3-nitrobenzoic acid

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:

not specified

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:

73.33

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 are reported in Tables 2 and 3, respectively. The BOD28 value of test chemical was observed to be 0.77 mgO2/mg. ThOD was determined by calculation as 1.05 mgO2/mg. % degradation was calculated using the values of BOD and ThOD for test item and was determined to be 73.33% 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	5.4	3.6
14	6.6	4	2.3
21	6.2	3.4	1.5
28	5.9	2.7	0.7

TABLE 2: BOD Values (mg O2/mg)

 

No. of Days	Test Suspension	Procedure Control (Reference Item)
0	0	0
7	0.35	0.77
14	0.62	1.02
21	0.67	1.12
28	0.77	1.25

 

TABLE 3: PERCENT BIODEGRDATION RESULTS

No. of Days	Test Suspension	Procedure Control (Reference Item)
0	0%	0%
7	33.33%	46.38%
14	59.04%	61.44%
21	65.8%	67.46%
28	73.33%	75.3%

 

Table 4: BOD28, THOD AND % BIODEGRADATION VALUES

 

Method details	BOD28 (mgO2/mg)	ThOD (mgO2/mg)	%Biodegradation
Test Item	0.77	1.05	73.33
Reference Item	1.25	1.66	75.3

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item undergoes 73.33% biodegradation after 28 days in the test condition. Thus, the test item was 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 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 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 0.77 mgO2/mg. ThOD was calculated as 1.05 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 73.33%. 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 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 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 0.77 mgO2/mg. ThOD was calculated as 1.05 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 73.33%. 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 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 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 0.77 mgO2/mg. ThOD was calculated as 1.05 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 73.33%. Based on the results, the test item, under the test conditions, was considered to be readily biodegradable in nature.

 

For the test chemical, a batch test in an open system was conducted for 20 days for evaluating the biodegradability of test chemical (from peer reviewed journal P. Pitter; 1976, handbook, authoritative databases and secondary source). Adapted activated sludge was used as a test inoculum obtained from a sewage plant is cultivated in a1000ml volumetric cylinder. The mixture is aerated with pressure air. Every day 200 ml of the mixture is driven off so that the sludge age is 5 days. After driving off the 200ml of the mixture aeration is interrupted, and after sedimentation ca.600ml of the liquid phase is driven off. The residue (200 ml of the thickened activated sludge) is diluted with tap water to the volume ofca.800 ml and 600 mg/l of starch or glucose, 600 mg/l of peptone, 25 ml of a phosphate buffer pH 7.2, and the solution of the tested compound are added. Then the mixture in the cylinder is made up to 1000ml with tap water and aerated for 23 h (the recirculation ratio is 0-25). After this period the procedure is repeated. Test chemical conc. used for the study was 200 mg/l based on COD. To 1000-1500ml of the biological medium such amount of the solution of the substance tested is added that the initial COD is 200 mg/l. Then such an amount of the adapted activated sludge, washed and thickened by sedimentation, is dosed to the medium that the concentration of the dry matter is 100 mg/l. Simultaneously, a blank test is prepared. The beaker is placed in a dark room with a roughly 3 constant temperature of 20±3°C on an electromagnetic stirrer and a pH of 7.2 for 120 hrs. The initial value of COD or organic carbon of the liquid phase are determined. Samples filtered or centrifuged before analysis, are taken at suitable intervals. The decrease of the tested substance in the liquid phase is evaluated by determining COD or organic carbon. The results are compared with those of a blank test and standard compound decomposition. With the degree of degradation also the average specific rate of degradation is determined, expressed in terms of mg COD (or organic carbon) removed by a gram of dry matter of the activated sludge per hour. The percentage degradation of test chemical was determined to be 93.4% by using COD removal parameter in < 20 days. Thus, based on percentage degradation, test chemical was considered to be readily biodegradable in nature.

 

Another biodegradation study was conducted for 5 days for evaluating the percentage biodegradability of test chemical (from peer reviewed journal, 2002 and authoritative databases, 2017). The study was performed according to standard iodometric titration method under aerobic conditions. River water was used as a test inoculum gathered from Jilin section in the Songhua River. Temperature of water sample was 15-20°C, pH 6.8-7.0, 800-3000/ml bacterial counts and dissolved oxygen was about 8.0 mg/l, respectively. Initial test substance conc. used in the study was 2 and 1 mg/l based on ThOD and residual DO, respectively. The test chemical was added to 150 ml water sample in 250 ml bottles. The bottles were then filled to capacity with the water sample, sealed and incubated for 5 days at 20 ± 1°C. There were two replicates for each chemical and each control (only inoculum), respectively. Inoculum blank was setup for the study. The DO concentrations were determined by the iodometric titration method. The test result was expressed as BOD% by comparing the measured BOD5 with ThOD, which is calculated from the molecular formula of the test compound. The percentage degradation of test chemical was determined to be 46.7% degradation by BOD parameter in 5 days. Thus, based on percentage degradation, test chemical is considered to be readily biodegradable in nature.

 

In a supporting study from peer reviewed journal (Lloyd J. Nadeau et. al. 1995),biodegradation experiment was conducted for evaluating the percentage biodegradability of test chemical. Pseudomonas sp. strain JS51, a spontaneous mutant obtained from Pseudomonas sp. strain JS6 that grows on 3-chlorobenzoate was used as a test inoculum for the study. Strain JS51 utilizes test chemical as the sole source of carbon and nitrogen. Minimal salt medium (MSB) containing 7.6 mM (NH4)2SO4 was used as a test medium for the study. Initial test chemical conc. used for the study was 0.6 mM, respectively. Test chemical undergoes complete degradation by using Pseudomonas sp. strain JS51 in 2 hrs by analyzing test chemical concentration by HPLC. During degradation of test chemical, with the release of nitrite protocatechuate intermediate was formed, which also undergoes complete degradation during the study. Thus, based on percentage degradation, test chemical is considered to be readily biodegradable in nature.

 

For the test chemical,biodegradation study was conducted for evaluating the percentage biodegradability of test chemical (Gsbl database, 2017). The study was performed according to OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test) under aerobic conditions.The activated sludge was used as a test inoculum obtained from the biological purification plant of the Hoechst AG's works were taken and dimensioned in such a way that in the test batch 1.1 ± 0.1 g / l activated sludge dry substance (BTS) were included. Initial test substance conc. used in the study was 50-400 mg/l based on DOC removal or 200-1000 mg/l based on COD, respectively. The percentage degradation of test chemical was determined to be 100% after 7 days. Thus, based on percentage degradation, test chemical is considered to be readily biodegradable in nature.

 

Another biodegradation study was conducted for 13 days for evaluating the percentage biodegradability of test chemical (SRC database, 2017). The study was performed under aerobic conditions at a temperature of29°C and at a pH 7.3 to 8.5, respectively. Sewage was used as a test inoculum for the study. Initial test substance conc. used in the study was 10 mg/l. Analysis method involve the use of UV. The percentage degradation of test chemical was determined to be 0, 25 and 88% degradation after 7, 10 and 13 days, respectively. Thus, based on percentage degradation, test chemical is 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 by bacteria as a test inoculum (Takashi Kameya et. al., 1995). Seeding bacteria was used as a test inoculum. The seeding bacteria had been cultured in a continuous anaerobic bioreactor at 37 + 1°C. Synthetic sewage, composed of glucose, peptone and corn steap liquor (carbon ratio = 1:1:2), is supplied into the reactor (3.0 g-C/l, 8.0 g-CS./.d). In this culture, > 95% acidic decomposition and > 90% methanogenic decomposition are achieved. A standard test and low conc. tests using 50 ml vials (total capacity: 68 ml) was employed. Ten test vials were prepared under the same conditions, and they were set in a water bath at 37°C ± 0.5”C. The original solution was added to the test inoculum and organic medium in oxygen-free water. At the starting time and after every week, two vials were opened simultaneously, and the concentration of organic compound was analyzed. The concentration of organic compound was determined by dissolved organic carbon (DOC) in the standard test and by chromatography in low conc. test. Biodegradation ratio is determined by analysing the decrease of DOC in the standard test. The percentage degradation of the test compound was determined to be < 30% degradation by DOC removal, chromatography in 28 days by using standard test and low conc. test. Thus, based on the percentage degradation, the test chemical was considered to be not readily biodegradable in nature.

 

Another biodegradation study was conducted for 28 days for evaluating the percentage biodegradability of test chemical by anaerobic bacteria as a test inoculum (Takashi Kameya et. al., 1995). Anaerobic bacteria was used as a test inoculum. The cultivated bacteria was prepared for seeding as follows. The bacteria suspension was drowned, homogenized, and separated with a centrifuge at 3000 rev./min for 10 min. The deposit was washed by the basal medium solution and separated again with a centrifuge. The deposit was dissolved again in the basal medium solution and its concentration was determined. This original suspension of bacteria was seeded in the test vials and its concentration was reconfirmed. The seeding bacteria had been cultured by a synthetic sewage in a continuous bioreactor at 37 + 1°C. 100 and 30 mg/l conc. was used for both inoculum and test substance conc., respectively. The test involve the use of 50 ml vials (total capacity: 68 ml). Ten test vials were prepared under the same conditions, and they were set in a water bath at 37°C ± 0.5”C. The original solution was added to the test inoculum and basal medium solution, which was prepared by organic medium and/or inorganic medium in oxygen-free water. At the starting time and after every week, two vials were opened simultaneously, and the concentration of organic compound was analyzed. The concentration of organic compound was determined by dissolved organic carbon (DOC) or chromatography. The concentrations of DOC were determined with a total organic carbon analyzer (Shimazu TOC 500) before and after filtration with a 0.45 pm membrane. Biodegradation ratio is determined by analyzisng the decrease of DOC. The percentage degradation of the test chemical was determined to be <30% by DOC removal parameter in 28 days. Thus, based on the percentage degradation, the test chemical was considered to be not readily biodegradable under the given test conditions.

 

Additional supporting biodegradation study (from peer reviewed journal, authoritative database and secondary source) was conducted with an electrolytic respirometer for 14 days for evaluating the percentage biodegradability of test chemical at a temperature of 20± 1°C and pH 7 ± 1. Activated sludge was used as a test inoculums for the study. Concentration of inoculum i.e, sludge used was 30 mg/l and initial test substance conc. used in the study was 100 mg/l, respectively. JIS inorganic medium (1ml/300 ml) was used as a test medium for the study.The measurements of the biochemical oxygen demand (BOD) curves and the concentrations of dissolved organic carbon (DOC) were repeated two or three times for the test compound, and the reproducibilities were confirmed. The biodegradability rank of test chemical was evaluated to be D, indicating that the test chemical is difficult to degrade. Thus, based on this, test chemical is considered to be not readily biodegradable in nature.

 

For the test chemical, biodegradation study was conducted for for evaluating the percentage biodegradability of test chemical (Nigel S. Battersby et. al., 1989). The study was performed under anaerobic conditions at a temperature of35°C.Primary digesting sludge collected from Reading Sewage Works (Berkshire, England), which receives a mixture of domestic and industrial (brewing, food processing, electronics) wastewaters was used as a test inoculum for the study. The average solids content of the sludge during the study period was 2.5% total, 65% volatile.The sludge was collected in air-tight polyethylene bottles and maintained at 35°C until use (within 2 h). Initial test substance conc. used in the study was 50 mg/l. Serum bottles under a headspace of 90% N2-10% CO2 was used as a test vessel for the study. Sterile controls was also setup during the study, to correct for abiotic gas production, contained autoclaved sludge and sterile test chemical. Total gas production was measured weekly with a hand-held pressure meter (John Watson and Smith, Leeds, UK). Ethanol and 4-Cresol were used as a reference substance for the study. Net (blank corrected) gas production (NGP) was expressed as a percentage of the theoretical gas production (ThGP) calculated from the stoichiometry of test chemical mineralization to CH4 and CO2. An NGP .80% of the theoretical gas production (ThGP) was taken to indicate complete mineralization of the test chemical, while an NGP <30% of the ThGP indicated a persistent compound. Inhibitory chemicals resulted in negative NGP, where gas production in test bottles was lower than that in blanks. Biodegradation potential of test chemical was inhibitory. The percentage degradation of test chemical was determined to be -25 ± 6.5% by theoretical gas production as parameter after more than 80 days under anaerobic conditions. Thus, based on percentage degradation, test chemical is considered to be not readily biodegradable in nature.

 

In an additional supporting study, biodegradation experiment was conducted for 14 days for evaluating the percentage biodegradability of test chemical (J-CHECK, 2018 and Envichem, 2014). The study was performed according to OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I). Concentration of inoculum i.e, sludge used was 30 mg/l and initial test substance conc. used in the study was 100 mg/l, respectively. The percentage degradation of test chemical was determined to 6, 0 and 3% by BOD, TOC removal and UV-Vis parameter in 14 days. Thus, based on percentage degradation, test chemical is considered to be not readily biodegradable in nature.

 

On the basis of above overall results for test chemical, it can be concluded that the test chemical can be expected to be readily biodegradable in nature.