2-Ethylhexyl Nitrate: Biodegradability and Microbial Degradation Pathways
Aug 4,2025
The 2-Ethylhexyl nitrate (2-EHN), the nitric acid ester of 2-ethyl-1-hexanol, is currently added in significant amounts (0.05%–0.4%) to diesel oil to improve ignition and boost cetane number (Guibet and Faure 1999; Bornemann et al. 2002). 2-EHN is a large-scale commodity, the worldwide production of which is estimated to be about 100,000 tons per year. It has long been considered as presenting no particular risk to human health. 2-ethylhexyl nitrate was also non-mutagenic according to the Ames test. Upon acute oral or dermal administration to mammals, it seemed relatively non-toxic (ATC 2006). Nevertheless, 2-EHN has been found to be significantly more harmful when administered in repeated doses.
Biodegradability of 2-ethylhexyl nitrate
The low NOAEL level indicates that biodegradability may be a key factor in the overall 2-ethylhexyl nitrate environmental risk assessment. Indeed, in the case of accidental release biodegradability will determine the residual concentration in the environment and exposure may become chronic if no degradation occurs. Under environmental conditions, many organic contaminants are biodegraded by local indigenous microorganisms. Removal of contaminants depends on the intrinsic biodegradability of molecules, on physical parameters existing on site, such as temperature and oxygen and on degradative capacity of local microbial populations. The biodegradation of contaminating molecules often involves cometabolism and cooperation between microorganisms. It is usually assessed using laboratory tests derived from those designed for commercial products. However, as indicated by Battersby et al., current test guidelines designed for water-soluble, organic compounds with low volatility are unsuitable for most oil products. As little information was available on aerobic biodegradation of 2-ethylhexyl nitrate, our study aimed at evaluating the intrinsic biodegradability of 2-EHN under different laboratory conditions. For this purpose, microbial populations from wastewater treatment plants and from soil were used. 2-ethylhexyl nitrate biodegradation by some Actinobacteria species was also investigated because of the recognized ability of these microorganisms to attack recalcitrant molecules.[1]
Because of its hydrophobic nature (log Ko/w of 5.24), and slight solubility in water (12.6 mg l?1 at 20°C), 2-EHN biodegradability could not be adequately estimated by the standard Sturm test which has been designed for freely-soluble substrates of low volatility. As currently performed for hydrophobic compounds, a NAPL system involving HMN was used. Microbial communities from urban WWTPs 1, 4 and 5 exhibited no biodegradation capacity since the CO2 evolved in test flasks after about four weeks did not significantly differ from that of 2-ethylhexyl nitrate-free flasks. The CO2 productions from the urban WWTPs 2 and 3 showed only a slight mineralization of 2-EHN. Urban WWTP microbial populations were therefore not competent for complete 2-EHN-biodegradation although they had been previously shown to be able to degrade both diesel oil and gasoline. Biodegradation capacities of 2-ethylhexyl nitrate were not extensively distributed among microbial populations, since only refinery WWTP microbial populations and some of the soil microbial populations were able to degrade 2-ethylhexyl nitrate. This molecule has a branched structure, the biodegradation of which probably requires particular microorganisms harbouring specific pathways such as the so-called citronellol pathway which removes anteiso methyl groups of branched alkanes.
Biodegradation of 2-Ethylhexyl Nitrate by Mycobacterium austroafricanum
As a result of the extensive use of diesel worldwide, the 2-EHN market is about 100,000 tons per year. Although biodegradability has for a long time been regarded as a relevant characteristic of chemicals, it was only recently incorporated into safety assessments. In the case of fuel oils, large volumes of oxygenates, such as methyl-tert-butyl ether (MTBE), have been added to gasoline since 1992. In case of accidental release of 2-ethylhexyl nitrate into the environment, the fate and impact of the pollution are unpredictable because of the scarcity of data on 2-EHN biodegradation. Screening tests have been recommended by both the U.S. Environmental Protection Agency and the Organization for Economic Cooperation and Development to evaluate the biodegradability of commercial substances. The backbone of 2-ethylhexyl nitrate is a branched alkane, a type of molecule that is more resistant to biodegradation than linear alkanes. The metabolism of both linear and branched hydrocarbons by bacteria involves enzymes of the β-oxidation pathway. In the case of branched alkanes, their degradation may lead to the formation of β-substituted acyl-coenzyme A intermediates that block β-oxidation. The isolation of pure strains able to utilize 2-ethylhexyl nitrate as a sole source of carbon and energy proved rather difficult.[2]
2-ethylhexyl nitrate is a recalcitrant compound that was considered not readily biodegradable according to standard procedures. However, we demonstrated in the present study that selected strains of mycobacteria were able to slowly utilize 2-EHN as a sole source of carbon under defined culture conditions. However, it is unclear whether the unique ability of strain IFP 2173 to grow on 2-ethylhexyl nitrate without NAPL is due to a cell wall composition slightly different from that of other strains or to some other strain-specific trait. The biodegradation of 2-EHN by M. austroafricanum IFP 2173 illustrates the remarkable metabolic capabilities of this strain for recalcitrant hydrocarbons. Indeed, it can degrade another methyl branched alkane, 2,2,4-trimethylpentane, suggesting that it produces enzymes specific for the degradation of anteiso-alkanes. Nevertheless, our results indicate that the degradation of 2-ethylhexyl nitrate by strain IFP 2173 is partial and gives rise to the release of an acyl with an ethyl substituent in the beta position. At least two reasons might explain the accumulation of this metabolite: (i) strain IFP 2173 lacks enzymes able to degrade it, and (ii) because of the ethyl group in the beta position, the metabolite might block the enzyme catalyzing the next step in the degradation of branched alkanes.
References
[1]Solano-Serena, Floriane et al. “Biodegradability of 2-ethylhexyl nitrate (2-EHN), a cetane improver of diesel oil.” Biodegradation vol. 20,1 (2009): 85-94. doi:10.1007/s10532-008-9202-6
[2]Nicolau E, Kerhoas L, Lettere M, Jouanneau Y, Marchal R. Biodegradation of 2-ethylhexyl nitrate by Mycobacterium austroafricanum IFP 2173. Appl Environ Microbiol. 2008 Oct;74(20):6187-93. doi: 10.1128/AEM.01142-08. Epub 2008 Aug 22. PMID: 18723659; PMCID: PMC2570280.
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