethylbenzamide, Benzamide, phenanthrene, DDT having Koc value ranging from 1.25 to 5.63. The Log Koc value of test chemical was determined to be 3.486 ± 0.002 at 25°C. This log Koc value indicates that the test chemical has a strong sorption to soil and sediment and therefore have negligible to slow migration potential to ground water.

Additional information

Hydrolysis

The half-life of the test chemical was estimatedusing the Bond SAR method (secondary source, 2017).The half-life value of test chemical in river and lake water was estimated to be 8.655 hrs and 168.9 hrs (7 days), respectively. Thus based on the value, test chemical is considered to undergoe moderate to slow hydrolysis in water.

Biodegradation in water

Various experimental studies of the test chemical were reviewed for the biodegradation end point which are summarized as below:

 

In an experimental key study report (2018) for the test chemical,28-days Closed Bottle test following the OECD guideline 301 D 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. Polyseed were used for this study. 1 polyseed capsule were added in 500 ml D.I water and then stirred for 1 hour for proper mixing and functioning of inoculum. This gave the bacterial count as 10E7 to 10E8 CFU/ml. At the regular interval microbial plating was also performed on agar to confirm the vitality and CFU count of microorganism. The concentration of test and reference item (Sodium Benzoate) chosen for both the study was 4 mg/L, while that of inoculum was 32 ml/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 % degradation of procedure control (reference item) was also calculated using BOD & ThOD and was determined to be 76.5%. Degradation of Sodium Benzoate exceeds 39.15% on 7 days & 70.48% on 14th day. The activity of the inoculum was thus verified and the test can be considered as valid. The BOD28 value of test chemical was observed to be 1.25 mgO2/mg. ThOD was calculated as 2.94 mgO2/mg. Accordingly, the % degradation of the test item after 28 days of incubation at 20 ± 1°C according to Closed Bottle test was determined to be 42.51%. Based on the results, the test item, under the test conditions, was considered to be ultimate inherently biodegradable in nature.

 

Another biodegradation study was conducted for 56 days for evaluating the percentage biodegradability of test chemical (from secondary source, 2017). The study was performed according to OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test) at a temperature of 14-22°C. Initial test chemical conc. Used for the study was10 mg DOC/L. Activated sludge (Unacclimatized) was used as a test inoculum for the study. The test material (10 mg DOC/L) and secondary effluent from an unacclimatized activated sludge were shaken in a sealed vessel at 14-22 °C for 28 days. The headspace carbon dioxide level and inorganic carbon in the test medium was analysed. The percentage degradation of test chemical was determined to be 61.8% by CO2 evolution parameter in 28 days. Thus, based on percentage degradation, test chemical is considered to be readily biodegradable in nature.

 

In a supporting study from secondary source (2017), biodegradation experiment was conducted according to OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II)) for evaluating the percentage biodegradability of test chemical. Activated sludge mixture(1:1) from city sewage plant and chemical sewage plant was used as a test inoculum for the study. Concentration of inoculum i.e, sludge used was 100 mg/l and initial test substance conc. used in the study was 30 mg/l, respectively. Sodium benzoate (100 mg/l) was used as reference substance.The test substance (30 mg/L) or reference substances (100 mg/L) sodium benzoate was inoculated with a mixture of activated sludge (100 mg dry sludge/L) in a closed oxygen consumption measuring device for 56 days at 25 °C. Oxygen concentration was monitored continuously using a BOD meter. Percent biodegradation values were arithmetic mean of 2 or 3 independent measurements. The reference substance was 100% degraded after 28 days. Test chemical undergoes 63.4% degradation after 56 days. Time required for 10% degradation of test substance was 12 days. Thus, the test chemical was considered as inherently biodegradable in nature.

 

For the test chemical,biodegradation study was conducted for 28 days for evaluating the percentage biodegradability of test chemical (Susumu Ishizaki et. al., 2004). Activated sludge was used as a test inoculum for the study. Basic culture medium was used as a test medium for the study. The medium consisted of the following components (per liter of distilled water): 0.063g of K2HPO4, 0.026g of KH2PO4, 0.134g of Na2HPO4.7H2O, 0.0005g of NH4Cl, 0.068f of MgSO4, 0.083g of CaCl2, and 0.0008g of FeCl3.6H2O. The study was performed at a temperature of 25°C.To the resulting standard medium (900 ml), 108 mg of test chemical and 120 mg of standard activated sludge were added. The mixture was incubated at 25°C for 28 days undercontinuous shaking. The metabolites were extracted with ether, and the organic layer was washed with brine, dried over MgSO4 and concentrated in vacuo. The residue was chromatographed on silica gel, eluting with n-hexane-ethyl acetate (10:1) to give the product A. An analytical sample was further purified by GLC analysis. The IR absorption band at 1730/cm indicated the presence of carbonyl group. The 1H-NMR spectrum showed the presence of a geminal dimethyl at 0.76 & 0.91 ppm, a methyl on a double bond at 1.62 ppm, a methyl of methyl ketone at 2.81 ppm, and a proton on a double bond at 5.33 ppm. The 13C-NMR spectrum indicated the presence of 12 carbons. Thus, the product A was identified as (±)-1-(2,6,6-trimethyl-2-cyclohexen-1-yl)propan-2-one. Although percentage degradation of test chemical was not known, but major degradation product of test chemical was isolated and identified as(±)-1-(2,6,6-trimethyl-2-cyclohexen-1-yl)propan-2-one, indicating that the test chemical is biodegradable in nature.

 

Additional supporting biodegradation study (from peer reviewed journal Staci L. Simonich et. al., 2000) was conducted in activated sludge & trickling filter wastewater treatment for 3 days for evaluating the percentage biodegradability of test chemical. Wastewater was used as a test inoculum for the study. Initial test chemical conc. used for the study were 0.00367 mg/l and 0.00267 mg/l, respectively. The percentage degradation of test chemical was determined to be 99.5 and 94.6% degradation in activated sludge & trickling filter wastewater treatment over a period of 3 days. Thus, based on percentage degradation, test chemical is considered to be readily biodegradable in nature.

 

On the basis of above overall results for test chemical, it can be concluded that the test chemical can be expected to be ultimate inherently biodegradablein nature.

Biodegradation in water & sediment

Estimation Programs Interface (2018) prediction model was run to predict the half-life in water and sediment for the test chemical. If released in to the environment, 12.5% of the chemical will partition into water according to the Mackay fugacity model level III and the half-life period of test chemical in water is estimated to be 37.5 days (900 hrs). The half-life (37.5 days estimated by EPI suite) indicates that the chemical is not persistent in water and the exposure risk to aquatic animals is moderate to low whereas the half-life period of test chemical in sediment is estimated to be 337.5 days (8100 hrs). However, as the percentage release of test chemical into the sediment is less than 1% (i.e, reported as 0.758%), indicates that test chemical is not persistent in sediment.

 

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, 86.7% 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 75 days (1800 hrs). Based on this half-life value of test chemical, 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.

On the basis of available information, the test chemical can be considered to be undergoe ultimate inherently biodegradable in nature.

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 (Experimental study report, 2018). The solutions of the test substance and reference substances were prepared in appropriate solvents. A test item solution was prepared by accurately weighing 5 microliter of test item and diluted with acetonitrile up to 10 ml. Thus, the test solution concentration was 465.5 mg/l. The pH of test substance was 4.7. 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 The reference substances were Acetanilide, 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-naphtol, 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, Nmethylbenzamide, Benzamide, phenanthrene, DDT having Koc value ranging from 1.25 to 5.63. The Log Koc value of test chemical was determined to be 3.486 ± 0.002 at 25°C. This log Koc value indicates that the test chemical has a strong sorption to soil and sediment and therefore have negligible to slow migration potential to ground water.