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Description of key information

Hydrolysis

The test chemical is not expected to undergo hydrolysis in the environment due to the lack of functional groups that hydrolyze under environmental conditions (authoritative database, 2017).

Biodegradation in water

35-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. The concentration of test and reference item (Sodium Benzoate) chosen for both the study was 4 mg/L, while that of inoculum was 30 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 82.53%. Degradation of Sodium Benzoate exceeds 42.16 % on 7 days & 63.25 % on 14th day. The activity of the inoculum was thus verified and the test can be considered as valid. The BOD35 value of test chemical was observed to be 1.3 mgO2/mg. ThOD was calculated as 2.87 mgO2/mg. Accordingly, the % degradation of the test item after 35 days of incubation at 20 ± 1°C according to Closed Bottle test was determined to be 45.29%. Based on the results, the test item, under the test conditions, was considered to be ultimate inherently biodegradable in nature.

Biodegradation in water and sediment

Estimation Programs Interface (2017) prediction model was run to predict the half-life in water and sediment for the test chemical. If released in to the environment, 30.2% 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 15 days (360 hrs). The half-life (15 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 135 days (3240 hrs). However, as the percentage release of test chemical into the sediment is less than 1% (i.e, reported as 0.117%), 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 (2017). If released into the environment, 65.2% 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 30 days (720 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.

Bioaccumulation: aquatic / sediment:

The bioaccumulation study was conducted for estimating the BCF (bioaccumulation factor) value of test chemical (HSDB and PubChem, 2017). The bioaccumulation factor (BCF) value was calculated using a logKow of 2.37 and a regression-derived equation. The estimated BCF (bioaccumulation factor) value of test chemical was determined to be 13 dimensionless, which does not exceed the bioconcentration threshold of 2000, indicating that the test chemical is considered to be non-accumulative in aquatic organisms.

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, 2017). A test item solution was prepared by accurately measuring 4μL of test item and diluted with methanol up to 10 ml. Thus, the test solution concentration was 327.6 mg/l. The pH of test substance was 6.95. The reference substances were chosen according to structural similarity with the test substance and calibration graph prepared. The reference substances were 2-nitrophenol, nitrobenzene, 4 -nitrobenzamide, N,N-dimethylbenzamide, N-methylbenzamide and benzamide having Koc value ranging from 1.239 to 2.47. The Log Koc value of test chemical was determined to be 1.8135 ± 0.001 at 25°C.This logKoc value indicates that the test chemical has a low sorption to soil and sediment and therefore have moderate migration potential to ground water.

Additional information

Hydrolysis

The test chemical is not expected to undergo hydrolysis in the environment due to the lack of functional groups that hydrolyze under environmental conditions (authoritative database, 2017).

Biodegradation in water

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

 

In an experimental key study from study report (2018),35-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. The concentration of test and reference item (Sodium Benzoate) chosen for both the study was 4 mg/L, while that of inoculum was 30 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 82.53%. Degradation of Sodium Benzoate exceeds 42.16 % on 7 days & 63.25 % on 14th day. The activity of the inoculum was thus verified and the test can be considered as valid. The BOD35 value of test chemical was observed to be 1.3 mgO2/mg. ThOD was calculated as 2.87 mgO2/mg. Accordingly, the % degradation of the test item after 35 days of incubation at 20 ± 1°C according to Closed Bottle test was determined to be 45.29%. Based on the results, the test item, under the test conditions, was considered to be ultimate inherently biodegradable in nature.

 

In a supporting study, the biodegradability of test chemical was determined by biodegradation inhibition test on micro-organism. Test chemical initial concentration of 100 uL/L (D. D. Vaishnav et. al, 1990).The biodegradation was determined by BIC50 (chemical concentration that would reduce the maximum biodegradation rate by 50%)value of micro-organism .The BIC50 of chemical of test chemical was determined to be 12.88 mmol/L, hence the test chemical can be considered to be inherently biodegradable.

 

In an another supporting study (DINESH D. VAISHNAV et. al, 1987), the biodegradability of test chemical was determined by BOD technique. Test chemical initial concentration of 3.2 mg/L or uL/L and 1 mL of acclimated seed were added to 20 mLof dilution water in 300-mL BOD bottles for 5 days. 5-day BOD values, in mmol BOD per mmol test chemical was determined to be 5.53 mmol. The percentage degradation of test chemical was determined to be 48.1% by ThOD parameter in 5 days. Thus, based on percentage degradation, test chemical was considered  to be readily biodegradable in nature.

 

Another biodegradation experiment was conducted for evaluating the percentage biodegradability of test chemical. Sewage was used as a test inoculum (HSDB, 2017). The percentage degradation of test chemical was determined to be 33% by ThOD parameter in 5 days. Thus, based on percentage degradation, test chemical is considered to be readily biodegradable in nature.

 

For the test chemical, biodegradation study was carried out for evaluating the percentage biodegradability of test chemical (Robert M. Gerhold et. al, 1962). Activated sludge was used as test inoculums obtained from 3 treatment plants of different sizes and designs and fed by different sewage systems. Initial test substance conc. used for the study was 500 mg/l and conc. of the inoculum used was 2,500 mg/l, respectively. Warburg constant temperature respirometer was used as a test vessel. The suspended solid conc. was adjusted to 2,500 mg/l by removal of supernatant liquid or by addition of tap water.The sludge was not washed. No mineral salts were added. The sludge suspension was blended for 10 sec and 20 ml were pipetted into 125 ml Warburg flasks containing the test substance (substrate).  A control flask for measurement of endogenous respiration was included with each run. Readings were made for 24 hr at 0.5 to 5 hr interval, depending on the rate of oxygen uptake. The experimental results were plotted as accumulative oxygen uptake corrected for endogenous respiration. Test chemical was observed to be more resistant to oxidation by activated sludge. %ThOD of the test chemical was determined to 4.6% by activated sludge in 24 hrs. Thus, the test chemical was considered to be not readily biodegradable in nature.

 

Biodegradation study from peer reviewed journal (Robert M. Gerhol, 1962) was carried out for evaluating the percentage biodegradability of test chemical. Activated sludge was used as test inoculums obtained from 3 treatment plants of different sizes and designs and fed by different sewage systems. Initial test substance conc. used for the study was 500 mg/l and conc. of the inoculum used was 2,500 mg/l, respectively.Test chemical (substrates) easily soluble in water were made up in 0.1 per cent concentration with distilled water and stored at 6°C until needed. Warburg constant temperature respirometer was used as a test vessel. They were modified 125 ml Erlenmeyer flasks fitted with 1.5 ml center-wells and female ground glass joints. Warburg flasks were cleaned by the following procedure: (a) flasks were rinsed once with tap water, and dried In the 103°C oven; (b) flasks were washed with two rinses of chloroform to remove fats and greases, then dried; (c) the flasks were submerged in potassium dichromate cleaning solution for 24 hr, rinsed In the same manner as the pipettes, and dried in an inverted position. Each flask received 10 ml of substrate solution or suspension delivered with a volumetric pipette. Next, 10 ml of blended sludge were added to each flask. The final concentration of substrate was 500 mg/liter. The final concentration of sludge solids was 2500 mg/liter. The control for endogenous respiration contained 10 ml of distilled water and 10 ml of adjusted sludge. Endogenous respiration was defined as the amount of accumulative O2uptake observed in the control flask containing sludge and distilled water. After 10-20 min of shaking for temperature equilibration the flasks were closed off to the atmosphere and shaken for 24 hr at 78 oscillations per min. From 9 to 16 readings were made during each experiment. The terms "percentage oxidized," or "percentage of oxidation," or "X per cent oxidized" mean the ratio of the amount of oxygen taken up by the sludge in the presence of that concentration of the substrate to the amount of oxygen required for complete oxidation of that concentration of substrate, i.e., oxidation to carbon dioxide, water, nitrate, and sulfate. This ratio is also referred to as the "percentage of total theoretical oxygen demand (ThOD)."Theoretical O2 uptake of the test chemical by activated sludge was determined to be1435 mg/l. Percentage degradation of the test chemical was determined to be 3.0% by Brookside culture and 10.9% by Columbus culture whereas no degradation of test chemical was observed in 24 hrs by Hilliard culture. Thus, no sufficient degradation of the test chemical was observed in 24 hr period. Thus, the test chemical was considered to be not readily biodegradable in nature.

 

Additional biodegradation study was carried out for a period of115 to 195 hrs evaluating the percentage biodegradability of test chemical (C. V. Marion et. al, 1963). Oxygen uptake study was carried out in the Warburg constant volume respirometer on the ability of Alcaligenes faecalis to oxidize aerobically test chemical at a temp. of 20°C and pH of 7.0. Alcaligenes faecalis was used as a test inoculum obtained from University Department of Microbiology, Ohio state. The cells were grown in a medium having the following composition: Dextrose, 2.0 g; tryptone, 10.0 g; beef extract, 6.0 g; distilled water upto 1 liter. Incubation was at 32°C for 48 hrs. Using aseptic techniques, the cells were harvested, washed four times with sterile distilled water, adjusted to 5,000 mg/l with sterile diluent. The suspension was blended for 10 sec in a sterile blender. 10 ml of the cell suspension were added to each Warburg flask. Initial test substance conc. used for the study was 500 mg/l and conc. of the inoculum used was 2,500 mg/l, respectively. Each flask contained 1.0 ml of 20% KOH solution in the center well. The reaction compartment of the flask held 20 ml of a mixture containing 500 mg/l of test chemical and 2,500 mg/l of inoculums, with mineral salts as the diluents. Incubation was at 20°C for 115 – 195 hrs. Aseptic techniques were used throughout the experiment. A flask containing 2,500 mg/l of test inoculum served as a control for measurement of endogenous respiration of the organism. Air oxidation or volatility of the substance was measured in a flask containing 500 mg/l of substrate in the mineral salts diluents. Corrections for air oxidation or volatility were made when necessary. These air oxidation flasks also served as a partial check of oxygen uptake by contaminants introduced in the unsterilized air or with unsterilized substrates. When the substrate uptake curve fell below the curve for endogenous respiration (control), the result was interpreted as indicating some measure of inhibition of the organism by the substrate. It is assumed that such an inhibitory compound is resistant to oxidation by Alcaligenes faecalis. After a long lag period, the organism was not able to utilize the test chemical. The test chemical remained uniformly resistant to oxidation by Alcaligenes faecalis. Thus, the test chemical was considered to be not readily biodegradable.

 

In a supporting study from peer reviewed journal (L. Babeu, et. al; 1987), biodegradation experiment was conducted for 20 days for evaluating the percentage biodegradability of test chemical.  Mixed microbial cultures capable of using 45 model organic chemicals (analytical grade) as sole carbon and energy sources were separately isolated by an enrichment culture technique. Microbial seeds for the BOD tests were prepared from the culture growth (105 -106 cells/ml) in mineral salts medium containing 100 mg/l (solid) or 100µl/l (liquid) chemical substrate. The culture was diluted (1:1) with physiological saline and incubated on a shaker (150 rpm) for 24 h prior to its use. Biodegradation of 45 model chemicals by acclimated microbes was measured separately by the BOD technique. Test chemical and 1 ml of the seed were added to 20 ml of dilution water contained in a 300-ml BOD bottle. Bottles were filled to capacity with the same water, and sealed and incubated for 20 days at 21±3°C. Each test was run in duplicate. A seed control and two or more chemical concentrations ranging from 0.4 to 3.2 mg or µl/l were employed in each test. Glucose-glutamic acid controls for assessing the dilution water quality were included. Initial and 5, 10 or 11, 14 or 15, and 20-day dissolved oxygen (DO) concentrations were determined using a YSI 54 oxygen meter equipped with a self-stirring probe. The DO concentrations in randomly selected bottles were measured by the azide modification of the iodometric method. The test BOD values showing DO depletions of at least 2 mg/1 and residual DO of at least 1mg/l were acceptable. These values were adjusted for the seed control and used for calculating the 5-day mmol BOD/mmol chemical at various chemical concentrations. Comparisons of 3 predicted BOD values with the corresponding experimental value for each chemical were made by computing percent relative standard deviation (%RSD) between predicted and measured values. Test chemical undergoes 11.5% degradation by ThBOD parameter in 20 days. The BOD5 value was determined to be 5.53±0.10 mmol/mmol of chemical. Thus, the test chemical was determined to be not readily biodegradable under the test conditions.

 

Although other studies from peer reviewed journals and authoritative database indicates that the test chemical is readily and not readily biodegradable, but as these studies were not performed as per the standard duration as that mentioned in the OECD guideline, thus, on the basis of experimental study result from peer reviewed journal and detailed experimental study report result (K1) performed as per the OECD guideline with duration of 35 days, it has been concluded that the test chemical is considered to be inherently biodegradable in nature.

Biodegradation in water and sediment

Estimation Programs Interface (2017) prediction model was run to predict the half-life in water and sediment for the test chemical. If released in to the environment, 30.2% 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 15 days (360 hrs). The half-life (15 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 135 days (3240 hrs). However, as the percentage release of test chemical into the sediment is less than 1% (i.e, reported as 0.117%), 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 (2017). If released into the environment, 65.2% 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 30 days (720 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 inherently biodegradable in nature.

Bioaccumulation: aquatic / sediment:

Experimental study and various predicted data of the test chemical were reviewed for the bioaccumulation end point which are summarized as below:

 

In an experimental key study from authoritative databases (2017),bioaccumulation experiment was conducted for estimating the BCF (bioaccumulation factor) value of test chemical. The bioaccumulation factor (BCF) value was calculated using a logKow of 2.37 and a regression-derived equation. The estimated BCF (bioaccumulation factor) value of test chemical was determined to be 13 dimensionless.

 

In a prediction done using the BCFBAF Program of Estimation Programs Interface was used to predict the bioconcentration factor (BCF) of test chemical. The bioconcentration factor (BCF) of test chemical was estimated to be 17.01 L/kg whole body w.w (at 25 deg C) which does not exceed the bio concentration threshold of 2000, indicating that the test chemical is not expected to bioaccumulate in the food chain.

 

Using Bio-concentration Factor module Bio-concentration Factor of the test chemical was estimated to be 46.7 at pH range 1-14, respectively (ACD (Advanced Chemistry Development)/I-Lab predictive module, 2017)). This value indicates that the test chemical was considered to be non-accumulative in aquatic organisms.

 

On the basis of above results for test chemical (from authoritative and modelling databases,2017), it can be concluded that the BCF value of test chemical ranges from 13 to 46.7, respectively, which does not exceed the bioconcentration threshold of 2000, indicating that the test chemical is not expected to bioaccumulate in the food chain.

Adsorption / desorption:

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

 

In an experimental key study from study report (2017),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. A test item solution was prepared by accurately measuring 4μL of test item and diluted with methanol up to 10 ml. Thus, the test solution concentration was 327.6 mg/l. The pH of test substance was 6.95. The reference substances were chosen according to structural similarity with the test substance and calibration graph prepared. The reference substances were 2-nitrophenol, nitrobenzene, 4 -nitrobenzamide, N,N-dimethylbenzamide, N-methylbenzamide and benzamide having Koc value ranging from 1.239 to 2.47. The Log Koc value of test chemical was determined to be 1.8135 ± 0.001 at 25°C.This logKoc value indicates that the test chemical has a low sorption to soil and sediment and therefore have moderate migration potential to ground water.

 

In a supporting study, adsorption experiment was conducted for estimating the adsorption coefficient (Koc) value of test chemical (authoritative database, 2017). The adsorption coefficient (Koc) value was calculated using a logKow of 2.37 and a regression derived equation. The adsorption coefficient (Koc) value of test chemical was estimated to be 460 (Log Koc = 2.66). This Koc value indicates that the test chemical has a moderate sorption to soil and sediment and therefore have slow migration potential to ground water.

 

On the basis of above overall results for test chemical (from study report and authoritative databases), it can be concluded that the logKoc value of test chemical ranges from 1.81–2.66indicating that the test chemical has a low to moderatesorption to soil and sediment and therefore have moderate to slow migration potential to ground water.