2-furoic 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 substance.

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:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

95.31

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 1.22 mgO2/mg. ThOD was determined by calculation as 1.28 mgO2/mg. % degradation was calculated using the values of BOD and ThOD for test chemical and was determined to be 95.31 % 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 49.39% on 7 days & 75.30% on 14th day. The activity of the inoculum is 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 49.39% on 7 days & 75.30% on 14th day. The activity of the inoculum is thus verified and the test can be considered as valid.

Table: D.O Values (mg/L)

No. of Days	Inoculum Blank (Control)	Test Suspension	Procedure Control (Reference Item)
0	6.7	6.3	6.5
7	6.5	2.9	3
14	6.2	1.3	1
21	6	1	0.8
28	5.7	0.4	0.5

 

Table: BOD Values (mgO2/mg)

No. of Days	Test Suspension	Procedure Control (Reference Item)
0	0	0
7	0.8	0.82
14	1.12	1.25
21	1.15	1.25
28	1.22	1.25

 

Table: Percentage degradation

No. of Days	Test Suspension	Procedure Control (Reference Item)
0	0%	0%
7	62.5%	49.39%
14	87.5%	75.3%
21	89.84%	75.3%
28	95.31%	75.3%

 

Table: BOD28, ThOD and %biodegradation values

Method details	BOD28 (mgO2/mg)	ThOD (mgO2/mg)	% Biodegradation
Test Item	1.22	1.28	95.31
Reference Item	1.25	1.66	75.3

 

Table: pH

No. of Days	Inoculum Blank (Control)	Test Suspension	Procedure Control (Reference Item)
0	6.9	6.6	6.5
7	6.7	6.8	6.7
14	6.6	6.5	6.3
21	6.8	6.6	6.4
28	6.5	6.3	6.5

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test chemical undergoes 95.31% biodegradation after 28 days in the test condition. Thus, the test chemical was considered to be readily biodegradable.

Executive summary:

Biodegradation study was conducted for 28-days by 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 chemical and reference substance. Polyseed were used as a test inoculum for the study. The concentration of test and reference chemical (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 chemical was determined by calculation. % degradation was calculated using the values of BOD and ThOD for test chemical and 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 49.39% on 7 days & 75.30% 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 1.22 mgO2/mg. ThOD was calculated as 1.28 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 95.31%. Based on the results, the test chemical, under the test conditions, was considered to be readily biodegradable in nature.

Description of key information

Biodegradation study was conducted for 28-days by Closed Bottle test following the OECD guideline 301 D to determine the ready biodegradability of the test chemical (Study report). The study was performed at a temperature of 20°C. The test system included control, test chemical and reference substance. Polyseed were used as a test inoculum for the study. The concentration of test and reference chemical (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 chemical was determined by calculation. % degradation was calculated using the values of BOD and ThOD for test chemical and 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 49.39% on 7 days & 75.30% 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 1.22 mgO2/mg. ThOD was calculated as 1.28 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 95.31%. Based on the results, the test chemical, 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

Experimental key and supporting study of the test chemical were reviewed for the biodegradation end point which are summarized as below:

 

In an experimental key study from study report, biodegradation study was conducted for 28-days by 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 chemical and reference substance. Polyseed were used as a test inoculum for the study. The concentration of test and reference chemical (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 chemical was determined by calculation. % degradation was calculated using the values of BOD and ThOD for test chemical and 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 49.39% on 7 days & 75.30% 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 1.22 mgO2/mg. ThOD was calculated as 1.28 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 95.31%. Based on the results, the test chemical, under the test conditions, was considered to be readily biodegradable in nature.

 

Another biodegradation study (Elmar P. Kuhn et. al., 1998) was conducted under anaerobic conditions for evaluating the percentage biodegradability of test chemical.Mixed culture was used as a test inoculum obtained from aquifer slurries. Aquifer solids and site water were sampled from two well-characterized sites adjacent to the Norman municipal landfill. The sampling was conducted in late spring. The water table at the methanogenic site was 1.35 m below the surface and had a temperature of 18.3°C, a pH of 6.8, a total organic carbon content (TOC) of 288 mg/L and a sulfate concentration of less than 0.1 mM. The water table at the sulfate-reducing site was 1.15 m below the surface and had a temperature of 15.6"C, a pH of 7.2, a TOC of 14.4 mg/L and a sulfate concentration of 2.1 mM. Aquifer samples were transported to the laboratory and placed in an anaerobic glove box where 50 ± 1 g aquifer material and 50 ml site water were aseptically transferred to sterile glass serum bottles (160 ml). The bottles were closed under an atmosphere of N2/C02 (80/20) and reducing conditions were controlled by the addition of Na2S to reach an initial concentration of 1 mM. Filter-sterilized resazurin (0.0002%) was added as a redox indicator. The bottles were sealed with butyl rubber stoppers and crimped with aluminum seals. The bottles were stored for at least 5 d prior to the addition of the test chemical. 10 mM anaerobic stock solutions of test chemical in distilled water were filter sterilized through 0.2-pm membrane filters. Test chemical concentration used for the study was 22.416mg/l(0.2 mM). Serum bottles of 160 ml volume was used as a test vessel. Test vessel was closed with butyl rubber stoppers and aluminum crimp seals. Controls were also run simultaneously during the study. Autoclaved aquifer slurries (121°C, 15 Ib/in2, 180 min) served as controls. The bottles were incubated in the dark at room temperature. 1 ml samples of the aquifer slurries were aseptically and anaerobically removed using a syringe and needle immediately after the addition of the substrates and periodically thereafter during the course of the experiment. The samples were stored at -10°C until analyzed for parent substrate depletion. The samples from the aquifer slurries were thawed and centrifuged (5 min, 8,000 g) prior to reversed-phase high-pressure liquid chromatography (HPLC) of the supernatant using a C18 column. A mixture of acetonitrile and acetate buffer (50 mM, pH 4.5) was used as the mobile phase at a flow rate of 1.5 ml/min. Compound detection was by UV absorbance with a variable-wavelength spectrophotometer (Beckman model 165). The analytical conditions (acetonitrile/buffer ratio, wavelength in nm, retention time in min) for test chemical (0/15, 248, 4.0). The headspace concentration of methane in the serum bottles was determined by flame ionization GC or with a Varian model 3300 gas chromatograph using a 1.8 m x 0.32 cm stainless steel column packed with 80/100 mesh poropack Q. Nitrogen was used as a carrier gas at a flow of 30 ml/min. The temperature of the injector, column and detector were 100, 105 and 120°C, respectively. External standards were employed and kept in stoppered glass serum bottles. Concentrations of methane in the headspace of methanogenic incubations were typically on the order of a few percent. Small pressure changes were not accounted for in the calculation of the methane formed. The aqueous phase samples of the aquifer slurries were prepared as reported above for the substrate analysis and analyzed for sulfate by anion exchange HPLC. The mobile phase (4.0 mM potassium hydrogen phthalate, pH 5.0) was pumped at 2.5 ml/min using an LDC/Milton Roy minipump (model 396) through an anion exchange column (Vydac, 250 x 4.6 mm, Alltech Associates, Inc., Deerfield, IL). Sulfate had a retention time of 6.3 min and was detected with a Wescan (Santa Clara, CA) conductivity detector (model 213-505). The detection limit was at least 0.1 mM. The total amount of methane formed in the serum bottles was corrected for the amount produced in substrate-unamended controls. The percentage degradation of test chemical was determined to be 100% after a period of 30 days. Methane was formed during the degradation study. Thus, test chemical was considered to be readily biodegradable in water.

In a supporting study, biodegradation study (Kerstin Koenig et. al., 1989) was carried out for 14 d for evaluating the biodegradation potential of test chemical. The organism isolated from soil samples was used as a test inoculum. Inocula were suspended in flasks with 5 ml of a mineral salts medium. This medium consisted of 0.18 mM CaCl2, 0.14 mM MnSO4, 5.6 mM NH4CI, 0.85 mM NaCl, 10 mM potassium phosphate buffer (pH 7.2), and 10 mM 2-furoic acid or succinate as the carbon and energy source. Cells were grown in 250-ml baffled flasks containing 50 ml of medium. The final pH was adjusted to 7.2. After sterilization, 5 ml of filter-sterilized vitamin solution and 1 ml of sterile trace element solution SL 10 were added per liter. The flasks were aerobically shaken at 30°C. After 2 weeks, serial dilutions were plated on mineral agar (mineral salts medium containing 2% agar) and incubated at 30°C. Several colonies were isolated and again were transferred onto mineral salts medium. The organism Pseudomonas putida Fu1 used in this study was isolated from soil samples. Isolated bacterial colonies were maintained on nutrient agar plates containing (per liter of demineralized water) 10 g of tryptone, 5 g of yeast extract, 2 g of succinate, and 20 g of agar. Test chemical conc. used for the study was 10 mM. The study was performed in a 250 ml baffled flasks at a temperature of 30°C and pH 7.5 for 14 days. Flasks were shaken aerobically. HPLC was used to detect test chemical while spectrophotometer for bacterial growth (bacterial growth was measured at 600 nm). HPLC performed by using a Kontron system with a pump model 420, an injector, a RP-18 reverse-phase column (4 by 250 mm, 10-p.m particle size), and a detector model 430. Isocratic elution was performed with 60% 2-propanol and 40% H20, pH 2.9 (with formic acid), at a flow rate of 1 ml/min. The detection was set at a 244-nm wavelength. The percentage degradation of test chemical was determined to be 100% using HPLC parameter after 14 days. Thus, test chemical was considered to be readily biodegradable in water.

 

Thus, on the basis of this available information, test chemical was considered as readily biodegradable in water