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

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experimental result using OECD guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Principles of method if other than guideline:

Biodegradation study was conducted according to OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test) for evaluating the percentage biodegradability of test chemical.

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

other: Mixed inoculum

Details on inoculum:

Mixed Inoculum Preparation:Polyseed were used for this study. 1 polyseed capsule was 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.

Duration of test (contact time):

28 d

Initial conc.:

4 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: OECD mineral medium was used for the study
- Test temperature: 20°C
- Continuous darkness: Yes
- Other: The water used in this study is deionized water.

TEST SYSTEM
- Culturing apparatus: The apparatus used in this study is BOD bottles; with glass stoppers (125 ml), BOD incubator & oxygen electrode and meter.

CONTROL AND BLANK SYSTEM
- Inoculum blank: it contains only test inoculum
- Procedure control: contains reference compound and inoculum

Reference substance:

other: Sodium Benzoate

Key result

Parameter:

% degradation (O2 consumption)

Value:

94.56

Sampling time:

28 d

Remarks on result:

other: Other details not known

Details on results:

The oxygen consumed by the test systems was corrected for oxygen consumption occurring in the blank test systems.The BOD Values (mgO2/mg) and percent biodegradation results for each test system arereported in Tables 2 and 3, respectively. The BOD28 value of test chemical was observed to be 0.87 mgO2/mg. ThOD was determined by calculation as 0.92 mgO2/mg. % degradation was calculated using the values of BOD and ThOD for test item and was determined to be 94.56% at 20 ± 1°C. 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.

Results with reference substance:

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.

TABLE 1: D.O Values (mg/L)

 

No. of Days	Inoculum Blank (Control)	Test Suspension	Procedure Control (Reference Item)
0	7.4	7.3	7.2
7	6.9	6.1	3.6
14	6.6	3.8	2.3
21	6.2	2.8	1.5
28	5.9	2.3	0.7

TABLE 2: BOD Values (mg O2/mg)

 

No. of Days	Test Suspension	Procedure Control (Reference Item)
0	0	0
7	0.17	0.77
14	0.67	1.02
21	0.82	1.12
28	0.87	1.25

 

TABLE 3: PERCENT BIODEGRDATION RESULTS

No. of Days	Test Suspension	Procedure Control (Reference Item)
0	0%	0%
7	18.47%	46.38%
14	72.82%	61.44%
21	89.13%	67.46%
28	94.56%	75.3%

 

Table 4: BOD28, THOD AND % BIODEGRADATION VALUES

 

Method details	BOD28(mgO2/mg)	ThOD (mgO2/mg)	%Biodegradation
Test Item	0.87	0.92	94.56
Reference Item	1.25	1.66	75.3

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item undergoes 94.56% biodegradation after 28 days in the test condition. Thus, the test item considered to be readily biodegradable in nature.

Executive summary:

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.

Description of key information

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.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

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.