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Hydrolysis

On the basis of the experimental studies of the read across chemical and applying the weight of evidence approach, the hydrolysis half-life value of the test chemical can be expected to be ranges from 61 to 65.9 days at pH 7.0 and 25°C temperature, respectively. Thus, based on this half-life value, test chemical can be considered to be hydrolytically stable.

Biodegradation in water

Biodegradation study was conducted for 28 -days following the OECD guideline 301 D for determining the ready biodegradability of the test chemical (Experimental study report, 2017). The test system included control, test item and reference item. Polyseed were used as a test inoculum. 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. 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 the values of BOD & ThOD and was determined to be 73.49% at 20 ± 1°C. Degradation of Sodium Benzoate exceeds 33.13% on 7 days & 40.36% on 14th day. The activity of the inoculum is thus verified and the test can be considered as valid. The BOD28 value of test chemical was observed to be 1.32 mgO2/mg. ThOD was calculated as 2.62 mgO2/mg. Accordingly, the % degradation of the test chemical after 28 days of incubation at 20 ± 1°C according to Closed Bottle test was determined to be 50.38%. Based on the results, the test chemical, under the test conditions was considered to be inherently biodegradable in water.

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, 19.2% 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 0.673%), 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, 79.9 % 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

BCFBAF model (v3.01) of Estimation Programs Interface (2018) was used to predict the bioconcentration factor (BCF) of test chemical . The bioconcentration factor (BCF) of test chemical was estimated to be 90.47 L/kg whole body w.w (at 25 deg C) which does not exceed the bio concentration threshold of 2000, indicating that the chemical is not expected to bioaccumulate in the food chain.

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, 2019). The solutions of the test substance and reference substances were prepared in appropriate solvents. A test chemical solution was prepared by dissolving 4 mg of test chemical in acetonitrile to prepare 400 mg/L and filtered through 0.22 nm nylon syringe filter. pH of the resulting solution was 5.7. 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.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 generalize calibration graph was prepared. Both were analyzed on HPLC considering the similar conditions. After analyzing on above conditions calibration curve of selected reference substances was prepared to obtain log Koc value. The reference substances were Formamide, 2-Nitrobenzamide, p-Toluamide , Aniline, 2 - Nitrophenol , Benzamide and Phenanthrene having Koc value ranging from 1.3 to 3.7. The Log Koc value of test chemical was determined to be 3.280±0.001 dimensionless at 25°C.This log Koc value indicates that the substance has a moderate sorption to soil and sediment and therefore have slow migration potential to ground water.

Additional information

Hydrolysis

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

 

The hydrolysis half-life of the test chemical was determined at different pH andat 25°C temperature.The study was performed following the the OECD Guideline 111 (Hydrolysis as a Function of pH).The half-lives of test chemical was determined to be 65.9 and 1.04 days at pH 7 and 9 & at a temperature of 25°C and was reported to be stable at pH 4.0. Based on this, it is concluded that the chemical was hydrolytically stable.

 

In a supporting weight of evidence study, the hydrolysis half-life of the test chemical was determined. The study was performed in accordance with the OECD Guideline 111 (Hydrolysis as a Function of pH). Preliminary test was carried out for 5 daysat different pH and at temperature of 50°C. Hydrolysis of test chemical did not reach > 10% at any pH system. Thus, the half-lives of test chemical was determined to be > 5 days at pH 4, 7 and 9 & at 50°C. Based on this, it is concluded that the chemical was hydrolytically stable.

 

For the test chemical, the half-life and base catalyzed second order hydrolysis rate constant was determined of the test chemical. The second order hydrolysis rate constant of test chemical was determined to be 1.3L/mol-sec with a corresponding half-lives of 61 and 6 days at pH 7 & 8 and 25°C temperature, respectively. On the basis of the half-life value at pH 7.0 (i.e, 61 days), it is concluded that the test chemical was stable in water.

 

On the basis of the experimental studies of the read across chemical and applying the weight of evidence approach, the hydrolysis half-life value of the test chemical can be expected to be ranges from 61 to 65.9 days at pH 7.0 and 25°C temperature, respectively. Thus, based on this half-life value, test chemical can be considered to be hydrolytically stable.

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 from study report (2017), biodegradation study was conducted for 28 -days following the OECD guideline 301 D for determining the ready biodegradability of the test chemical. The test system included control, test item and reference item. Polyseed were used as a test inoculum. 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. 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 the values of BOD & ThOD and was determined to be 73.49% at 20 ± 1°C. Degradation of Sodium Benzoate exceeds 33.13% on 7 days & 40.36% on 14th day. The activity of the inoculum is thus verified and the test can be considered as valid. The BOD28 value of test chemical was observed to be 1.32 mgO2/mg. ThOD was calculated as 2.62 mgO2/mg. Accordingly, the % degradation of the test chemical after 28 days of incubation at 20 ± 1°C according to Closed Bottle test was determined to be 50.38%. Based on the results, the test chemical, under the test conditions was considered to be inherently biodegradable in water.

 

Another biodegradation study of test substance was carried out for 96 hrs using various bacterial organisms each containing different naphthalene degrading enzyme (NAH) systems in plasmids (Jeffrey D. Leblond et al, 2001). Following strains were used Pseudomonas fluorescens 5R and 5RL, Pseudomonas spp. strain DFC49 and DFC50, Pseudomonas putida PpG7 and E. coli DH5α, respectively.The purity of test substance used in the study is greater than 95%. The analysis of test chemical by gas chromatography (GC)/ mass spectrometry was carried out. ThePAH mixture containing the test substance under study was prepared fresh prior to each experiment by dissolving 0.05 g of each compound together in 5 ml of acetone. This concentrated solution was then added to 1 l of minimal salts medium (pH 7), and allowed to sit in the dark for 3 days in order to achieve full saturation. After equilibration, 5 ml aliquots of this solution were filltered through a 0.2µm PTFE syringe top filter and added to sterilized 12 ml screw cap tubes with teflon-coated caps. The other bacterial strains utilized in the biodegradation experiment were pre-grown overnight to late-log phase in a yeast extract peptone- sodium succinate-sodium salicylate (YEPSS) medium. The bacterial strain in use was centrifuged and washed three times in minimal salts medium, and then re-suspended in 20 ml of minimal salts medium to achieve a 100-fold concentration of cells.50 µl of 100-fold concentrated solution of bacterial cells were added to tubes containing the polyaromatic hydrocarbons (PAHs) mixture (which contains the test substance ) to achieve a cell density of 108cells/ml. Serial dilutions and plating on yeast extract-peptone-glucose (YEPG) medium were performed approximately every 24 h. On the average, the number of colony forming units (cfu) was maintained at 108cells/ml through the 48 h time point. From 48 to 96 h, the cfu concentration dropped one order of magnitude. The tubes were then placed on a  Glass-col rotary shaker at room temperature. Triplicate samples were taken every half an hour from time 0 up to 7 h by adding 5 ml of hexane to the appropriate sacrificed tubes and then shaking horizontally for 2 h at 150 rpm. After this time, approximately 2 ml of the hexane phase was taken from the tube, crimp-sealed in an autosampler vial with a teflon-lined cap, and then stored at  -20°C until analysis. Samples were also usually taken at 12, 24, 48, 72, and 96 h during the experiment. Negative controls, killed with concentrated sulfuric acid prior to time zero, were treated identically. Gas chromatography (GC) analysis of the hexane extracts were carried out. GC analysis of hexane extracts showed 100% disappearance of test substance by test organism Pseudomonas fluorescens 5R within 72 hrs. The first order rate constant and r2value was also noted and determined to be 0.05 h-1and 0.822, respectively. Thus, indicating that the test substance to be readily biodegradable.

 

In a supporting study from peer reviewed journal (EFFREY D. LEBLOND et al, 2000), biodegradation experiment of test chemical was carried out for 18hrs using Pseudomonas fluorescens 5RL.The purity of test substance used is greater than 95% indicating that no further purification was necessary. Bacterial culture used for the study is Pseudomonas fluorescens 5RL.Strain 5RL was pre-grown to late log phase at 28ᵒC and 150 rpm in 100 ml of a yeast extract–peptone– glucose medium amended with 14 mg/L of tetracycline. Ten milliliters of cells was then transferred to two 4.0-L Erlenmeyer flasks, each of which contained 1 L of a yeast extract– peptone–salicylic acid–succinate medium with the same concentration of tetracycline. Cells were harvested in the exponential phase of growth by centrifugation for 10 min at 22,095gand 4ᵒC. The cells were washed three times and resuspended in 200 ml of 50 mM sodium phosphate buffer (pH 7.0) to an approximate concentration of 1.0 g of cells per 100 ml.The bacterial cells which were washed in 200 ml of 50 mM sodium phosphate buffer was then dividedin half into two 500- ml Erlenmeyer flasks. Into one flask was added 20 mg of substrate dissolved in 0.5 ml of N,N-dimethylformamide. The negative control consisted of addingN,N-dimethylformamide to 100 ml of cells. Similar negative control experiments were performed usingEscherichia colicontaining pUTK202 grown on Luria-Bertani broth in the presence of 50 mg/L of ampicillin. All flasks were then incubated at 28ᵒC and 150 rpm for approximately 18 h. After centrifugation, the cell-free suspensions were extracted with ethyl acetate (neutral extract; three 100-ml volumes). The aqueous layer was then acidified (pH < 2.0) with concentrated H2SO4 and extracted with ethyl acetate (acid extract). Both the neutral and acid ethyl acetate extracts were dried over anhydrous sodium sulfate, and the solvent was removed in vacuo at 30ᵒC. The residues were dissolved in 2.0 ml of acetone and then prefiltered through a 0.2-µm-pore-size polytetrafluoroethylene filter before analysis.Gas chromatography (GC) analysis of neutral extracts were carried out.GC-MS analysis of neutral extracts showed that the test substance undergoes transformation by Pseudomonas fluorescens 5RL and transformation product was determined to be 4-methoxysalicylic acid. Thus, based on this, test chemical can considered to be biodegradable in water.

 

On the basis of the study result conducted as per the OECD guideline 301 D, it is concluded that the test chemical was considered to be inherently biodegradable in water.

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, 19.2% 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 0.673%), 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, 79.9 % 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 is considered to be inherently biodegradable in water.

Bioaccumulation: aquatic / sediment

Various predicted data for the test chemical and supporting weight of evidence studies for its structurally and functionally similar read across substance were reviewed for the bioaccumulation end point which are summarized as below:

 

In aprediction done using the BCFBAF Program(v3.01) of Estimation Programs Interface (2018) prediction program was used to predict the bioconcentration factor (BCF) of test chemical. The bioconcentration factor (BCF) of test chemical was estimated to be 90.47 L/kg whole body w.w (at 25 deg C).

Another prediction was done by using OECD QSAR toolbox version 3.4 (2019) to estimate the bioaccumulation value (BCF) of test chemical in fish. The BCF value was estimated to be 366 dimensionless whole body w.w.

 

In an another prediction done by using Bio-concentration Factor (v12.1.0.50374) moduleACD (Advanced Chemistry Development)/I-Lab predictive module, 2017), thebio-concentration factor over the entire pH scale (pH 1 -14) of the test chemical was estimated to be 212.

 

From CompTox Chemistry Dashboard using OPERA (OPEn (quantitative) structure-activity Relationship Application)  V1.02 model in which calculation based on PaDEL descriptors (calculate molecular descriptors and fingerprints of chemical)  the bioaccumulation i.e BCF for test chemical was estimated to be 293 dimensionless . The predicted BCF result based on the 5 OECD principles.

 

In a supporting weight of evidence study from authoritative database (2017) and secondary source, bioaccumulation study was conducted on test organism Cyprinus carpio for 8 weeks for evaluating the bioconcentration factor (BCF value) of test chemical. The study was performed according to other guideline "Bioaccumulation test of a chemical substance in fish or shellfish" provided in "the Notice on the Test Method Concerning New Chemical Substances", respectively. Cyprinus carpio was used as a test organism for the study. Test chemical nominal conc. used for the study were 0.05 mg/l and 0.005 mg/l, respectively. Test chemical solution was prepared in HCO-40. Analytical method involve the recovery ratio: Test water : 1st concentration area : 87.3 %, 2nd concentration area : 90.0 %, Fish : 91.0 %, - Limit of detection : Test water: 1st concentration area : 2.4 ng/ml, 2nd concentration area : 0.23 ng/ml, Fish : 69 ng/g. Range finding study involve theLC50(48h) 11.1 mg/Lon Rice fish (Oryzias latipes). Lipid content of the test organism Cyprinus carpio was determined to be 4.1% at the start of exposure. The bioconcentration factor (BCF value) of substance on Cyprinus carpio was determined to be in the range of 431-1180 L/Kg at a conc. of 0.05 mg/l and 398-873 L/Kg at a conc. of 0.005 mg/l, respectively, which does not exceed the bioconcentration threshold of 2000, indicating that the test chemical is not expected to bioaccumulate in the food chain.

 

For the test chemical,bioaccumulation study was conducted on test organism Cyprinus carpio for 6 weeks for evaluating the bioconcentration factor (BCF value) of test chemical. The study was performed according to OECD Guideline 305 C (Bioaccumulation: Test for the Degree of Bioconcentration in Fish) and other guideline "Bioaccumulation test of a chemical substance in fish or shellfish" provided in "the Notice on the Test Method Concerning New Chemical Substances", respectively at 25°C. Cyprinus carpio was used as a test organism for the study. Test chemical nominal conc. used for the study were 0.1mg/l and 0.01 mg/l, respectively. Test chemical solution was prepared in HCO-20. Analytical method involve the recovery ratio: Test water : 96.7 %, Fish : 88.5 %, - Limit of quantitation : Test water : 1st concentration area : 2.7 microg/L, 2nd concentration area : 0.27 microg/L, Fish : 33 ng/g. Range finding study involve the LC50(48h) ≥ 26 mg/Lon Rice fish (Oryzias latipes). Lipid content of the test organism Cyprinus carpio was determined to be 4.0% at the start of exposure. The bioconcentration factor (BCF value) of test chemical on Cyprinus carpio was determined to be in the range of 6.1 to 63 L/Kg at a conc. of 0.1 mg/l and 5.9 to 23 L/Kg at a conc. of 0.01 mg/l, respectively, which does not exceed the bioconcentration threshold of 2000, indicating that the test chemical is not expected to bioaccumulate in the food chain.

 

On the basis of above results of the test chemical, it can be concluded that the BCF value of test chemical was evaluated to be ranges from 5.9 to 1180, 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

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, 2019). The solutions of the test substance and reference substances were prepared in appropriate solvents. A test chemical solution was prepared by dissolving 4 mg of test chemical in acetonitrile to prepare 400 mg/L and filtered through 0.22 nm nylon syringe filter. pH of the resulting solution was 5.7. 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.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 generalize calibration graph was prepared. Both were analyzed on HPLC considering the similar conditions. After analyzing on above conditions calibration curve of selected reference substances was prepared to obtain log Koc value. The reference substances were Formamide, 2-Nitrobenzamide, p-Toluamide , Aniline, 2 - Nitrophenol , Benzamide and Phenanthrene having Koc value ranging from 1.3 to 3.7. The Log Koc value of test chemical was determined to be 3.280±0.001 dimensionless at 25°C.This log Koc value indicates that the substance has a moderate sorption to soil and sediment and therefore have slow migration potential to ground water.