Uptake and transformation of phenol and chlorophenols by hairy root cultures of Daucus carota, Ipomoea batatas and Solanum aviculare.

Hairy root cultures of Daucus carota L., Ipomoea batatas L. and Solanum aviculare Forst were investigated for their susceptibility to the highly toxic pollutants phenol and chlorophenols and for the involvement of inherent peroxidases in the removal of phenols from liquid media. Roots of D. carota grew normally in medium containing 1000 micromol l(-1) of phenol, whilst normal growth of roots of I. batatas and S. aviculare was only possible at levels up to 500 micromol l(-1). In the presence of chlorophenols, normal root growth was possible only in concentrations not exceeding 50 micromol l(-1), except for I. batatas which was severely affected at all concentrations. Despite the reduction in biomass, the growth of S. aviculare cultures was sustained in medium containing up to 2000 micromol l(-1) of phenol or 2-chlorophenol, and up to 500 micromol l(-1) of 2,6-dichlorophenol. The amounts of phenol removed by the roots within 72 h of treatment were 72.7%, 90.7% and 98.6% of the initial concentration for D. carota, I. batatas and S. aviculare, respectively. For the removal of 2,6-dichlorophenol the values were, respectively, 83.0%, 57.7% and 73.1%. Phenols labelled with 14C were absorbed by the root tissues and condensed with highly polar cellular substances as well as being incorporated into the cell walls or membranes. The results suggest that S. aviculare, an ornamental plant, would be best suited for remediation trials under field conditions.

Physicochemical characteristics of animal and municipal wastes decomposed in arid soils.

The application of anaerobically processed animal manure to maintain adequate levels of organic matter in arid soils is becoming a common practice. The purpose of this study was to characterize two farm manure products as compared with municipal waste compost (MWC). The anaerobic processing to obtain a biogas manure (BM) product was much faster (25 d) than the aerobic composting of farmyard manure (FYM) (90 d). Drying by three different methods (solar-drying, vacuum-drying at 45 degrees C, and freeze-drying) did not affect the quality of BM. Based on the chemical characteristics, FYM and BM products were comparable, and, containing less ash (30%) and heavy metals (50 mg Pb kg(-1)), seemed superior to MWC that contained 65% ash and 108 mg Pb kg(-1). On the other hand, MWC had higher C content (69.9%), lower acidity (15.04 mol kg(-1)), and higher exothermic peaks (300-460 degrees C) than BM and FYM (50% C, 20 mol kg(-1), and 275-450 degrees C, respectively), thus showing a greater extent of humification. Also, when the organic materials were incubated with arid soils and monitored for mean residence times (MRT), MWC was slightly more resistant to decomposition (MRT 175-180 d) than BM or FYM (MRT 161-166 d). The observed differences, however, were too small to dismiss any of the products as a valuable material for land applications to improve soil quality. In view of the results obtained, MWC may be considered an adequate substitute for BM or FYM, whenever the latter are in short supply.

Effect of phenolic mediators and humic acid on cyprodinil transformation in presence of birnessite.

Naturally occurring phenols were evaluated as mediators for the transformation of the fungicide cyprodinil by birnessite. With birnessite alone, cyprodinil transformation was negligible (0.6%). In the presence of mediators, however, it increased considerably (1.5-60.9%). With some exceptions (2,6-dimethoxyphenol, syringic acid), methoxylated phenols showed a substantial capacity for enhancing the transformation. Mass spectrometry indicated that cyprodinil cross-coupled with free radicals of phenols formed at birnessite surfaces. The extent of cyprodinil transformation in the presence of syringaldehyde, m-methoxyphenol, or vanillin increased with the amount of birnessite. In reactions with o- and p-methoxyphenol and vanillic acid, cyprodinil transformation was unaffected by the amount of birnessite, but it increased with increasing phenol concentration. The addition of various humic acids at low concentrations (5-10 mg/L) had little effect on cyprodinil transformation in the presence of o-methoxyphenol or syringaldehyde. At higher concentrations, however, humic acids inhibited the transformation (by 5-20%) when o-methoxyphenol was a mediator, but showed no effect in the presence of syringaldehyde.

Using 19F NMR spectroscopy to determine trifluralin binding to soil.

Trifluralin is a widely used herbicide for the control of broad leaf weeds in a variety of crops. Its binding to soil may result in significant losses in herbicidal activity and a delayed pollution problem. To investigate the nature of soil-bound trifluralin residues, 14C-labeled herbicide was incubated for 7 weeks with four soils under anoxic conditions. As determined by radiocounting, trifluralin binding ranged between 10 and 53% of the initial 14C depending on the soil tested. 19F NMR analyses of the methanol extracts and different fractions of the extracted soil suggested that bound residue formation largely involved reduced metabolites of the herbicide. A 2,6-diamino product of trifluralin reduction with zero-valent iron (Fe-TR), and the standard of a 1,2-diaminotrifluralin derivative (TR6) formed covalent bonds with fulvic acid (FA), as indicated by the 19F NMR spectra taken periodically over a 3-week contact time. At short contact times, TR6 and Fe-TR formed weak physical bonds with FA, as the respective spin-lattice relaxation times (T1) decreased from the range 1300-1831 ms for TR6 or Fe-TR analyzed in the absence of FA to the range 150-410 ms for TR6/FA or Fe-TR/FA mixtures. In general, the results indicated that trifluralin immobilization involved a variety of mechanisms (covalent binding, adsorption, sequestration), and with time it became increasingly stable.

Use of additives to enhance the removal of phenols from water treated with horseradish and hydrogen peroxide.

Use of additives, such as polyethylene glycol (PEG), selected surfactants, chitosan gel, or activated carbon, has been shown to enhance enzymatic treatment of water polluted with organic compounds. In this study, additives were used to facilitate the removal of 2,4-dichlorophenol (2,4-DCP) from water using minced horseradish (Armoracia rusticana P. Gaertn. et al.) as a carrier of peroxidase activity. The specific objectives of the study were to (i) enhance the pollutant removal activity of minced horseradish by the addition of PEG and other additives (e.g., Tween 20, Triton X-100, and rhamnolipid); (ii) eliminate colored reaction products by the addition of chitosan; and (iii) eliminate color by amending treated water with activated carbon. The disappearance of 2,4-DCP in horseradish-treated water samples amended with PEG or various surfactants (75-90%) was greatly increased over that observed in nonamended samples (29%). The effect of PEG depended on its average molecular weight. As indicated by visible spectrophotometry, enclosing horseradish pieces between two sealed chitosan films completely eliminated colored reaction products; however, the decolorization was accompanied by a reduction in 2,4-DCP removal (from 95 to 60%). On the other hand, commercially available activated carbon completely removed colored reaction products from the treated water without reducing the removal efficiency. Based on the results obtained, it can be concluded that the use of additives may considerably improve the quality of wastewater treated by plant materials.

Release of substituents from phenolic compounds during oxidative coupling reactions.

Phenolic compounds originating from plant residue decomposition or microbial metabolism form humic-like polymers during oxidative coupling reactions mediated by various phenoloxidases or metal oxides. Xenobiotic phenols participating in these reactions undergo either polymerization or binding to soil organic matter. Another effect of oxidative coupling is dehalogenation, decarboxylation or demethoxylation of the substrates. To investigate these phenomena, several naturally occurring and xenobiotic phenols were incubated with various phenoloxidases (peroxidase, laccase, tyrosinase) or with birnessite (delta-MnO(2)), and monitored for chloride release, CO(2) evolution, and methanol or methane production. The release of chloride ions during polymerization and binding ranged between 0.2% and 41.4%. Using the test compounds labeled with 14C in three different locations (carboxyl group, aromatic ring, or aliphatic chain), it was demonstrated that 14CO(2) evolution was mainly associated with the release of carboxyl groups (17.8-54.8% of the initial radioactivity). Little mineralization of 14C-labeled aromatic rings or aliphatic carbons occurred in catechol, ferulic or p-coumaric acids (0.1-0.7%). Demethoxylation ranged from 0.5% to 13.9% for 2,6-dimethoxyphenol and syringic acid, respectively. Methylphenols showed no demethylation. In conclusion, dehalogenation, decarboxylation and demethoxylation of phenolic substrates appear to be controlled by a common mechanism, in which various substituents are released if they are attached to carbon atoms involved in coupling. Electron-withdrawing substituents, such as -COOH and -Cl, are more susceptible to release than electron-donating ones, such as -OCH(3) and -CH(3). The release of organic substituents during polymerization and binding of phenols may add to CO(2) production in soil.

Transformation of the fungicide cyprodinil by a laccase of Trametes villosa in the presence of phenolic mediators and humic acid.

Xenobiotic chemicals can be transformed or covalently bound to humic materials by oxidoreductive enzymes present in terrestrial systems. Chemicals that are not substrates for oxidoreductive enzymes may undergo transformation in the presence of certain reactive compounds, which are often referred to as mediators. In this study, cyprodinil, a broad-spectrum fungicide, did not show any transformation when incubated alone with a laccase from Trametes villosa. It was transformed to a significant extent, however, when a mediator was present. All of the 13 tested mediators belonged to the group of naturally occurring phenols. With some exceptions (2,6-dimethoxyphenol, syringic acid, and ferulic acid), phenols substituted with one or two methoxy groups were very effective mediators. In experiments with 14C-labeled cyprodinil, the radioactive label was largely associated with brown transformation products that precipitated out of the aqueous solution. As determined by mass spectrometry, the products were mixed oligomers resulting from cross-coupling between cyprodinil and a mediator. The addition of large amounts of humic acid (HA) (400 mg/L) to the reaction mixtures involving the most effective mediators reduced cyprodinil transformation (42.6-68.6%) by 12-48%, probably due to an inhibitory effect. The inhibition decreased with decreasing concentration of HA. The addition of HA (400 mg/L) to the reaction mixtures involving the least effective mediators or no mediators (control) enhanced cyprodinil transformation (0.3-17.6%) by 2.9-17.1%, probably as a result of binding to HA.

Treatment of 2,4-dichlorophenol polluted soil with free and immobilized laccase.

Enzyme treatment is currently considered for remediation of terrestrial systems polluted with organic compounds. In this study, two soils from Pennsylvania with 2.8 or 7.4% organic matter contents (Soils 1 and 2, respectively) were amended with 14C-labeled 2,4-dichlorophenol (2,4-DCP) and incubated with a laccase from Trametes villosa (free or immobilized on montmorillonite). 2,4-DCP was either transformed to methanol-soluble polymeric products (11-32%) or covalently bound to soil organic matter (53-85%); unaltered 2,4-DCP could be recovered from soil by methanol extraction (0-38%) at the completion of a 14-d incubation period. In Soil 1, both free and immobilized laccase removed 100% of 2,4-DCP without regard for moisture conditions. In Soil 2, immobilized laccase removed more 2,4-DCP (about 95%, regardless of moisture conditions) than free enzyme (55, 75, and 90% at 30, 55, and 100% of maximum water-holding capacity, respectively). Binding of 2,4-DCP in the humin fraction was nearly the same for free and immobilized laccase. More 2,4-DCP, however, was bound to humic and fulvic acids in the presence of immobilized laccase than in the presence of free laccase. In general, immobilized laccase performed better than free laccase. However, for practical applications, the higher activity of immobilized laccase is offset by a 23% loss in enzyme activity during immobilization, which approximates the 30% increase in free laccase needed to achieve the same level of remediation. Furthermore, immobilized laccase is more costly than free T. villosa laccase.