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Description of key information

Hydrolysis

The half-life of the test chemical was determined (Rensheng Zhuang et. al., 2013). The study was performed at a temperature of 25°C and pH 6.9, respectively. Initial test chemical conc. used was 10 mg/l. Experiment was performed with solutions of test chemical (10 mg L–1 in 0.01 M KH2PO4 + Na2HPO4 buffer (pH = 6.9). Firstly, 0.29 mL of 30% of NaOCl (sodium hypochlorite) or 0.4 mg of TCCA (trichloroisocyanuric acid) were added to 100 mL solution of test chemical and stirred in darkness at room temperature. Reaction mixtures were left to stand at room temperature for 24 h, after certain period of time reactions were stopped by addition of Na2SO3 and were afterwards analyzed by HPLC-DAD and UV-Vis spectrophotometer. The HPLC analyses were made on an Agilent 1100 Series chromatograph, coupled with DAD detector. The chromatographic separations were run on a Zorbax C8 column (4.6 mm ID × 250 mm, 5 μm) using a 60: 40 mixture of acetonitrile and acetic acid (pH 3) as the mobile phase. The column temperature was kept at 25 °C with the flow rate of 1.0 mL/min, injection volume 75 μL and the duration was 25 min with 5 min of post run. Test chemical and their chlorinated products were monitored at 240 nm. All the analyses were done in triplicates and are presented as mean values. Mass spectra were recorded on a 6224 Agilent Accurate-Mass TOF mass spectrometer. As the starting material was completely consumed in less than 15 min and there is almost no difference in the composition of reaction mixture in 15 min, 1 h or 24 h after the setup of the reaction. Thus, the half-life value of test chemical can be considered to be< 15 min. Based on this, it can be concluded that the test chemical was rapidly hydrolysable 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. 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 79.51%. Degradation of Sodium Benzoate exceeds 64.45 % on 7 days & 75.30 % 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 1.07 mgO2/mg. ThOD was calculated as 1.60 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 66.87%. Based on the results, the test item, under the test conditions, was considered to be readily biodegradable in nature.

Biodegradation in water and 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,19.5% 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 0.0997%), 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, 80.4% 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 isnot 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 (authoritative database, 2017). The bioaccumulation factor (BCF) value was calculated using a logKow of 0.37 and a regression-derived equation. The estimated BCF (bioaccumulation factor) value of test chemical was determined to be 3.0 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, 2018). 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 4.8. 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-naphtol, Direct Red 81, Benzoic acid methylester, Carbendazim, Benzoic acid phenylester, Xylene, Ethylbenzene, Toluene, Naphthalene, 1,2,3-trichlorobenzene, Pentachlorophenol, 3,5-dinitrobenzamide, Benzamide, phenanthrene having Koc value ranging from 1.25 to 4.09. The Log Koc value of test chemical was determined to be 0.992± 0.053 dimensionless at 25°C.This log Koc value indicates that the substance has a negligiblesorption to soil and sediment and therefore have rapid migration potential to ground water.

Additional information

Hydrolysis

The half-life of the test chemical was determined (Rensheng Zhuang et. al., 2013). The study was performed at a temperature of 25°C and pH 6.9, respectively. Initial test chemical conc. used was 10 mg/l. Experiment was performed with solutions of test chemical (10 mg L–1 in 0.01 M KH2PO4 + Na2HPO4 buffer (pH = 6.9). Firstly, 0.29 mL of 30% of NaOCl (sodium hypochlorite) or 0.4 mg of TCCA (trichloroisocyanuric acid) were added to 100 mL solution of test chemical and stirred in darkness at room temperature. Reaction mixtures were left to stand at room temperature for 24 h, after certain period of time reactions were stopped by addition of Na2SO3 and were afterwards analyzed by HPLC-DAD and UV-Vis spectrophotometer. The HPLC analyses were made on an Agilent 1100 Series chromatograph, coupled with DAD detector. The chromatographic separations were run on a Zorbax C8 column (4.6 mm ID × 250 mm, 5 μm) using a 60: 40 mixture of acetonitrile and acetic acid (pH 3) as the mobile phase. The column temperature was kept at 25 °C with the flow rate of 1.0 mL/min, injection volume 75 μL and the duration was 25 min with 5 min of post run. Test chemical and their chlorinated products were monitored at 240 nm. All the analyses were done in triplicates and are presented as mean values. Mass spectra were recorded on a 6224 Agilent Accurate-Mass TOF mass spectrometer. As the starting material was completely consumed in less than 15 min and there is almost no difference in the composition of reaction mixture in 15 min, 1 h or 24 h after the setup of the reaction. Thus, the half-life value of test chemical can be considered to be< 15 min. Based on this, it can be concluded that the test chemical was rapidly hydrolysable in water.

Hydrolysis endpoint can also be considered for waiver as per in accordance with column 2 of Annex VIII of the REACH regulation, testing for this endpoint is scientifically not necessary and does not need to be conducted since the test chemical is readily biodegradable.

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),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. 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 79.51%. Degradation of Sodium Benzoate exceeds 64.45 % on 7 days & 75.30 % 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 1.07 mgO2/mg. ThOD was calculated as 1.60 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 66.87%. Based on the results, the test item, under the test conditions, was considered to be readily biodegradable in nature.

 

Another biodegradation study was conducted for 35 days for evaluating the percentage biodegradability of test chemical (Experimental study report, 1991). The study was performed in accordance with the OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test) under aerobic conditions. Activated sludge obtained from a laboratory plant operated with concomitant wastewater was used as a test inoculum. Conc. of the inoculum used was 1 g/l dry substance in the test batch. Test inoculum was first pre-adapted with the test substance. After completion of this test, the pre-adapted inoculum was separated and washed twice. With the pre-adapted inoculum, the Zahn-Wellens test was performed. Test chemical conc. used for the study was 765 mg/l based on test material analysis / 400 mg/l based on DOC removal. Measuring equipment used during the DOC / TOC analysis with the analyzer was Shimadzu. Diethylene glycol was used as a reference substance for the study. The percentage degradation of test chemical was determined to be 71% based on DOC removal parameter after a period of 35 days. Thus, based on the percentage degradation, test chemical can be considered to be readily biodegradable in nature.

 

In a supporting study,biodegradation experiment was conducted for 28 days for evaluating the percentage biodegradability of test chemical (Experimental study report, 1991). The study was performed in accordance with the OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test) under aerobic conditions. Municipal wastewater obtained from a laboratory treatment plant operated with concomitant wastewater was used as a test inoculum. Test chemical conc. used for the study was 37 mg/l based on test material analysis. Sodium benzoate was used as a reference substance for the study. Reference substance sodium benzoate undergoes 100% degradation after 4 days. The percentage degradation of test chemical was determined to be 7% based on DOC removal parameter after a period of 28 days. Thus, based on the percentage degradation, test chemical can be considered to be not readily biodegradable in nature.

 

For the test chemical from peer reviewed journal (Rita Beel et. Al, 2013), biodegradation study was conducted for 20 days for evaluating the percentage biodegradability of test chemical. The study was performed under aerobic conditions. Activated sludge was used as a test inoculums for the study. Initial test substance conc. used in the study was 20 mg/l. Analytical methods involve the use of both MS and HPLC. The percentage degradation of test chemical was determined to be 100% by after 7 days. Thus, based on percentage degradation, test chemical is considered to be readily biodegradable in nature.

 

Although result from one screening test (performed as per the OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test)) indicates that the test chemical is not readily biodegradable; to confirm this another study as per the OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test) was carried out indicating that the test chemical is readily biodegradable which is supported further by the results of other screening test (from study reports (K1) performed as per the OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test))) and peer reviewed journal. On the basis of this, it has been concluded that the test chemical is considered to be readily biodegradable in nature.

Biodegradation in water and 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,19.5% 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 0.0997%), 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, 80.4% 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 isnot 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 readily biodegradable in nature.

Bioaccumulation: aquatic / sediment

Various studies and 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), the bioaccumulation study was conducted for estimating the BCF (bioaccumulation factor) value of test chemical. The bioaccumulation factor (BCF) value was calculated using a logKow of 0.37 and a regression-derived equation. The estimated BCF (bioaccumulation factor) value of test chemical was determined to be 3.0 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 3.162 L/kg whole body w.w (at 25 deg C).

 

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 4.29 dimensionless . The predicted BCF result based on the 5 OECD principles.

 

On the basis of above results for target chemical, it can be concluded that the BCF value of test chemicalwas determined to be ranges from 3.0– 4.29, respectively, which does not exceed the bioconcentration threshold of 2000, indicating that the test chemical is not expected to bioaccumulate in the food chain.

Bioaccumulation endpoint can also be considered for waiver as per in accordance with column 2 of Annex IX of the REACH regulation, testing for this endpoint is scientifically not necessary and does not need to be conducted since the test chemical has a low potential for bioaccumulation based on logKow ≤ 3.

Adsorption / desorption

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

In an experimental study from study report (2018),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 4.8. 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-naphtol, Direct Red 81, Benzoic acid methylester, Carbendazim, Benzoic acid phenylester, Xylene, Ethylbenzene, Toluene, Naphthalene, 1,2,3-trichlorobenzene, Pentachlorophenol, 3,5-dinitrobenzamide, Benzamide, phenanthrene having Koc value ranging from 1.25 to 4.09. The Log Koc value of test chemical was determined to be 0.992± 0.053 dimensionless at 25°C.This log Koc value indicates that the substance has a negligible sorption to soil and sediment and therefore have rapid migration potential to ground water.

 

Another adsorption study was conducted for estimating the adsorption coefficient (Koc) value of test chemical (authoritative databases, 2017). The adsorption coefficient (Koc) value was calculated using a structure estimation method based on molecular connectivity indices. The adsorption coefficient (Koc) value of test chemical was estimated to be 67 (Log Koc = 1.826). This Koc value indicates that the test chemical has a low sorption to soil and sediment and therefore have moderate migration potential to ground water.

 

On the basis of above results of test chemical (from study report and authoritative databases), it can be concluded that the logKoc value of test chemical was evaluated to be ranges from 0.992 to1.826, respectively, indicating that the test chemical has a negligible to low sorption to soil and sediment and therefore have rapid to moderate migration potential to ground water.