Disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate

Endpoint summary

Description of key information

Hydrolysis

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

28-days Closed Bottle test following the OECD guideline 301 D to determine the ready biodegradability of the test item (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 32ml/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 75.3%. Degradation of Sodium Benzoate exceeds 46.38% on 7 days & 61.44% 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 0.87 mgO2/mg. ThOD was calculated as 0.92 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 94.56%. Based on the results, the test item, under the test conditions, was considered to be readily 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. 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 5 ml water and make the volume with Acetonitrile up to 10 ml. Thus, the test solution concentration was 400 mg/l. The pH of test substance was 6.4. 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, Benzamide, 4-methylaniline(p-Tolouidine), Nmethylaniline, 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, N,N-dimethylbenzamide, 3,5-dinitrobenzamide, phenanthrene, DDT having Koc value ranging from 1.25 to 5.63. The Log Koc value of test chemical was determined to be 1.024±0.010 at 25°C. This log Koc value indicates that the test chemical has a negligible sorption to soil and sediment and therefore have rapid migration potential to ground water.

Additional information

Hydrolysis

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 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 item. 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 32ml/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 75.3%. Degradation of Sodium Benzoate exceeds 46.38% on 7 days & 61.44% 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 0.87 mgO2/mg. ThOD was calculated as 0.92 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 94.56%. Based on the results, the test item, under the test conditions, was considered to be readily biodegradable in nature.

 

Another biodegradation study was carried out for 42 days for evaluating the percentage biodegradation of the test chemical using modified OECD Guideline 302B (U. Pagga et. al., 1986). Activated sludge was used as a test inoculum. The sources of the activated sludge were treatment plants conveniently located to the laboratories carrying out the test. These treatment plants received communal and/or industrial wastewater. Concentration of inoculum i.e, activated sludge used was 0.5 g/l and initial test substance conc. used in the study was 100 mg/l. Analytical methods involve the measurement of extinction at absorption maximum 412 nm and DOC (dissolved organic carbon). The percentage degradation of the test chemical was determined to be -3% by using DOC removal parameter in 42 days. Thus, based on percentage degradation, the chemical was considered to be not readily biodegradable in nature.

 

For the test chemical, biodegradation study was carried out for evaluating the percentage biodegradation of the test chemical (Laura Carmen Apostol et. al., 2012). Anaerobic granular sludge (non-adapted) collected from wastewater treatment plant of ‘‘Central de Cervejas’’, Vialonga, Portugal was used as a test inoculum for the study. The study was performed under anaerobic conditions at a temperature of 37°C and pH7 ± 0.2, respectively. The volatile suspended solids content of the biomass was determined as 0.0943 g VSS g-1.120 ml serum bottles was used as a test vessel for the study. Batch assays were conducted in 120 mL serum bottles with butyl rubber stopper, containing the biomass, 0.94 g VSS L-1, the substrate and macronutrients in a total volume of 50 mL of medium, that was buffered at a pH of 7±0.2 with NaHCO3 (2.5 g L-1).The headspace of the serum bottles was flushed with the N2:CO2 (80/20 v/v) and pre-incubation of the sludge was done overnight at 37°C, in a rotary shaker at 120 rpm. As macronutrients, 2.8 g/l NH4Cl, 2.5 g/LKH2PO4, 1 g/l MgSO4.7H2O and 0.06 g/L CaCl2 were added. Volatile fatty acids (VFAs: acetic, propionic, and butyric acid, 1:10:10) were supplemented as electron source for the reduction (2 g COD L-1). Test chemical conc. used for the study was207.55 mg/l (0.3 mM).The serum bottles were further incubated at 37°C in a rotary shaker at 120 rpm for 1 day. All the experiments were performed in triplicates.Color decrease was monitored spectrophotometrically in a 96-well plate reader (ELISA BIO-TEK, Izasa). At select intervals, samples were withdrawn (300lL), centrifuged at 5000 rpm for 10 min to remove the biomass and/orACand diluted, with the same buffer as of the reaction, to obtain less than one absorbance unit (AU), due to the high absorbance of the dye, even at low concentrations. The visible spectra (300–900 nm) were recorded and dye concentration calculated atλmax. HPLC analyses were also performed in a HPLC (JASCO AS-2057 Plus) equipped with a DAD (Diode Array Detector) detector. A C18 reverse phase Nucleodur MNC18 (25094.0 mm, 5lM particle size and pore of 100 A ° from Machenerey-Nagel, Switzerland) column was used. The following solvent systems were used as mobile phase: solvent A (ACN) and solvent B (Sodium acetate buffer, pH 5.3). Compounds were eluted at a flow rate of 0.7 mL min-1 and at room temperature, with a linear gradient of mobile phase from 10 to 100 % of solvent A, over 15 min, followed by isocratic condition with 100% of solvent A over 10 min. Compounds elution was monitored atλmax of each dye and 230 nm. First-order reduction rate constants were calculated in OriginPro 6.1 software. The percentage decolorization of the test chemical was determined to be 7% after 1 days. Thus, based on percentage degradation, test chemical was considered to be not readily biodegradable in nature.

 

Second study results from peer reviewed journal indicate the chemical to be not readily biodegradable which was not performed as per the standard guideline and in third study, duration of the study was very less (i.e, 1 day), so considering the experimental study result from study report (K1) performed as per the standard OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test), it can be concluded that the test chemical is readily biodegradable in nature.

Adsorption / desorption

Various experimental key and supporting 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 (2018),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 5 ml water and make the volume with Acetonitrile up to 10 ml. Thus, the test solution concentration was 400 mg/l. The pH of test substance was 6.4. 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, Benzamide, 4-methylaniline(p-Tolouidine), Nmethylaniline, 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, N,N-dimethylbenzamide, 3,5-dinitrobenzamide, phenanthrene, DDT having Koc value ranging from 1.25 to 5.63. The Log Koc value of test chemical was determined to be 1.024±0.010 at 25°C. This log Koc value indicates that the test chemical has a negligible sorption to soil and sediment and therefore have rapid migration potential to ground water.

 

Another adsorption study (from peer reviewed journal Yi Wang et. al., 2006) was conducted for150 mins for evaluating the adsorption capacity of test chemical on anaerobic sludge. The study was performed using the batch equilibrium method under anaerobic conditions at a temperature of 38 °C and pH 7.0, respectively. Test chemical concentration used for the study was50 µmol/l. The anaerobic sludge used in this study was collected from a full-scale upflow anaerobic sludge blanket reactor treating citrate-producing wastewater in Bengpu, China. Prior to use, the sludge was first washed with tap water twice, and was then sieved to remove stone, sand and other coarse matters. Adsorption experiments were conducted using 250-ml screwtopped flasks to which 150 ml of dye-containing wastewater and biomass were added. One flask with dosage of dye solution but no biomass was used as control .Before shaking, nitrogen was sparged into the flasks and then the flasks were sealed with rubber plugs to ensure the anaerobic condition in the biosorption process. These flasks were then reciprocated in a water-bath shaker with a shaking rate of 150 rpm. Samples were taken at given time intervals and were then centrifuged at 12,000 rpm for 10 min. The supernatant was used for analysis of the residual test chemical concentration. Each run of the experiments was replicated at least three times. The concentrations of the test chemical was determined using a UV–vis spectrophotometer (UV751GD) at an absorbance wavelength of 516 nm. The concentration of volatile suspended solids (VSS) was measured according to the StandardMethods. Since anaerobic sludge has negative charges, the repulsive electrostatic interaction between test chemical with negative charges and sludge might be responsible for the lower adsorption density (q).Adsorption density (q) of the test chemical was determined to be 2.4 mg/g at a temperature of 38 °C. Anaerobic sludge had a much lower equilibrium adsorption density for test chemical. Based on this adsorption density, it indicates that test chemical has a low sorption and therefore have moderate migration to groundwater.

 

For the test chemical, adsorption experiment was conducted for evaluating the adsorption capacity of test chemical on Iraqi clay. Iraqi clay used in the study was obtained from river in Baghdad and supplied from general company of geological survey and mining Iraq. Soil was prepared by mechanical grinding and sieving to obtain a relatively narrow range of particles passing a no. 500 μm. After sieving Iraqi clay was washed with acidic solution then was washed with distilled water to remove dust and fines (impurities compounds), and dried to constant weight at 100°C.The adsorption isotherms have been determined by allowing eosin solution of known initial concentration to be mixed with accurately weighted amount of Iraqi clay in a tightly closed flask at a certain temperature and pH. The amount of Iraqi clay in the slurry has been 0.2 gm/10 ml solution. A constant mixing at a constant temperature and PH was achieved using a shaker water bath. The Iraqi clay–solution have been then equilibrated for two hours , clay suspensions have been then filtered and the supernatant solution was subjected to analysis using ultra violet –visible technique at (516)nm. Freundlich adsorption constant (Kf) of the test chemical was determined. The high Kf values indicate that the saturation time for adsorption of a test chemical is attained quickly due to high affinity of Iraqi clay towards adsorbate, while low Kf values indicate low adsorption rate of the test chemical. The Freundlich isotherm constant (Kf) value of the test chemical was determined to be 0.025, 0.024, 0.021 and 0.0152 at temperature 20.85 °C, 29.85 °C, 39.85 °C and 49.85 °C, respectively. This low kf indicates that the test chemical has a low adsorption to soil and therefore have moderate migration potential to ground water.

 

On the basis of above overall results for test chemical (from study report and peer reviewed journals), it can be concluded that thetest chemicalhas a negligible to low sorption to soil and sediment and therefore have rapid to moderate migration potential to ground water.