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Toxicity of mono-, di- and tri-chlorophenols to lux marked terrestrial bacteria, Burkholderia species Rasc c2 and Pseudomonas fluorescens.

Burkholderia species RASC and Pseudomonas fluorescens were marked with lux genes, encoding for bioluminescence and used to assess the toxicity of mono-, di- and tri-chlorophenols by determining the decline in bioluminescence following exposure to the compounds in aqueous solution. Toxicity was expressed as a 50% effective concentration value (EC50, equating to the concentration of compound which caused a 50% decline in bioluminescence. Comparing the toxicity values of the compounds showed that, in general, increasing the degree of chlorination, increased toxicity. By carrying out forward multiple linear regressions with log10 EC50 values and physio-chemical descriptors, it was shown that molecular parameters describing the hydrogen bonding nature of a chlorophenol provided a better fit than regressions between toxicity data and log10 Kow alone. Utilising these descriptor variables in equations, it was shown that the toxicity of chlorophenols to the lux marked bacteria could be predicted from the compounds physio-chemical characteristics. By correlating lux marked RASC c2 and P. fluorescens EC50 values with toxicity values using Pimephales promelas (fathead minnow), Tetrahymena pyriformis (ciliate) and marine bacterium Vibriofischeri, it was apparent that lux marked RASC c2 correlated well with the freshwater aquatic species (P. promelas and T. pyriformis). This implied that for predictions of toxicity of organic xenobiotic compounds to higher organisms, lux marked RASC c2 could be utilised as a rapid surrogate.

Bacterial Proteins↗

Acute toxicity of chlorophenols to earthworms using a simple paper contact method and comparison with toxicities to fresh water organisms.

An acute toxicity test of chlorophenols on earthworms (Eisenia fetida) was performed using a simple paper contact method proposed by OECD testing guideline no. 207, that were applied as an earthworm toxicity test. The median lethal concentration, EC50, had significant correlation with logP(ow) (1-octanol/water partition coefficient) of the chemicals. The toxicity of chlorophenols on E. fetida was compared with toxicities for other species: an algae (Selenostrum capricornutum), a crustacean (Daphnia magna), and a fish (Oryzias latipes). It was found that the toxicity of chlorophenols was almost same for E. fetida and for fresh water organisms. These results suggest the possibility of drawing correlations between the effects of pollutants on living things in different environments, fresh water and soil.

Animals↗

Effect of liquid inhibitors on PCDD/F formation. Prediction of particle-phase PCDD/F concentrations using PLS modelling with gas-phase chlorophenol concentrations as independent variables.

Emissions of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) from municipal waste incineration are currently a subject of considerable public concern because of their extreme toxicity. PCDD/F formation in incineration processes is being studied widely, but little work has been done on their inhibition. We studied the effect of two liquid inhibitors, sodium ammonium hydrogen phosphate (NAHF) and urea (H2NCONH2), on PCDD/F formation in the combustion of liquid fuel doped with copper and chlorine using a pilot-scale plant. The inhibitors were injected into the flue gas stream at a temperature of 725 degrees C, whereupon both the chlorophenol and PCDD/F concentrations decreased. Particle-phase PCDD/F concentrations in particular decreased by up to 90% with NAHF and 70% with urea, but gas phase reduction took place only with urea. The results suggest that the formation of PCDD/Fs is hindered in the particle phase at the early stages of the PCDD/F formation chain, probably even before precursors such as chlorophenols have been formed. As a consequence, particle-phase PCDD/F concentrations can be predicted by a PLS (partial least-squares) approach with the gas-phase chlorophenol concentrations as independent variables. The structure and partial charges of Cu(+)-urea complex were calculated by the HF/3-21G basis set.

Benzofurans↗

Quantitative structure-activity relationships for the toxicity of chlorophenols to mammalian submitochondrial particles.

The toxicity of a series of chlorophenols, determined by a short-term in vitro assay utilizing mammalian submitochondrial particles, was related to the physicochemical and structural properties of these compounds. Quantitative Structure-Activity Relationships were defined by correlating EC50 values with six molecular descriptors, chosen to represent lipophilic, electronic and steric effects: the n-octanol/water partition coefficient (log Kow), the constant of Hammett (sigma sigma), the acid dissociation constant (pKa), the first order valence molecular connectivity index (1 chi v), the perimeter of the efficacious section (sigma D) and the melting point (m.p.). The results of regression analysis showed that log Kow is the most successful descriptor, indicating that the ability of chlorophenols to partition into the lipid bilayer of the mitochondrial membrane has an important role in determining their toxic effects. These results are consistent with a molecular mechanism of uncoupling action based on the chemiosmotic theory and on the protonophoric properties of chlorophenols. The quality of the QSAR models confirms the suitability of the SMP assay as a short-term prediction tool for aquatic toxicity of environmental pollutants acting on respiratory functions.

Animals↗

Microbial degradation of 4-chlorophenol by microorganisms entrapped in carrageenan-chitosan gels.

A new cell immobilization method based on the replacement of KCl by KCl + chitosan as gelling agent was developed. The results showed that through addition of chitosan into gelling agent, the mechanical strength and thermal stability of the carrageenan gel were greatly improved. The new immobilization method was used to entrap a chlorophenol-degrading microorganism. The immobilized microbial cells were applied for chlorophenol biodegradation. The experiments demonstrated that immobilized cells exhibit a higher bioactivity in the degradation of chlorophenol than free cells.

Biodegradation, Environmental↗

Contribution of free radicals to chlorophenols decomposition by several advanced oxidation processes.

The chemical decomposition of aqueous solutions of various chlorophenols (4-chlorophenol (4-CP), 2,4-dichlorophenol (2-DCP), 2,4,6-trichlorophenol (2,4,6-TCP) and 2,3,4,6-tetrachlorophenol (2,3,4,6-TeCP)), which are environmental priority pollutants, is studied by means of single oxidants (hydrogen peroxide, UV radiation, Fenton's reagent and ozone at pH 2 and 9), and by the Advanced Oxidation Processes (AOPs) constituted by combinations of these oxidants (UV/H2O2 UV/Fenton's reagent and O3/UV). For all these reactions the degradation rates are evaluated by determining their first-order rate constants and the half-life times. Ozone is more reactive with higher substituted CPs while OH* radicals react faster with those chlorophenols having lower number of chlorine atoms. The improvement in the decomposition levels reached by the combined processes, due to the generation of the very reactive hydroxyl radicals. in relation to the single oxidants is clearly demonstrated and evaluated by kinetic modeling.

Chlorophenols↗

Sensitive high-performance liquid chromatographic determination with fluorescence detection of phenol and chlorophenols with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride as a labeling reagent.

A sensitive HPLC method for the determination of phenol and chlorophenols was developed. The fluorescence labeling reaction of phenols with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) was completed in 30 min at 60 degrees C. The separation of DIB-derivatives of five representative phenols, i.e., phenol, o-, p-chlorophenol, 2,4-dichlorophenol, 2,4,6-trichlorophenol, was achieved within 35 min with an ODS column using isocratic elution. The detection limits of these DIB derivatives at a signal-to-noise ratio (SIN) of 3 were in the range of 0.024 to 0.08 microM (0.12-0.45 pmol/20 microl injection). Twelve kinds of DIB derivatives with phenols containing mono-, di-, tri-, tetra- and penta-chlorophenol were also well separated within 208 min by changing the elution conditions. The derivatives were stable for at least for 24 h when they were placed at room temperature in the dark. The proposed method was applied to the assay of human urine samples and free and total phenol were determined. The relative standard deviations (RSDs) of the proposed method for within and between-day assay were <7.0% and <14.2%, respectively. The average concentrations of free and total phenol found in urine (n=6) were 4.3+/-2.5 and 29.5+/-14.0 microM, respectively.

Benzoates↗

Ethanol/Water extraction combined with solid-phase extraction and solid-phase microextraction concentration for the determination of chlorophenols in cork stoppers.

The appearance of 2,4,6-trichloroanisole (TCA) in cork stoppers is of great concern because it can cause off-flavors in bottled wine. To prevent this sensorial defect, there should not be any traces of 2,4,6-trichlorophenol (TCP), 2,3,4,6-tetrachlorophenol (TeCP), or pentachlorophenol (PCP) in the finished corks, because they are the direct precursors of TCA. In the course of this study two methodologies based upon an extraction with ethanol/water mixtures to determine the chlorophenolic content in cork matrices were developed. The cork extract is preconcentrated using both solid-phase extraction and solid-phase microextraction methodologies. The latter was optimized by applying a full two-level factorial design. Finally, spiked ground corks at nanogram per gram levels of each chlorophenol were analyzed under optimal conditions and by applying both procedures. The obtained results demonstrate that chlorophenols can be detected in corks contaminated at the nanogram per gram level and, thus, these approaches can be successfully applied as quality control measures in the cork industry.

Chlorophenols↗

Increase of specific symptoms after long-term use of chlorophenol polluted drinking water in a community.

Chlorophenols contaminated the drinking water system and a local lake in the village of Järvelä in southern Finland. Period prevalence rates of symptoms, signs and diseases among the residents 15 years or older who responded (69%) to a survey in the contaminated area (1773 subjects) were compared with the rates of three uncontaminated areas (2018 subjects). Gastrointestinal and skin symptoms, in particular, were significantly (p < 0.05) more common in the contaminated area than in each control area. Nausea, general malaise, headache, anorexia, exceptional tiredness, and respiratory infections were significantly increased compared to the control areas combined. A dose-response was also observed: higher consumption of drinking water and contaminated fish further significantly increased (p < 0.05) reported symptoms. In conclusion, long-term use of chlorophenol polluted household water and fish can cause symptoms already familiar in connection with occupational chlorophenol exposures.

Chlorophenols↗

Cytotoxic effects of environmentally relevant chlorophenols on L929 cells and their mechanisms.

The chlorophenol chemicals (CPs) are a major class of widely distributed and frequently occurring persistent environmental pollutants. Pentachlorophenol (PCP) has been proposed to be procarcinogen in rodents and in possibly human beings. Human beings also easily expose to other chlorophenol chemicals, including 4-chlorophenol (CP), 2,4-dichlorophenol (DCP), 2,3,4-trichlorophenol (TCP), prompting this investigation of their comparative cytotoxic effects and cell death mechanisms, assayed in fibroblast L929 cells. The effective concentration for half-maximal response (EC50 values at 24 h for CP, DCP, TCP, and PCP are 2.18, 0.83, 0.46, and 0.11 mmol/L respectively and the EC50 values at 48 h are 1.18, 0.13, 0.08, and 0.06 mmol/L respectively by using 3-(4,5-dimethylthiazd-2-yl)-2,5-diphenyltentrazolium bromide (MTT) reduction assay. A clear structure-activity relationship was observed between toxicity of CPs and their octanol-water partition coefficients. The further studies indicate that CP, DCP, and TCP induce apoptosis in L929 cells in a concentration or time-dependent manner, but PCP mediates cell death more characteristic of necrosis than apoptosis. These results not only demonstrate that L929 cell growth inhibition bioassay may be useful to provide the comparative evaluation of toxicity of CPs in vitro, but also implicate that CP, DCP, TCP, in comparison with PCP, can induce L929 cell death by apoptosis, resulting in lower procarcinogensis, which may help to elucidate the molecular basis for the adverse health effects associated with CPs exposure.

Animals↗

Phenoxy herbicides and chlorophenols: a case control study on soft tissue sarcoma and malignant lymphoma.

A case control study on patients with soft tissue sarcoma and malignant lymphoma was undertaken to test whether there was any association between these diseases and past exposure to chlorinated phenoxy acid herbicides or chlorophenols. It was carried out over the period 1982-1988 in Victoria, Australia. Thirty males with soft tissue sarcoma and 52 males with malignant lymphoma were matched by age, place of residence and sex with one population control and one cancer control each. Exposure was assessed by personal interviews conducted by an occupational hygienist. Exposures within 5 years prior to diagnosis of each matched case were ignored, both for the cases and their matched controls. The estimated relative risks for definite or probable exposure to chlorinated phenoxy compounds or chlorophenols for at least 1 day were 1.0 (95% confidence interval (CI): 0.3-3.1) for soft tissue sarcoma and 1.5 (95% CI: 0.6-3.7) for malignant lymphoma. When the criterion for exposure was raised to more than 30 days, the estimated relative risks were 2.0 (95% CI: 0.5-8.0) for soft tissue sarcoma and 2.7 (95% CI: 0.7-9.6) for malignant lymphoma. Additional analyses were carried out for exposure of at least 1 day to phenoxy herbicides alone or chlorophenols alone. None of the estimated relative risks was significantly greater than unity.

Adult↗

Serum dioxin levels in former chlorophenol workers.

Using gas chromatography/mass spectrometry, we measured lipid-adjusted serum levels for all 2,3,7,8-substituted dioxins and furans, and four coplanar polychlorinated biphenyls in 62 workers with chlorophenol exposure and 36 workers without chlorophenol exposures working at the same plant during the same time. We oversampled among workers diagnosed with chloracne. Mean dioxin background levels from 36 nonchlorophenol workers were estimated as 6.0 parts-per-trillion (ppt) for 2,3,7,8 tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and 67.5 ppt for 1,2,3,4,6,7,8 heptachlorodibenzo-p-dioxin (Hepta-CDD). We found different dioxin and furan profiles for trichlorophenol and pentachlorophenol (PCP) workers. Among trichlorophenol workers with chloracne, we found 2,3,7,8-TCDD (mean=30.5 ppt) above background levels and among PCP workers with chloracne, we found high levels of Hepta-CDD (mean=312.5 ppt) and other higher chlorinated dioxins and furans. Cumulative exposure estimates for dioxins for both 2,3,7,8-TCDD and the higher chlorinated dioxins created in the early 1980s for our epidemiology studies were highly correlated with serum dioxin levels when age and body mass index were taken into account. While workers previously diagnosed with chloracne had high serum dioxin levels, some workers without diagnosed chloracne also had high levels. Among tradesworkers with plant-wide responsibilities, we observed serum dioxins and PCB levels higher than background indicating workplace exposures. We estimate that the mean level of 2,3,7,8-TCDD present in the serum of workers on the date workplace exposure terminated was 267 ppt (ranging from 8 to 1184 ppt) assuming a 9-year half-life, 582 ppt (ranging from 10 to 2,641 ppt) assuming a 7-year half-life, and 1928 ppt (ranging from 22 to 17,847) when a toxicokinetic model is used. We conclude that our findings are consistent with other studies reporting high serum dioxin levels among chlorophenol workers after occupational exposures.

Aged↗

Cancer incidence following chlorophenol exposure in a community in southern Finland.

Chlorophenols have contaminated the drinking water system and the local lake in the village of Järvelä in southern Finland. Local geology, ground water streams, and chemical analyses incriminated a local sawmill as the only plausible source of exposure. Cancer incidence in the municipality of Kärkölä (half of the population lives in Järvelä), compared with the rest of the local health-care district and with the greater cancer control region, indicated an excess of soft-tissue sarcomas and non-Hodgkin's lymphomas. A case-control study, which focused on cancers of the colon, bladder and soft tissues, lymphomas, and leukemia, demonstrated a significantly elevated risk ratio for non-Hodgkin's lymphomas among persons who consumed fish from the local lake, which was contaminated with chlorophenols. Probable exposure to chlorophenol-contaminated drinking water played a role in the increased incidence of non-Hodgkin's lymphomas and possibly was a factor in the development of soft-tissue sarcoma.

Animals↗

Evaluation of the sorption properties of a Mexican organo clinoptilolite-rich tuff for phenol and 4-chlorophenol.

A Mexican clinoptilolite-rich tuff was modified with the hexadecyltrimethyl ammonium (HDTMA) and used for the sorption of phenol and 4-chlorophenol. Whereas the zeolitic tuff had no affinity for phenol and 4-chlorophenol, the modified zeolite removed these organic compounds. The sorption behavior from aqueous solutions was studied by means of batch type and column experiments and isotherms were determined. It was found that the uptake was higher for 4-chlorophenol than for phenol.

Adsorption↗

Quantitative structure-toxicity relationships for chlorophenols to bioluminescent lux-marked bacteria using atom-based semi-empirical molecular-orbital descriptors.

Literature data on the toxicity of chlorophenols for three luminescent bacteria (Vibrio fischeri, and the lux-marked Pseudomonas fluorescens 10586s pUCD607 and Burkholderia spp. RASC c2 (Tn4431)) have been analyzed in relation to a set of computed molecular physico-chemical properties. The quantitative structure-toxicity relationships of the compounds in each species showed marked differences when based upon semi-empirical molecular-orbital molecular and atom based properties. For mono-, di- and tri-chlorophenols multiple linear regression analysis of V. fischeri toxicity showed a good correlation with the solvent accessible surface area and the charge on the oxygen atom. This correlation successfully predicted the toxicity of the heavily chlorinated phenols, suggesting in V. fischeri only one overall mechanism is present for all chlorophenols. Good correlations were also found for RASC c2 with molecular properties, such as the surface area and the nucleophilic super-delocalizability of the oxygen. In contrast the best QSTR for P. fluorescens contained the 2nd order connectivity index and ELUMO suggesting a different, more reactive mechanism. Cross-species correlations were examined, and between V. fischeri and RASC c2 the inclusion of the minimum value of the nucleophilic susceptibility on the ring carbons produced good results. Poorer correlations were found with P. fluorescens highlighting the relative similarity of V. fischeri and RASC c2, in contrast to that of P. fluorescens.

Burkholderia↗

By-side chlorobenzenes and chlorophenols in technical chlorobiphenyl formulations of Aroclor 1268, Chlorofen, Clophen T 64, Kanechlor 600, and Kanechlor 1000.

All 19 possible chlorophenol (CPh) and 12 chlorobenzene (CBz) congeners as potential impurities or additive were quantified in a relatively highly chlorinated type of technical chlorobiphenyl (CB) mixtures of Aroclor 1268, Chlorofen, Clophen T 64, Kanechlor 600, and Kanechlor 1000 using isotope dilution technique and HRGC/HRMS. The total CBzs content of Aroclor 1268, Chlorofen and Clophen T 64, Kanechlor 600, and Kanechlor 1000 was 0.039, 0.5, 230, 0.068, and 400 mg/g, respectively, while of CPhs was < 0.007, 0.48, 10, 0.093, and 0.98 microg/g. All 12 congeners of chlorobenzene could be quantified in all the formulations examined, but their proportions varied largely. It seems that stockpiles of technical chlorobiphenyl formulations and hazardous wastes containing CBs, both with added and/or by-side CBzs are also a somehow forgotten source of environmental contamination with those environmentally relevant compounds. No CPhs were found in Aroclor 1268 (< 7 ng/g). 2-MoCPh, 2,6-DiCPh, 3,5-DiCPh, 3,4,5-TrCPh, and 2,3,4,5,6-PeCPh were absent (< 1- < 20 ng/g) in Chlorofen and Clophen T 64, while other chlorophenol congeners were found at concentration from 7.4 to 130 and from 9.9 to 8,800 ng/g, respectively. Then, 3- and 4-chlorophenol, which co-eluted, were main contributors (88%) to the total CPhs content of Clophen T 64, while 2,3,4,6-TeCPh with 27% abundance was a major congener among CPhs in Chlorofen. Then, 2,4,6-TrCPh was the most abundant congener in Kanechlor 600, while 2,4,5-TrCPh was the most abundant congener in Kanechlor 1000.

Chlorobenzenes↗

Bioaugmentation of activated sludge with two Pseudomonas putida strains for the degradation of 4-chlorophenol.

The augmentation of activated sludge with two strains of Pseudomonas putida, CP1 and A(a) was investigated. Both strains of bacteria degraded 1.56 mM 4-chlorophenol. P. putida CP1 degraded the chemical using a modified ortho-cleavage pathway while P. putida A(a) used the meta-cleavage pathway. When activated sludge incapable of 4-chlorophenol degradation was augmented with either strain, substrate degradation occurred and followed the same biochemical pathways as when the bacteria were grown in pure culture. Insertion, in tandem, of the genes for gentamycin resistance and green fluorescent protein into the chromosomes of the two strains, enabled the survival and spatial location of the bacteria in the mixed microbial population to be monitored. Labelling the bacteria did not alter their degradative capabilities. P. putida CP1::Tn7-gfp survived in higher numbers than P. putida A(a)::Tn7-gfp following addition to the activated sludge. This was attributed to the ability of this strain to flocculate and become integrated in the activated sludge floc. Addition of P. putida CP1::Tn7-gfp or A(a)::Tn7-gfp to activated sludge resulted in smaller decreases in total cell numbers indicating a protective effect of the introduced P. putida strains on the overall microbial population from the harmful effects of 4-chlorophenol. The non-flocculant strain A(a) did not survive as well as CP1 in the activated sludge system and required a higher inoculum size to effect substrate degradation.

Biodegradation, Environmental↗

Biodegradation of 3-chlorophenol in a sequencing batch reactor.

The present paper shows the results obtained through a study on the biodegradation of 3-chlorophenol (3-CP) in a Sequencing Batch Reactor (SBR). To such a purpose a lab-scale SBR was fed a synthetic wastewater containing 3-CP and nutrients (nitrogen and phosphorus) diluted in tap water. The operating strategy, in terms of both the duration of either the cycle or the react phase, was changed throughout the experimental activity in order to find out the optimal one allowing to ensure constant and high removal efficiency despite the increasing 3-chlorophenol concentration in the feed. Biomass collected from a full-scale continuous flow activated sludge facility treating domestic wastewater was used as seed, after being acclimated to 3-CP by means of several batch tests. The results showed that a periodically operated activated sludge system can be successfully used for the biodegradation of chlorophenol compounds, after the needed members of the microbiological consortium are selected and enriched.

Biodegradation, Environmental↗