Fluoranthene, a volatile mutagenic compound, present in creosote and coal tar

Creosote, a coal-tar distillation product, contains mutagens which are volatile at 37 degrees C. After distillation of creosote we found that these volatile mutagens were present in the distillation fraction with the highest boiling range (greater than 360 degrees C). The "volatile mutagenic activity" was connected with the presence of fluoranthene, a polycyclic aromatic hydrocarbon. Commercially available fluoranthene was positive in the so-called "taped-plate assay" (the test system used for the detection of volatile mutagens) towards the strains TA98 and TA100 in the presence of S9 mix. The tested creosote and coal tar contained fluoranthene in concentrations of 5.2 and 2.2%, respectively.     

Action of a Fluoranthene-Utilizing Bacterial Community on Polycyclic Aromatic Hydrocarbon Components of Creosote

Cultures enriched by serial transfer through a mineral salts medium containing fluoranthene were used to establish a stable, seven-member bacterial community from a sandy soil highly contaminated with coal tar creosote. This community exhibited an ability to utilize fluoranthene as the sole carbon source for growth, as demonstrated by increases in protein concentration and changes in absorption spectra when grown on fluoranthene in liquid culture. Biotransformation of other polycyclic aromatic hydrocarbons (PAHs) was verified by demonstrating their disappearance from an artificial PAH mixture by capillary gas chromatography. When grown on fluoranthene as the sole carbon source and subsequently exposed to fluoranthene plus 16 additional PAHs typical of those found in creosote, this community transformed all PAHs present in this defined mixture. After 3 days of incubation, 13 of the original 17 PAH components were degraded to levels below the limit of detection (10 ng/liter). Continued incubation resulted in extensive degradation of the remaining four compounds. The ability of this community to utilize a high-molecular-weight PAH as the sole carbon source, in conjunction with its ability to transform a diverse array of PAHs, suggests that it may be of value in the bioremediation of environments contaminated with PAHs, such as those impacted by creosote.     

Detection of volatile mutagens in creosote and coal tar

Creosote and coal tar were examined for the presence of volatile mutagens by use of the so-called "taped plate assay". Application of this method, recently published by Distlerath et al., reveals that vapour escaping from creosote and coal tar at 37 degrees C was able to revert the Salmonella typhimurium strains TA98 and TA100 in the presence of S9 mix. The simplicity of this method makes it useful for routine screening of industrial fluids or solid products for the presence of volatile mutagens.     

The presence of the mutagenic polycyclic aromatic hydrocarbons benzo[a]pyrene and benz[a]anthracene in creosote P1

Several fractions of creosote P1 separated by TLC showed mutagenicity towards Salmonella typhimurium TA98. Thus mutagenicity is probably caused by the presence of mutagenic aromatic hydrocarbons. The mutagenic polycyclic aromatic hydrocarbons, benzo[a]pyrene and benz[a]anthracene, were detected in concentrations of 0.18 and 1.1% respectively. Because these compounds are probably not essential for the wood-preserving properties of creosote , a more selective composition of the product should be considered.     

Physiologo-biochemical properties of a strain of Beijerinckia mobilis 1phi Phn+--a degrader of polycyclic aromatic hydrocarbons

Beijerinckia mobilis 1f capable of degrading polycyclic aromatic hydrocarbons (PAHs) was isolated from a soil contaminated with creosote. Strain 1f could utilize phenanthrene and naphthalene as the sole sources of carbon. The mean rate of phenanthrene degradation during culture growth was 7-8 micrograms/(ml h). After cultivation under nonselective conditions, strain 1f retained its ability to degrade phenanthrene. Cometabolism considerably widened the range of PAHs that could be transformed by strain 1f. The strain was able to grow in a mineral medium with creosote as the sole source of carbon. After 30 days of cultivation in this medium, the total concentration of PAHs decreased from 665.5 mg/l to 170 mg/l.     

Contribution of primary aromatic amines to the mutagenicity of gasifier tars and coal oils

Two reactions that chemically alter primary aromatic amines (PAA) were used to assess the contribution of these compounds to the indirect bacterial mutagenicity of tar from an experimental low Btu gasifier. The first reaction, nitrosation, effectively eliminated the mutagenicity of several PAA standards and a coal oil when run in a low pH media (1.2). When applied to gasifier tar, extensive direct (not requiring metabolic activity) mutagenicity was generated. This direct mutagenicity limited the interpretation of results. When the pH of the reaction media was raised to 2.5, the mutagenicity of PAA standards and the coal oil were still greater than 90% eliminated, however, no direct mutagenicity was observed for the gasifier tar. Furthermore, only 61% of the indirect (requiring metabolic activation) mutagenicity was eliminated. Acetylation reduced the indirect activity of most primary amine standards by greater than 79%. Acetylation of the tar likewise eliminated part, but not all, of the activity, whereas most of the activity of the coal oil was eliminated. These results indicated that a much lower percentage of the mutagenic activity of low Btu coal tar samples was due to primary aromatic amines than was the case for coal oil.     

Carcinogen metabolism in human skin grafted onto athymic nude mice: a model system for the study of human skin carcinogenesis

Human skin grafted onto athymic nude mice maintains its major histological features and may provide a useful system with which to assess the carcinogen interaction with human skin. Significant differences were observed in basal levels of cytochrome P-450 and cytochrome P-448-dependent monooxygenase activities between human grafted and nude mouse epidermis. Topical application of crude coal tar (CCT) to human skin transplanted onto nude mice resulted in 3.9 & 3.5; 3.2 & 2.9 and 1.1 & 1.2 fold increases in mouse and human epidermal aryl hydrocarbon hydroxylase (AHH), ethoxyresorufin deethylase (ERD) and ethoxycoumarin deethylase (ECD) activities, respectively. CCT applied topically to mouse skin resulted in 27.8 & 6.4; 12.8 & 3.3 and 1.7 & 2.6 fold increases in mouse and human epidermal AHH, ERD and ECD activities, respectively. Topical application of coal tar either onto human transplanted skin or to mouse skin also resulted in substantial induction of hepatic and pulmonary AHH and ERD activities. These studies indicate that human skin grafted onto nude mice preserves its metabolic capacity and offers a useful model system with which to assess the effects of polycyclic aromatic hydrocarbons and CCT on cutaneous xenobiotic metabolism in the human population.     

Coal Tar‐Containing Asphalt Resource or Hazardous Waste?

Abstract: Coal tar was used in Sweden for the production of asphalt and for the drenching of stabilization gravel until 1973. The tar has high concentrations of polycyclic aromatic hydrocarbons (PAH), some of which may be strongly carcinogenic. Approximately 20 million tonnes of tar-containing asphalt is present in the public roads in Sweden. Used asphalt from rebuilding can be classified as hazardous waste according to the Swedish Waste Act. The cost of treating the material removed as hazardous waste can be very high due to the large amount that has to be treated, and the total environmental benefit is unclear. The transport of used asphalt to landfill or combustion will affect other environmental targets. The present project, based on three case studies of road projects in Sweden, evaluates the consequences of four scenarios for handling the material: reuse, landfill, biological treatment, and incineration. The results show that reuse of the coal tar-containing materials in new road construction is the most favorable alternative in terms of cost, material use, land use, energy consumption, and air emissions.     

A Biosurfactant/Polystyrene Polymer Partition System for Remediating Coal Tar-Contaminated Sediment

A sustainable, green chemistry process is proposed for the cleanup of coal tar impacted sediment in under 2 hr. A mixture of proteins and polypeptides, extracted from corn gluten meal and hemp, when mixed with sediment and polystyrene foam pellets (PFPs), serves to mobilize tar, which sorbs onto PFP. Since the sorbent floats, coal tar is easily extracted from the agitation vessel. An empirically derived 4-dimensional surface response model accurately predicts removal rates of the tar and operational costs of the system under various experimental conditions. At optimum relative to cost, 81% of the two to six ring polycyclic aromatic hydrocarbons (PAHs) and 73% of the total tar mass are removed despite high sediment organic carbon content (16.4%) and silty fines (～85%). Multiple PFP extractions (n = 2) of the same sediment/biosurfactant mixture yielded 94% extraction of PAH. Scanning electron microscope images illustrate free-phase tar (globule) sorption onto the foam. A field pilot was conducted in which 25 kg of sediment was processed. Results were in excellent agreement with both lab (10 g) experiments and model predictions. The process is considered sustainable and green because the active ingredients are derived from renewable crop materials, recycled polystyrene is used, and the biosurfactant is recyclable which reduces water demand and treatment costs, with the recovered tar used as fuel and sediment as beneficial reuse material.     

Multiple biomarkers study in painters in a shipyard in Korea

Shipbuilding workers are exposed to a variety of genotoxic compounds including polycyclic aromatic hydrocarbons (PAHs). A limited number of studies have been conducted to evaluate biomarkers related to PAH exposure in painters in the shipyard industry. We examined this in 208 workers recruited from a shipyard located in South Korea. Employees were grouped into three exposure groups: (1) 111 painters using coal tar paints, (2) 70 painters using general paints, and (3) 27 on-site controls using no paints. Levels of urinary 1-hydroxypyrene glucuronide (1-OHPG), as internal dose of PAH exposure, were measured by synchronous fluorescence spectroscopy. Glutathione S-transferase (GST) M1 and T1 genotypes were assessed by a multiplex polymerase chain reaction (PCR)-based method, aromatic-DNA adducts in peripheral white blood cells were measured by 32P-postlabeling, and glycophorin A (GPA) variant frequencies in red blood cells were assessed by flow cytometry. Information on demographic characteristics, smoking habits, diet, job title and use of personal protective equipment (e.g. respiratory and dermal) were collected by self-administered questionnaire. Average urinary 1-OHPG levels in coal tar paint (2.24 micromol/mol creatinine) and general paint (1.38 micromol/mol creatinine) users were significantly higher than in on-site controls (0.62 micromol/mol creatinine) (P<0.001). Paint use, irrespective of the type of paints, and smoking (yes/no) were positively associated with urinary 1-OHPG levels, whereas green tea consumption (yes/no) was negatively associated with the 1-OHPG levels. No significant effect in the 1-OHPG levels were observed for the GSTM1 and GSTT1 genotypes. Aromatic-DNA adduct levels tended to be higher in coal tar paint users (P = 0.06) and painters (P = 0.07) compared to on-site controls. No differences in adduct levels were observed, between the two groups of painters, and the combined group showed greater adduct levels than on-site controls (P = 0.05). GPA mutation frequencies measured in 55 individuals with MN heterozygote genotypes were not significantly different among the three exposure groups, and no correlation was observed between urinary 1-OHPG levels and aromatic-DNA adducts or GPA mutation frequency. These results suggest that painters in the shipyard were exposed to significant amounts of PAHs and possibly to other genotoxic aromatic compounds, and that urinary 1-OHPG may be a potential biomarker of PAH exposure in this population.     

Fungal cultures of tar bush and creosote bush for production of two phenolic antioxidants (Pyrocatechol and Gallic acid)

‘Tar bush’ and ‘creosote bush’ were substrates of fungal cultivation for tannase production and gallic acid and pyrocatechol accumulation. Aspergillus niger GH1 grew similarly on both plant materials under solid state culture conditions, reaching maximal levels after 4 d. Fungal strain degraded all tannin content of creosote bush after 4 d of fermentation and >75 % of tar bush after 5 d. Higher level of tannase activity was detected in tar bush fermentation. Biotransformation of tannins to gallic acid was high (93 % in creosote bush and 89 % in tar bush). Pyrocatechol was released poorly. Kinetic parameters of tannin conversion were calculated.     

Optimization of soil physical and chemical conditions for the bioremediation of creosote-contaminated soil

Mispah type soil (FAO : Lithosol) contaminated with >250 000 mg kg^-1 creosote was collected from the yard of a creosote treatment plant. The soils carbon, nitrogen and phosphorus contents were determined. Due to creosote contamination, thecarbon content of the soil was found to be 130,000 mg C kg^-1. This concentration was found to greatly affect the nitrogen content (0.08%). The phosphorus content was less affected (4.5%). It was estimated that a nutrient amendment to bring the soil to a C : N 10 : 1 would be adequate to stimulate microbial growth and creosote degradation. The soil was amended with a range of C : N ratios below and above the estimated ratio. In one of the treatments, the phosphorus content was amended. Sterile and natural controls were also set up. The soil was incubated at 30 °C on a rotaryshaker at 150 rpm in the dark for six weeks. Water content was maintained at 70% field capacity. The lowest nitrogen supplementation (C : N = 25 : 1) was more effective in enhancing microbial growth (3.12E + 05) and creosote removal (68.7%) from the soil. Additional phosphorus was not very effective in enhancing the growth of microorganisms and removal of creosote. The highest nitrogen supplementation(C : N = 5 : 1) did not enhance microbial growth and creosote removal.A relationship between mass loss and creosote removal was also observed. Phenolics and lower molecular mass polycyclic aromatic hydrocarbons (PAHs) were observed to be more susceptible to microbial degradation than higher molecular mass compounds. Nutrient concentration, moisture content and pH were thus observed to play very significant roles in the utilization of creosote in soil. These results are being used for the development of a bioremediation technology for the remediation of creosote contaminated soils in a treatment plant in South Africa.     

Interindividual variation in the levels of certain urinary polycyclic aromatic hydrocarbon metabolites following medicinal exposure to coal tar ointment

Determination of human exposure to polycyclic aromatic hydrocarbons PAHs is challenging because they are so broadly distributed in the environment and often difficult to quantitate using questionnaire methods. To enhance the ability to non invasively evaluate markers of both internal dose and biologically effective dose we have developed methods for the identification and quantitation of 1 hydroxypyrene-glucuronide and r 7, t 8, t 9, c 10 tetrahydroxy 7,8,9,10 tetrahydrobenzo a pyrene BP 7,10 8,9 tetrol in human urine. In the current study we applied these assays to urine samples collected from 43 hospitalized psoriasis patients treated with coal tar medication and 39 non treated volunteer controls. BP 7,10 8,9 tetrol was detected in 20 of 43 47 patients, ranging from 1 not detected to 124 fmol mumol-1 creatinine. In contrast, BP 7,10 8,9 tetrol was detected in only 4 of 39 10 controls, range 1 to 20.6 fmol mumol-1 creatinine p = 0.0006, Wilcoxon rank sum test . A second, more polar PAH metabolite, identified as 1 hydroxypyrene-glucuronide, was present in all urine samples. Mean 1 hydroxypyreneglucuronide levels were 40.96 72.62 pmol mumol-1 creatinine in patients and 0.38 0.32 pmol mumol-1 creatinine in control subjects p 0.0001 . The ratio of urinary levels of BP 7,10 8,9 tetrol to 1 hydroxypyrene-glucuronide was examined in the coal tar treated patients. This ratio was found to vary by approximately 6000 fold. This parameter cannot be explained by measurement error because the coefficients of variation for these assays are only 12 and 10 respectively, nor can it be explained by use of different coal tar products. These results provide further evidence that substantial interindividual variation in activation of benzo a pyrene and other PAHs exists, which may have implications for disease risk.     

Microbial-assisted remediation of creosote- and pentachlorophenol-treated wood products

A recycling process designed to recover wood fiber from discarded utility poles and cross ties was tested. Laboratory and field studies were conducted using a combined physical, chemical and microbiological protocol designed for the removal of creosote and pentachlorophenol wood preservatives from wood fiber. Woodchips produced in an industrial type wood chipper were batch extracted in methanol. The extractions successfully removed more than 95% of eight major creosote compounds contained within the woodchips. An initial combined concentration of 29 262 ppm during the extraction phase was reduced to 95 ppm in the laboratory study and to 1364 ppm in the field study. Biopolishing with a microbial consortium containing adapted strains from the generaPseudomonas, Flavobacterium andAcinetobacter further reduced the preservative concentration to 8 ppm and 200 ppm, respectively, with anthracene being the most recalcitrant compound in both studies. Pentachlorophenol-treated wood with an initial concentration of 1190 ppm, when subjected to the recycling process, yielded end product wood containing less than 2 ppm of the preservative. The solvent/preservative mixture (miscella) produced during the extraction process yielded a pure methanol fraction and a still bottom mixture when subjected to flash distillation. Fractional (vacuum) distillation of the still bottom mixture produced methanol, creosote, pentachlorophenol, and coal tar fractions.     

Bioremediation of Polycyclic Aromatic Hydrocarbons in Creosote-Contaminated Soil by Peniophora incarnata KUC8836

ABSTRACT Polycyclic aromatic hydrocarbons (PAHs) are present in products made from creosote, coal tar, and asphalt. When wood pile treated with creosote is placed in soil, PAHs can contaminate it. Creosote has been used for wood preservation in the past and is composed of approximately 85% PAHs and 15% phenolic compounds. PAHs cause harmful effects to humans and the environment because of their carcinogenic and mutagenic properties. White rot fungi can degrade not only lignin, but also recalcitrant organic compounds such as PAHs. Among numerous white rot fungi used in previous studies, four species were selected to degrade PAHs in a liquid medium. From this evaluation of the degradation of PAHs by the four fungal isolates, two species were ultimately selected for the highest rates of removal. Following 2 weeks of incubation with Peniophora incarnata KUC8836, the degradation rates of phenanthrene, fluoranthene, and pyrene were 86.5%, 77.4%, and 82.6%, respectively. Mycoaciella bispora KUC8201 showed the highest degradation rate for anthracene (61.8%). Hence, bioremediation of creosote-contaminated soil with an initial concentration of 229.49 mg kg^?1 PAHs was carried out using the two selected fungi because they could simultaneously degrade 13 more PAHs than the comparison species. More importantly, isolates of P. incarnata KUC8836 were discovered as powerful degraders of PAHs by producing laccase and manganese-dependent peroxidase (MnP), with 1.7- and 1.1-fold higher than the comparison species, respectively. Therefore, the white rot fungus may be proposed for the removal of PAHs and xenobiotic compounds in contaminated environments.     

Comprehensive GC²/MS for the monitoring of aromatic tar oil constituents during biodegradation in a historically contaminated soil

The constituents of tar oil comprise a wide range of physico-chemically heterogeneous pollutants of environmental concern. Besides the sixteen polycyclic aromatic hydrocarbons defined as priority pollutants by the US-EPA (EPA-PAHs), a wide range of substituted (NSO-PAC) and alkylated (alkyl-PAC) aromatic tar oil compounds are gaining increased attention for their toxic, carcinogenic, mutagenic and/or teratogenic properties. Investigations on tar oil biodegradation in soil are in part hampered by the absence of an efficient analytical tool for the simultaneous analysis of this wide range of compounds with dissimilar analytical properties. Therefore, the present study sets out to explore the applicability of comprehensive two-dimensional gas chromatography (GC2/MS) for the simultaneous measurement of compounds with differing polarity or that are co-eluting in one-dimensional systems. Aerobic tar oil biodegradation in a historically contaminated soil was analyzed over 56 days in lab-scale bioslurry tests. Forty-three aromatic compounds were identified with GC2/MS in one single analysis. The number of alkyl chains on a molecule was found to prime over alkyl chain length in hampering compound biodegradation. In most cases, substitution of carbon with nitrogen and oxygen was related to increased compound degradation in comparison to unalkylated and sulphur- or unsubstituted PAH with a similar ring number.The obtained results indicate that GC2/MS can be employed for the rapid assessment of a large variety of structurally heterogeneous environmental contaminants. Its application can contribute to facilitate site assessment, development and control of microbial cleanup technologies for tar oil contaminated sites.     

Use of 13C Nuclear Magnetic Resonance To Assess Fossil Fuel Biodegradation: Fate of [1-13C]Acenaphthene in Creosote Polycyclic Aromatic Compound Mixtures Degraded by Bacteria

[1-¹³C]acenaphthene, a tracer compound with a nuclear magnetic resonance (NMR)-active nucleus at the C-1 position, has been employed in conjunction with a standard broad-band-decoupled ¹³C-NMR spectroscopy technique to study the biodegradation of acenaphthene by various bacterial cultures degrading aromatic hydrocarbons of creosote. Site-specific labeling at the benzylic position of acenaphthene allows ¹³C-NMR detection of chemical changes due to initial oxidations catalyzed by bacterial enzymes of aromatic hydrocarbon catabolism. Biodegradation of [1-¹³C]acenaphthene in the presence of naphthalene or creosote polycyclic aromatic compounds (PACs) was examined with an undefined mixed bacterial culture (established by enrichment on creosote PACs) and with isolates of individual naphthalene- and phenanthrene-degrading strains from this culture. From ¹³C-NMR spectra of extractable materials obtained in time course biodegradation experiments under optimized conditions, a number of signals were assigned to accumulated products such as 1-acenaphthenol, 1-acenaphthenone, acenaphthene-1,2-diol and naphthalene 1,8-dicarboxylic acid, formed by benzylic oxidation of acenaphthene and subsequent reactions. Limited degradation of acenaphthene could be attributed to its oxidation by naphthalene 1,2-dioxygenase or related dioxygenases, indicative of certain limitations of the undefined mixed culture with respect to acenaphthene catabolism. Coinoculation of the mixed culture with cells of acenaphthene-grown strain Pseudomonas sp. strain A2279 mitigated the accumulation of partial transformation products and resulted in more complete degradation of acenaphthene. This study demonstrates the value of the stable isotope labeling approach and its ability to reveal incomplete mineralization even when as little as 2 to 3% of the substrate is incompletely oxidized, yielding products of partial transformation. The approach outlined may prove useful in assessing bioremediation performance.     

Review on proteomic analyses of benzo[a]pyrene toxicity

Benzo[a]pyrene (BaP), a five-ring polycyclic aromatic hydrocarbon, is a well-recognized environmental pollutant. Coal-processing waste products, petroleum sludge, asphalt, creosote, and tobacco smoke, all contain high levels of BaP. Exposure to BaP elicits many adverse biological effects, including tumor formation, immunosuppression, teratogenicity, and hormonal effects. In addition to the genetic damage caused by BaP exposure, several studies have indicated the disruption of protein-protein signaling pathways. However, contrary to the large number of studies on BaP-induced DNA damage, only few data have been gathered on its effects at the protein level. This review highlights all proteomic studies to date used for assessing the toxicity of BaP and its metabolites in various organ systems. It will also give an overview on the role proteomics may play to elucidate the mechanisms underlying BaP toxicity.     

Standard reference materials for chemical and biological studies of complex environmental samples.

Standard Reference Materials (SRMs) from the National Institute of Standards and Technology (NIST) are often used in methods development and interlaboratory comparison studies since they are homogeneous and readily available to the scientific community. SRM 1649 (urban dust/organics), SRM 1650 (diesel particulate matter), and SRM 1597 (complex mixture of polycyclic aromatic hydrocarbons from coal tar) are three environmental samples which have been used by the scientific community for these purposes. These SRMs were originally developed to assist laboratories in validating analytical procedures for the determination of polycyclic organic compounds in complex mixtures. In addition, these SRMs have been valuable for the comparison of methodologies for bacterial bioassays and the development of bioassay-directed fractionation and bioassay-directed chemical analysis techniques. Most recently these SRMs were chosen for use as test samples in a collaborative study coordinated by the World Health Organization--International Programme on Chemical Safety. This paper provides a summary of much of the work to date (published and unpublished) on the chemical and biological characterization of these three SRMs. Information regarding the availability of other NIST SRMs that might be useful for these types of studies are provided also.     

Combined ozonation and biodegradation for remediationof mixtures of polycyclic aromatic hydrocarbons in soil

A study was conducted to investigate the feasibilityof a combined treatment (i.e., ozonation andbiodegradation) to overcome the inherent bacterialbioavailability limitation, and hence bioremediationlimitation, of polycyclic aromatic hydrocarbons insoil. Ozonation was very efficient in the removal ofnaphthalene, fluorene, phenanthrene, and anthracene,but not for pyrene, chrysene, and benzo(a)pyrene fromsoil freshly spiked with the hydrocarbons. A similarresult was obtained from coal tar-contaminated soil.Elimination of polycyclic aromatic hydrocarbonsincreased appreciably in sand containing 0.03%organic carbon, indicating the adverse effect oforganic carbon on the efficiency of ozone treatment.In spiked and coal tar-contaminated soils, ozonationfollowed by biodegradation significantly increased thedegradation of various polycyclic aromatichydrocarbons including chrysene and benzo(a)pyrenewhich were not degraded by the test bacterialconsortium alone. In particular, the effect of thecombined treatment was more pronounced in coaltar-contaminated soil than in sterile soil spiked withhydrocarbons, probably due to the augmented biologicalactivity of the introduced consortium. The resultssuggest that a combined treatment including ozonationand biodegradation may be a promising bioremediationtechnology in soil contaminated with mixtures ofpolycyclic aromatic hydrocarbons such as formermanufactured gas plant sites.