Quantification of the influence of extracellular laccase and intracellular reactions on the isomer-specific biotransformation of the xenoestrogen technical nonylphenol by the aquatic hyphomycete Clavariopsis aquatica.

The aquatic hyphomycete Clavariopsis aquatica was used to quantify the effects of extracellular laccase and intracellular reactions on the isomer-specific biotransformation of technical nonylphenol (t-NP). In laccase-producing cultures, maximal removal rates of t-NP and the isomer 4-(1-ethyl-1,4-dimethylpentyl)phenol (NP112) were about 1.6- and 2.4-fold higher, respectively, than in laccase-lacking cultures. The selective suppression of either laccase or intracellular reactions resulted in essentially comparable maximal removal rates for both compounds. Evidence for an unspecific oxidation of t-NP isomers was consistently obtained from laccase-expressing fungal cultures when intracellular biotransformation was suppressed and from reaction mixtures containing isolated laccase. This observation contrasts with the selective degradation of t-NP isomers by bacteria and should prevent the enrichment of highly estrogenic isomers in remaining t-NP. In contrast with laccase reactions, intracellular fungal biotransformation caused a significant shift in the isomeric composition of remaining t-NP. As a result, certain t-NP constituents related to more estrogenic isomers were less efficiently degraded than others. In contrast to bacterial degradation via ipso-hydroxylation, the substitution pattern of the quaternary alpha-carbon of t-NP isomers does not seem to be very important for intracellular transformation in C. aquatica. As-yet-unknown intracellular enzymes are obviously induced by nonylphenols. Mass spectral data of the metabolites resulting from the intracellular oxidation of t-NP, NP112, and 4-(1-ethyl-1,3-dimethylpentyl)phenol indicate nonyl chain hydroxylation, further oxidation into keto or aldehyde compounds, and the subsequent formation of carboxylic acid derivatives. Further metabolites suggest nonyl chain desaturation and methylation of carboxylic acids. The phenolic moieties of the nonylphenols remained unchanged.

Role of dissolved humic acids in the biodegradation of a single isomer of nonylphenol by Sphingomonas sp.

This study shows the important role of humic acids in the degradation of (14)C and (13)C labeled isomer of NP by Sphingomonas sp. strain TTNP3 and the detoxification of the resulting metabolites. Due to the association of NP with humic acids, its solubility in the medium was enhanced and the extent of mineralization of nonylphenol increased from 20% to above 35%. This was accompanied by the formation of significant amounts of NP residues bound to the humic acids, which also occurred via abiotic reactions of the major NP metabolite hydroquinone with the humic acids. Gel permeation chromatography showed a non-homogenous distribution of NP residues with humic acids molecules, with preference towards molecules with high-molecular-weight. Solid state (13)C nuclear magnetic resonance spectroscopy indicated that the nonextractable residues resulted exclusively from the metabolites. The chemical shifts of the labeled carbon indicated the possible covalent binding of hydroquinone to the humic acids via ester and possibly ether bonds, and the incorporation of degradation products of hydroquinone into the humic acids. This study provided evidences for the mediatory role of humic acids in the fate of NP as a sink for bacterial degradation intermediates of this compound.

Synthesis of branched para-nonylphenol isomers: occurrence and quantification in two commercial mixtures.

Eight tertiary nonanols were synthesized via Grignard reaction and coupled by Friedel-Crafts alkylation with phenol to the corresponding nonylphenols. Six branched para-nonylphenols (NP) were obtained: 4-(3'-methyl-3'-octyl)phenol (33NP), 4-(2'-methyl-2'-octyl)phenol (22NP), 4-(2',5'-dimethyl-2'-heptyl)phenol (252NP), 4-(2',5',5'-trimethyl-2'-hexyl)phenol (2552NP), 4-(2',4'-dimethyl-2'-heptyl)phenol (242NP) and 4-(4'-ethyl-2'-methyl-2'-hexyl)phenol (4E22NP). Their structures were confirmed by GC-MS and NMR spectroscopy. These six isomers as well as the earlier synthesized 4-(3',5'-dimethyl-3'-heptyl)phenol (353NP), 4-(3',6'-dimethyl-3'-heptyl)phenol (363NP) and 4-(2',6'-dimethyl-2'-heptyl)phenol (262NP) were compared with commercial NP mixtures purchased from Acros and Fluka by GC-MS (equipped with a 100 m polysiloxane column). The analyses revealed that all obtained isomers are occurring in different quantities in both commercial NP mixtures.

Use of 15N-depleted artificial compost in bound residue studies.

Association of bound residues to soil humic matter may be accomplished by different binding mechanisms such as sequestration in hydrophobic interiors of the organic material or covalent linkage to the organic matter. The structures and chemical environments of compounds can be observed by NMR spectroscopy. We applied 15N-NMR spectroscopy to study the soil-bound residues of 15N-labeled simazine. As the 15N-isotope has a low sensitivity and natural abundance 15N-NMR experiments require long measurement times and often result in low signal-to-noise (S/N) ratios. Therefore, in addition to the use of 15N-labeled simazine, 15N-depleted compost was used to reduce the amount of background signal and enhance the sensitivity. The compost was produced from maize and wheat plants grown on sand with 15N-depleted NH4NO3 as sole nitrogen source. The plants were freeze-dried, ground and mixed with sand for composting. After a composting period of 224 days analysis of the compost revealed a 15N-content of 267 ppm as opposed to a natural abundance of 3650 ppm. Characterization of this artificial compost produced parameter values similar to those of a natural compost. The 13C-NMR-spectra of the humic and fulvic acids during different stages of maturity showed that there was a shift from single-bond functional groups to more complex double-bond and aromatic structures. Experiments with this compost showed an increased signal intensity. The improved sensitivity made it possible to obtain interpretable NMR signals in contrast to experiments with 15N-simazine on native soil where no signals were detectable. The data indicated that the bound residues of simazine are composed of metabolites resulting from N-dealkylation and triazine ring destruction. Silylation of the bound residues showed a very strong binding of the residues to the matrix as only a small fraction could be solubilized.

Sorption and dialysis experiments to assess the binding of phenolic xenobiotics to dissolved organic matter in soil.

The binding of two phenolic compounds, nonylphenol (NP) and bisphenol A (BPA), and the herbicide metabolite hydroxydesethyl terbuthylazine (HDT) to dissolved organic matter (DOM) as well as its impact on the environmental fate of the compounds in soil have been investigated. In sorption studies with an acidic silty loam soil and a neutral loam soil, no impact of added DOM on adsorption and desorption coefficients was observed. Adsorption coefficients normalized to soil organic carbon (Koc) of the compounds on the loamy silt soil spanned three orders of magnitude: 90, 890, and 9,140 L/kg for HDT, BPA, and NP, respectively. Significantly lower Koc values for HDT on the neutral loam soil as compared to the acidic loamy silt soil indicated additional polar interactions between the latter and the basic herbicide metabolite. In dialysis experiments with fulvic acid from silty loam soil (FA) and peat humic acid (HA), a formation of analyte-DOM associates was observed only for HA. The degree of association was characterized by distribution coefficients normalized to the dissolved organic carbon concentration (KDOC). Values for NP (8,970 L/kg) and BPA (860 L/kg) corresponded with the respective soil sorption coefficients, suggesting similar binding mechanisms in both cases.