Pentachlorophenol causes redox imbalance, inhibition of brush border membrane and metabolic enzymes, DNA damage and histological alterations in rat kidney.

Pentachlorophenol (PCP) is a synthetic organochlorine compound that is widely used in biocide and pesticide industries, and in preservation of wood, fence posts, cross arms and power line poles. Humans are usually exposed to PCP through air, contaminated water and food. PCP enters the body and adversely affects liver, gastrointestinal tract, kidney and lungs. PCP is a highly toxic class 2B or probable human carcinogen that produces large amount of reactive oxygen species (ROS) within cells. This work aimed to determine PCP-induced oxidative damage in rat kidney. Adult rats were given PCP (25, 50, 100, 150 mg/kg body weight), in corn oil, once a day for 5 days while control rats were given similar amount of corn oil by oral gavage. PCP increased hydrogen peroxide level and oxidation of thiols, proteins and lipids. The antioxidant status of kidney cells was compromised in PCP treated rats while enzymes of brush border membrane (BBM) and carbohydrate metabolism were inhibited. Plasma level of creatinine and urea was also increased. Administration of PCP increased DNA fragmentation, cross-linking of DNA to proteins and DNA strand scission in kidney. Histological studies supported biochemical findings and showed significant damage in the kidneys of PCP-treated rats. These changes could be due to redox imbalance or direct chemical modification by PCP or its metabolites. These results signify that PCP-induced oxidative stress causes nephrotoxicity, dysfunction of BBM enzymes and DNA damage.

Concurrent anaerobic chromate bio-reduction and pentachlorophenol bio-degradation in a synthetic aquifer.

Chromate [Cr(VI)] and pentachlorophenol (PCP) coexist widely in the environment and are highly toxic to public health. However, whether Cr(VI) bio-reduction is accompanied by PCP bio-degradation and how microbial communities can keep long-term stability to mediate these bioprocesses in aquifer remain elusive. Herein, we conducted a 365-day continuous column experiment, during which the concurrent removals of Cr(VI) and PCP were realized under anaerobic condition. This process allowed for complete Cr(VI) bio-reduction and PCP bio-degradation at an efficiency of 92.8 ± 4.2% using ethanol as a co-metabolic substrate. More specifically, Cr(VI) was reduced to insoluble chromium (III) and PCP was efficiently dechlorinated with chloride ion release. Collectively, Acinetobacter and Spirochaeta regulated Cr(VI) bio-reduction heterotrophically, while Pseudomonas mediated not only Cr(VI) bio-reduction but also PCP bio-dechlorination. The bio-dechlorinated products were further mineralized by Azospira and Longilinea. Genes encoding proteins for Cr(VI) bio-reduction (chrA and yieF) and PCP bio-degradation (pceA) were upregulated. Cytochrome c and intracellular nicotinamide adenine dinucleotide were involved in Cr(VI) and PCP detoxification by promoting electron transfer. Taken together, our findings provide a promising bioremediation strategy for concurrent removal of Cr(VI) and PCP in aquifers through bio-stimulation with supplementation of appropriate substrates.

Role of tetrachloro-1,4-benzoquinone reductase in phenylalanine hydroxylation system and pentachlorophenol degradation in Bacillus cereus AOA-CPS1.

This study reports a ≅12.5 kDa protein tetrachloro-1,4-benzoquinone reductase (CpsD) from Bacillus cereus strain AOA-CPS1 (BcAOA). CpsD is purified to homogeneity with a total yield of 35% and specific activity of 160 U·mg-1 of protein. CpsD showed optimal activity at pH 7.5 and 40 °C. The enzyme was found to be functionally stable between pH 7.0-7.5 and temperature between 30 °C and 35 °C. CpsD activity was enhanced by Fe2+ and inhibited by sodium azide and SDS. CpsD followed Michaelis-Menten kinetic exhibiting an apparent vmax, Km, kcat and kcat/Km values of 0.071 µmol·s-1, 94 µmol, 0.029 s-1 and 3.13 × 10-4 s-1·µmol-1, respectively, for substrate tetrachloro-1,4-benzoquinone. The bioinformatics analysis indicated that CpsD belongs to the PCD/DCoH superfamily, with specific conserved protein domains of pterin-4α-carbinolamine  dehydratase (PCD). This study proposed that CpsD catalysed the reduction of tetrachloro-1,4-benzoquinone to tetrachloro-p-hydroquinone and released the products found in phenylalanine hydroxylation system (PheOHS) via a Ping-Pong or atypical ternary mechanism; and regulate expression of phenylalanine 4-monooxygenase by blocking reverse flux in BcAOA PheOHS using a probable Yin-Yang mechanism. The study also concluded that CpsD may play a catalytic and regulatory role in BcAOA PheOHS and pentachlorophenol degradation pathway.

Nitrate supply and sulfate-reducing suppression facilitate the removal of pentachlorophenol in a flooded mangrove soil.

An anaerobic incubation was launched with varying nitrate (1, 5, 10 and 20 mM exogenous NaNO3) and molybdate (20 mM Na2MoO4, a sulfate-reducing inhibitor) additions to investigate the characteristics of PCP dechlorination, as well as the reduction of natural co-occurring electron acceptors, including NO3-, Fe(III) and SO42-, and the responses of microbial community structures under a unique reductive mangrove soil. Regardless of exogenous addition, nitrate was rapidly eliminated in the first 12 days. The reduction process of Fe(III) was inhibited, while that of SO42- reduction depended on addition concentration as compared to the control. PCP was mainly degraded from orth-position, forming the only intermediate 2,3,4,5-TeCP by anaerobic microbes, with the highest PCP removal rate of average 21.9% achieved in 1 and 5 mM NaNO3 as well as 20 mM Na2MoO4 treatments and the lowest of 7.5% in 20 mM NaNO3 treatment. The effects of nitrate on PCP dechlorination depended on addition concentration, while molybdate promoted PCP attenuation significantly. Analyses of the Illumina sequencing data and the relative abundance of dominant microorganisms indicated that the core functional groups regulated PCP removal at genera level likely included Bacillus, Pesudomonas, Dethiobacter, Desulfoporosinus and Desulfovbrio in the nitrate treatments; while that was likely Sedimentibacter and Geosporobacter_Thermotalea in the molybdate treatment. Nitrate supplement but not over supplement, or addition of molybdate are suggested as alternative strategies for better remediation in the nitrate-deficient and sulfur-accumulated soil ecosystem contaminated by PCP, through regulating the growth of core functional groups and thereby coordinating the interaction between dechlorination and its coupled soil redox processes due to shifts of more available electrons to dechlorination. Our results broadened the knowledge regarding microbial PCP degradation and their interactions with natural soil redox processes under anaerobic soil ecosystems.

Aerobic Biodegradation of OCDD by P. Mendocina NSYSU: Effectiveness and Gene Inducement Studies.

The goals of this study were to assess the effectiveness of (1) enhancing octachlorinated dibenzo-p-dioxin (OCDD) biodegradation under aerobic conditions by Pseudomonas mendocina NSYSU (P. Mendocina NSYSU) with the addition of lecithin, and (2) inducing OCDD ring-cleavage genes by pentachlorophenol (PCP) and OCDD addition. P. Mendocina NSYSU could biodegrade OCDD via aerobic cometabolism and lecithin was used as a primary substrate. Approximately 74 and 67% of OCDD biodegradation was observed after 60 days of incubation with lecithin and glucose supplement, respectively. Lecithin was also used as the solubilization additive resulting in OCDD solubilization and enhanced bioavailability of OCDD to P. Mendocina NSYSU. Two intradiol and extradiol ring-cleavage dioxygenase genes (Pmen_0474 and Pmen_2526) were identified from gene analyses. Gene concentration was significantly enhanced after the inducement by PCP and OCDD. Higher gene inducement efficiency was obtained using PCP as the inducer, and Pmen_2526 played a more important role in OCDD biodegradation.

Pentachlorophenol degradation by Janibacter sp., a new actinobacterium isolated from saline sediment of arid land.

Many pentachlorophenol- (PCP-) contaminated environments are characterized by low or elevated temperatures, acidic or alkaline pH, and high salt concentrations. PCP-degrading microorganisms, adapted to grow and prosper in these environments, play an important role in the biological treatment of polluted extreme habitats. A PCP-degrading bacterium was isolated and characterized from arid and saline soil in southern Tunisia and was enriched in mineral salts medium supplemented with PCP as source of carbon and energy. Based on 16S rRNA coding gene sequence analysis, the strain FAS23 was identified as Janibacter sp. As revealed by high performance liquid chromatography (HPLC) analysis, FAS23 strain was found to be efficient for PCP removal in the presence of 1% of glucose. The conditions of growth and PCP removal by FAS23 strain were found to be optimal in neutral pH and at a temperature of 30 °C. Moreover, this strain was found to be halotolerant at a range of 1-10% of NaCl and able to degrade PCP at a concentration up to 300 mg/L, while the addition of nonionic surfactant (Tween 80) enhanced the PCP removal capacity.

Evaluating the phytoremediation potential of Phragmites australis grown in pentachlorophenol and cadmium co-contaminated soils.

Pot-culture experiments were conducted to evaluate the phytoremediation potential of a wetland plant species, Phragmites australis in cadmium (Cd) and pentachlorophenol (PCP) co-contaminated soil under glasshouse conditions for 70 days. The treatments included Cd (0, 5 and 50 mg kg(-1)) without or with PCP (50 and 250 mg kg(-1)). The results showed that growth of P. australis was significantly influenced by interaction of Cd and PCP, decreasing with either Cd or PCP additions. Plant biomass was inhibited and reduced by the rate of 89 and 92% in the low and high Cd treatments and by 20 and 40% in the low and high PCP treatments compared to the control. The mixture of low Cd and low PCP lessened Cd toxicity to plants, resulting in improved plant growth (by 144%). Under the joint stress of the two contaminants, the ability of Cd uptake and translocation by P. australis was weak, and the BF and TF values were inferior to 1.0. A low proportion of the metal is found aboveground in comparison to roots, indicating a restriction on transport upwards and an excluding effect on Cd uptake. Thus, P. australis cannot be useful for phytoextraction. The removal rate of PCP increased significantly (70%) in planted soil. Significant positive correlations were found between the DHA and the removal of PCP in planted soils which implied that plant root exudates promote the rhizosphere microorganisms and enzyme activity, thereby improving biodegradation of PCP. Based on results, P. australis cannot be effective for phytoremediation of soil co-contaminated with Cd and PCP. Further, high levels of pollutant hamper and eventually inhibit plant growth. Therefore, developing supplementary methods (e.g. exploring the partnership of plant-microbe) for either enhancing (phytoextraction) or reducing the bioavailability of contaminants in the rhizosphere (phytostabilization) as well as plant growth promoting could significantly improve the process of phytoremediation in co-contaminated soil.

Polyphasic characterization of two microbial consortia with wide dechlorination spectra for chlorophenols.

Two soil-free anaerobic dechlorinating cultures (3-CP and 35-DCP) were enriched from a pentachlorophenol (PCP)-to-phenol dechlorinating soil-dependent culture, using 3-chlorophenol (3-CP) and 3,5-dichlorophenol (3,5-DCP) as specific respective substrates, and characterized polyphasically. Physiological characterization indicated that the 3-CP and 35-DCP cultures had similar features, but with some variations. Both cultures utilized formate or acetate preferably as optimum electron donors for reductive dechlorination, and they shared similar patterns of dechlorination spectra for chlorophenols ranging from mono-CPs to a tetra-CP, with preferred dechlorination pathways in the ortho and meta positions. Alternative electron acceptors such as NO(3)(-) but not SO(4)(2-) inhibited the dechlorination activity in both cultures, while amorphous iron oxides (FeOOH) suppressed dechlorination activity only in the 35-DCP culture. Complete inhibition of dechlorination was observed in both cultures supplemented with chloramphenicol and vancomycin. The addition of 2-bromoethanesulfonate resulted in delayed dechlorination activity in the 35-DCP culture but not in the 3-CP culture; molybdate did not exert any inhibitory effect in either culture. Phylogenetic analysis based on 16S rRNA genes confirmed that the two cultures exhibited similar bacterial species but with varied responsible dechlorinators. Dehalobacter spp. were the likely dechlorinators in the 3-CP culture versus Sulfurospirillum spp. in the 35-DCP culture, with Clostridium and Clostridium-like spp. as candidate dechlorinators in both cultures.

Pentachlorophenol degradation by Pseudomonas stutzeri CL7 in the secondary sludge of pulp and paper mill.

A pentachlorophenol (PCP) mineralizing bacterium was isolated from the secondary sludge of pulp and paper mill and identified as Pseudomonas stutzeri strain CL7. This isolate used PCP as its sole source of carbon and energy and was capable of degrading this compound as indicated by stoichiometric release of chloride and biomass formation. P. stutzeri (CL7) was able to mineralize a high concentration of PCP (600 mg/L) than any previously reported Pseudomonad with PCP as sole carbon source. As the concentration of PCP increased from 50 to 600 mg/L, the reduction in the cell growth was observed and the PCP degradation was more than 90% in all studied concentrations. This isolate was able to remove 66.8% of PCP from the secondary sludge of pulp and paper mill when supplemented with 100 mg/L of PCP and grown for two weeks. This study showed that the removal efficiency of PCP by CL7 was found to be very effective and can be used in PCP remediation of pulp paper mill waste in the environment.

Identification and characterization of integron mediated antibiotic resistance in pentachlorophenol degrading bacterium isolated from the chemostat.

A bacterial consortium was developed by continuous enrichment of microbial population isolated from sediment core of pulp and paper mill effluent in mineral salts medium (MSM) supplemented with pentachlorophenol (PCP) as sole source of carbon and energy in the chemostat. The consortia contained three bacterial strains. They were identified as Escherichia coli, Pseudomonas aeruginosa and Acinetobacter sp. by 16S rRNA gene sequence analysis. Acinetobacter sp. readily degraded PCP through the formation of tetrachloro-p-hydroquinone (TecH), 2-chloro-1,4-benzenediol and products of ortho ring cleavage detected by gas chromatograph/mass spectrometer (GC-MS). Out of the three acclimated PCP degrading bacterial strains only one strain, Acinetobacter sp. showed the presence of integron gene cassette as a marker of its stability and antibiotic resistance. The strain possessed a 4.17 kb amplicon with 22 ORF's. The plasmid isolated from the Acinetobacter sp. was subjected to shotgun cloning through restriction digestion by BamHI, HindIII and SalI, ligated to pUC19 vector and transformed into E. coli XLBlue1alpha, and finally selected on MSM containing PCP as sole source of carbon and energy with ampicillin as antibiotic marker. DNA sequence analysis of recombinant clones indicated homology with integron gene cassette and multiple antibiotic resistance genes.

Increased PCP removal by Amylomyces rouxii transformants with heterologous Phanerochaete chrysosporium peroxidases supplementing their natural degradative pathway.

Fungal peroxidases and phenoloxidases are widely used in aromatic toxic compounds degradation. Peroxidases, such as lignin peroxidase and manganese peroxidase, as well as laccases are mainly produced by basidiomycetes and to a lower extent by other fungi, such as ascomycetes. Peroxidase-encoding genes have been described and homologous expression has been achieved in basidiomycetes. Heterologous expression has also been achieved in some non-producing peroxidase ascomycetes, like Penicillium and Aspergillus. In this work, heterologous expression of peroxidase-encoding genes, lignin peroxidase, and manganese peroxidase was achieved in a zygomycete producing only phenoloxidases (Amylomyces rouxii), aimed at coupling two different pathways used in nature for PCP removal in only one microbial strain. The ability of PCP removal was assayed with one of the obtained transformants, resulting in increased activity with respect to the ability of the parental strain cultured free of the inducer tyrosine (95% and 45%, respectively, of the initial PCP (12.5 mg L(-1)) in 120 h, or 100% and 49%, respectively, of the initial PCP after 144 h of liquid culture).

Enrichment, isolation and characterization of pentachlorophenol degrading bacterium Acinetobacter sp. ISTPCP-3 from effluent discharge site.

Three pentachlorophenol (PCP) degrading bacterial strains were isolated from sediment core of pulp and paper mill effluent discharge site. The strains were continuously enriched in mineral salts medium supplemented with PCP as sole source of carbon and energy. One of the acclimated strains with relatively high PCP degradation capability was selected and characterized in this study. Based on morphology, biochemical tests, 16S rDNA sequence analysis and phylogenetic characteristics, the strains showed greatest similarity with Acinetobacter spp. The strain was identified as Acinetobacter sp. ISTPCP-3. The physiological characteristics and optimum growth conditions of the bacterial strain were investigated. The results of optimum growth temperature revealed that it was a mesophile. The optimum growth temperature for the strain was 30 degrees C. The preferential initial pH for the strain was ranging at 6.5-7.5, the optimum pH was 7. The bacterium was able to tolerate and degrade PCP up to a concentration of 200 mg/l. Increase in PCP concentration had a negative effect on biodegradation rate and PCP concentration above 250 mg/l was inhibitory to its growth. Acinetobacter sp. ISTPCP-3 was able to utilize PCP through an oxidative route with ortho ring-cleavage with the formation of 2,3,5,6-tetrachlorohydroquinone and 2-chloro-1,4-benzenediol, identified using gas chromatograph-mass spectrometric (GC-MS) analysis. The degradation pathway followed by isolated bacterium is different from previously characterized pathway.

Characterization of pentachlorophenol degrading bacterial consortium from chemostat.

A microbial consortium was developed by continuous enrichment of bacterial population isolated from sediment core of pulp and paper mill effluent in mineral salts medium (MSM) supplemented with pentachlorophenol (PCP) as sole source of carbon and energy in the chemostat. The enriched consortium contained three bacterial strains identified as Escherichia coli (PCP1), Pseudomonas aeruginosa (PCP2) and Acinetobacter sp. (PCP3) by morphological and biochemical tests, further confirmation was done using 16S rDNA sequence analysis. The potency of bacterial isolates in degradation of PCP was monitored in terms of growth and utilization of PCP as substrate with spectrophotometer and gas chromatograph-mass spectrometer (GC-MS) analysis. The strains were tested for their utilization of various organic compounds. The strain PCP3, showed higher potency to utilize PCP as sole source of carbon and energy than PCP1 and PCP2. The bacterial strain were able to utilize PCP through an oxidative and reductive route as indicated with the formation of tetrachloro-p-hydroquinone (TeCH), 2-chloro-1,4-benzenediol and 2,3,4,6-tetrachlorophenol, respectively.

Extended bioremediation of PAH/PCP contaminated soils from the POPILE wood treatment facility.

A study was conducted using two pilot-scale land-treatment units (LTUs) to evaluate the efficacy of different cultivation and maintenance schedules during bioremediation of contaminated soil from a wood treatment facility using landfarming technology. The soil contained high concentrations of polycyclic aromatic hydrocarbons (PAHs, approximately 13000 ppm) as well as of pentachlorophenol (PCP, approximately 1500 ppm). An initial 6-month intensive-treatment phase was followed by 24 months of less-intensive treatment. During the first phase, traditional landfarming practice of regular cultivation was compared with a gas-phase composition based cultivation strategy, and both the landfarming units were intensively monitored and maintained with respect to moisture control and delivery of nutrients. The two strategies resulted in similar contaminant concentration profiles with time during this phase, although different microbial populations developed in the two-landfarming units. The second (less-intensive) treatment phase involved no moisture control and nutrient delivery beyond the initial adjustments, and compared natural attenuation (no cultivation) with quarterly cultivation of soil. Both the strategies showed similar behavior again. GC/MS analysis of the soil samples showed PAH removal including four-ring homologues. Leachability tests at zero time and after 6 and 22 months of operation showed significant reductions in leaching of PCP and low molecular weight PAHs. Extended treatment resulted in some leaching of high molecular weight PAHs. Significant biological activity was demonstrated, even at the high contaminant concentrations. Phospholipid ester-linked fatty acid (PLFA) analysis showed an increase in biomass and a divergence in community composition in soils depending on the treatment conducted.

Expression of a gene for Mn-peroxidase from Coriolus versicolor in transgenic tobacco generates potential tools for phytoremediation.

In efforts aimed at the detoxification of contaminated areas, plants have many advantages over bacteria and fungi. We are attempting to enhance the environmental decontamination functions of plants by transferring relevant genes from microorganisms. When the gene for Mn-peroxidase (MnP) from Coriolus versicolor was expressed in transgenic tobacco plants, one line (designated fMnP21) expressed MnP activity at levels 54-fold higher than in control lines. When undamaged roots of transgenic plants were applied to liquid medium supplemented with 250 microM pentachlorophenol (PCP), the decrease in the level of PCP in fMnP21 (86% reduction) was about 2-fold higher than that in control lines (38% reduction). Expression of the gene for MnP in the transgenic plants had no obvious negative effects on their vegetative and sexual growth. Our system should contribute to the development of novel methods for the removal of hazardous chemicals from contaminated environments using transgenic plants.

An investigation of the in vivo formation of octachlorodibenzo-p-dioxin.

The in vivo formation of dioxins from chemical precursors was investigated in rats. Sprague-Dawley rats were fed pentachlorophenol or a predioxin in peanut oil for 14 days. Mass balance calculations indicated that pentachlorophenol was not converted to dioxins; however, the predioxin, nonachloro-2-phenoxyphenol, was converted to OCDD. Conversion of the predioxin ranged from 0.5% to 153% and depended on the amount of predioxin and OCDD present in the diet. The analytical procedures used for sample preparation did not appear to cause conversion of the predioxin to OCDD. The mechanism for biological conversion may be enzymatic or spontaneous.

Degradation of pentachlorophenol by a Flavobacterium species grown in continuous culture under various nutrient limitations.

A Flavobacterium sp. was grown in continuous culture limited for growth with ammonium, phosphate, sulfate, glucose, glucose + pentachlorophenol (PCP) (0.065 h -1), or PCP. Cells ere harvested, washed, and suspended to 3 x 10(7) cells ml (-1) in shake flasks containing a complete mineral salts medium without added carbon or supplemented with 50 mg of PCP ml(-1) or 50 mg of PCP ml(-1) + 100 mg of glucose ml(-1). The PCP concentration and the viable cell density were determined periodically. Cells that were grown under phosphate, glucose, or glucose + PCP limitation were more sensitive to PCP and took longer to degrade 50 mg of PCP ml(-1) than did cells that very were grown under ammonium, sulfate, or PCP limitation. Glucose stimulated viability and PCP degradation in all cases except when the cells were grown under carbon limitation with glucose and PCP added together as the carbon source. These results indicate that there is a relationship between nutrient limitation, phenotypic variation, and the sensitivity to and degradation of PCP by this organism.

Influence of readily metabolizable carbon on pentachlorophenol metabolism by a pentachlorophenol-degrading Flavobacterium sp.

The influence of high concentrations of pentachlorophenol (PCP) and readily metabolizable carbon on the activity and viability of a PCP-degrading Flavobacterium sp. was examined in a mineral salts medium. Lags preceding PCP removal by glutamate-grown Flavobacterium cells were greatly attenuated by the addition of glutamate, aspartate, succinate, acetate, glucose, or cellobiose. The effect of these supplementary carbon sources on the apparent lag was not mediated entirely through the stimulation of growth since PCP metabolism accompanied the onset of growth. The specific activity of PCP-degrading cells in the absence of supplementary carbon was 1.51 x 10(-13) +/- 0.08 x 10(-13) g of PCP per cell per h and in the presence of supplementary carbon was 0.92 x 10(-13) +/- 0.09 x 10(-13) g of PCP per cell per h. Glutamate in combination with glucose or cellobiose partially repressed PCP metabolism. PCP removal by PCP-induced, glutamate-grown cells suspended in the presence of 4 g of sodium glutamate per liter was sensitive to shock loads of PCP, with a Ki of about 86.8 micrograms/ml. Subsequent removal rates, however, were more resistant to PCP. Optimal stimulation of PCP removal by sodium glutamate required 3.0 g/liter, about the same concentration as that which saturated growth in the absence of PCP. PCP removal rates decayed within minutes following the transfer of PCP-induced, glutamate-grown cells to media containing PCP without supplementary carbon, and increasing PCP concentrations accelerated the decay.(ABSTRACT TRUNCATED AT 250 WORDS)

Dielectric properties of adsorption/ionization site of pentachlorophenol in lipid membranes.

The results of three complementary studies focused on characterization of the local environment of the common pesticide pentachlorophenol (PCP) adsorbed to phosphatidylcholine (PC) and phosphatidylglycerol (PG) membranes are reported. The effect of cholesterol (Chol) was examined. These studies included: Measurements of solvatochromic shifts of the ultraviolet absorption spectra of PCP in membranes and in polar non-hydrogen-bonding (a red shift) and hydrogen-bonding (a blue shift) solvents. Pi-pi transition energies were analyzed in terms of the dielectric cavity models of Onsager, Block-Walker, which includes dielectric saturation, and a soft dipole model of Suppan, which accounts for PCP's polarizability. The estimates of dielectric constant of the PCP adsorption site yielded 8.1-8.7 for the PC and 16.8-20.1 for PG membranes. Solvatochromic effects indicate hydrogen bonding between the membrane-bound ionized PCP molecule and water, which is enhanced by the presence of cholesterol. Determinations of the pKa of PCP adsorbed to PC, PG, PC/Chol, PG/Chol membranes and dissolved in dioxane-water solutions of a known dielectric constant. The pKa value of PCP adsorbed to membranes was always greater than the standard pKa value and it increased with the membrane's negative charge. The pKa value sequence in 0.1 M KCl was 6.68 (PG), 6.32 (PG/Chol = 70:30 mole fractions), 5.97 (PC), and 5.75 (PC/Chol = 70:30). The intrinsic pKa values of PCP in membranes were 5.2-5.4 (PG) and 5.5-6.0 (PC). Estimates of the dielectric constant of PCP's ionization site in membranes yielded 10-22 (PC) and 27-37 (PG). Cholesterol facilitated the release of the hydrogen ion from membrane-bound PCP. Measurements of pH dependence of PCP-induced membrane electrical conductivity. pH values of conductivity maxima were always greater than the standard pKa of PCP, and their sequence corresponded to that of the pKa values of membrane-bound PCP. The anomalous properties of PCP as a Class 2 uncoupler are due to PCP's lipophilic character. In response to a low dielectric constant of the adsorption/ionization site, the physicochemical characteristics of PCP adsorbed to membranes are different from the standard values--a fact that needs to be taken into account in the development of models of PCP's toxicity.