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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:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Data is from peer reviewed journal
Justification for type of information:
Data is from peer reviewed journal
Qualifier:
according to
Guideline:
other: as mentioned below
Principles of method if other than guideline:
Biodegradation study was conducted for evaluating the percentage biodegradability of test substance 2,4-Pyridinedicarboxylic acid. The study was carried out using Micrococcus luteus as the test inoculum.
GLP compliance:
not specified
Specific details on test material used for the study:
- Name of test material (IUPAC name): 2,4-Pyridinedicarboxylic acid
- Common name: Pyridine-2,4-dicarboxylic acid
- Molecular formula: C7H5NO4
- Molecular weight: 167.12 g/mol
- Smiles notation: c1(cc(ncc1)C(O)=O)C(O)=O
- InChl: 1S/C7H5NO4/c9-6(10)4-1-2-8-5(3-4)7(11)12/h1-3H,(H,9,10)(H,11,12)
- Substance type: Organic
- Physical state: Solid
- Other: Test chemical 2,4-Pyridinedicarboxylic acid was obtained from Aldrich Chemical Co., Inc.
Oxygen conditions:
not specified
Inoculum or test system:
other: Micrococcus luteus (Micro-organisms)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Test inoculum Micrococcus luteus was isolated from Chalmers silt loam soil, not previously exposed to pyridine, using enrichment culture techniques.

- Preparation of inoculum for exposure: A Chalmers silt loam soil, not previously exposed to pyridine, was used as an inoculum for enrichment cultures. The medium contained basal salts, yeast extract (150 mg/liter), and pyridine (0.01 M). The soil was perfused with pyridine enrichment medium in a rotary perfusion device. Perfusion of the soil resulted in enrichment of a gram-positive, aerobic, furazolidone-resistant coccus which was identified as a Micrococcus species. Cells were maintained on pyridine agar slants at 4°C.
Initial conc.:
1 671.2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
test mat. analysis
Remarks:
(%degradation )
Details on study design:
TEST CONDITIONS
- Composition of medium: The medium contained basal salts, yeast extract (150 mg/liter), and pyridine (0.01 M). Media containing substituted pyridines were filter sterilized.
Defined media used in enzyme induction experiments thus contained thiamine (2 µM), arginine (0.3 mM), valine (0.4 mM), leucine (0.4 mM), and methionine (0.2 mM) to satisfy growth factor requirements and produce a high yield of cells.

- Additional substrate: M. luteus required thiamine for growth on pyridine or succinate, and growth was enhanced by the addition of arginine, valine, leucine, and methionine.

- Test temperature: 24°C

TEST SYSTEM
- Measuring equipment: Degradation of substituted pyridines was measured as the decrease in absorbance at an appropriate wavelength in the UV. Measurements at fixed wavelengths were made with a Gilford 250.
Parameter:
% degradation (test mat. analysis)
Value:
0
Remarks on result:
other: M. luteus was unable to grow at the expense of the substituted pyridine tested i.e. 2,4-Pyridinedicarboxylic acid.
Details on results:
No degradation of test substance 2,4-Pyridinedicarboxylic acid was determined by M. luteus.
Validity criteria fulfilled:
not specified
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The percentage degradation of test substance 2,4-Pyridinedicarboxylic acid was determined to be 0% by M. luteus. Thus, based on percentage degradation, 2,4-Pyridinedicarboxylic acid is considered to be not readily biodegradable in water.
Executive summary:

Biodegradation study was conducted for evaluating the percentage biodegradability of test substance 2,4-Pyridinedicarboxylic acid (CAS no. 499-80-9). The study was carried out usingMicrococcus luteus as the test inoculum isolated from Chalmers silt loam soil, not previously exposed to pyridine, using enrichment culture techniques. A Chalmers silt loam soil, not previously exposed to pyridine, was used as an inoculum for enrichment cultures. The medium contained basal salts, yeast extract (150 mg/liter), and pyridine (0.01 M).The soil was perfused with pyridine enrichment medium in a rotary perfusion device. Perfusion of the soil resulted in enrichment of a gram-positive, aerobic, furazolidone-resistant coccus which was identified as a Micrococcus species.Cells were maintained on pyridine agar slants at 4°C. Growth was measured as optical density at 540 nm and expressed as cell density (dry weight) based on standard curves. Measurement at 540 nm avoided interference from a soluble yellow pigment produced by M. luteus when grown on pyridine. The test medium contained basal salts, yeast extract (150 mg/liter), and pyridine (0.01 M).Media containing substituted pyridines were filter sterilized.Defined media used in enzyme induction experiments thus contained thiamine (2 µM), arginine (0.3 mM), valine (0.4 mM), leucine (0.4 mM), and methionine (0.2 mM) to satisfy growth factor requirements and produce a high yield of cells. M. luteus required thiamine for growth on pyridine or succinate, and growth was enhanced by the addition of arginine, valine, leucine, and methionine.Cells were cultured in the salt medium with yeast extract and various carbon sources at 24°C.Degradation of substituted pyridines was measured as the decrease in absorbance at an appropriate wavelength in the UV, and samples were periodically scanned from 300 to 200 nm to detect the formation of UV-absorbing intermediates. Scans were performed with a Cary 17-D spectrophotometer, whereas measurements at fixed wavelengths were made with a Gilford 250.M. luteus was unable to grow at the expense of the substituted pyridine tested i.e.2,4-Pyridinedicarboxylic acid. Thus,no degradation of test substance 2,4-Pyridinedicarboxylic acidwas determined by M. luteus. Based on percentage degradation (0%), 2,4-Pyridinedicarboxylic acid is considered to be not readily biodegradable in nature.

Description of key information

Biodegradation study was conducted for evaluating the percentage biodegradability of test substance 2,4-Pyridinedicarboxylic acid (CAS no. 499-80-9) (Gerald K. Sims, et. al; 1986).

The study was carried out usingMicrococcus luteus as the test inoculum isolated from Chalmers silt loam soil, not previously exposed to pyridine, using enrichment culture techniques. A Chalmers silt loam soil, not previously exposed to pyridine, was used as an inoculum for enrichment cultures. The medium contained basal salts, yeast extract (150 mg/liter), and pyridine (0.01 M).The soil was perfused with pyridine enrichment medium in a rotary perfusion device. Perfusion of the soil resulted in enrichment of a gram-positive, aerobic, furazolidone-resistant coccus which was identified as a Micrococcus species.Cells were maintained on pyridine agar slants at 4°C. Growth was measured as optical density at 540 nm and expressed as cell density (dry weight) based on standard curves. Measurement at 540 nm avoided interference from a soluble yellow pigment produced by M. luteus when grown on pyridine. The test medium contained basal salts, yeast extract (150 mg/liter), and pyridine (0.01 M).Media containing substituted pyridines were filter sterilized.Defined media used in enzyme induction experiments thus contained thiamine (2 µM), arginine (0.3 mM), valine (0.4 mM), leucine (0.4 mM), and methionine (0.2 mM) to satisfy growth factor requirements and produce a high yield of cells. M. luteus required thiamine for growth on pyridine or succinate, and growth was enhanced by the addition of arginine, valine, leucine, and methionine.Cells were cultured in the salt medium with yeast extract and various carbon sources at 24°C.Degradation of substituted pyridines was measured as the decrease in absorbance at an appropriate wavelength in the UV, and samples were periodically scanned from 300 to 200 nm to detect the formation of UV-absorbing intermediates. Scans were performed with a Cary 17-D spectrophotometer, whereas measurements at fixed wavelengths were made with a Gilford 250.M. luteus was unable to grow at the expense of the substituted pyridine tested i.e.2,4-Pyridinedicarboxylic acid. Thus,no degradation of test substance 2,4-Pyridinedicarboxylic acidwas determined by M. luteus. Based on percentage degradation (0%), 2,4-Pyridinedicarboxylic acid is considered to be not readily biodegradable in nature.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

Various experimental studies for the target compound 2,4-Pyridinedicarboxylic acid (CAS No. 499-80-9) and supporting weight of evidence study for its structurally similar read across substance were reviewed for the biodegradation end point which are summarized as below:

 

In an experimental key study from peer reviewed journal (Gerald K. Sims, et. al; 1986),biodegradation experimemt was conducted for evaluating the percentage biodegradability of test substance 2,4-Pyridinedicarboxylic acid (CAS no. 499-80-9). The study was carried out using Micrococcus luteus as the test inoculum isolated from Chalmers silt loam soil, not previously exposed to pyridine, using enrichment culture techniques. A Chalmers silt loam soil, not previously exposed to pyridine, was used as an inoculum for enrichment cultures. The medium contained basal salts, yeast extract (150 mg/liter), and pyridine (0.01 M).The soil was perfused with pyridine enrichment medium in a rotary perfusion device. Perfusion of the soil resulted in enrichment of a gram-positive, aerobic, furazolidone-resistant coccus which was identified as a Micrococcus species. Cells were maintained on pyridine agar slants at 4°C. Growth was measured as optical density at 540 nm and expressed as cell density (dry weight) based on standard curves. Measurement at 540 nm avoided interference from a soluble yellow pigment produced by M. luteus when grown on pyridine. The test medium contained basal salts, yeast extract (150 mg/liter), and pyridine (0.01 M).Media containing substituted pyridines were filter sterilized. Defined media used in enzyme induction experiments thus contained thiamine (2 µM), arginine (0.3 mM), valine (0.4 mM), leucine (0.4 mM), and methionine (0.2 mM) to satisfy growth factor requirements and produce a high yield of cells. M. luteus required thiamine for growth on pyridine or succinate, and growth was enhanced by the addition of arginine, valine, leucine, and methionine. Cells were cultured in the salt medium with yeast extract and various carbon sources at 24°C.Degradation of substituted pyridines was measured as the decrease in absorbance at an appropriate wavelength in the UV, and samples were periodically scanned from 300 to 200 nm to detect the formation of UV-absorbing intermediates. Scans were performed with a Cary 17-D spectrophotometer, whereas measurements at fixed wavelengths were made with a Gilford 250.M. luteus was unable to grow at the expense of the substituted pyridine tested i.e.2,4-Pyridinedicarboxylic acid. Thus, no degradation of test substance 2,4-Pyridinedicarboxylic acid was determined by M. luteus. Based on percentage degradation (0%), 2,4-Pyridinedicarboxylic acid is considered to be not readily biodegradable in nature.

 

In a supporting study, biodegradability of test substance 2,4-Pyridinedicarboxylic acid (CAS no. 499-80-9) was estimated using the MULTICASE method which involve the use of two QSAR models (Bodo Phillip, et. al; 2007). The models have been applied are Quantitative Structure-Activity Relationships.QSAR models for biodegradation are created with experimental biodegradation data and structures of the corresponding chemicals. The MULTICASE method has been described.This software is designed to automatically perform statistical analysis of learning sets containing structures and biological activity data of organic compounds, detect the molecular fragments significantly linked to a biological endpoint, and derive predictive QSAR models there from.The MULTICASE method has been described.This software is designed to automatically perform statistical analysis of learning sets containing structures and biological activity data of organic compounds, detect the molecular fragments significantly linked to a biological endpoint, and derive predictive QSAR models there from. Order to run statistical algorithms on the learning set, the program calculates all the possible fragments for each molecule, ranging from 2 to 10 non-hydrogen atoms along with their associated hydrogen atoms. Each of these fragments as well as automatically calculated 2-dimensional distance descriptors (based upon the presence of lipophilic centers and heteroatoms in the molecule) is associated with a confidence level and a probability of activity that is derived from its distribution among biological active and inactive molecules. Molecules containing the same activating fragments but having slightly different activity values are then searched for modulators of activity. These modulators may be chemical properties (eg. Structural fragments), physicochemical properties (eg. logPow), or quantum chemical parameters (eg. HOMO and LUMO energies), which are calculated by the program. Provided that the learning set contained sufficient data on potential SAR, a QSAR-model is established which enables the program to run in a predictive mode and evaluate untested molecules. Experimental biodegradation data were taken from different databases.On the basis of this evaluation, we classified the N-heterocycles into three categories, namely readily/inherently, moderately, and poorly degradable, according to OECD Standards. MULTICASE analysis was performed with the 194 data set to determine fragments that activate (QSAR1) or inactivate (QSAR 2) the aerobic biodegradation of N-heterocycles. In QSAR1, biodegradable compounds were considered as active and were assumed to contain biodegradation-related structural fragments (activating fragments). Poorly biodegradable compounds were considered as inactive and were assumed not to contain structural fragments activating biodegradation or containing inactivating fragments. To identify inactivating fragments (QSAR 2), the scale of activity was inversed, labelling biodegradable molecules as inactive and poorly biodegradable molecules as active before analysis with MULTICASE. If MULTICASE detects unknown fragments, or fragments with a low confidence level or with a low probability, the respective molecules are rejected as inconclusive and no prediction is made. Test chemical was estimated to contain fragment 4 and 9, respectively. Molecules carrying activating fragment 4 are aromatic N-heterocycles, mainly pyridines and quinolines.Molecules with activating fragment 9 overlap largely with aromatic compounds carrying fragment 4 (68%) or with nonaromatic molecules carrying fragments 1 or 2. Chemical biodegradability was reported as (+) readily/inherently biodegradable, (0) moderately biodegradable or (-) poorly biodegradable. As the biodegradability prediction of chemical 2,4-Pyridinedicarboxylic acid by the two QSAR models (used within MULTICASE analysis) comes out to be +, test chemical 2,4-Pyridinedicarboxylic acid is estimated to be readily biodegradable in water.

 

For the read across chemical 2,5-Pyridinedicarboxylic acid (CAS no. 100-26-5) from authoritative database (J-CHECK, 2017), biodegradation study was conducted for 28 days for evaluating the percentage biodegradability of read across substance 2,5-Pyridinedicarboxylic acid. 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 2,5-Pyridinedicarboxylic acid was determined to be 0, 1 and 9% by O2 consumption, BOD, TOC removal, Test mat. analysis and HPLC parameter in 28 days.. Thus, based on percentage degradation, 2,5-Pyridinedicarboxylic acid is considered to be not readily biodegradable in nature.

 

Although one study frompeer reviewed journalwhich is the estimated result indicates that the test chemical 2,4-Pyridinedicarboxylic acid is readily biodegradable, but based on the other experimental study from peer reviewed journal (K2) and on the basis ofread across substance (from authoritative database J-CHECK), it can be concluded that the test substance 2,4-Pyridinedicarboxylic acid can be expected to be not readily biodegradable in nature.