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[Pentachlorophenol--results of a Bavarian human monitoring program].

In connection with an investigation of state-owned buildings for wood preservatives, human monitoring on pentachlorophenol (PCP) was performed on 484 subjects from Northern Bavaria and 299 persons from Southern Bavaria during the period November 1994 to October 1995. Altogether, a similar pattern of concentrations resulted, the 95th percentile amounting to 15 (Northern Bavaria) and 20 micrograms PCP/l blood plasma (Southern Bavaria), respectively. An inquiry of the exposure conditions of the subjects studied in Southern Bavaria revealed that 28% had been exposed to wood containing 5-100 mg PCP/kg (slightly elevated) and 61 % to wood with > 100 mg PCP/kg (definitely treated). One-third each of the subjects had been exposed to dust containing 5 mg PCP/kg (normal), 5-10 mg/kg (slightly elevated) and > 10 mg/kg (clearly elevated), respectively. The mean exposure period was approx. 10 years with a mean daily stay of 8 hrs. No statistically significant correlations were found between the human monitoring data and PCP concentrations in wood or dust or the stated complaints.

Adolescent↗

Gene sequences of the pcpB gene of pentachlorophenol-degrading Sphingomonas chlorophenolica found in nondegrading bacteria.

Bacteria isolated from a pentachlorophenol (PCP) contaminated site grew in the presence of 50 micrograms PCP/mL but were not able to degrade it in either liquid medium or the presence of 1% sterile potting soil as a solid support. Probes developed using the gene sequence of PCP-4-monooxygenase (pcpB) from Sphingomonas chlorophenolica sp.nov hybridized to two separate isolates. Identification based on fatty acid methyl ester profiles (Sherlock), substrate utilization (BIOLOG), and 16S rRNA showed that the two strains were different from each other and from Sphingomonas chlorophenolica. Sequences from these isolates, amplified by polymerase chain reaction, confirmed the homology with pcpB. The presence of pcpB sequences in these nondegraders indicated that growth and hybridization data alone were insufficient for predicting degradation capability.

Bacteria, Aerobic↗

The effect of redox potential changes on reductive dechlorination of pentachlorophenol and the degradation of acetate by a mixed, methanogenic culture

The effect of changes in redox potential on methanogenesis from acetate, and on the reductive dechlorination of pentachlorophenol (PCP), was evaluated using a computer-monitored and feedback-controlled bioreactor. PCP was transformed via 2,3,4, 5-tetrachlorophenol (2,3,4,5-TeCP) to 3,4,5-trichlorophenol (3,4, 5-TCP). In 6- to 12-d experiments, pH, acetate concentration, and temperature were held constant; the redox potential, defined here as the potential measured at a platinum electrode (EPt), was maintained at different set points, while transformation of multiple PCP additions was monitored. Without redox potential control, the value of EPt for the culture was approximately -0.26 V (vs. SHE). The value of EPt was elevated from -0.26 V for periods up to 10 h by computer-controlled addition of H2O2 or K3Fe(CN)6. Methanogenesis continued during a relatively mild shift of EPt to -0.2 V with H2O2, but was halted when EPt was raised to -0.1 V with either H2O2 or K3Fe(CN)6. Methanogenesis resumed when EPt returned to -0.26 V. During periods in which EPt was elevated significantly and methanogenesis stopped, transformation of PCP and 2,3,4,5-TeCP continued at progressively slower rates, but the rate of 2,3,4, 5-TeCP transformation was diminished to a greater extent. When a small volume of pure H2 was added to the reactor headspace, while EPt was maintained at -0.1 V, reductive dechlorination rates increased dramatically. Lower H2 concentrations during periods of oxidant addition, perhaps due to the effect of the oxidant on H2-producing bacteria, may contribute to decreased reductive dechlorination rates. Copyright 1999 John Wiley & Sons, Inc.

Journal Article↗

Poly(ethylene glycol)-modified ligninase enhances pentachlorophenol biodegradation in water-solvent mixtures

Polychlorinated hydrocarbons are prevalent environmental contaminants whose rates of biodegradation are limited by their minimal solubilities in aqueous solutions where the biological reactions take place. In this study, ligninase (LiP) from Phanerochaete chrysosporium was modified by poly(ethylene glycol) to enhance its activity and stability for the biodegradation of pentachlorophenol (PCP) in the presence of acetonitrile (MeCN), a water-miscible solvent. The modified enzyme retained 100% of its activity in aqueous solutions and showed enhanced tolerance against the organic solvent. The activity of the modified enzyme was found to be over twice that of the native enzyme in the presence of 10% (v/v) MeCN. The solubility of PCP was enhanced significantly by the addition of MeCN to aqueous solutions, such that it was over 10-fold more soluble in the presence of 15% (v/v) MeCN than in pure aqueous buffer solution (from 0.06 to 0.65 mM). Capitalizing on the enhanced substrate solubility and the increased activity of the modified enzyme, the catalytic efficiency of the modified LiP in solutions containing 15% MeCN was over 11-fold higher than that of the native enzyme in buffer solutions (pH 4.2) in unoptimized reactor systems (from 44 to 480 mol PCP/mol LiP.h). Continued research both in the use of organic solvents to increase the availability of recalcitrant contaminants and in the modification of enzymes to enhance their activity and stability in such solvents promises to dramatically affect our ability to remediate contaminated sites. Published by John Wiley & Sons.

Journal Article↗

Production and characterization of the recombinant Sphingomonas chlorophenolica pentachlorophenol 4-monooxygenase.

Pentachlorophenol 4-monooxygenase (PCP4MO) from Sphingomonas chlorophenolica is a flavoprotein that hydroxylates PCP in the presence of NADPH and oxygen. In order to investigate the structure and function of active site, recombinant PCP4MO (rePCP4MO) was produced in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Moreover, a tobacco etch virus (TEV) protease cleavage site (EKLYFQG) was introduced into GST-PCP4MO and a his-tagged TEV protease was employed. Hence, a two-step purification protocol was developed which allowed obtaining 15-20 mg of rePCP4MO from 1 L culture. The rePCP4MO revealed identity with native enzyme by SDS-PAGE and N-terminal sequence analyses. Furthermore, a polyclonal PCP4MO antibody was produced with GST-PCP4MO and purified by immunoaffinity chromatography, where both the native and recombinant forms of PCP4MO showed interaction. However, rePCP4MO was identified as apoprotein with no evidence for a typical flavoprotein spectrum. The catalytic activity could be detected in the presence of FAD. The K(m) and V(max) values for PCP were 50 microM and 30 nmol/min/mg, respectively.

Amino Acid Sequence↗

Toxicity and tissue distribution of pentachlorophenol and permethrin in pipistrelle bats experimentally exposed to treated timber.

The dependence of bats in Britain on houses as roosts may result in them being exposed to pesticides used in remedial timber treatments. Pentachlorophenol (PCP) and permethrin are used as a fungicide and an insecticide for timber treatment, respectively. The present study investigated toxicity and distribution in body tissues of these two pesticides in pipistrelle bats. Four groups of nine to ten bats were kept in separate outdoor flight enclosures and were provided with roost boxes treated with either PCP only, permethrin, PCP/permethrin mixture or solvent only (control). At the start of the experiment, mean (+/-SE) PCP and permethrin concentrations on the surface of wooden blocks that had been treated in the same way as roost boxes were 69.32+/-6.76 mg g(-1) (n=6) and 3.3+/-1.6 mg g(-1) (n=3), respectively. All bats exposed to PCP and PCP/permethrin treated boxes died within 24 and 120 h, respectively; nine out of the ten controls survived the 32 day experimental period (P<0.001; both groups compared with control). Bats exposed to permethrin treated boxes survived as well as controls. Mean (+/-SE) carcass PCP concentration (excluding deposits on fur) of bats exposed to PCP and PCP/permethrin treated boxes was 13.11+/-2.52 microg g(-1)BW (n=20). PCP burdens on fur were positively correlated with total weight of PCP in the carcass (P<0.001). PCP was present in fat depots, liver, kidney and the remainder of the body which, despite containing low PCP concentrations, was the main PCP reservoir (66.4+/-5.0% of carcass PCP load; n=20). Total PCP in the carcass was significantly correlated with lipid weight (P<0.005). Permethrin was not detectable in body washes and tissues of bats exposed to PCP/permethrin mixture or permethrin.

Journal Article↗

Identification, characterization, and site-directed mutagenesis of recombinant pentachlorophenol 4-monooxygenase.

In a previous study, we constructed a three-dimensional (3D) structure of pentachlorophenol 4-monooxygenase (PcpB). In this study, further analyses are performed to examine the important amino acid residues in the catalytic reaction by identification of the proteins with mass spectrometry, circular dichroism (CD) and UV spectrometry, and determination of kinetic parameters. Recombinant histidine-tagged PcpB protein was produced and shown to have a similar activity to the native protein. Mutant proteins of PcpB were then produced (F85A, Y216A, Y216F, R235A, R235E, R235K, Y397A and Y397F) on the basis of the proposed 3D structure. The CD spectra of the proteins showed that there were no major changes in the structures of the mutant proteins, with the exception of R235E. Steady-state kinetics showed a 20-fold reduction in k(cat)/K(m) and a ninefold increase in K(m) for Y216F and a threefold reduction in k(cat)/K(m) and a sixfold increase in K(m) for Y397F compared to the wild type. On the other hand, the value of k(cat)/K(m) of R235K mutant was the same as that of wild type. As a result, it was confirmed that Y216 and Y397 play an important role with respect to the recognition of the substrate.

Amino Acid Substitution↗

Porous organoclay composite for the sorption of polycyclic aromatic hydrocarbons and pentachlorophenol from groundwater.

Complex mixtures of hazardous chemicals such as polycyclic aromatic hydrocarbons (PAHs) in contaminated soil and groundwater can have severe and long-lasting effects on health. The evidence that these contaminants can cause adverse health effects in animals and humans is rapidly expanding. The frequent and wide-spread occurrence of PAHs in groundwater makes appropriate intervention strategies for their remediation highly desirable. The core objective of this research was to assess the ability of a clay-based composite to sorb and remove toxic contaminants from groundwater at a wood-preserving chemical waste site. Treatment efficiencies were evaluated using either effluent from an oil-water separator (OWS) or a bioreactor (B2). The effluent water from these units was passed through fixed bed columns containing either an organoclay composite or granular activated carbon. The sorbent columns were placed in-line using existing sampling ports at the effluent of the OWS or B2. Individual one-liter samples of treated and untreated effluent were collected in Kimax bottles over the course of 78 h (total of 50 samples). Subsequently each sample was extracted by solid phase extraction methodology, and pentachlorophenol (PCP) and PAH concentrations were quantitated via GC/MS. Columns containing porous organoclay composite, i.e. sand-immobilized cetylpyridinium-exchanged low-pH montmorillonite clay (CP/LPHM), were shown to reduce the contaminant load from the OWS effluent stream by 97%. The concentrations of benzo[a]pyrene (BaP) and PCP were considerably reduced (i.e. >99%). An effluent stream from the bioreactor was also filtered through columns packed with composite or an equivalent amount of GAC. Although the composite reduced the majority of contaminants (including BaP and PCP), it was less effective in diminishing the levels of lower ring versus higher ring PAHs. Conversely, GAC was more effective in removing the lower ring PAHs, except for naphthalene and PCP. The effectiveness of sorption of PCP from the OWS effluent by the composite was confirmed using a PCP-sensitive adult hydra bioassay previously described in our laboratory. The findings of this initial study have delineated differences between CP/LPHM and GAC for groundwater remediation, and suggest that GAC (instead of sand) as the solid support for organoclay may be more effective for the treatment of contaminated groundwater under field conditions than GAC or CP/LPHM alone. Further work is ongoing to confirm this conclusion.

Adsorption↗

Combined effects of pentachlorophenol and salinity stress on phagocytic and chemotactic function in two species of abalone.

The effect of pentachlorophenol (PCP) combined with salinity stress on specific aspects of cellular immunity was examined in two species of abalone. Chemotactic and phagocytic ability (percent phagocytosis, %P; and phagocytic index, PI), as well as gross morphological characteristics, of hemocytes withdrawn from red (Haliotis rufescens) and black abalone (H. cracherodii) after in vivo exposure to 25, 35, or 45 per thousand seawater salinity plus 1.2 mg/l PCP were determined. Abalone exposures of 3.5 and 6.5 h, respectively, were based on species-specific metabolic endpoints (MEPs) derived from previous NMR data. Hemocyte chemotaxis was found to be augmented by low salinity in red but not black abalone, while high salinity seemed to reduce chemotactic ability in both species. PCP did not potentiate the effects demonstrated by salinity variations alone in red abalone, although chemotaxis was further compromised in black abalone exposed to high salinity stress combined with PCP. Although chemotactic ability was similar among both species, both %P and PI was greater among black abalone. Low salinity may offset the effects of PCP in red but not black abalone. Whereas red abalone demonstrated a reduction in phagocytic ability upon exposure to high salinity alone, black abalone appeared more resilient to high salinity stress alone. Furthermore, while the effect of high salinity plus PCP is subadditive among red abalone, high salinity seems to potentiate the effect of PCP on black abalone. Finally, hemocytes exposed to acute salinity variations plus PCP showed varying morphological characteristics when compared to controls. Over 30% of hemocytes of high salinity plus PCP exposed abalone remained rounded and generally unattached to glass slides with only a few pseudopod extensions. Hemocyte preparations incubated in rhodamine-conjugated phalloidin were observed with fluorescence microscopy and reveal an altered actin filament pattern. Overall, black abalone demonstrate greater phagocytic ability than red abalone both in the presence and absence of PCP. Furthermore, increases in salinity are accompanied with reduced phagocytic ability with red abalone demonstrating particular sensitivity to salinity variations. While PCP inhibits ATP synthesis, salinity variations may impose an additional stress on intertidal animals exposed to transient or seasonal environmental changes.

Journal Article↗

Plant electrophysiology: pentachlorophenol induces fast action potentials in soybean.

In the present work, we show that pentachlorophenol (PCP) induces ultra fast action potentials and decreases the variation potential in a soybean. The speed of the propagation of action potentials in a soybean induced by PCP reaches up to 30 m/s, similar to excitation propagation in animal nerves. Measured velocities for transmission of action potentials in green plants reported by different authors were registered in the range of a few mm/s. Action potentials induced in soybeans by PCP are many times faster. Seventy hours after adding PCP to soil variation (resting) potential decreases to zero level. This is the first attempt of high-speed automatic measurements of fast action potentials in green plants.

Journal Article↗

Treated wood in livestock facilities: relationships among residues of pentachlorophenol, dioxins, and furans in wood and beef.

Wood and other environmental samples were collected from sites that produced beef with higher than average residues of dibenzo-p-dioxin (PCDD) and dibenzofuran (PCDF). Analyses of these samples for PCDD/Fs and pentachlorophenol (PCP) indicated that the high beef residues were associated with PCP-treated wood in the animal facilities. Concentrations of PCDD/Fs in wood as toxic equivalents ranged from 10 to 320,000 pg/g. These concentrations were closely related to the concentrations of PCP, indicating that analysis for PCP provides an economical method to identify wood with high concentrations of PCDD/Fs. Further evidence for the PCP-treated wood as the source of the beef residues is provided by the similarity of the congener profiles in beef from the sites and those profiles predicted from the profiles in wood.

Animals↗

UV-B exposure increases acute toxicity of pentachlorophenol and mercury to the rotifer Brachionus calyciflorus.

Adverse biological effects of ultraviolet-B (UV-B) radiation have been well documented for phytoplankton and zooplankton in both marine and freshwater ecosystems. However, investigations of interactions between UV-B and anthropogenic toxicants have focused primarily on the chemical interactions between UV-B and the toxicant. Here we investigate the potential for UV-B to increase the sensitivity of the rotifer Brachionus calyciflorus to either acute pentachlorophenol (PCP) or mercury toxicity, independent of UV-B effects on these toxicants. UV-B increased the toxicity of PCP and mercury to B. calyciflorus as much as five-fold, depending on duration of UV-B exposure and toxicant concentration. Reductions in the LC(50) of up to 60% were also seen for both toxicants. UV-B alone effectively eliminated B. calyciflorus reproduction and reduced ingestion by up to 90%. These results demonstrate the potential for UV-B to increase rotifer sensitivity to anthropogenic stressors independent of photochemical reactions with toxicants.

Journal Article↗

Steady-state oxidation model by horseradish peroxidase for the estimation of the non-inactivation zone in the enzymatic removal of pentachlorophenol.

A theoretical model for the rate of oxidation of pentachlorophenol (PCP) catalyzed by horseradish peroxidase (HRP), was investigated to account for the influence of hydrogen peroxide (H2O2) concentration on the catalytic activity. To evaluate the maximum allowable H2O2 concentration, a relatively simple steady-state model was developed based on the Ping-Pong Bi-Bi mechanism considering the effect of excess H2O2. Several sets of experimental data obtained from batch reactions using an equimolar concentration of H2O2 and PCP were used to estimate the kinetic parameters by a nonlinear regression method. The model profiles acquired using the estimated parameters were in good agreement with experimental data at different initial enzyme and substrate concentrations. The best-fitted parameters were used to predict the initial rate of the enzyme reaction. The model prediction was coincident with the experimental results of other studies, indicating that the proposed model could be used for the optimization of reaction conditions. The maximum allowable H2O2 concentration to prevent H2O2 inhibition was calculated from the proposed model equation: [H2O2](0,max) = (square root)KmH2O2Ki[PCP]0/KmPCP+[PCP]0. Using this equation, a curve depicting the non-inactivation zone for the two substrates (hydrogen peroxide and PCP) was plotted and it could be used for experimental design and optimal process operation. To minimize enzyme inactivation by H2O2, it was determined that the concentration of H2O2 should be lower than 2.78 mM, regardless of the stoichiometric ratio.

Journal Article↗

Colonization of contaminated soil by an introduced bacterium: effects of initial pentachlorophenol levels on the survival of Sphingomonas chlorophenolica strain RA2.

The survival of a Sphingomonas species that was introduced into pentachlorophenol (PCP)-contaminated soil was monitored with two complementary methods, a respiration-based assay and a most probable number (MPN) technique. Sphingomonas chlorophenolicastrain RA2 is a PCP-mineralizing bacterium that was introduced into soil contaminated with a range of PCP concentrations (0-300 &mgr;g PCP g(-1) soil). The population of introduced microorganisms was followed for 170 days using a substrate-induced growth-response method and a MPN assay that specifically targets PCP-mineralizing bacteria. Varying the initial PCP concentration resulted in the emergence of three distinct patterns of survival. In soil contaminated with 300 &mgr;g PCP g(-1) the population of S. chlorophenolica strain RA2 immediately declined following introduction, increased by 200-fold and leveled off by the end of the 170-day incubation. In contrast, populations of S. chlorophenolica strain RA2 declined to levels below detection limits in uncontaminated soil by the end of the experiment. Intermediate PCP concentrations (10-100 &mgr;g PCP g(-1) soil) resulted in the establishment of S. chlorophenolica strain RA2 that slowly declined in numbers. These results indicate that Sphingomonas chlorophenolica strain RA2 is an effective colonizer of PCP-contaminated soil but will not persist in the absence of PCP.

Journal Article↗

Phylogeny of Sphingomonas species that degrade pentachlorophenol.

Four pentachlorophenol (PCP)-degrading bacteria isolated from geographically diverse areas have been examined in detail as regards their physiology and phylogeny. According to traditional biochemical methods, these strains had been classified as members of the genera Arthrobacter, Flavobacterium, Pseudomonas, and Sphingomonas. The PCP degradation pathway has been studied extensively in Sphingomonas (Flavobacterium) sp strain ATCC 39723 and the first three degradation steps catalyzed by a PCP-4-monooxygenase (PcpB) and a reductive dehalogenase (PcpC) that functions twice are well established. A fourth step appears to involve ring-fission of the aromatic nucleus (PcpA). Molecular analyses revealed that the PCP degradation pathway in these four strains was rather conserved, leading to a phylogenetic analysis using 16S rDNA. The results revealed a much closer phylogenetic relationship between these organisms than traditional classification indicated, placing them into the more recently established genus Sphingomonas where they may even represent a single species. With 16S rDNA analysis, many bacterial isolates involved in degradation of xenobiotic compounds that were previously classified into diverse genera have been reclassified into the genus Sphingomonas.

Journal Article↗

Oxidative damage and direct adducts in calf thymus DNA induced by the pentachlorophenol metabolites, tetrachlorohydroquinone and tetrachloro-1,4-benzoquinone.

DNA damage induced by quinoid metabolites of pentachlorophenol (PCP), i.e. tetrachloro-1,4-benzoquinone (Cl(4)BQ) and tetrachlorohydroquinone (Cl(4)HQ), was investigated in calf thymus DNA. The (32)P-post-labeling assay revealed four major and several minor adducts (3.5 adducts per 10(5) total nucleotides) that were produced in calf thymus DNA treated with Cl(4)BQ (5 mM). These DNA adducts were chemically stable even after conditions that induce thermal depurination and are unlikely to undergo depurination/depyrimidination to form apurinic/apyrimidinic (AP) sites. In addition, increases in 8-hydroxy-deoxyguanosine (8-HO-dG) (5 8-HO-dG per 10(5) nucleotides) and AP sites (0.5 AP sites per 10(5) nucleotides) were observed in Cl(4)BQ-modified calf thymus DNA. Further investigation indicated that in the presence of Cu(II) and NADPH, low concentrations of Cl(4)BQ (1 microM) induced a doubling of 8-HO-dG (10 8-HO-dG per 10(5) nucleotides) and dramatic increases in AP sites (20 AP sites per 10(5) nucleotides) and DNA single-strand breaks. The types of DNA damage induced by Cl(4)HQ plus Cu(II) were similar to those by Cl(4)BQ plus Cu(II) and NADPH, whereas catalase inhibited the formation of DNA damage. These data suggest that oxidative damage is causally involved in the formation of AP sites. Concentration-dependent increases in 8-HO-dG induced by Cl(4)HQ plus Cu(II) and Cl(4)BQ plus Cu(II) and NADPH were correlated with the formation of AP sites (r(2) = 0.977) with a ratio of 8-HO-dG to AP sites at 1:1.6. The AP site-cleavage assay confirmed that approximately 85% of the AP sites induced by Cl(4)HQ and Cu(II) were detected as 5'-cleaved AP sites. Since hydrogen peroxide alone causes similar DNA damage, these results suggest the involvement of Cu(II) and hydrogen peroxide in the induction of oxidative DNA damage by Cl(4)HQ/Cl(4)BQ. The data demonstrate that PCP quinone and hydroquinone induce direct and oxidative base modifications as well as the formation of 5'-cleaved AP sites in genomic DNA. These lesions may have important implications for PCP clastogenicity and carcinogenicity.

8-Hydroxy-2'-Deoxyguanosine↗

Microbial treatment of soil to remove pentachlorophenol.

Direct inoculation of bacteria capable of degrading pentachlorophenol (PCP) into PCP-contaminated soil was investigated as a prophylactic measure to reduce the hazards of runoffs when spills occur or when wooden poles freshly treated with PCP-containing preservatives are located near streams and lakes. In laboratory tests at 30 degrees C, the direct addition of 10 PCP-utilizing Arthrobacter cells per g of dry soil reduced the half-life of the pesticide from 2 weeks to <1 day. Soil inoculation also was shown to be an effective way to increase the PCP disappearance rate in a test conducted in an outdoor shed.

Journal Article↗

In Situ Depletion of Pentachlorophenol from Contaminated Soil by Phanerochaete spp.

The ability of two white rot fungi to deplete pentachlorophenol (PCP) from soil, which was contaminated with a commercial wood preservative, was examined in a field study. Inoculation of soil containing 250 to 400 mug of PCP g with either Phanerochaete chrysosporium or P. sordida resulted in an overall decrease of 88 to 91% of PCP in the soil in 6.5 weeks. This decrease was achieved under suboptimal temperatures for the growth and activity of these fungi, and without the addition of inorganic nutrients. Since the soil had a very low organic matter content, peat was included as a source of organic carbon for fungal growth and activity. A small percentage (8 to 13%) of the decrease in the amount of PCP was a result of fungal methylation to pentachloroanisole. Gas chromatographic analysis of sample extracts did not reveal the presence of extractable transformation products other than pentachloroanisole. Thus, when losses of PCP via mineralization and volatilization were negligible, as they were in laboratory-scale studies (R. T. Lamar, J. A. Glaser, and T. K. Kirk, Soil Biol. Biochem. 22:433-440, 1990), most of the PCP was converted to nonextractable soil-bound products. The nature, stability, and toxicity of soil-bound transformation products, under a variety of conditions, must be elucidated before use of these fungi in soil remediation efforts can be considered a viable treatment method.

Journal Article↗