The degradation of radiolabeled 4(3',5'-dimethyl-3'-heptyl)-phenol [nonylphenol (NP)] was tested with resting cells of Sphingomonas sp. strain TTNP3. Concomitantly to the degradation of NP, a metabolite identified as hydroquinone transiently accumulated and short-chain organic acids were then produced at the expense of hydroquinone. Two other radiolabeled isomers of NP, 4(2',6'-dimethyl-2'-heptyl)-phenol and 4(3',6'-dimethyl-3'-heptyl)-phenol, were synthesized. In parallel experiments, the 4(2',6'-dimethyl-2'-heptyl)-phenol was degraded more slowly than the other isomers of NP by strain TTNP3, possibly because of effects of the side-chain structure on the kinetics of degradation. Alkylbenzenediol and alkoxyphenol derivatives identified as metabolites during previous studies were synthesized and tested as substrates. The derivatives were not degraded, which indicated that the mineralization of NP does not proceed via alkoxyphenol as the principal intermediate. The results obtained led to the elucidation of the degradation pathway of NP isomers with a quaternary alpha-carbon. The proposed mechanism is a type II ipso substitution, leading to hydroquinone and nonanol as the main metabolites and to the dead-end metabolites alkylbenzenediol or alkoxyphenol, depending on the substitution at the alpha-carbon of the carbocationic intermediate formed.

Sphingomonas sp. strain TTNP3 degrades 4(3,5-dimethyl-3-heptyl)-phenol and unidentified metabolites that were described previously. The chromatographic analyses of the synthesized reference compound and the metabolites led to their identification as 2(3,5-dimethyl-3-heptyl)-1,4-benzenediol. This finding indicates that the nonylphenol metabolism of this bacterium involves unconventional degradation pathways where an NIH shift mechanism occurs.Nonylphenol (NP) is a ubiquitous pollutant which mainly results from the biodegradation of widely used NP polyethoxylate surfactants, and it is well known for its endocrine systemdisrupting potential (5,8,11,13,20,21). NP is used as part of a technical mixture which consists of more than 20 branched isomers of the nonyl chain (23, 28). Although some studies report an easy degradation of the NP with a linear alkyl chain (4-n-NP) through oxidation of the side chain, the situation is different for branched isomers of NP (2,25,26). The main reason for the poor degradability of NP is a quaternary carbon atom (␣-carbon) of the branched alkyl chain, which is involved in the attachment of the nonyl group to the phenol moiety. More than 85% of the isomers of the technical mixture contain such quaternary ␣-carbon atoms (26, 28). Some strains belonging to the genera Sphingomonas, Sphingobium, and Pseudomonas have been reported to be able to degrade the branched isomers of NP, but the catabolic pathways in these bacteria remain poorly characterized (6,7,18,19,21,24).Previously, it was proven that the dead-end product of the degradation of 4(3Ј,5Ј-dimethyl-3Ј-heptyl)-phenol (p353NP) diastereomers is 3,5-dimethyl-3-heptanol (nonanol). This metabolite corresponds to the nonyl chain of p353NP, where the aromatic ring has been replaced by a hydroxyl group (3). It remains to be elucidated how the very stable quaternary C atom structure of NP can lead to the formation of nonanol. Nonanol has been found only in cultures of Sphingomonas sp. strain TTNP3 and Sphingomonas cloacae, so these bacteria seem to possess particular degradation pathways for NP (3,6,21). Recently, it was reported that neither 4-n-NP nor shortchain alkylphenols were degraded by Sphingomonas sp. strain TTNP3, whereas the diastereomers of the branched single isomer p353NP (Fig. 1) led to the formation of ring-oxidized metabolites (4). Ring-oxidized metabolites were detected only in the intracellular fraction and were shown to be different from 4(3Ј,5Ј-dimethyl-3Ј-heptyl)-catechol (Fig. 1b) and 4(3Ј,5Ј-dimethyl-3Ј-heptyl)-resorcinol (Fig. 1c), hydroxylated at positions C-2 and C-3 (Fig. 1), respectively. We hypothesize the formation of metabolites which undergo either hydroxylation with an alkyl chain internal rearrangement, as is the case for bisphenol A (14), or hydroxylation-induced migration of the alkyl chain immediately after hydroxylation at position C-4. The latter process would lead to the formation of 2(3Ј,5Ј-dimethyl-3Ј-heptyl)-1,4-benzenediol (o353NHQ) (Fig. 1d). In order to identify definitively these metabolites ...

The degradation of the 4(3',5'-dimethyl-3'-heptyl)-phenol (p353NP) nonylphenol isomer in cultures of Sphingomonas TTNP3 supplemented with the technical mixture of nonylphenol was first assessed. Then the radioactive and non-labelled form of these diastereomers were both synthesised. The radioactive isomers were synthesised using [ring-U-14C]-labelled phenol and 3,5-dimethyl-3-heptanol by Friedel and Crafts alkylation. The time-course of degradation was performed with and without 14C-p353NP; balancing of radioactivity was calculated from different soluble fractions (organic, aqueous), bacterial biomass, and 14CO2 evolved as mineralization product. The noticeable portion of 14C bound to biomass showed that at least the aromatic ring of 14C-p353NP was degraded and served as energy source and probably as carbon source for bacterial growth. In addition, the appearance of 3,5-dimethyl-3-heptanol, the nonanol corresponding with the side-chain of p353NP, was demonstrated in the bacterial media, and its concentration determined during the course of fermentation. Besides the parent 14C-p353NP, no other radioactive compounds, i.e. metabolites of 14C-p353NP were detected in the media.

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