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Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from thesis submitted to JNTU
Qualifier:
according to
Guideline:
other: as mentioned below
Principles of method if other than guideline:
Biodegradation study was conducted for determining the biodegradability of pyrazine-2-carboxylic acid by various bacterial isolates.
GLP compliance:
not specified
Specific details on test material used for the study:
- Name of test material: Pyrazine-2-carboxylic acid
- Molecular formula: C5H4N2O2
- Molecular weight: 124.099 g/mol
- Substance type: organic
- Physical state: solid
- Smiles: c1ncc(C(=O)O)nc1
- InChI: 1S/C5H4N2O2/c8-5(9)4-3-6-1-2-7-4/h1-3H,(H,8,9)
Oxygen conditions:
aerobic/anaerobic
Inoculum or test system:
other: Bacteria
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Bacterial cultures were isolated from environmental samples like cow dung, domestic sewage sample, industrial effluents, effluents from common effluent treatment plant etc.
- Method of cultivation: 0.5 ml of various bacterial cultures were inoculated into 5 ml of pyrazine-2-carboxylic acid (nitrogen source) and glucose (carbon source) in both test tubes and screw cap tubes. It is then incubated under both aerobic and anaerobic conditions for 24 hrs.
- Preparation of inoculum for exposure: The bacterial isolates were grown in mineral salt medium containing pyrazine-2-carboxylic acid (1 mM) as sole nitrogen source and glucose (3%) as sole source of carbon.
Duration of test (contact time):
5 d
Initial conc.:
124.099 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
other: Biodegradation was determined by measuring the U.V absorption maxima at 268 nm and decrease in O.D at that lambda i.e; at 268 nm and at 540 nm.
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral salt medium is used in the study. Composition of the medium is not known.
- Additional substrate: pyrazine-2-carboxylic acid and glucose (3%).

TEST SYSTEM
- Culturing apparatus: Test tubes and screw cap tubes were taken for the study.
- Other:Aerobic bioreactor is used as a test vessel in the study when Pseudomonas aeruginosa strain “Cd” is taken for cultivation.

CONTROL AND BLANK SYSTEM
- Other: Test vessel containing the test substance pyrazine-2-carboxylic acid, glucose but not the bacterial culture is used as a control.
Reference substance:
not specified
Key result
Parameter:
other: % degradation
Value:
90
Sampling time:
5 d
Remarks on result:
other: Measurement of absorption maxima at 268 nm and 540 nm.
Details on results:
90% degradation of pyrazine-2-carboxylic acid was occurred by bacteria Pseudomonas aeruginosa strain “Cd”. No biodegradation was occurred by the remaining bacterial cultures.

Table: Biodegradation of pyrazine-2-carboxylic acid by various bacterial cultures.

 

Organism

Aerobic condition

Anaerobic condition

 

OD

(540 nm)

OD

(268 nm)

Biodegradation

OD

(540 nm)

OD

(268 nm)

Biodegradation

Blank

0.00

3.648

-

0.00

3.583

-

S1

0.10

3.681

-

0.05

3.673

-

S2

0.10

3.663

-

0.04

3.774

-

P1

0.05

3.785

-

0.11

3.764

-

P2

0.05

3.874

-

0.07

3.642

-

C1

0.03

3.690

-

0.05

3.674

-

C2

0.03

3.770

-

0.07

3.651

-

In

0.09

3.687

-

0.08

3.763

-

Id

0.06

3.742

-

0.06

3.660

-

Cd

0.13

1.571

+

0.04

3.783

-

Pold

0.14

3.890

-

0.06

3.653

-

Pold a

0.11

3.786

-

0.04

3.641

-

Pold b

0.09

3.674

-

0.05

3.776

-

Pold c

0.10

3.794

-

0.06

3.650

-

Pold d

0.08

3.661

-

0.05

3.595

-

 

Note:”+” sign indicates that pyrazine-2-carboxylic acid was degraded.

           ”-” sign indicates that pyrazine-2-carboxylic acid was not degraded

Validity criteria fulfilled:
not specified
Interpretation of results:
readily biodegradable
Conclusions:
Among the tested bacterial strains, only Pseudomonas aeruginosa strain “Cd” was able to degrade the test substance pyrazine-2-carboxylic acid in aerobic conditions. The percent degradation was determined to be 90% within 5 days after which no further degradation was observed. Thus, based on percentage degradation, test chemical pyrazine-2-carboxylic acid was considered to be readily biodegradable in water.
Executive summary:

Biodegradation study of test substance pyrazine-2-carboxylic was performed in both aerobic and anaerobic conditions. Bacterial cultures were used as a test inoculum isolated from environmental samples like cow dung, domestic sewage sample, industrial effluents, effluents from common effluent treatment plant etc. The bacterial isolates were grown in mineral salt medium containing pyrazine-2-carboxylic acid (1 mM) as sole nitrogen source and glucose (3%) as sole source of carbon. Concentration of the test substance used for the study was 124.099 mg/l. Aerobic bioreactor is used as a test vessel in the study when Pseudomonas aeruginosa strain “Cd” is taken for cultivation. 0.5 ml of various bacterial cultures were inoculated into 5 ml of pyrazine-2-carboxylic acid (nitrogen source) and glucose (carbon source) in both test tubes and screw cap tubes. It is then incubated under both aerobic and anaerobic conditions for 24 hrs. Test vessel containing the test substance pyrazine-2-carboxylic acid, glucose but not the bacterial culture was used as a control. Biodegradation was determined by measuring the U.V absorption maxima at 268 nm and decrease in O.D at that lambda i.e; at 268 nm and at 540 nm. Among the tested bacterial strains, only Pseudomonas aeruginosa strain “Cd” was able to degrade the test substance pyrazine-2-carboxylic acid in aerobic conditions. The percent degradation was determined to be 90% within 5 days after which no further degradation was observed. Thus, based on percentage degradation, test chemical pyrazine-2-carboxylic acid was considered to be readily biodegradable in nature.

Description of key information

Biodegradation study of test substance pyrazine-2-carboxylic was performed in both aerobic and anaerobic conditions (K. R. Girija et. al; 2002). Bacterial cultures were used as a test inoculum isolated from environmental samples like cow dung, domestic sewage sample, industrial effluents, effluents from common effluent treatment plant etc. The bacterial isolates were grown in mineral salt medium containing pyrazine-2-carboxylic acid (1 mM) as sole nitrogen source and glucose (3%) as sole source of carbon. Concentration of the test substance used for the study was 124.099 mg/l. Aerobic bioreactor is used as a test vessel in the study when Pseudomonas aeruginosa strain “Cd” is taken for cultivation. 0.5 ml of various bacterial cultures were inoculated into 5 ml of pyrazine-2-carboxylic acid (nitrogen source) and glucose (carbon source) in both test tubes and screw cap tubes. It is then incubated under both aerobic and anaerobic conditions for 24 hrs. Test vessel containing the test substance pyrazine-2-carboxylic acid, glucose but not the bacterial culture was used as a control. Biodegradation was determined by measuring the U.V absorption maxima at 268 nm and decrease in O.D at that lambda i.e; at 268 nm and at 540 nm. Among the tested bacterial strains, onlyPseudomonas aeruginosastrain “Cd” was able to degrade the test substance pyrazine-2-carboxylic acid in aerobic conditions. The percent degradation was determined to be 90% within 5 days after which no further degradation was observed. Thus, based on percentage degradation, test chemical pyrazine-2-carboxylic acid was considered to be readily biodegradable in nature.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Various experimental studies and predicted data for the target compound Pyrazine-2-carboxylic acid (CAS No. 98-97-5) and supporting weight of evidence studies for its closest read across substance with logKow as the primary descriptor were reviewed for the biodegradation end point which are summarized as below:

 

In a weight of evidence study from review article (K. R. Girija et. al; 2002), biodegradation study of test substance pyrazine-2-carboxylic was performed in both aerobic and anaerobic conditions. Bacterial cultures were used as a test inoculum isolated from environmental samples like cow dung, domestic sewage sample, industrial effluents, effluents from common effluent treatment plant etc. The bacterial isolates were grown in mineral salt medium containing pyrazine-2-carboxylic acid (1 mM) as sole nitrogen source and glucose (3%) as sole source of carbon. Concentration of the test substance used for the study was 124.099 mg/l. Aerobic bioreactor is used as a test vessel in the study when Pseudomonas aeruginosa strain “Cd” is taken for cultivation. 0.5 ml of various bacterial cultures were inoculated into 5 ml of pyrazine-2-carboxylic acid (nitrogen source) and glucose (carbon source) in both test tubes and screw cap tubes. It is then incubated under both aerobic and anaerobic conditions for 24 hrs. Test vessel containing the test substance pyrazine-2-carboxylic acid, glucose but not the bacterial culture was used as a control. Biodegradation was determined by measuring the U.V absorption maxima at 268 nm and decrease in O.D at that lambda i.e; at 268 nm and at 540 nm. Among the tested bacterial strains, only Pseudomonas aeruginosa strain “Cd” was able to degrade the test substance pyrazine-2-carboxylic acid in aerobic conditions. The percent degradation was determined to be 90% within 5 days after which no further degradation was observed. Thus, based on percentage degradation, test chemical pyrazine-2-carboxylic acid was considered to be readily biodegradable in nature.

 

Another biodegradation study was conducted for evaluating the percentage biodegradability of test substance Pyrazine-2-carboxylic acid (CAS no. 98-97-5) (K. S. Rajini, Ch. Sasikala, Ch. V. Ramana, 2010). Stenotrophomonas sp. HCU1 was used as a test inoculum. Strain HCU1 was serendipitously identified from a pyrazine-2-carboxylate contaminated broth growing in lab. Conc. of test chemical used for the study was24.81 mg/l (200µM).Pyrazine-2-carboxylate (used as an additional supplement) adapted strain HCU1 was harvested by centrifugation (16,0009gfor 10 min) and the pellet was washed (twice) with 0.05 M Tris–HCl buffer (pH 7.4) and resuspended in 1 ml of the same buffer. Cells were sonicated with MS-72 probe (Bandelin, model-UW 2070) to complete cell lysis after 8–9 cycles. The sonicated cell lysate was used for assay of pyrazine-2-carboxylate reduction and degradation. The assay mixture (1 ml) contained; 200µM of pyrazine-2-carboxylate, 200µM of NADPH, 5 mM MgCl2 and 40µl (130–140µg ml-1 protein) of the cell free extract in Tris HCl (pH 7.4). The reaction was stopped at an appropriate time by adding 10µl of 5 N HCl and filtered through 0.22µm polyvinyldienefluoride membrane. The samples were stored at 4°C and were analyzed in HPLC. Ring reduction of pyrazine-2-carboxylate was enzymatically assayed using 1 mM PCA, 200µM NADPH and increase in absorbance of PCA was calculated in terms of % peak height increase/microgram gram protein/minute. Km and Vmax of the enzyme were calculated using the software graph pad prism 5.0.

Mid-logarithmic phase culture (8 h) of strain HCU1 was harvested by centrifugation (12,0009gfor 20 min) and the supernatant was directly concentrated in flash evaporator to dryness. The concentrate was extracted with 50 ml methanol and the precipitated impurities were removed by centrifugation (12,0009gfor 15 min). The methanolic supernatant was diluted with methanol, water (1:0.2) where further precipitation occurred, which was separated by centrifugation (12,0009gfor 15 min). The precipitate was redissolved in methanol (fraction A) and the methanolic water supernatant was concentrated to dryness (fraction B). Both fractions were used for further metabolite purification using semi-preparative HPLC. Analysis of substrates and products was performed in HPLC at room temperature using a Shimadzu SPD-10AT isocratic or gradient system. HPLC instrument details are: solvent flow rate—1.5 ml min-1, Luna 5µC18 100A column (250×4.6 mm) and the heterocyclic compounds were detected using PDA detector at 268 nm. Pyrazine-2-carboxylate, (tR=1.77 min) were identified using standards. Increase in the absorbance (m AU) at 268 nm in HPLC was attributed to aromatic ring reduction of the compounds and loss as its utilization. Purity of the metabolites were confirmed using HPLC with three different solvent systems (methanol: water [1:1]; methanol: water: acetonitrile [1:1: 0.25]; water: acetonitrile [7:3]) at 230, 268, 280 and 350 nm for independent analysis. In addition to this, LC-MS, NMR and Gel exclusion chromatography in FPLC (Fast Protein Liquid Chromatography) was also carried out.  

One metabolite was isolated through semi-preparative HPLC from fraction A. This metabolite had a tR of 1.55 min. The metabolite was characterized based on IR, 1H, 13C NMR and mass spectroscopic analyses. The metabolite is a white amorphous solid and has absorption maximum at 268 nm. Based on the spectral data the metabolite is proposed as 1,2,5,6-tetrahydropyrazine-2-carboxylate. Eight chromatographically (HPLC) distinct peaks were observed from fraction B. This metabolite had a tR of 1.55 min. The metabolite is an orangish sticky compound and has an absorption maximum at 400 nm. The metabolite was characterized based on IR, 1H, 13C NMR and mass spectroscopic analyses. Based on the spectral data analyses, the metabolite is proposed as 2-amino-2-hydroxy-3-(methylamino)propanoic acid. The percentage degradation of test substance Pyrazine-2-carboxylic acid was determined to be 86% by HPLC parameter. Thus, based on percentage degradation, Pyrazine-2-carboxylic acid is considered to be readily biodegradable in nature.

 

In a prediction using the Estimation Programs Interface Suite (EPI suite, 2017), the biodegradation potential of the test compoundPyrazine-2-carboxylic acid(CAS No. 98-97-5) in the presence of mixed populations of environmental microorganisms was estimated. The biodegradability of the substance was calculated using seven different models such as Linear Model, Non-Linear Model, Ultimate Biodegradation Timeframe, Primary Biodegradation Timeframe, MITI Linear Model, MITI Non-Linear Model and Anaerobic Model (called as Biowin 1-7, respectively) of the BIOWIN v4.10 software. The results indicate that chemical Pyrazine-2-carboxylic acid is expected to be readily biodegradable.

 

In a supporting weight of evidence study from authoritative database (HSDB, 2017) and secondary source (2003) for the read across chemical 3-methylpyridine (CAS no. 108-99-6),an aerobic river die-away test was performed for test chemical 3-methylpyridine (CAS No: 108-99-6) to determine its percentage biodegradability for 18 days. Sewage inoculum and polluted river water were used as a test inoculum obtained from Ohio and Little Miami River waters. Initial test substance conc. used for the study was 1 mg/l. Experiment consisted of adding test substance to carboys (size/volume not stated) containing Ohio River water and stored at 20 °C. Samples were removed periodically and analyzed for test substance. After the test substance disappeared, additional test substance was added and disappearance of that portion was monitored. After an initial lag phase of approximately 9 days, biodegradation was rapid. Subsequent additions of test compound resulted in rapid degradation of 100% with no or little lag phase. Degradation attained approximately 100% after 14 days. Additions of test substance after the disappearance of the initial dose were rapidly biodegraded within two days. Thus, based on percentage degradation, chemical 3 -methylpyridine can be considered to be readily biodegradable in nature.

 

For the same read across chemical 3-methylpyridine (CAS no. 108-99-6) from secondary source (2003), a standard BOD5 test of 3-methylpyridine was conducted to evaluate its biodegradability in wastewater. Activated sludge organisms was used as a test inoculum obtained from wastewater. In addition, removal during a pilot plant biological treatment system consisting of a recirculating trickling filter, followed by activated sludge aeration was evaluated. Evaluations were performed at 163, 413 and 746 mg/l of 3-methylpyridine and oxygen demand by BOD5 and test substance removal was calculated. The calculated oxygen demand by BOD5 was 31.0% and 93.8% removal of the read across substance 3-methylpyridine was observed under continuous flow conditions with retention times of up to 312 hours (13 days). Thus, based on percentage degradation, chemical 3-methylpyridine can be considered to be readily biodegradable in nature.

 

For the another read across chemical Benzoic acid (CAS no. 65-85-0) from authoritative database (J-CHECK, 2017), biodegradation experiment was conducted for 14 days for evaluating the percentage biodegradability of read across substance Benzoic acid (CAS no. 65-85-0). 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 substance Benzoic acid was determined to be 85, 98 and 100% by BOD, TOC removal and UV-Vis parameter in 14 days. Thus, based on percentage degradation, Benzoic acid is considered to be readily biodegradable in nature.

 

On the basis of above results for target chemicalPyrazine-2-carboxylic acid(fromreview article, peer reviewed journaland EPI suite, 2017) and for its read across substance (from authoritative database J-CHECK, HSDB and secondary source), it can be concluded that the test substancePyrazine-2-carboxylic acidcan be expected to be readily biodegradable in nature.