Short-term toxic effects of naphthalene and pyrene on the common prawn (Palaemon serratus) assessed by a multi-parameter laboratorial approach: mechanisms of toxicity and impairment of individual fitness

The short-term (96 h) toxic effects of two polycyclic aromatic hydrocarbons (PAHs), naphthalene (NAP) and pyrene (PYR), on the common prawn (Palaemon serratus) were investigated in laboratory bioassays, including a fitness related assay based on the post-exposure swimming velocity. Other effect criteria were biomarkers of neurotoxicity, oxidative stress and bioenergetics, and mortality. In the range of concentrations tested (NAP: 0.13-8 mg/L; PYR: 0.006-0.4 mg/L), both PAHs impaired the swimming velocity, induced oxidative stress and damage, and changed the activity of lactate dehydrogenase and isocitrate dehydrogenase. NAP also caused mortality (96 h-LC50=3.5 mg/L). Thus, both PAHs were able to cause toxic effects on P. serratus after a short period of exposure through the water, including the reduction of individual fitness. PYR was five folds more effective in reducing the swimming velocity of P. serratus than NAP. These findings are of interest for the marine ecological risk assessment of oil spills.     

Determination of dichloromethane, trichloroethylene and perchloroethylene in urine samples by headspace solid phase microextraction gas chromatography-mass spectrometry

A method for the determination of volatile chlorinated hydrocarbons, namely dichloromethane (DCM), trichloroethylene (TCE), and perchloroethylene (PCE), in urine samples was developed using headspace solid phase microextraction (HS-SPME) gas chromatography-mass spectrometry (GC-MS). HS-SPME was performed using a 75 microm Carboxen-polydimethylsiloxane fiber. Factors, which affect the HS-SPME process, such as adsorption and desorption times, stirring, salting-out effect, and temperature of sampling have been evaluated and optimized. The highest extraction efficiency was obtained when sampling was performed at room temperature (22 degrees C), from samples saturated with salt and under agitation. Linearity of the HS-SPME-GC-MS method was established over four orders of magnitude and the limit of detection was 0.005 microg/l for all the compounds. Precision, calculated as %R.S.D. at three different concentration levels, was within 1-8% for all intra- and inter-day determinations. The method was applied to the quantitative determination of TCE and PCE in human urine samples from exposed (TCE, n=5; median, 9.32 microg/l and PCE, n=39; median, 0.58 microg/l) and non-exposed individuals (n=120; median concentrations, 0.64, 0.22 and 0.11 microg/l for DCM, TCE and PCE, respectively. In addition, two cases of acute accidental exposure to DCM are reported, and the elimination kinetics in blood and urine was followed up. The calculated half-lives of urinary and blood DCM were, respectively, 7.5 and 8.1 h for one subject and 3.8 and 4.3 h for the other.     

Development of a gas chromatography/mass spectrometry method to quantify several urinary monohydroxy metabolites of polycyclic aromatic hydrocarbons in occupationally exposed subjects

The aim of this study was the development of a method for the determination of 12 urinary monohydroxy metabolites of PAHs, namely 1-hydroxynaphthalene, 2-hydroxynaphthalene, 2-hydroxyfluorene, 9-hydroxyfluorene, 1-hydroxyphenanthrene, 2-hydroxyphenanthrene, 3-hydroxyphenanthrene, 4-hydroxyphenanthrene, 9-hydroxyphenanthrene, 1-hydroxypyrene, 6-hydroxychrysene, and 3-hydroxybenzo[a]pyrene. Analytes were determined in the presence of deuterated analogues as internal standards, by GC/MS operating in the electron impact mode. Sample preparation was performed by enzymatic hydrolysis of glucoronate and sulphate conjugates of hydroxy metabolites of PAHs, liquid-liquid extraction with n-hexane, and derivatization with a silylating reagent. The method is very specific, limits of quantification are in the range 0.1-1.4 microg/l, and range of linearity is from limit of detection to 208 microg/l. Within- and between-run precision, expressed as coefficient of variation, are <20%; accuracy for most analytes is within 20% of the theoretical value. An application of the proposed method to the analysis of 10 urine samples from coke-oven workers shows that 1-hydroxynaphthalene and 2-hydroxyfluorene were the most abundant compounds (median 61.4 and 69.7 microg/l, respectively), while 6-hydroxychrysene, and 3-hydroxybenzo[a]pyrene were always below the quantification limit.     

Short-term toxic effects of naphthalene and pyrene on the common prawn (Palaemon serratus) assessed by a multi-parameter laboratorial approach: mechanisms of toxicity and impairment of individual fitness

The short-term (96?h) toxic effects of two polycyclic aromatic hydrocarbons (PAHs), naphthalene (NAP) and pyrene (PYR), on the common prawn (Palaemon serratus) were investigated in laboratory bioassays, including a fitness related assay based on the post-exposure swimming velocity. Other effect criteria were biomarkers of neurotoxicity, oxidative stress and bioenergetics, and mortality. In the range of concentrations tested (NAP: 0.13–8?mg/L; PYR: 0.006–0.4?mg/L), both PAHs impaired the swimming velocity, induced oxidative stress and damage, and changed the activity of lactate dehydrogenase and isocitrate dehydrogenase. NAP also caused mortality (96 h-LC50?=?3.5?mg/L). Thus, both PAHs were able to cause toxic effects on P. serratus after a short period of exposure through the water, including the reduction of individual fitness. PYR was five folds more effective in reducing the swimming velocity of P. serratus than NAP. These findings are of interest for the marine ecological risk assessment of oil spills.     

Sensitive biomarker of polycyclic aromatic hydrocarbons (PAHs): urinary 1-hydroxyprene glucuronide in relation to smoking and low ambient levels of exposure.

The study was conducted in a Chinese population with occupational or environmental exposures to polycyclic aromatic hydrocarbons (PAHs). A total of 106 subjects were recruited from coke-oven workers (workers), residents in a metropolitan area (residents) and suburban gardeners (gardeners). All subjects were monitored twice for their personal exposures to PAHs. The biological samples were collected for measurements of 1-hydroxypyrene (1-OHP) and cotinine in urine. The geometric means of personal exposure levels of pyrene, benz(a)anthracene (BaA) and benzo(a)pyrene (BaP) in workers were 1.470, 0.978 and 0.805 microg m-3, respectively. The corresponding levels in residents were 0.050, 0.034 and 0.025 microg m-3; and those in gardeners were 0.011, 0.020 and 0.008 microg m-3, respectively. The conjugate of 1-OHP with glucuronide (1-OHP-G) is the predominant form of pyrene metabolite in urine and it showed strong associations with exposures not only to pyrene, but also to BaA, BaP and total PAHs. Most importantly, a significant difference in 1-OHP-G was even detected between the subgroups with exposures to BaP at < 0.010 and > 0.010 but < 0.020 microg m-3, suggesting that 1-OHP-G is a good marker that can be used for the risk assessment of BaP exposure at levels currently encountered in ambient air. Furthermore, multiple regression analyses of 1-OHP-G on PAHs exposure indicated that cigarette smoke was a major confounding factor and should be considered and adjusted for while using 1-OHP to estimate PAHs exposure.     

Impact of Clay Minerals and DOM on the Competitive Sorption/Desorption of PAHs

Two model compounds were used to investigate sorptive phenomena of a silty-sand soil under single and binary solute systems at different concentrations. In the sorption isotherms, the presence of phenan-threne (PHE) exhibited a statistically significant (P<0.05) sorption competition over pyrene (PYR), regardless of the concentration. PYR influenced the PHE isotherms only when it was present at 15?mg/L. The concentration dependence in sorption competition was only evident for the more hy-drophobic PYR. In the presence of PHE, the fraction of desorbed PYR was significantly increased with an increase in PYR concentration. PHE desorption enhancement was the most observable with the higher initial concentration (15?mg/L). However, the presence of PYR did not affect PHE desorption. This study found that, based on equivalent solid mass, soils containing only clay minerals sorbed 12.2% more PHE than soils with only soil organic matter (SOM) for 3?mg/L PHE. Clay minerals also impacted desorption as evidenced by a 65% decrease in desorbed PYR fraction compared with when the soil only contained SOM. The dissolved organic matter (DOM) amendment did not increase desorption. Instead, PHE desorption was significantly inhibited by the added DOM. For this study, co-sorption was found to be the mechanism for the inhibited desorption.     

Degradation of polycyclic aromatic hydrocarbons dissolved in Tween 80 surfactant solutions by Sphingomonas paucimobilis EPA 505

The degradation rates of mixtures of pyrene (PYR), fluoranthene (FLA), and phenanthrene (PHE) by Sphingomonas paucimobilis EPA 505 were measured in the presence of the nonionic surfactant Tween 80. For strain EPA 505, FLA and PHE are growth substrates, while PYR is not. Linear degradation rates ranging from 0.05 to 2.2 mg x L(-1) x h(-1) were observed for FLA, PYR, and PHE at approximately 10(7) colony-forming units (CFU)/mL. At lower biomass, PYR degradation exhibited lognormal degradation. The degradation rates of PYR, FLA, and PHE increased with increasing biomass and substrate concentration. At high FLA concentrations, FLA degradation rates were faster in the presence of surfactant than in the absence of surfactant, suggesting that some of the FLA was transported directly into the cell from the micellar phase. In mixtures, PHE was the preferred substrate and was utilized first, followed by FLA and then PYR. Once the competing substrates were degraded, the remaining substrate was degraded at the same rate or faster than the rate found in the single-substrate system. Based on the results with Tween 80, it appears that PHE, PYR, and FLA are competing for the same enzymatic sites.     

Biodegradation of phenanthrene by Pseudomonas sp. BZ-3, isolated from crude oil contaminated soil

A phenanthrene (PHE) degrading bacterium strain BZ-3 was isolated from the crude oil contaminated soil in Binzhou, China. The isolate was identified as Pseudomonas sp. BZ-3 on the basis of 16S rRNA gene sequence. Various experiments were conducted to investigate the effect of pH, salinity and PHE concentration on the degradation efficiency of PHE. The degradation efficiency and degradation metabolites of PHE were detected by using GC–MS and HPLC-MS analyses. The strain BZ-3 could degrade 75% of PHE at an initial concentration of 50 mg/L under 20 g/L salinity in 7 days. PHE degradation kinetics was estimated in a first-order degradation rate model and the rate coefficient was calculated as 0.108 d⁻¹. On the basis of the identified degradation metabolites, the strain BZ-3 could degrade PHE in the salicylate metabolic pathway. In a mixture system consisting of PHE and other PAHs including naphthalene (NA), anthracene (ANTH), and pyrene (PYR), the strain BZ-3 showed an efficiently degradation capability. Further study showed that the strain BZ-3 could also use NA, ANTH, PYR, xylene, 1-hydroxy-2-naphthoic acid, and hexane as the sole carbon and energy source, but did not grow on nitrobenzene-containing medium.     

Degradation of a mixture of high‐molecular‐weight polycyclic aromatic hydrocarbons by a Mycobacterium strain PYR‐1

Mycobacterium sp. PYR‐1, which was previously shown to mineralize several individual polycyclic aromatic hydrocarbons (PAHs), simultaneously degraded phenanthrene, anthracene, fluoranthene, pyrene and benzo[a]pyrene in a six‐component synthetic mixture. Chrysene was not degraded significantly. When provided with a complex carbon source, Mycobacterium sp. PYR‐1 degraded greater than 74% of the total PAH mixture during 6 d of incubation. Mycobacterium sp. PYR‐1 appeared to preferentially degrade phenanthrene. No significant difference in degradation rates was observed between fluoranthene and pyrene. Anthracene degradation was slightly delayed but, once initiated, proceeded at a constant rate. Benzo[a]pyrene was degraded slowly. Degradation of a crude mixture of benzene‐soluble PAHs from contaminated sediments resulted in a 47% reduction of the material in 6 d compared with that of autoclaved controls. Experiments using an environmental microcosm test system indicated that mineralization rates of individual ¹⁴C‐labeled compounds were significantly lower in the mixtures than in equivalent doses of these compounds alone. Mineralization of the complete mixture was estimated conservatively to be between 49.7 and 53.6% and was nearly 50% in 30 d of incubation when all compounds were radiolabeled. These results strengthen the argument for the potential application of Mycobacterium sp. PYR‐1 for bioremediation of PAH‐contaminated wastes.     

Urinary hydroxylated metabolites of polycyclic aromatic hydrocarbons as biomarkers of exposure in asphalt workers

Background. Fumes and vapours released during laying of hot asphalt mix have been recognised as a major source of exposure for asphalt workers. Objectives. We investigated the relationships between inhalation exposure to asphalt emissions and urinary biomarkers of polycyclic aromatic hydrocarbons (PAHs) in asphalt workers (AW, n=75) and in ground construction workers (CW, n=37). Methods. Total polyaromatic compounds (PAC) and 15 priority PAHs in inhaled air were measured by personal sampling. Hydroxylated PAH metabolites (OH-PAHs) (2-naphthol, 2-hydroxyfluorene, 3-hydroxyphenanthrene, 1-hydroxypyrene, 6-hydroxychrysene and 3-hydroxybenzo[a]pyrene) were determined in urine spot samples collected in three different times during the work week. Results. Median vapour-phase PAC (5.5 µg m^-3), PAHs (≤50 ng m^-3) and OH-PAHs (0.08-1.11 µg l^-1) were significantly higher in AW than in CW, except in the cases of air naphthalene and 2-naphthol. Airborne levels of particle-phase contaminants were similar in the two groups and much lower than vapour-phase levels; metabolites of particulate PAHs were never found in quantifiable amounts. An appreciable increase in OH-PAH levels during the work day and work week was found in AW; median levels for 2-hydroxyfluorene, 3-hydroxyphenanthrene and 1-hydroxypyrene were, respectively, 0.29, 0.08 and 0.18 at baseline; 0.50, 0.18 and 0.29, pre-shift; 1.11, 0.44 and 0.44 µg l^-1, post-shift. Each OH-PAH exhibited a characteristic profile of increase, reflecting differences in half-lives of the parent compounds. In non-smoking subjects, positive correlations were found between vapour-phase PAC or PAHs and OH-PAHs both in pre- and post-shift samples (0.34 ≤ r≤69). Smokers exhibited 2-5-fold higher OH-PAHs than non-smokers, at any time and at both workplaces. Conclusions. Our results suggest that OH-PAHs are useful biomarkers for monitoring exposure to asphalt emissions. The work-related exposure to PAC and PAHs was low in all AW, but urinary metabolites reflected exposure satisfactorily.     

Inoculation of PAH-degrading strains of Fusarium solani and Arthrobacter oxydans in rhizospheric sand and soil microcosms: microbial interactions and PAH dissipation

Very little is known about the influence of bacterial-fungal ecological interactions on polycyclic aromatic hydrocarbon (PAH) dissipation in soils. Fusarium solani MM1 and Arthrobacter oxydans MsHM11 can dissipate PAHs in vitro. We investigated their interactions and their effect on the dissipation of three PAHs—phenanthrene (PHE), pyrene (PYR) and dibenz(a,h)anthracene (DBA)—in planted microcosms, in sterile sand or non-sterile soil. In sterile sand microcosms planted with alfalfa, the two microbes survived and grew, without any significant effect of co-inoculation. Co-inoculation led to the dissipation of 46 % of PHE after 21 days. In soil microcosms, whether planted with alfalfa or not, both strains persisted throughout the 46 days of the experiment, without any effect of co-inoculation or of alfalfa, as assessed by real-time PCR targeting taxon-level indicators, i.e. Actinobacteria 16S rDNA and the intergenic transcribed spacer specific to the genus Fusarium. The microbial community was analyzed by temporal temperature gradient electrophoresis and real-time PCR targeting bacterial and fungal rDNA and PAH-ring hydroxylating dioxygenase genes. These communities were modified by PAH pollution, which selected PAH-degrading bacteria, by the presence of alfalfa and, concerning the bacterial community, by inoculation. PHE and PYR concentrations significantly decreased (91 and 46 %, respectively) whatever the treatment, but DBA concentration significantly decreased (30 %) in planted and co-inoculated microcosms only.     

Phytoremediation and removal mechanisms in Bouteloua curtipendula growing in sterile hydrocarbon spiked cultures

Tolerance index and phytoremediation factors of side oats grama (Bouteloua curtipendula) with recalcitrant polycyclic aromatic hydrocarbons (PAH) phenanthrene (PHE), pyrene (PYR), and benzo[a]pyrene (BaP) and the resulting impact on phenotypic response, were evaluated in sterile conditions with whole plant growing in test-tube cultures with MS medium with PAH and compared with Tall fescue (Festuca arundinacea), control for this study. PAH mixture of PHE, PYR and BaP (1:1:1 w/w/w) blended with Maya crude oil (1:1 w/w), final concentration of 1500 mg kg(-1) was used. After 40 days, BaP removal, in the presence of Maya crude was superior compared with PHE and PYR removal Although the presence of PAH negatively affects the phenotypic response of the plants; sterile conditions experiments were helpful to evaluate phytoremediation factors to elucidate some important questions regarding phytoremediation mechanisms; in this study, B. curtipendula was able to phytostabilizate BaP associated to a significant hydrocarbon removal (57.4%) with high root accumulation but attenuated transport to stems, here reported as translocation factor. To our knowledge, this is the first time that quantifiable phytoremediation factors were used to evaluate the tolerance and removal capacity of a native semi-arid climate plant which is probably able to phytoremediate hydrocarbon contaminated soils.     

Characterization of fluoranthene- and pyrene-degrading bacteria isolated from PAH-contaminated soils and sediments

Sixteen environmental samples, from the United States, Germany and Norway, with histories of previous exposure to either creosote, diesel fuel or coal tar materials, were screened for bacteria which could degrade high molecular weight (HMW) polycyclic aromatic hydrocarbons (PAHs). A modified version of the spray plate technique was used for the isolations. Using fluoranthene (FLA) and pyrene (PYR) as model HMW PAHs, we isolated 28 strains on FLA and 21 strains on PYR. FLA degraders were defined as able to grow on FLA but not PYR. PYR degraders grew on both PAHs. All PYR degraders were found to be Gram-positive and all FLA degraders were Gram-negative. GC-FAME analysis showed that many of the PYR degraders were Mycobacterium spp and many of the FLA degraders were Sphingomonas spp. Comparison of the metabolic characteristics of the strains using the spray plate technique and direct growth studies revealed that more than half of the FLA degraders (59%) were able to cometabolize PYR (ie, they produced clearing zones or colored metabolites on spray plates but did not grow on the PAH) and the ability of many of these strains to cometabolize fluorene, anthracene, benzo[b]fluorene, benzo[a]anthracene and benzo[a]pyrene was significantly affected by pre-exposure to phenanthrene. Studies on the metabolic products produced from PYR cometabolism by strain EPA 505 suggested the possibility of attack at two different sites on the PYR molecule. However, the inability to derive degradable carbon from initial opening of one of the PYR rings probably accounted for the lack of growth on this PAH by the FLA-degrading strains. The PYR degraders on the other hand, were less able to cometabolize HMW PAHs, even following pre-exposure to PHE. Characterization of the FLA degradation pathway for several of the Sphingomonas isolates indicated oxidation and ring opening through to acenaphthenone as the principle metabolite. Strain CO6, however, also oxidized FLA through fluorenone, suggesting a dual attack on the FLA molecule, similar to that observed by others in Mycobacterium spp. Journal of Industrial Microbiology & Biotechnology (2000) 24, 100–112. Received 01 May 1999/ Accepted in revised form 01 November 1999     

Cadmium exposure in tobacco workers: possible renal effects.

Cadmium is a nephrotoxic metal widely used in industry and the main source of Cd in general population is smoking. Considering that the source of Cd in cigarettes is the tobacco leaf, the exposure to Cd was evaluated in workers employed at a tobacco leaf processing factory. Blood and urinary Cd levels were measured by flameless atomic absorption spectrometry in 87 workers and 35 controls. Urinary enzymes, total protein, albumin and uric acid were also determined to investigate the possible nephrotoxic effects of Cd. Blood Cd levels were significantly higher in workers (1.63 +/- 1.95 microg/L) than in controls (0.91 +/- 1.15 microg/L) (p = 0.044). The increase observed in urinary Cd levels of workers was non significant (0.56 +/- 0.5 microg/g creatinine in workers and 0.46 +/- 0.5 microg/g creatinine in controls). Both in workers and in controls, subjects smoking >10 cigarettes/day showed significantly increased blood Cd levels compared to non-smokers (p = 0.000 and p = 0.011, respectively). In workers, urinary alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), total protein, and uric acid were observed to be significantly increased (p = 0.013, p = 0.000, p = 0.000, p = 0.025, respectively), ALP, GGT and total protein being positively correlated with Cd in urine. In conclusion, the workers in the tobacco leaf processing factory were found to be exposed to Cd compared to the general population. The increase in the urinary enzymes and proteins suggests that an exposure to Cd affects kidney functions even below the toxic limits generally accepted.     

Simultaneous determination of urinary 1- and 2-naphthols, 3- and 9-phenanthrols, and 1-pyrenol in coke oven workers

A method was developed for simultaneous quantification of urinary 1- and 2-naphthols, 3- and 9-phenanthrols and 1-pyrenol using gas chromatography with mass spectrometry (GC-MS). This method was applied to urine samples from coke oven workers (n =28) and controls (n =22) from Northern China. Geometric mean levels of urinary 1-naphthol (58.8 μg l^-1), 2-naphthol (34.1 μg l^-1), 3-phenanthrol (7.35 μg l^-1), 9-phenanthrol (1.28 μg l^-1) and 1-pyrenol (25.4 μg l^-1) were significantly higher among coke oven workers than controls. All the substances tested were highest among top-of-oven workers, who had 15-fold higher 1-naphthol, eight-fold higher 2-naphthol and 20-fold higher 1-pyrenol levels compared with controls. Using multiple linear regression models, 72.5% of the variation in 1- and 2-naphthol and 82.8% of the variation in 1-pyrenol were explained by the concentration of naphthalene or pyrene in the urine, the work category and the smoking intensity. Cigarette consumption significantly contributed to levels of urinary 1-pyrenol and naphthols, particularly 2-naphthol. A negative relationship between work category and the ratio of naphthols/1-pyrenol was observed among smokers. Our results suggest that urinary naphthols and phenanthrols reflect polycyclic aromatic hydrocarbon (PAH) exposure as well as the widely used 1-pyrenol, and that interactions between cigarette smoking and PAH exposure result in different patterns of metabolism for individual PAHs.     

Determination of dihydroxynaphthalenes in human urine by gas chromatography-mass spectrometry

A gas chromatography-mass spectrometry (GC-MS) method was developed for measuring 1,2-dihydroxynaphthalene (1,2-DHN) and 1,4-dihydroxynaphthalene (1,4-DHN) in urine. The method involves enzymatic digestion of urinary conjugates to release the DHNs which were then analyzed as trimethylsilyl derivatives by GC-MS. For 1,2-DHN and 1,4-DHN, respectively, the assay limits of detection were 0.21 and 0.15 microg/l, the assay limits of quantitation were 0.69 and 0.44 microg/l, and the coefficients of variation were 14.7 and 10.9%. This method was successfully applied to determine urinary levels of 1,2-DHN and 1,4-DHN in coke workers (14 top workers and 13 side-bottom workers) and 21 matching control workers from the steel industry of northern China. The geometric mean (GM) levels of 1,2-DHN were approximately 100 and 30 times higher than those of 1,4-DHN in exposed and control subjects, respectively. The GM levels 1,2-DHN and 1,4-DHN were significantly higher for coke workers (1,2-DHN: top workers--552 microg/l, side-bottom workers--260 microg/l; 1,4-DHN: top workers--3.42 microg/l, side-bottom workers--3.56 microg/l) than for controls (1,2-DHN: 38.8 microg/l; 1,4-DHN: 1.21 microg/l) (por=0.623; p<0.0001). Also, levels of 1,2-DHN were significantly correlated with those of serum albumin adducts of l,2-naphthoquinone (rs=0.492, p=0.0004). These results indicate that 1,2- and 1,4-DHN are good biomarkers for assessment of naphthalene exposure in coke workers. Since the DHNs are precursors of the naphthoquinones, which have been implicated as toxic products of naphthalene metabolism, measurements of urinary DHNs may have toxicological significance.     

Biodegrading of pyrene by a newly isolated Pseudomonas putida PL2

Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic compounds derived from natural sources and anthropogenic processes, which have been recommended as priority pollutants. Degradation of PAHs in the environment is becoming more necessary and urgent. In the current study, strain PL2, which is capable of growing aerobically on pyrene (PYR) as the sole carbon source, was isolated from hydrocarbons-contaminated soil and then identified as Pseudomonas putida by morphological and physiological characteristics as well as 16S rDNA sequence. The strain PL2 was able to degrade 50.0% of the pyrene at 28°C within 6 days in the presence of 50 mg/L pyrene, while the strain PL2 degraded 50.0% of the pyrene within 2 days when a solution of 50 mg/L pyrene and 50 mg/L phenanthrene was used. In addition, phenanthrene was shown to increase the biodegradation efficiency of pyrene by the strain PL2. The order of degradation by the strain PL2 was pH 6.0 > pH 7.0 > pH 5.0 > pH 8.0. The degradation rate of PYR in the soil by the strain PL2 reached 70.0% at the 10^th day. The dynamics of PYR degradation in soil by PL2 was fit to the first order model and the strain PL2 was shown to efficiently degrade PYR in soil. The current study showed that P. putida PL2 was a novel bacterium that could degrade pyrene and holds great promise for use in PAHs bioremediation in soil.     

Microbial degradation of street dust polycyclic aromatic hydrocarbons in microcosms simulating diffuse pollution of urban soil

Diffuse pollution with polycyclic aromatic hydrocarbons (PAHs) of topsoil in urban regions has caused increasing concerns in recent years. We simulated diffuse pollution of soil in microcosms by spiking sandy topsoil (A-horizon) and coarse, mineral subsoil (C-horizon) with street dust (PM63) isolated from municipal street sweepings from central Copenhagen. The microbial communities adapted to PAH degradation in microcosms spiked with street dust in both A-horizon and C-horizon soils, in spite of low PAH-concentrations. The increased potential for PAH degradation was demonstrated on several levels: by slowly diminishing PAH-concentrations, increased mineralization of 14C-PAHs, increasing numbers of PAH degraders and increased prevalence of nah and pdo1 PAH degradation genes, i.e. the microbial communities quickly adapted to PAH degradation. Three- and 4-ring PAHs from the street dust were biodegraded to some extent (10-20%), but 5- and 6-ring PAHs were not biodegraded in spite of frequent soil mixing and high PAH degradation potentials. In addition to biodegradation, leaching of 2-, 3- and 4-ring PAHs from the A-horizon to the C-horizon seems to reduce PAH-levels in surface soil. Over time, levels of 2-, 3- and 4-ring PAHs in surface soil may reach equilibrium between input and the combination of biodegradation and leaching. However, levels of the environmentally critical 5- and 6-ring PAHs will probably continue to rise. We presume that sorption to black carbon particles is responsible for the persistence and low bioaccessibility of 5- and 6-ring PAHs in diffusely polluted soil.     

Simultaneous determination of urinary 1- and 2-naphthols, 3- and 9-phenanthrols,and 1-pyrenol in coke oven workers

A method was developed for simultaneous quantification of urinary 1- and 2-naphthols, 3- and 9-phenanthrols and 1-pyrenol using gas chromatography with mass spectrometry (GC-MS). This method was applied to urine samples from coke oven workers (n =28) and controls (n =22) from Northern China. Geometric mean levels of urinary 1-naphthol (58.8 μg l^?1), 2-naphthol (34.1 μg l^?1), 3-phenanthrol (7.35 μg l^?1), 9-phenanthrol (1.28 μg l^?1) and 1-pyrenol (25.4 μg l^?1) were significantly higher among coke oven workers than controls. All the substances tested were highest among top-of-oven workers, who had 15-fold higher 1-naphthol, eight-fold higher 2-naphthol and 20-fold higher 1-pyrenol levels compared with controls. Using multiple linear regression models, 72.5% of the variation in 1- and 2-naphthol and 82.8% of the variation in 1-pyrenol were explained by the concentration of naphthalene or pyrene in the urine, the work category and the smoking intensity. Cigarette consumption significantly contributed to levels of urinary 1-pyrenol and naphthols, particularly 2-naphthol. A negative relationship between work category and the ratio of naphthols/1-pyrenol was observed among smokers. Our results suggest that urinary naphthols and phenanthrols reflect polycyclic aromatic hydrocarbon (PAH) exposure as well as the widely used 1-pyrenol, and that interactions between cigarette smoking and PAH exposure result in different patterns of metabolism for individual PAHs.     

Genome-to-function characterization of novel fungal P450 monooxygenases oxidizing polycyclic aromatic hydrocarbons (PAHs)

Fungi, particularly the white rot basidiomycetes, have an extraordinary capability to degrade and/or mineralize (to CO₂) the recalcitrant fused-ring high molecular weight (?4 aromatic-rings) polycyclic aromatic hydrocarbons (HMW PAHs). Despite over 30 years of research demonstrating involvement of P450 monooxygenation reactions in fungal metabolism of HMW PAHs, specific P450 monooxygenases responsible for oxidation of these compounds are not yet known. Here we report the first comprehensive identification and functional characterization of P450 monooxygenases capable of oxidizing different ring-size PAHs in the model white rot fungus Phanerochaete chrysosporium using a successful genome-to-function strategy. In a genome-wide P450 microarray screen, we identified six PAH-responsive P450 genes (Pc-pah1-Pc-pah6) inducible by PAHs of varying ring size, namely naphthalene, phenanthrene, pyrene, and benzo(a)pyrene (BaP). Using a co-expression strategy, cDNAs of the six Pc-Pah P450s were cloned and expressed in Pichia pastoris in conjunction with the homologous P450 oxidoreductase (Pc-POR). Each of the six recombinant P450 monooxygenases showed PAH-oxidizing activity albeit with varying substrate specificity towards PAHs (3-5 rings). All six P450s oxidized pyrene (4-ring) into two monohydroxylated products. Pc-Pah1 and Pc-Pah3 oxidized BaP (5-ring) to 3-hydroxyBaP whereas Pc-Pah4 and Pc-Pah6 oxidized phenanthrene (3-ring) to 3-, 4-, and 9-phenanthrol. These PAH-oxidizing P450s (493-547 aa) are structurally diverse and novel considering their low overall homology (12-23%) to mammalian counterparts. To our knowledge, this is the first report on specific fungal P450 monooxygenases with catalytic activity toward environmentally persistent and highly toxic HMW PAHs.