Mixtures Of Polycyclic Aromatic Compounds Research Articles

A complex mixture of polycyclic aromatic compounds causes embryotoxic, behavioral, and molecular effects in zebrafish larvae (Danio rerio), and in vitro bioassays

Polycyclic aromatic compounds (PACs) are prevalent in the environment, typically found in complex mixtures and high concentrations. Our understanding of the effects of PACs, excluding the 16 priority polycyclic aromatic hydrocarbons (16 PAHs), remains limited. Zebrafish embryos and in vitro bioassays were utilized to investigate the embryotoxic, behavioral, and molecular effects of a soil sample from a former gasworks site in Sweden. Additionally, targeted chemical analysis was conducted to analyze 87 PACs in the soil, fish, water, and plate material. CALUX® assays were used to assess the activation of aryl hydrocarbon and estrogen receptors, as well as the inhibition of the androgen receptor. Larval behavior was measured by analyzing activity during light and darkness and in response to mechanical stimulation. Furthermore, qPCR analyses were performed on a subset of 36 genes associated with specific adverse outcomes, and the total lipid content in the larvae was measured. Exposure to the sample resulted in embryotoxic effects (LC50 = 0.480 mg dry matter soil/mL water). The mixture also induced hyperactivity in darkness and hypoactivity in light and in response to the mechanical stimulus. qPCR analysis revealed differential regulation of 15 genes, including downregulation of opn1sw1 (eye pigmentation) and upregulation of fpgs (heart failure). The sample caused significant responses in three bioassays (ERα-, DR-, and PAH-CALUX), and the exposed larvae exhibited elevated lipid levels. Chemical analysis identified benzo[a]pyrene as the predominant compound in the soil and approximately half of the total PAC concentration was attributed to the 16 PAHs. This study highlights the value of combining in vitro and in vivo methods with chemical analysis to assess toxic mechanisms at specific targets and to elucidate the possible interactions between various pathways in an organism. It also enhances our understanding of the risks associated with environmental mixtures of PACs and their distribution during toxicity testing.

Handle Matrix Rank Deficiency, Noise, and Interferences in 3D Emission-Excitation Matrices: Effective Truncated Singular-Value Decomposition in Chemometrics Applied to the Analysis of Polycyclic Aromatic Compounds.

The characterization of organic compounds in polluted matrices by eco-friendly three-dimensional (3D) fluorescence spectroscopy coupled with chemometric algorithms constitutes a powerful alternative to the separation techniques conventionally used. However, the systematic presence of Rayleigh and Raman scattering signals in the excitation–emission matrices (EEMs) complicates the spectral decomposition via PARAllel FACtor analysis (PARAFAC) due to the nontrilinear structure of these signals. Likewise, the specific problem of selectivity in spectroscopy for unexpected chemical components in a complex sample may render its chemical interpretation difficult at first glance. The relevant chemical information can then be complicated to extract, especially if the raw data is noisy. There are several strategies to overcome these drawbacks, but weaknesses remain. As a consequence, a new alternative method is proposed to handle these interferences, the noise, and the rank deficiencies in the data and applied for the characterization of polycyclic aromatic compound (PAC) mixtures. It is based on effective truncated singular-value decomposition (MT-SVD) that does not require any prior knowledge of the raw data. The algorithm provides a valuable estimation of the global rank to choose on complex samples where selectivity problems are observed. It is a real alternative compared to other existing methods applied to the fluorescence matrix to filter the signal from noise or light scattering effects. The first exploratory results of the proposed algorithm are promising to handle matrix rank deficiencies as well as the effects of noise and light scattering on complex PAC mixtures.

Low-level embryonic crude oil exposure disrupts ventricular ballooning and subsequent trabeculation in Pacific herring

There is a growing awareness that transient, sublethal embryonic exposure to crude oils cause subtle but important forms of delayed toxicity in fish. While the precise mechanisms for this loss of individual fitness are not well understood, they involve the disruption of early cardiogenesis and a subsequent pathological remodeling of the heart much later in juveniles. This developmental cardiotoxicity is attributable, in turn, to the inhibitory actions of crude oil-derived mixtures of polycyclic aromatic compounds (PACs) on specific ion channels and other proteins that collectively drive the rhythmic contractions of heart muscle cells via excitation-contraction coupling. Here we exposed Pacific herring (Clupea pallasi) embryos to oiled gravel effluent yielding ΣPAC concentrations as low as ~ 1 μg/L (64 ng/g in tissues). Upon hatching in clean seawater, and following the depuration of tissue PACs (as evidenced by basal levels of cyp1a gene expression), the ventricles of larval herring hearts showed a concentration-dependent reduction in posterior growth (ballooning). This was followed weeks later in feeding larvae by abnormal trabeculation, or formation of the finger-like projections of interior spongy myocardium, and months later with hypertrophy (overgrowth) of the spongy myocardium in early juveniles. Given that heart muscle cell differentiation and migration are driven by Ca2+-dependent intracellular signaling, the observed disruption of ventricular morphogenesis was likely a secondary (downstream) consequence of reduced calcium cycling and contractility in embryonic cardiomyocytes. We propose defective trabeculation as a promising phenotypic anchor for novel morphometric indicators of latent cardiac injury in oil-exposed herring, including an abnormal persistence of cardiac jelly in the ventricle wall and cardiomyocyte hyperproliferation. At a corresponding molecular level, quantitative expression assays in the present study also support biomarker roles for genes known to be involved in muscle contractility (atp2a2, myl7, myh7), cardiomyocyte precursor fate (nkx2.5) and ventricular trabeculation (nrg2, and hbegfa). Overall, our findings reinforce both proximal and indirect roles for dysregulated intracellular calcium cycling in the canonical fish early life stage crude oil toxicity syndrome. More work on Ca2+-mediated cellular dynamics and transcription in developing cardiomyocytes is needed. Nevertheless, the highly specific actions of ΣPAC mixtures on the heart at low, parts-per-billion tissue concentrations directly contravene classical assumptions of baseline (i.e., non-specific) crude oil toxicity.

Oral exposure to commercially available coal tar-based pavement sealcoat induces murine genetic damage and mutations.

Coal tar (CT) is a thick black liquid produced as a by‐product of coal carbonization to produce coke or manufactured gas. It is comprised a complex mixture of polycyclic aromatic compounds, including a wide range of polycyclic aromatic hydrocarbons (PAHs), many of which are genotoxic and carcinogenic. CT is used in some pavement sealants (also known as sealcoat), which are applied to pavement in order to seal and beautify the surface. Human exposure is known to occur not only during application, but also as a result of the weathering process, as elevated levels of PAHs have been found in settled house dust in residences adjacent to CT‐sealed surfaces. In this study we examined the genotoxicity of an extract of a commercially available CT‐based sealcoat in the transgenic Muta™Mouse model. Mice were orally exposed to 3 doses of sealcoat extract daily for 28 days. We evaluated genotoxicity by examining: (1) stable DNA adducts and (2) lacZ mutations in bone marrow, liver, lung, small intestine, and glandular stomach, as well as (3) micronucleated red blood cells. Significant increases were seen for each endpoint and in all tissues. The potency of the response differed across tissues, with the highest frequency of adducts occurring in liver and lung, and the highest frequency of mutations occurring in small intestine. The results of this study are the first demonstration of mammalian genotoxicity following exposure to CT‐containing pavement sealcoat. This work provides in vivo evidence to support the contention that there may be adverse health effects in mammals, and potentially in humans, from exposure to coal tar. Environ. Mol. Mutagen. 57:535–545, 2016. © 2016 Her Majesty the Queen in Right of Canada

Deviations from ideal sublimation vapor pressure behavior in mixtures of polycyclic aromatic compounds with interacting heteroatoms

Despite the relatively small atomic fraction of a given heteroatom in a binary mixture of polycyclic aromatic compounds (PAC), the presence of heteroatoms can significantly impact mixture vapor pressure behavior over a wide range of temperatures. The vapor pressures of several binary PAC mixtures containing various heteroatoms show a range of behavior, from practically ideal behavior following Raoult’s law to significant deviations from ideality depending on the heteroatoms present. Mixtures were synthesized using the quench-cool technique with equimolar amounts of two PAC, both containing heteroatoms such as aldehyde, carboxyl, nitrogen, and sulfur substituent groups. For some mixtures, deviation from ideality is inversely related to temperature, though in other cases we see deviation from ideality increasing with temperature, whereas some appear independent of temperature. Most commonly we see lower vapor pressures than predicted by Raoult’s Law, which indicates that the interacting heteroatoms prefer the solid mixture phase as opposed to the vapor phase. Although negative deviations predominate from Raoult’s Law, the varying mixtures investigated show both higher and lower enthalpies and entropies of sublimation than predicted. In each mixture, a higher enthalpy of sublimation leads to higher entropy of sublimation than predicted, and vice versa.

Characterization and Matching of Oil Samples Using Fluorescence Spectroscopy and Parallel Factor Analysis

A novel approach for matching oil samples by fluorescence spectroscopy combined with three-way decomposition of spectra is presented. It offers an objective fingerprinting based on the relative composition of polycyclic aromatic compounds (PACs) in oils. The method is complementary to GC-FID for initial screening of oil samples but can also be used for prescreening in the field, onboard ships, using a portable fluorescence spectrometer. Parallel factor analysis (PARAFAC) was applied to fluorescence excitation-emission matrixes (EEMs) of heavy fuel oils (HFOs), light fuel oils, lubricating oils, crude oils, unknown oils, and a sample collected in the spill area two weeks after the Baltic Carrier oil spill (Denmark, 2001). A total of 112 EEMs were decomposed into a five-factor PARAFAC model using excitation wavelengths from 245 to 400 nm and emission wavelengths from 280 to 550 nm. The PARAFAC factors were compared to EEMs of PAC standards with two to five rings, and the comparisons indicate that each of the factors can be related to a mixture of PACs with similar fluorescence characteristics: a mixture of naphthalenes and dibenzothiophenes, fluorenes, phenanthrenes, chrysenes, and five-ring PACs, respectively. Oils were grouped in score plots according to oil type. Except for HFOs and crude oils, the method easily discriminated between the four oil types. Minor overlaps of HFOs and crude oils were observed along all five PARAFAC factors, and the variability of crude oils was large along factor 2 due to a varying content of five-ring PACs. The spill sample was correctly assigned as a HFO with similar PAC pattern as oil from the cargo tank of the Baltic Carrier by comparing the correlation coefficient of scores for the oil spill sample and possible source oils (i.e., oils in the database).

An antibiotic with activity against gram-positive bacteria from the gentamicin-producing strain of Micromonospora purpurea.

A mixture of polycyclic aromatic compounds with activity against gram-positive bacteria was isolated from a gentamicin-producing species of Micromonospora.

Resolution of GC-MS Data of Complex PAC Mixtures and Regression Modeling of Mutagenicity by PLS

The present work describes a strategy to predict the mutagenicity of very complex mixtures of polycyclic aromatic compounds (PAC) from gas chromatography-mass spectrometry (GC-MS) patterns of the mixtures, each containing 260 compounds on average. The mixtures, 13 organic extracts of exhaust particles, were characterized by full scan GC-MS. The data were resolved into peaks and spectra for individual compounds by an automated curve resolution procedure. Similarity between spectra was evaluated for peaks that appeared within a time interval of 4 min, using a similarity index of 0.8 to ascertain that the same compound was represented by the same variable name (retention time) in all samples. The resolved chromatograms were integrated, resulting in a predictor matrix of size 13 x 721, which was used as input to a multivariate regression model. Partial least-squares projections to latent structures (PLS) were used to correlate the GC-MS chromatograms to mutagenicity as measured in the Ames Salmonella assay. The best model (high r2 and Q2) was obtained with 52 variables. These variables covary with the observed mutagenicity, and may subsequently be identified chemically. Furthermore, the regression model can be used to predict mutagenicity from GC-MS chromatograms of other organic extracts.

Evaluation of total luminescence data with chemometrical methods:: a tool for environmental monitoring

The detection of polycyclic aromatic compound (PAC) containing petroleum products in the environment is often accompanied by the problem of multicomponent mixture analysis. If stationary fluorescence techniques are used, total luminescence (TL) spectroscopy in combination with rank annihilation factor analysis (RAFA) is the most powerful analytical tool. TL–RAFA has been successfully applied in homogeneous solutions, but no application to environmental analysis has been presented. Therefore, in this paper the analysis of a simulated 15-component PAC mixture will be presented to demonstrate the potential of TL spectroscopy in combination with RAFA in general. Rank analysis turned out to be the most difficult step in RAFA, and will be discussed in more detail. The analysis of 5 six-component PAC mixtures in toluene will be shown to demonstrate the effects of different experimental conditions. From the simulated and the real experiments it can be concluded that TL–RAFA is capable of analyzing multicomponent PAC mixtures in solution. However, if the analysis is carried out under environmental conditions, e.g., in natural waters or on soils, two major interferences arise: the ubiquitous humic substances disturb the TL spectra and the amount of scattered light is highly increased. Therefore, the influence of the humic substance absorption and emission properties and of a strong scattered light band was investigated in solutions and is discussed in detail.

Supercritical Fluid Chromatography of Polycyclic Aromatic Compounds: A Review

An overview of supercritical fluid chromatography as a method of analysing complex mixtures of polycyclic aromatic compounds is given.

Evaporative Light-Scattering Detection in the Quantitative Analysis of Semivolatile Polycyclic Aromatic Compounds by High-Performance Liquid Chromatography

Polycyclic aromatic compounds (PACs) with molecular weights lower than 300 were not considered in the past to be analyzable using an evaporative light-scattering detector (ELSD) due exclusively to their supposed volatility. We demonstrate that a variety of small, low-boiling, 3-6-ringed PACs (which include condensed PAHs, heteronuclear polycyclic compounds, and hydroxy-PAHs) can be analyzed using ELSD under mild working conditions without significant volatilization. Although area counts versus sample load can be linearized with adequate regression coefficients in the mass range studied, results obtained from the injection of pure and binary mixtures of PACs indicate that ELSD behaves nonlinearly at low sample loads, regardless of the volatility or involatility of each compound. Quite uniform response factors are obtained on the linear zone for the studied PACs, and analyses of mixtures are carried out with errors generally lower than 6%. Experimental data are better fitted to logarithmic regressions, which provide similar slopes for the studied PACs, which in turn suggest that ELSD operates in similar scattering domains for these compounds under our working conditions. The use of a mathematical simulation of particle size distributions after nebulization and evaporation confirms this and explains why response factors of PACs are similar.

The Use of Copper Immobilized on Silica for Compound Class Fractionation of Polycyclic Aromatic Compounds in Environmental Samples

Abstract Copper immobilized on silica has been investigated as an adsorbent for the fractionation of polycyclic aromatic compounds (PACs) into different compound classes. an open column chromatographic packing based on copper (II) bound to a silica substrate was used as a means of subdividing the PACs into less complex groups of similar compound types. the process has been applied to the fractionation of PACs extracted from a marine harbour sediment, for which the complex mixture of PACs could be separated into three subgroups, the contents of which were: (1) polycyclic aromatic hydrocarbons (PAHs), alkylated PAHs, and thiophenes; (2) compounds containing pyrrole-type (sp 3-hybridized) nitrogen (carbazoles), and also quinones and other oxygen containing species; and (3) compounds containing pyridine-type(sp 2-hybridized) nitrogen (acridine and its homologues). the fractions thus obtained have been characterized by gas chromatography-mass spectrometry (GC-MS). Extracted mass spectra, obtained from the crests of GC-MS peaks observed in reconstructed single ion chromatograms of selected m/z values, enabled unambiguous identification of many of the minor components of the mixture.

Comparison of liquid chromatography/mass spectrometry interfaces for the analysis of polycyclic aromatic compounds

Three liquid chromatography/mass spectrometry interfaces were evaluated for their suitability for the analysis of complex mixtures of polycyclic aromatic compounds (PACs). Preliminary qualitative experiments, which used a carbon black extract as test material, confirmed that the moving belt interface is mechanically awkward, is limited with respect to the mobile phase composition which it can tolerate, and is not efficient in detecting the more volatile compounds. For these reasons it was not examined further, although it performed well for larger PACs and provided electron ionization (EI) mass spectra. The particle beam (PB) interface also provides EI spectra, but detection limits are poor (low nanogram range) and calibration curves are nonlinear. Only seven of the 16 PACs targeted for quantification in a complex coal tar reference material could be detected because of the difficulty the PB interface has with the analysis of compounds with very high or very low volatility. The heated pneumatic nebulizer (HPN) interface, which uses atmospheric pressure chemical ionization, produces both molecular ions (M .+ ) and protonated molecules (MH + ) of PACs. Detection limits were in the low picogram range, and calibration curves were linear. Using the HPN interface, 17 target PACs in the coal tar reference material could be detected and quantified within satisfactory agreement with certified values when perdeuterated internal standards were employed.

Use of immobilized copper ion chromatography and on-line mass spectrometry with atmospheric pressure chemical ionization for the profiling of complex mixtures of polycyclic aromatic compounds

Immobilized copper ion chromatography was combined with either diode array detection or atmospheric pressure chemical ionization mass spectrometry for the profiling of complex mixtures of polycyclic aromatic compounds (PACs). A heated pneumatic nebulizer interface was used to convey the eluent from the column into the ionization region of the mass spectrometer. Several modifications were made to the heated pneumatic nebulizer interface originally designed for liquid chromatography-mass spectrometry so that the mass spectrometer could be safely operated with large quantities of organic solvent passing into the ionization region of the instrument. A copper-coated Zorbax 300 SCX column was used in conjunction with a hexane, chloroform and acetonitrile gradient to accomplish fractionation of three extracts containing PACs. The column was able to separate portions of the extracts into a number of fractions, each of which contained homologous series of different PAC classes. Using this technique, it is possible to gain considerable insight into the types and relative amounts of minor components present in the PAC mixtures.

Capillary column supercritical fluid chromatography/mass spectrometry of polycyclic aromatic compounds using atmospheric pressure chemical ionization

AbstractCapillary column supercritical fluid chromatography (SCF) was combined with atmospheric pressure chemical ionization (APCI) mass spectrometry through a heated pneumatic‐nebulizer interface, originally developed for liquid chromatography/mass spectrometry (LC/MS), and subsequently modified for use with supercritical fluid chromatography/mass spectrometry (SFC/MS). A high pressure syringe pump was used to pass SFC‐grade carbon dioxide, used as the mobile phase, through a capillary SFC column which was contained in an oven, maintained at 100°C. The eluent passed through the heated pneumatic‐nebulizer interface into the ionization region of the mass spectrometer with the aid of the flow of nebulizing gas. The system was optimized using benz[a]anthracene, and then applied to analyze a standard mixture of polycyclic aromatic compounds (PACs) as well as complex mixtures of PACs obtained by the fractionation of a pond sediment contaminated by coke‐oven residues. A detection limit of 40 pg was established for chrysene.

Analysis of polycyclic aromatic compounds by supercritical fluid charomatography/mass spectrometry using atmospheric‐pressure chemical ionization

AbstractThe development of microbore packet‐column supercritical fluid chromatography/atmospheric pressure chemical ionization mass spectrometry (SFC/APCI‐MS) for the analysis of polycyclic aromatic compounds (PAC) is described. The SFC system was coupled to the mass spectrometer using an improved interface designed to accommodate commercially available fused silica restrictors. The atmospheric pressure chemical ionization characteristics of PAC are discussed and the use of chemical ionization reagents to modify the type of spectra obtained is described. With selected‐ion monitoring techniques the interface provided detection limits in the low picogram range for individual PAC standards. Using a photoionization detector for the off‐line optimization of chromatographic conditions, SFC/APCI‐MS was applied to the analysis of complex mixtures of PAC in coal tar and tar sand oil extracts. Tandem mass spectrometry was used to provide structural information.

On-line multidimensional open tubular column supercritical fluid chromatography using a valve-switching interface

An on-line two-dimensional open tubular column supercritical fluid chromatograph was constructed and evaluated. A rotary valve interface allowed independent flow control of two 50-μm i.d. open tubular columns, providing maximum versatility for heartcutting. A solvent-venting injection technique was incorporated in the system that enabled single or multiple 2.0-μL volumes to be injected into an uncoated, yet deactivated, length of capillary precolumn without flooding of the analytical column. A cold trap was employed to refocussolutes from single or multiple fractional cuts after being transferred to the second dimension. The performance of the system was demonstrated with the analysis of mixtures of polycyclic aromatic compounds and steroids. Efficiencies of 4,500 plates m−1 were preserved in the second column after heartcutting.

Determination of sulphur heterocycles in selected synfuels

A fractionation method for isolating the polycyclic aromatic sulphur heterocycles from complex mixtures of polycyclic aromatic compounds was evaluated with respect to recovery for selected standard compounds. It was found that recovery varied from 0 to 70% depending on the structure of the compound. Although quantitative recovery for all components was not obtained, isolation of the sulphur heterocycle fractions has allowed the identification of many new sulphur compounds previously not identified in coal liquids and shale oils. Capillary column gas chromatography with flame photometric detection and mass spectrometry were used for the identification of individual sulphur heterocycles.

Mixtures of polycyclic aromatic compounds inhibit mutagenesis in the Salmonella/microsome assay by inhibition of metabolic activation

We observed that complex mixtures of aromatic compounds isolated from a coal-derived oil suppressed the mutagenic activity of the indirect mutagens benzo[ a]pyrene, 7,12-dimethylbenz[ a]anthracene, 2-aminofluorene, and 2-acetylaminofluorene as measured in the Salmonella/microsome mutagenicity assay, using strain TA98 and metabolic activation with Aroclor-induced rat-liver S9 or microsomes. The mixture also inhibited S9-dependent benzo[ a]pyrene metabolism and covalent binding to DNA in a cell-free system. The mixture did not suppress the activity of either the direct acting mutagens 2-nitrofluorene and benzo[a]pyrene diol-epoxide, or of the indirect mutagen N-hydroxy-2-acetylaminofluorene which requires a microsomal deacetylase for metabolic activation. Spectrophotometric measurements showed that components of the mixture bound to microsomal cytochrome P-450. The mixture did not inhibit microsomal NADPH-cytochrome c (P-450) reductase. These observations show that the mixtures inhibited metabolic activation by the microsomal monooxygenase system, probably by binding of unidentified components to cytochrome P-450. The resulting inhibition of mutagenesis may have implications for risk estimates for the mixtures we examined as well as for other types of complex mixtures for which similar inhibitory effects have been observed.

Linear retention index system for polycyclic aromatic compounds : Critical evaluation and additional indices

A linear retention index system for temperature-programmed capillary gas chromatography of polycyclic aromatic compounds using naphthalene, phenanthrene, chrysene, and picene as internal standards has been used in this laboratory for routine analysis since 1979. Conclusions from a data set of 29 routine capillary gas chromatographic analyses of a complex mixture of polycyclic aromatic compounds are used to evaluate, describe, and justify the polycyclic aromatic compound retention index system. The number, width, and linearity of the interpolation intervals are described. The effects on the retention indices of variations in programming rate, column internal diameter, and initial temperature are discussed. The polycyclic aromatic compound retention index system is shown to be a consistent, useful tool for preliminary identification of polycyclic aromatic compounds in complex mixtures. Tables listing standard retention indices for polycyclic aromatic hydrocarbons, polycyclic aromatic sulfur heterocycles, and nitrogen-containing polycyclic aromatic compounds are included.