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Effects of phenolic compounds on 5-nucleotidase activity in some tissues of Notopterus notopterus (Pallas): a biochemical study.

In the present study, effects of phenol (P), 2,4-dinitrophenol (DNP) and pentachlorophenol (PCP) individually, and in three combinations viz., (PCP + DNP)/P (highly antagonistic), (DNP + P)/PCP (additive) and (P + DNP)/PCP (highly synergistic) at three sublethal levels (1/10, 1/15, and 1/20 fractions of 96 h LC50) on 5-nucleotidase activity have been studied in brain, liver, kidney and gills of a freshwater teleost, Notopterus notopterus. The results indicate that 5-nucleotidase activity was inhibited significantly (P less than 0.05; P less than 0.01; P less than 0.001) in these tissues at most of the concentrations after 15 and 30 days of exposure. However, significant stimulation (P less than 0.05) of this enzyme was also notice in kidney at 1/20 of P, and (PCP + DNP)/P combination after 15 days of exposure. Inhibition of this enzyme in tissues possibly represents the uncoupling of oxidative phosphorylation, while stimulation of the enzyme indicates that the concerned phenolic compounds might be inducing the cells of the kidney to produce more of this enzyme.

2,4-Dinitrophenol↗

Developmental toxicity and structure-activity relationships of chlorophenols using human embryonic palatal mesenchymal cells.

The chlorophenols (CPs) comprise a major class of widely distributed and frequently occurring environmental contaminants. Previous studies have demonstrated the adverse effects of CPs on embryonic and fetal development. HEPM (human embryonic palatal mesenchymal) and MOT (mouse ovarian tumor) cell lines have been utilized in complementary bioassays for the detection of teratogens, but not the CPs. In this study, our objectives were 2-fold: (1) to determine if the HEPM assay could be used to complement other bioassay systems of nonhuman origin, i.e., Hydra attenuata (HA) and rat whole embryo culture (WEC), in the evaluation of the developmental toxicity of CPs, and (2) to delineate the ability of the HEPM assay to evaluate structure-activity relationships of pentachlorophenol (C5P), 2,3,4,5-tetrachlorophenol (C4P), 2,3,5-trichlorophenol (C3P), 3,5-dichlorophenol (C2P), 4-monochlorophenol (CP), phenol, and CP derivatives (i.e., acetates, sodium phenates and anisoles). HEPM cells were seeded into each well of a 24-well plate and cultivated for 24 h. The medium was replaced with fresh medium containing various concentrations of test chemicals dissolved in dimethyl sulfoxide (DMSO, 0.1%). After culturing for 72 h, the medium was removed, cells were trypsinized, and cell number determined. The HEPM cell growth inhibition assay demonstrated a linear relationship between the IC50 values of the CPs and degree of chlorine substitution. The IC50 values of C5P, C4P, C3P, C2P, CP, and phenol were 18.8, 21.5, 27.5, 63.0, 150.0 and 470.0 microM, respectively. A clear structure-activity relationship was observed between toxicity of CPs and the degree of chlorine substitution. The rank order of CP toxicity from the HEPM assay (i.e., C5P > C4P > C3P > C2P > CP > phenol) is in excellent agreement with previous in vitro and in vivo studies. However, contrary to published reports, the HEPM assay predicted that all CPs were teratogenic (false positives). These findings suggest that the HEPM cell growth inhibition bioassay may be useful to discriminate between subtle differences in structure-activity and, in combination with other bioassays, might facilitate the rapid detection and prioritization of diverse cytotoxins, including various developmental toxicants. Importantly, conclusions about the teratogenicity of a test chemical (via HEPM testing) should be approached with caution and confirmed with other teratogen-sensitive systems.

Animals↗

Study of protein-ligand binding effects by direct chromatographic on-column injection.

The direct zonal on-column injection method was applied to a high-performance liquid chromatographic study of pollutant-protein binding interactions in solution. The protein and the protein-ligand complex are excluded on the basis of the size from the diol support, and the free ligand penetrates into the pores and is more retained. The pattern of the ligand elution profile depends on the protein-ligand dissociation constant. This effect was quantitatively analysed by developing a numerical simulation algorithm in which the column is divided into slices of given thickness. The column length, flow-rate and shape of the injection signal are given as input parameters. A global dispersion coefficient accounts for peak broadening. A rapid equilibrium is assumed with the hypothesis that a monovalent ligand interacts with a single binding site on the protein. The interaction of bovine serum albumin with pentachlorophenol was studied, and an apparent dissociation constant for the protein-ligand complex was determined by fitting the theoretical profile to the experimental one. The effect of the acetonitrile content in the solvent was studied. An important decrease of the dissociation constant is observed that affects the chromatographic elution pattern.

Acetonitriles↗

Changes of the liver UDP-glucuronosyltransferase activity in trout (Salmo gairdneri Rich.) acutely exposed to selected aquatic toxicants.

Rainbow trout were exposed for 4 or 8 days to various types of toxicants, each applied to the test water at a high sublethal concentration. The activity of liver UDP-glucuronosyltransferase (UDP-GT) was assayed from the submitochondrial fraction using p-nitrophenol as an aglycone. Activity of UDP-GT was inhibited by 2,4,6-trichlorophenol, pentachlorophenol and dehydroabietic acid, all toxicants regularly found in effluents of the pulp and paper industry. The heavy metals cadmium and zinc, the polychlorinated biphenyl, Pyralene 3010, and chloroform did not affect UDP-GT activity. The slimicide N-methyl-dithiocarbamate (Vapam) significantly increased the enzyme activity.

Abietanes↗

Application of Alamar blue/5-carboxyfluorescein diacetate acetoxymethyl ester as a noninvasive cell viability assay in primary hepatocytes from rainbow trout.

We have adopted the application of two fluorescent indicator dyes to studying the viability of monolayers of primary rainbow trout hepatocytes. The two fluorescent dyes--Alamar blue, which indicates metabolic activity of a cell, and 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM), which is an indirect measure of cell membrane integrity-are noninvasive and can be monitored conveniently directly in multiwell plates. According to these dyes, L-15 culture medium supported hepatocyte viability over 96 h more stably than did M199. The two dyes proved to be capable of detecting a concentration-dependent toxic insult to hepatocytes caused by the model compound, pentachlorophenol. In contrast, a lack of impact on cell viability was indicated for up to 10(-5) M 17beta-estradiol, and that observation was supported by the induction of vitellogenin (VTG) mRNA/protein as indicator of hepatocyte-differentiated function. Application of the Alamar blue/CFDA-AM for 30 min did not alter gene expression either specifically as reflected by VTG or generally as reflected by a random selection of gene sequences that were amplified by differential display reverse transcription PCR (dd-rt-PCR). Thus, the assay represents a resource-efficient way of integrating measures of cell viability and gene expression that should aid in the interpretation of in vitro results. The assay can be applied repeatedly to the same set of cells and can be performed just prior to analysis of gene expression.

Actins↗

Toxic effects of thiol-reactive compounds on anaerobic biomass.

The objective of this study was to characterize the toxic effects of three well known thiol-reactive electrophilic compounds, N-ethylmaleimide (NEM), pentachlorophenol (PCP) and 1-chloro-2,4-dinitrobenzene (CDNB) on anaerobic biotransformation process. The work was part of a larger investigation on potassium efflux as a possible response mechanism of anaerobic microorganisms to the presence of thiol-reactive organic compounds and the interference of such compounds on the reductive dehalogenation process. Using anaerobic toxicity assay (ATA) and granular anaerobic biomass from a full-scale upflow anaerobic sludge blanket (UASB) reactor, inhibitory concentrations of these compounds that reduced the microbial activity of granular biomass to 50% of a control (IC50) were determined to be 592, 0.97, and 450 mg/l for NEM, PCP, and CDNB, respectively. Toxicity of NEM was also tested on anaerobic biomass from a municipal wastewater treatment plant digester and slightly lower IC50 of 532 mg/l was obtained. The results presented here indicate that anaerobic biomass can acclimate to the three thiol-reactive compounds studied and recover from inhibition as long as the toxicant concentration is below a threshold level. That threshold concentration was found to be 500 mg/l for NEM on biomass from the municipal digester, 1 mg/l for PCP, and 500 mg/l for CDNB, both on granular biomass. Granular anaerobic biomass showed recovery even at NEM concentrations of 1000 mg/l.

Bacteria, Anaerobic↗

Displacement imprinted polymer receptor analysis (DIPRA) for chlorophenolic contaminants in drinking water and packaging materials.

The preparation of a molecularly imprinted polymer (MIP) for pentachlorophenol is described together with two alternative reporter derivatives for use in a displacement imprinted polymer receptor analysis (DIPRA) format procedure. In this procedure, alternative reporter molecules were rebound to the synthetic receptor sites and their displacement by the target analyte was employed as the basis of a simple procedure for the measurement of chlorophenols in water and packaging material samples. Water samples were extracted using the standard procedure (EPA 528) and a detection limit of 0.5 microg l(-1) was achieved using the DIPRA detection method, with good agreement between the displacement technique and GC-ECD analysis. A variety of packaging materials, extracted using a buffered detergent solution were also analysed using the DIPRA procedure and showed good agreement with GC results. In addition, investigation of the cross-reactivity of a range of pesticides and materials commonly encountered in environmental analysis indicated the procedure gave good discrimination between pesticides bearing a chlorophenolic moiety and other materials. The procedure is considered highly suitable for use as a rapid field-test method or for incorporation into a test kit device.

Food Analysis↗

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.

Biodegradation, Environmental↗

Studies on lethal concentrations and toxicity stress of some xenobiotics on aquatic organisms.

Three widely used xenobiotics pentachlorophenol (PCP), 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-chloro-2,6-diethyl-N-(butoxymethyl) acetanilide (Butachlor) are evaluated for acute toxicity and stress behavior on freshwater fish (Heteropneustes fossilis, Clarias batrachus, Channa punctatus) and mosquito larvae (Culex pipiens fatigans). The experiment was carried out by medium treatment using intermittent flow-through system. Median lethal concentrations (LC50) were calculated by probit analysis. The LC50 values and 95% confidence intervals showed variable range for tested chemicals. Mosquito larvae generally appeared resistant than fish, while H. fossilis was found to be most sensitive. Stress signs in the form of behavioral changes are also observed. Both types of organisms are recommended as good bioindicator for the risk assessment of aquatic environment due to chemicals tested.

2,4-Dichlorophenoxyacetic Acid↗

Mechanochemical removal of organo-chlorinated compounds by inorganic components of soil.

The purpose of this work is to evaluate the catalytic efficiency of two metal oxides, ferrihydrite and birnessite and of a ferruginous smectite, towards organic molecules such as 4-chloroaniline (4-CA), pentachlorophenol (PCP), and five polychlorinated biphenyls (PCBs) characterised by different number and position of chlorine atoms. Mechanochemical dry contacts with light grinding between catalytic surfaces and pollutants have been carried out. The efficiency of the mechanochemical removal was compared with batch experiments for the soluble compounds (PCP and 4-CA). The removal of 4-CA and PCP by the mechanochemical procedure resulted more effective than by batch contact in the presence of birnessite and ferrihydrite, particularly at higher pH (100% removal of 4-CA by birnessite in 30 min at pH 8.6 after the mechanochemical contact compared to 20% removal using the batch interaction at the same pH). The mechanochemical contact of PCBs and birnessite produced a removal of pollutant that was a function of the number of chlorine atoms (complete removal of 2,2'-dichlorobiphenyl in 10 days and a removal of 30% and 20% of 2',3,4-trichlorobiphenyl and 3,3',4,4'-tetrachlorobiphenyl, respectively in 90 days) and of the position of chlorine atoms about the biphenyl rings (100% of 2,2'-dichlorobiphenyl in 10 days, 84% of 3,3'-dichlorobiphenyl in 15 days and 40% of 4,4'-dichlorobiphenyl in 27 days).

Aniline Compounds↗

Dechlorination ability of municipal waste incineration fly ash for polychlorinated phenols.

Pathways of pentachlorophenol dechlorination have been investigated on municipal waste incineration fly ash at 200 degrees C under nitrogen atmosphere. Thermodynamic calculations have been carried out for these dechlorination conditions using the method of total Gibbs energy minimization for the whole system consisting of gaseous components, i.e., chlorinated phenols, phenol, hydrogen chloride and the Cu3Cl3 trimer and of solid Cu2O and CuCl2 components. The effects of water, temperature and of the amounts of the reaction components on the thermodynamic equilibrium have been discussed and the experimental results compared with the calculated thermodynamic data.

Carbon↗

Bioconcentration factor of relatively low concentrations of chlorophenols in Japanese medaka.

Bioconcentration factors (BCF) for pentachlorophenol (PCP) and 2,4-dichlorophenol (2,4-DCP) in Japanese medaka (Oryzias latipes) were determined at five different concentrations of the chemicals, between 0.1 and 10 microg/l (PCP), 0.3 and 30 microg/l (2,4-DCP), in the ambient water. Medaka were exposed to each chemicals in a continuous-flow system during the embryonic development period and 60 days after hatching from eggs collected in the laboratory. Both the exposure time and the aqueous concentrations are much more realistic and closer to natural aquatic environments than those used in conventional BCF studies. The BCF values of PCP were from (4.9+/-2.8)x10(3) at the aqueous concentration of 0.074+/-0.028 microg/l to (2.1+/-1.4)x10(3) at 9.70+/-0.56 microg/l. The BCF value of 2,4-DCP were from (3.4+/-3.0)x10(2) at 0.235+/-0.060 microg/l to 92+/-27 at 27.3+/-1.6 microg/l. Generally, BCF values increased as the aqueous concentrations of PCP or 2,4-DCP decreased. This finding suggests that a relatively low and realistic aqueous concentration of these compounds is necessary to more accurately determine their BCF values in natural aquatic environments. Conventional BCF experiments at higher aqueous concentrations may underestimate the BCF values.

Animals↗

Sorption-desorption behavior of PCP on soil organic matter and clay minerals.

Pentachlorophenol (PCP) contamination is a severe environmental problem due to its widespread occurrence, toxicity and recalcitrance. In order to gain a better understanding of the fate of PCP in soils, the role of the soil organic matter (SOM) and clay minerals in the PCP sorption-desorption was studied on two bulk field soils, two subsoils (i.e., SOM or clay-removed soil) and two artificial soils. The two field soils used were a silty loam from New Mexico (NM) containing 10% clay and a sandy-clay-loam from Colombia (CO) South America comprised of 18% clay minerals. The bulk CO soil containing kaolinite sorbed significantly less PCP than the NM soil. All soils depicted an apparent hysteresis during sorption. The CO bulk and subsoils desorbed 14-20% and 15-26% of the sorbed PCP respectively whereas the NM bulk and subsoils desorbed only 4-12% and 5-16%, respectively. Experiments conducted with pure clay and artificial soils indicated that the expandable clay minerals were key sorbent material. Additional studies to investigate the interaction between SOM and clay minerals are needed to fully understand sorptive phenomena.

Aluminum Silicates↗

Joint anti-estrogenic effects of PCP and TCDD in primary cultures of juvenile goldfish hepatocytes using vitellogenin as a biomarker.

This work evaluated the joint anti-estrogenic effects of pentachlorophenol (PCP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) against 17beta-estradiol (E2) in juvenile goldfish (Carassius auratus) hepatocyte cultures. The level of vitellogenin (VTG) as a biomarker was determined by exposing hepatocytes to individual E2, PCP and TCDD, as well as to E2 in the presence of PCP, TCDD or their mixtures of various concentrations. PCP and TCDD did not exhibit estrogenicity. Both chemicals reduced the estrogenicity of E2, indicating the anti-estrogenic effects of PCP and TCDD. Their anti-estrogenic EC(50) values were calculated. The joint anti-estrogenic effects against E2 increased with increasing the PCP-to-TCDD ratio of mixture. Marking's indices were <0, suggesting an antagonism in anti-estrogenic effects between PCP and TCDD. The anti-estrogenic effects of PCP appeared to result primarily from the competitive binding to estrogen receptor. While TCDD may undergo an indirect binding process for its anti-estrogenic effects, the accurate mechanisms remain to be understood. The observed antagonism in anti-estrogenic effects resulted apparently from the mutual inhibition by PCP and TCDD.

Animals↗

Rapid and complete dechlorination of PCP in aqueous solution using Ni-Fe nanoparticles under assistance of ultrasound.

The Ni-Fe bimetallic particles have been laboratory prepared using sodium borohydride (NaBH4) as the reductant to reduce Ni2+ and Fe2+ in aqueous solution simultaneously, and characterized by TEM, XRD, BET and XPS. The particles were proved to be nanoscale amorphous alloy with an average diameter of about 30 nm and a BET surface area of 20.9 m2 g(-1). Experiments for dechlorination of pentachlorophenol (PCP) by the Ni-Fe bimetallic nanoscale particles in aqueous solutions were carried out under the enhancement of ultrasound. Major factors that influence the dechlorination efficiency, such as initial pH value, Ni content in the Ni-Fe particles, and output power of ultrasonic irradiation, were investigated. The results indicated that Ni-Fe nanoscale bimetallic particles were very effective for the dechlorination of PCP. Dechlorination efficiency was 46% in 30 min under the optimal condition without assistance of ultrasound, whereas it increased to 96% when ultrasonic irradiation was present. Initial pH value showed apparent effect on the dechlorination. As the pH varied from acidic condition to neutral condition, the dechlorination efficiency decreased dramatically. In addition, the dechlorination efficiency was improved with increased Ni/Fe ratio and ultrasonic output power. Less chlorinated phenols including tetrachlorophenol, trichlorophenol, dichlorophenol, monochlorophenol were formed during the initial reaction, and phenol was determined by GC-MS as sole product in the end of reaction.

Environmental Pollutants↗

Microbial activities in soils of a former sawmill area.

To find out microbial metabolic functioning and toxicity in a former sawmill area, carbon dioxide evolution, methane oxidation potential, 10 hydrolytic enzyme activities, Vibrio fischeri test, fluorescein diacetate hydrolysis activity (FDA), soil pH, carbon, nitrogen and pentachlorophenol (PCP) content were measured at four sites. The area is contaminated with aged chlorophenols. Chlorophenol content of soil was analyzed with a novel HPLC-MS technique, which allowed to measure chlorophenols without derivatization. The sites had a pollution gradient from 0.5 to 15 microg PCP g dw of soil(-1). Endogenous carbon dioxide evolution, methane oxidation potential, butyrate-esterase, acetate-esterase, sulphatase and aminopeptidase activities were lower at the site 2 than 3, although the site 2 and 3 had similar content of carbon and nitrogen. The soil was toxic in V. fischeri test at the site 2, which had high content of PCP (3.93+/-1.00 microg PCP g dw of soil(-1)). The results indicated that endogenous carbon dioxide evolution, methane oxidation potential, butyrate-esterase, acetate-esterase, sulphatase and aminopeptidase activities were sensitive to PCP in the soil. The results indicated that alpha-glucosidase, beta-glucosidase, beta-xylosidase, beta-cellobiosidase, phosphomonoesterase, N-acetyl-glucosaminidase activity and FDA hydrolysis activity were not sensitive to PCP in the soil. Soil processes involved in the cycling of carbon, nitrogen, sulphur and phosphorus were only slightly vulnerable in the former sawmill area and most sensitive microbial species were probably replaced with more tolerant ones to maintain and recover functioning of the former sawmill soils.

Acetylesterase↗

Determination of endocrine-disrupting compounds in cereals by pressurized liquid extraction and liquid chromatography-mass spectrometry. Study of background contamination.

A sensitive method based on pressurized liquid extraction (PLE) and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) has been developed for the determination in cereal samples of seven endocrine-disrupting compounds: bisphenol A (BPA), 4-tert-butylbenzoic acid (BBA), 4-nonylphenol (NP), 4-tert-butylphenol (t-BP), 2,4-dichlorophenol (DCP), 2,4,5-trichlorophenol (TCP) and pentachlorophenol (PCP). For the PLE procedure, methanol was selected as the extraction solvent. An experimental design approach was applied to optimize other PLE parameters. The recoveries achieved for the all seven compounds were in the 81-104% range, with relative standard deviations of 4-9%. An additional preconcentration step, based on solid-phase extraction (SPE), after the PLE step proved to be a successful way for obtaining a more sensitive method. The detection limits achieved in corn breakfast cereals were in the 0.003-0.013 microg g(-1) range, except for BPA, with a detection limit of 0.043 microg g(-1), for a sample size of 2.5 g. These values are similar to or even lower than currently legislated limits for pesticides in cereals and cereal-based foodstuffs. We also investigated possible contamination during the experimental process by the target compounds released from purified water, plastics, syringes, peristaltic pump tubes, glassware and other laboratory materials in contact with the samples along the analytical process.

Analytic Sample Preparation Methods↗

Impact of electrokinetic remediation on microbial communities within PCP contaminated soil.

Electrokinetic techniques have been used to stimulate the removal of organic pollutants within soil, by directing contaminant migration to where remediation may be more easily achieved. The effect of this and other physical remediation techniques on the health of soil microbial communities has been poorly studied and indeed, largely ignored. This study reports the impact on soil microbial communities during the application of an electric field within ex situ laboratory soil microcosms contaminated with pentachlorophenol (PCP; 100mg kg(-1) oven dry soil). Electrokinetics reduced counts of culturable bacteria and fungi, soil microbial respiration and carbon substrate utilisation, especially close to the acidic anode where PCP accumulated (36d), perhaps exacerbated by the greater toxicity of PCP at lower soil pH. There is little doubt that a better awareness of the interactions between soil electrokinetic processes and microbial communities is key to improving the efficacy and sustainability of this remediation strategy.

Electrolysis↗