Effects of Cu2+ and humic acids on degradation and fate of TBBPA in pure culture of Pseudomonas sp. strain CDT

Soil contamination with tetrabromobisphenol A(TBBPA) has caused great concerns;however, the presence of heavy metals and soil organic matter on the biodegradation of TBBPA is still unclear. We isolated Pseudomonas sp. strain CDT, a TBBPA-degrading bacterium, from activated sludge and incubated it with ~(14)C-labeled TBBPA for 87 days in the absence and presence of Cu~(2+)and humic acids(HA). TBBPA was degraded to organic-solvent extractable(59.4% ± 2.2%) and non-extractable(25.1% ± 1.3%) metabolites,mineralized to CO_2(4.8% ± 0.8%), and assimilated into cells(10.6% ± 0.9%) at the end of incubation. When Cu~(2+)was present, the transformation of extractable metabolites into non-extractable metabolites and mineralization were inhibited, possibly due to the toxicity of Cu~(2+)to cells. HA significantly inhibited both dissipation and mineralization of TBBPA and altered the fate of TBBPA in the culture by formation of HA-bound residues that amounted to 22.1% ± 3.7% of the transformed TBBPA. The inhibition from HA was attributed to adsorption of TBBPA and formation of bound residues with HA via reaction of reactive metabolites with HA molecules, which decreased bioavailability of TBBPA and metabolites in the culture. When Cu~(2+)and HA were both present, Cu~(2+)significantly promoted the HA inhibition on TBBPA dissipation but not on metabolite degradation. The results provide insights into individual and interactive effects of Cu~(2+)and soil organic matter on the biotransformation of TBBPA and indicate that soil organic matter plays an essential role in determining the fate of organic pollutants in soil and mitigating heavy metal toxicity.     

Metabolism of a nonylphenol isomer by Sphingomonas sp. strain TTNP3

For elucidation of the metabolism of the endocrine disruptor nonylphenol by Sphingomonas sp. strain TTNP3, the degradation of an isomer of nonylphenol, 4(2,6-dimethyl-2-heptyl)-phenol, has been studied. As in the case of 4(3,5-dimethyl-3-heptyl)-phenol, the metabolism of this nonylphenol isomer leads to the formation of the NIH-shifted product 2(2,6-dimethyl-2-heptyl)-1,4-benzenediol (NIH: National Institute of Health), but also to the alkoxy derivative 4(2,6-dimethylheptan-2-yloxy)phenol as additional metabolite. To the best of our knowledge, this is the first report describing the formation of alkoxyphenol as a degradation product of nonylphenol. Additionally, these results provide for the first time evidence for slight differences in the biodegradation of the isomers of nonylphenol.     

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 ¹⁴C and ¹³C 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 ¹³C 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 [13C]- and [14C]-labeled phenolic humus and lignin monomers

Natural phenolic monomers are ubiquitous in the environment and are involved in the stabilization of atmospheric carbon and the transformation of xenobiotics. Investigations on the stabilization of phenolic carbons and their environmental fate are hampered by the unavailability of commercial [13C]- and [14C]-labeled phenols. Here we report the complete chemical synthesis of the lignin and humus structural monomers p-coumaric, ferulic, and caffeic acids, p-hydroxybenzaldehyde, protocatechualdehyde, vanillin, catechol, and guaiacol, uniformly [13C]- or [14C]-labeled in the aromatic ring, starting from commercially available [U-ring-13C]- or [U-ring-14C]-labeled phenol. The synthesis of these compounds involved selective ortho-hydroxylation of the aromatic ring, Friedel-Crafts alkylation, and Knoevenagel condensation. [U-ring-13C]- or [U-ring-14C]-p-coumaric acid was synthesized via p-hydroxybenzaldehyde with a 75% yield with respect to phenol. Synthesis of [U-ring-13C]- or [U-ring-14C]-ferulic acid, consisting of six single steps via guaiacol and vanillin, had an overall yield of up to 45%. Uniformly ring-labeled caffeic acid was synthesized either via catechol and protocatechualdehyde in five single steps, yielding [U-ring-14C]-caffeic acid with a 37% yield, or via guaiacol, vanillin, and ferulic acid in seven steps, yielding [U-ring-13C]-caffeic acid with an 18% yield. Ferulic acid, [14C]-labeled at beta-C of the propenoic side chain, was synthesized from [2-14C]-malonic acid under Knoevenagel conditions with a 67% yield with respect to malonic acid. Demethylation of the [beta-14C]-ferulic acid with BBr3 in CH3CN resulted in [beta-14C]-caffeic acid with a 62% yield. All [U-ring-13C]-labeled phenolic products were analyzed by 13C nuclear magnetic resonance (13C-NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS).     

Rapid incorporation and short-term distribution of a nonylphenol isomer and the herbicide MCPA in soil-derived organo-clay complexes

Organo-clay complexes in soil are a major sink for xenobiotics and, thus, often enhance their persistence dramatically. However, the knowledge on environmental processes of non-extractable residue formation on a short time scale is very restricted. Therefore, this study examined the distribution of 4-(3,5-dimethylhept-3-yl)phenol (NP) and 4-chloro-2-methylphenoxyacetic acid (MCPA) in soil over a short time period of 48 h and in different soil sub-fractions. The overall proportion of organo-clay-associated bound residues was not only abundant but also in the same range for both substances (MCPA: 8%; NP: 11% of applied ¹⁴C-radioactivity). However, a more detailed view revealed two different distribution patterns: a higher proportion of clay-associated NP was accompanied by a lower content of bound residues, whereas a smaller fraction of clay-associated MCPA was characterized by a higher proportion of non-extractable residues. Further on, a selective accumulation of bound residues among clay-associated humic fractions was observed. NP residues were linked predominantly to humic acids, whereas MCPA residues tended to be incorporated more into fulvic acids. It was evident that the overall distribution was influenced primarily by the physico-chemical properties of the contaminants. This study demonstrates in detail a rapid initial incorporation accompanied by a specific distribution into soil sub-fractions for selected xenobiotics in soil and points to a complex interaction of clay-associated organic matter with low molecular weight compounds.     

Fate in soil of 14C-sulfadiazine residues contained in the manure of young pigs treated with a veterinary antibiotic

The fate of (14)C-labeled sulfadiazine ((14)C-SDZ) residues was studied in time-course experiments for 218 days of incubation using two soils (A(p) horizon of loamy sand, orthic luvisol; A(p) horizon of silt loam, cambisol) amended with fresh and aged (6 months) (14)C-manure [40 g kg(-1) of soil; 6.36 mg of sulfadiazine (SDZ) equivalents per kg of soil], which was derived from two shoats treated with (14)C-SDZ. Mineralization of (14)C-SDZ residues was below 2% after 218 days depending little on soil type. Portions of extractable (14)C (ethanol-water, 9:1, v/v) decreased with time to 4-13% after 218 days of incubation with fresh and aged (14)C-manure and both soils. Non-extractable residues were the main route of the fate of the (14)C-SDZ residues (above 90% of total recovered (14)C after 218 days). These residues were high immediately after amendment depending on soil type and aging of the (14)C-manure, and were stable and not remobilized throughout 218 days of incubation. Bioavailable portions (extraction using CaCl(2) solution) also decreased with increasing incubation period (5-7% after 218 days). Due to thin-layer chromatography (TLC), 500 microg of (14)C-SDZ per kg soil were found in the ethanol-water extracts immediately after amendment with fresh (14)C-manure, and about 50 microg kg(-1) after 218 days. Bioavailable (14)C-SDZ portions present in the CaCl(2) extracts were about 350 microg kg(-1) with amendment. Higher concentrations were initially detected with aged (14)C-manure (ethanol-water extracts: 1,920 microg kg(-1); CaCl(2) extracts: 1,020 microg kg(-1)), probably due to release of (14)C-SDZ from bound forms during storage. Consistent results were obtained by extraction of the (14)C-manure-soil samples with ethyl acetate; portions of N-acetylated SDZ were additionally determined. All soluble (14)C-SDZ residues contained in (14)C-manure contributed to the formation of non-extractable residues; a tendency for persistence or accumulation was not observed. SDZ's non-extractable soil residues were associated with the soluble HCl, fulvic acids and humic acids fractions, and the insoluble humin fraction. The majority of the non-extractable residues appeared to be due to stable covalent binding to soil organic matter.     

Exploring the potential of applying proteomics for tracking bisphenol A and nonylphenol degradation in activated sludge

A significant percentage of bisphenol A and nonylphenol removal in municipal wastewater treatment plants relies on biodegradation. Nonetheless, incomplete information is available concerning their degradation pathways performed by microbial communities in activated sludge systems. Hydroquinone dioxygenase (HQDO) is a specific degradation marker enzyme, involved in bisphenol A and nonylphenol biodegradation, and it can be produced by axenic cultures of the bacterium Sphingomonas sp. strain TTNP3. Proteomics, a technique based on the analysis of microbial community proteins, was applied to this strain. The bacterium proteome map was obtained and a HQDO subunit was successfully identified. Additionally, the reliability of the applied proteomics protocol was evaluated in activated sludge samples. Proteins belonging to Sphingomonas were searched at decreasing biomass ratios, i.e. serially diluting the bacterium in activated sludge. The protein patterns were compared and Sphingomonas proteins were discriminated against the ones from sludge itself on 2D-gels. The detection limit of the applied protocol was defined as 10^?3 g TTNP3 g^?1 total suspended solids (TSSs). The results proved that proteomics can be a promising methodology to assess the presence of specific enzymes in activated sludge samples, however improvements of its sensitivity are still needed.     

Effect of sludge treatment on the bioaccumulation of nonylphenol in grass grown on sludge-amended soil

We studied the accumulation of p353-nonylphenol residues in the biomass of grass grown in soil amended with sewage sludge submitted to various conditioning/dewatering treatments. Incubation experiments were conducted growing Poa pratensis in sludge-amended soils and applying one ¹⁴C-labelled isomer of nonylphenol in the different systems. More metabolites than parent compounds were recovered in both roots and leaves of the grass. The type of sludge conditioning and dewatering treatment had a slight effect on the bioaccumulation of nonylphenol and its metabolites. When the grass was cultivated in soils amended with dewatered sludge without conditioning pretreatment, an increased accumulation was observed in the roots, while the final biomass of the grass was lower.     

Selenium speciation in acidic environmental samples: application to acid rain-soil interaction at Mount Etna volcano

Speciation plays a crucial role in elemental mobility. However, trace level selenium (Se) speciation analyses in aqueous samples from acidic environments are hampered due to adsorption of the analytes (i.e. selenate, selenite) on precipitates. Such solid phases can form during pH adaptation up till now necessary for chromatographic separation. Thermodynamic calculations in this study predicted that a pH < 4 is needed to prevent precipitation of Al and Fe phases. Therefore, a speciation method with a low pH eluent that matches the natural sample pH of acid rain-soil interaction samples from Etna volcano was developed. With a mobile phase containing 20 mM ammonium citrate at pH 3, selenate and selenite could be separated in different acidic media (spiked water, rain, soil leachates) in <10 min with a LOQ of 0.2 μg L⁻¹ using ⁷⁸Se for detection. Applying this speciation analysis to study acid rain-soil interaction using synthetic rain based on H₂SO₄ and soil samples collected at the flanks of Etna volcano demonstrated the dominance of selenate over selenite in leachates from samples collected close to the volcanic craters. This suggests that competitive behavior with sulfate present in acid rain might be a key factor in Se mobilization. The developed speciation method can significantly contribute to understand Se cycling in acidic, Al/Fe rich environments.     

Radio (<Superscript>14</Superscript>C)- and fluorescent-doubly labeled silica nanoparticles for biological and environmental toxicity assessment

A new and efficient synthetic route to fluorescent and ¹⁴C-double-labeled silica-based nanoparticles (NPs) is described. The synthesis has been carried out using the “oil-in-water” micro-emulsion technique. Fluorescent and radioactive labeling have been achieved entrapping labeled poly(ethylene glycol) (PEG) molecules in the NPs. The produced particles have been analyzed by means of scanning electron microscopy, photon correlation spectroscopy, confocal microscopy, scintillation counting and oxidation/combustion experiments. Fluorescence quenching experiments confirm that the label is entrapped in the particles. The results presented suggest that the silica matrix does not block the β-radiations emitted from the labeled PEG molecules entrapped in the NPs.