4-methylpyridine

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

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:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Well-conducted, peer-reviewed academic study which demonstrates the biodegradation of the substance.

Qualifier:

no guideline followed

Principles of method if other than guideline:

A strain of Gordonia terrea was isolated from soil and established. Pyridine and 4-methylpyridine were added as sole carbon and nitrogen sources to either growing or resting cultures. Growth of cultures and disappearance of substrate (by UV spectroscopy) were monitored at various time periods.

GLP compliance:

not specified

Remarks:

No data available.

Oxygen conditions:

aerobic

Inoculum or test system:

other: newly isolated Gordonia terrea IIPN1

Details on inoculum:

The actinomycete Gordonia was isolated from soil sampled located near petroleum drilling sites in Gujarat, India.
One gram of soil sample was thoroughly mixed in 10 ml distilled water with intermitted shaking and filtered
through Whatman No. 1 filter paper. Filtrate (1 ml) was used for inoculating 25 ml basal medium enriched with 5 mM pyridine as sole carbon and nitrogen source.

Duration of test (contact time):

330 h

Initial conc.:

70 mmol/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

test mat. analysis

Remarks:

HPLC

Parameter followed for biodegradation estimation:

other: bacterial culture expansion with test material as sole carbon and nitrogen source

Details on study design:

TEST CONDITIONS
- Composition of medium:
The basal medium contained (g/l) of 6.3 KH2PO4, 8.0 K2HPO4, 0.2 MgSO4 and 10 ml of metal solution. The metal solution contained (g/l) of 2.0 CaCl2, 1.0 NaCl, 0.5 FeCl2, 0.5 ZnCl2, 0.5 MnCl2, 0.1 Na2MoO4, 0.05 CuCl2, 0.05 Na2WO4.2H2O and 10 ml 10 MHCl per liter of deionized water.

TEST SYSTEM
Pyridine degradation by growing cells: Exponentially growing cells were inoculated into conical flasks (250 ml) containing 40 ml basal medium at initial cell concentration of 200 mg dry cell weight (DCW) per liter. Pyridine (10–70 mM) was used as carbon and nitrogen source in the medium. The culture was incubated in incubator shaker (Innova 4430, USA) at 170 rpm and 30ºC.
Pyridine and 4-methylpyridine degradation by resting cells: Bacterial cells were grown in basal medium with pyridine, 4-methylpyridine as carbon and nitrogen source, and glucose with ammonium sulphate as carbon and nitrogen source in different shake flask culture up to exponential phase (OD540 = 1). Cells were harvested by centrifugation at 10,000g (Sorvall evolution RC, USA) and the cell pellet was washed three times with 50 mM potassium phosphate buffer (pH 7.0) before resuspending 20 mg cells in the same buffer (20 ml) supplemented with 1 mM pyridine or 1 mM 4-methylpyridine. The culture was incubated (Innova 4430, USA) at 170 rpm and 30°C and tested for their ability to degrade pyridine and 4-methylpyridine in resting state.

CONTROL AND BLANK SYSTEM
Medium without adding inoculum or pyridine served as control.

Key result

Parameter:

% degradation (test mat. analysis)

Value:

100

Sampling time:

10 d

Remarks on result:

other: 70 mM 4-Me-pyridine; cell expansion 7.5-fold

Key result

Parameter:

% degradation (test mat. analysis)

Value:

100

Sampling time:

5 d

Remarks on result:

other: 30 mM 4-Me-Pyr; accompanying cell growth yield of 0.29 g/g.

Details on results:

Pyridine degradation by strain Gordonia terrea IIPN1 was observed in mineral salt medium with pyridine as sole source of carbon and nitrogen. Medium containing 30 mM pyridine was completely degraded in 120 h with growth yield of 0.29 g g–1. The growth response of the strain to pyridine as sole nitrogen source increased with increasing pyridine concentration reaching a maximum at 70 mM. Exposure to pyridine concentrations above 120 mM inhibited growth. Cells grown on pyridine degraded 4-methylpyridine without a lag time and vice versa. The overall degradation rate of pyridine and 4-methylpyridine by the resting cells of Gordonia terrea IIPN1 was much faster than that of by the growing cells.

Interpretation of results:

readily biodegradable

Conclusions:

In a biodegradation test in aerobic aqueous medium, the test substance was demonstrated as readily biodegradable.

Executive summary:

4-Methylpyridine is capable of biodegradation by natural actinomyces bacteria, specifically Gordonia terrea IIPN1. Biodegradation of 70 mM concentrations occurred within 10 days, suggesting ready biodegradability.

Reference 2

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:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Well-conducted, peer-reviewed academic study which demonstrates the biodegradation of the substance.

Qualifier:

no guideline followed

Principles of method if other than guideline:

A strain of Pseudonacardia was isolated from soil and established. 4-Methylpyridine, 4-ethylpyridine, and pyridine were added as sole carbon and nitrogen sources to either growing or resting cultures. Disappearance of substrate (by UV spectroscopy) and appearance of metabolites were monitored at various time periods. Identify of metabolites was confirmed by comparison to standards.

GLP compliance:

not specified

Remarks:

No data available.

Oxygen conditions:

aerobic

Inoculum or test system:

other: Pseudonocardia sp. strain M43

Details on inoculum:

Identification of the Pseudonocardia species involved comparison of the published 16S RNA gene sequence for strain M43 and the type strains of other validly described Pseudonocardia species.

Duration of test (contact time):

240 h

Initial conc.:

1.88 mmol/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

test mat. analysis

Remarks:

HPLC

Parameter followed for biodegradation estimation:

other: formation of metabolites

Details on study design:

TEST CONDITIONS
- Composition of medium:
Minimal salts medium (MSM) used for the enrichment culture of microorganisms was prepared as described previously (Lee et al., 2001).
MSM used for the experiment contained the the test item as the sole source of carbon and nitrogen.
Enrichment cultures were initiated by mixing 45mL of MSM with 5mL of excess sludge collected from the wastewater treatment facility in the Taegu dyeing industrial complex, Korea.

TEST SYSTEM
Cultures were carried out in 250mL Erlenmeyer flasks and appended with 0.5mM of test item and incubated aerobically at 30ºC on a rotary shaking incubator (170 rpm).

CONTROL AND BLANK SYSTEM
Medium with a substrate and without an inoculum was used as control.

Key result

Parameter:

% degradation (test mat. analysis)

Value:

100

Sampling time:

240 h

Remarks on result:

other: starting concentration 1.88 mM. Up to 6.4mM of 4-MP was degraded within 240 h.

Details on results:

Strain M43 degraded 4-MP without additional nitrogen sources. 1.88mM of 4-MP was completely degraded in 121 h. Ammonia concentrations were increased in the broth during the course of 4-MP degradation. About 60% of ring nitrogen was released as ammonia. As there was no additional nitrogen source, detection of ammonia indicates that the aromatic ring was cleaved and the nitrogen was released. A total of 86 mg L_1 of cell mass was produced by inoculating 2.1mM of 4-MP. Up to 6.4mM of 4-MP was degraded within 240 h of incubation in the aerobic batch culture. Incubation with 4-MP concentrations higher than 6.4mM resulted in more than 60 h of lag time and reduced the rate of degradation. The optimum initial pH for the degradation of 4-MP was 7.5, but a pH range of 6.5–8.0 was tolerated.

Changes in the UV absorption pattern were noted: the alkylpyridine peak at 254nm decreased and new peaks emerged at wavelengths around 289 and 224 nm. The metabolite from 4-MP was separated by the HPLC with a retention time of 2.8 min. The UV absorption spectrum of commercially available 2-hydroxy-4-methylpyridine was identical to that of the metabolite and when both compounds were coinjected for the HPLC analysis, a single symmetric peak appeared. Mass analysis of the metabolite produced from 4-MP gave a molecular ion [M+] of m/z 109. The molecular ion corresponds to the molecular formula C6H7NO. The metabolite and commercial 2-hydroxy-4-methylpyridine showed similar fragmentation patterns.

On the basis of UV absorption spectra, the mass spectra and the data obtained for the 2-hydroxy-4-methylpyridine intermediate, the metabolite produced from 4-MP was identified as 2-hydroxy-4-methylpyridine. In summary, during the degradation of 4-MP (1.88mM) 2-hydroxy-4-MP corresponding to 37% of 4-MP was transiently accumulated in the culture media.

Interpretation of results:

readily biodegradable

Conclusions:

In a biodegradation test in aerobic aqueous medium, the test substance was demonstrated as readily biodegradable.

Executive summary:

4-Methylpyridine was investigated for the ability to be degraded by a Pseudonocardia sp. strain M43. The substance was completely degraded within 7 days of incubation with the bacterium. Metabolites were identified as 2-hydroxy-4-methylpyridine and ammonia (NH3), indicating that the pyridine ring is broken during metabolic degradation.

Description of key information

Key study: Non-guideline study but well-conducted, peer-reviewed study by Lee et al. (2006). This study showed the complete degradation of 4-methylpyridine in activated sludge, by a filamentous bacterium isolated from the culture identified as Pseudonocardia sp.

Key study: Non-guideline study but well-conducted, peer-reviewed study by Stoban et al. (2008). Results of this study in actinomyces bacteria suggest that 4-methylpyridine is likely to be readily biodegradable.  

 

 

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater