Validation of a HILIC/ESI-MS/MS method for the wood burning marker levoglucosan and its isomers in airborne particulate matter.

In the present study, a methodology involving hydrophilic interaction liquid chromatography (HILIC) and electrospray (ESI) tandem mass spectrometry (MS/MS) was developed for measurement of anhydrous monosaccharides as markers for wood burning in atmospheric aerosols, PM10. No extensive sample preparation, other than ultrasound-assisted solvent extraction and evaporation, was applied. A pentahydroxysilica column enabled separation of levoglucosan from mannosan and galactosan within 5 min and the quantitative performance was validated using the standard reference materials (SRM) 1649a and 1649b. The experimentally obtained results for SRMs were in agreement with values previously reported in other studies. Achieved instrumental limits of detection (LODs) were below 10 pg injected on column, corresponding to LODs in air lower than 0.10 ng/m3 for all measured isomers for 2-3 day sampling with 1.0 m-3 h-1 sampling rate. The validated method was used for the determination of levoglucosan and its isomers in atmospheric aerosols collected in three different Swedish urban areas during the winter and summer time in 2017. The total measured concentrations for levoglucosan and galactosan + mannosan were determined to be between 78 and 167 ng/m3 in January 2017, which is approximately 10-times higher compared to the levels detected in July, reflecting the higher frequency of wood burning for heating during the cold season. Calculated concentration ratios between levoglucosan and its isomers in the urban area samples indicated mostly mixed softwood/hardwood combustion in winter time; on the other hand, softwood burning was observed as the major emission in summer time.

Nanomolar levels of PAHs in extracts from urban air induce MAPK signaling in HepG2 cells.

Polycyclic aromatic hydrocarbons (PAHs) are common environmental pollutants that occur naturally in complex mixtures. Many of the adverse health effects of PAHs including cancer are linked to the activation of intracellular stress response signaling. This study has investigated intracellular MAPK signaling in response to PAHs in extracts from urban air collected in Stockholm, Sweden and Limeira, Brazil, in comparison to BP in HepG2 cells. Nanomolar concentrations of PAHs in the extracts induced activation of MEK4 signaling with down-stream increased gene expression of several important stress response mediators. Involvement of the MEK4/JNK pathway was confirmed using siRNA and an inhibitor of JNK signaling resulting in significantly reduced MAPK signaling transactivated by the AP-1 transcription factors ATF2 and c-Jun. ATF2 was also identified as a sensitive stress responsive protein with activation observed at extract concentrations equivalent to 0.1 nM BP. We show that exposure to low levels of environmental PAH mixtures more strongly activates these signaling pathways compared to BP alone suggesting effects due to interactions. Taken together, this is the first study showing the involvement of MEK4/JNK/AP-1 pathway in regulating the intracellular stress response after exposure to nanomolar levels of PAHs in environmental mixtures.

Particulate PAH emissions from residential biomass combustion: time-resolved analysis with aerosol mass spectrometry.

Time-resolved emissions of particulate polycyclic aromatic hydrocarbons (PAHs) and total organic particulate matter (OA) from a wood log stove and an adjusted pellet stove were investigated with high-resolution time-of-flight aerosol mass spectrometry (AMS). The highest OA emissions were found during the addition of log wood on glowing embers, that is, slow burning pyrolysis conditions. These emissions contained about 1% PAHs (of OA). The highest PAH emissions were found during fast burning under hot air starved combustion conditions, in both stoves. In the latter case, PAHs contributed up to 40% of OA, likely due to thermal degradation of other condensable species. The distribution of PAHs was also shifted toward larger molecules in these emissions. AMS signals attributed to PAHs were found at molecular weights up to 600 Da. The vacuum aerodynamic size distribution was found to be bimodal with a smaller mode (Dva ∼ 200 nm) dominating under hot air starved combustion and a larger sized mode dominating under slow burning pyrolysis (Dva ∼ 600 nm). Simultaneous reduction of PAHs, OA and total particulate matter from residential biomass combustion may prove to be a challenge for environmental legislation efforts as these classes of emissions are elevated at different combustion conditions.

Measurement of regulated and unregulated exhaust emissions from a lawn mower with and without an oxidizing catalyst: a comparison of two different fuels.

Relatively few emission characterization studies have been made on small engines used in garden equipment. The present investigation focuses on exhaust characterization from a lawn mower engine fueled with two different fuels in combination with and without an oxidizing catalyst. The compounds measured in the exhaust are carbon monoxide, hydrocarbons, nitrogen oxides, particulates, polycyclic aromatic hydrocarbons, methane, ethane, ethene, ethanol, and nitrous oxide. A significant reduction can be achieved by the use of a catalyst. By selection of the fuel, a significant reduction of certain carcinogenic compounds ("probably carcinogenic to humans" according to the IARC; benzo[a]pyrene and benzo[a]anthracene) may be achieved. The highest reduction improvement is achieved through the combination of an environmentally improved fuel, i.e., alkylate fuel, and a catalyst system. The data presented show that emissions from lawn mower engines are still relatively large although there is the potential for further improvements.

Comparison of exhaust emissions from Swedish environmental classified diesel fuel (MK1) and European Program on Emissions, Fuels and Engine Technologies (EPEFE) reference fuel: a chemical and biological characterization, with viewpoints on cancer risk.

Diesel fuels, classified as environmentally friendly, have been available on the Swedish market since 1991. The Swedish diesel fuel classification is based upon the specification of selected fuel composition and physical properties to reduce potential environmental and health effects from direct human exposure to exhaust. The objective of the present investigation was to compare the most stringent, environmentally classified Swedish diesel fuel (MK1) to the reference diesel fuel used in the "European Program on Emissions, Fuels and Engine Technologies" (EPEFE) program. The study compares measurements of regulated emissions, unregulated emissions, and biological tests from a Volvo truck using these fuels. The regulated emissions from these two fuels (MK1 vs EPEFE) were CO (-2.2%), HC (12%), NOx (-11%), and particulates (-11%). The emissions of aldehydes, alkenes, and carbon dioxide were basically equivalent. The emissions of particle-associated polycyclic aromatic hydrocarbons (PAHs) and 1-nitropyrene were 88% and 98% lower than those of the EPEFE fuel, respectively. The emissions of semi-volatile PAHs and 1-nitropyrene were 77% and 80% lower than those from the EPEFE fuel, respectively. The reduction in mutagenicity of the particle extract varied from -75 to -90%, depending on the tester strain. The reduction of mutagenicity of the semi-volatile extract varied between -40 and -60%. Furthermore, the dioxin receptor binding activity was a factor of 8 times lower in the particle extracts and a factor of 4 times lower in the semi-volatile extract than that of the EPEFE fuel. In conclusion, the MK1 fuel was found to be more environmentally friendly than the EPEFE fuel.

Short-term exposure of rodents to diesel exhausts: usefulness for studies of genotoxic and immunotoxic effects.

An exposure facility was tested with regard to the information obtainable from short-term animal experiments for the assessment of health hazards from automotive engine exhausts. Indicators of immunotoxicity and genotoxicity were studied in guinea pigs and mice, respectively, exposed for 2 weeks, 8 h/day, to ten times diluted exhausts from a one-cylinder research diesel engine running at constant load. Regulated and non-regulated pollutants were determined. Besides increased number of lavageable cells in the airways, exposed guinea pigs exhibited, after immunization and challenge to ovalbumin, reduced leukotrienes B4 and C4 in lavage fluid and reduced anti-ovalbumin IgG in serum. Absence of increased CYP1A activity indicated that the exposure was below the threshold for induction of these enzymes. Instead a certain reduction of this activity indicated interaction with active enzyme sites. In vivo doses of some reactive metabolites of low molecular mass were measured by adducts to hemoglobin. Doses from aliphatic epoxides were low, in accordance with low hydrocarbon levels in the exhaust. The levels of hemoglobin adducts from aldehydes showed no clearcut influences of exposure. Genetic effects determined by DNA fingerprint analysis were indicated. It is concluded that repeated dose inhalation exposure of small numbers of animals is a useful mode of exposure for studying parameters that may elucidate toxic effects of air pollutants emitted from automotive engines, with a possibility to evaluate engine and fuel with regard to health hazards.

Neurotoxic effects of fractionated diesel exhausts following microinjections in rat hippocampus and striatum.

Exhaust emissions from a heavy-duty diesel vehicle, separated into particulate and semivolatile phases and thereafter fractionated according to polarity, were studied in the adult rat brain after intracranial microinjections using cresyl violet staining and immunohistochemistry. Intrastriatal as well as intrahippocampal injections of particulate fractions III [containing mononitro-polycyclic aromatic hydrocarbon (PAH)], IV (dinitro-PAH and quinones) and V (polar material) and of semivolatile fractions IV and V, in amounts corresponding to a driven length of 19.5 m, caused major lesions with tissue loss and disappearance of immunoreactivity for glial fibrillary acidic protein, tyrosine hydroxylase, and acetylcholine esterase. Particulate fractions I ("light" aliphatic hydrocarbons) and II ("heavy" aliphatic hydrocarbons and PAH) and semivolatile fraction III produced smaller lesions; semivolatile fractions I and II led to lesions equivalent to those of the vehicle dimethyl sulfoxide alone. Microinjected doses of particulate fractions III or IV corresponding to driven lengths of 2.0 and 9.8 m produced a variable lesion. Thus, fractions containing nitro-derivatives of PAH, quinones, and polar material caused the greatest damage after intracranial injections. It is concluded that intracerebral microinjections of fractionated motor vehicle exhausts provide a method for systematic testing of direct neurotoxicity.

Influence of physical and chemical characteristics of diesel fuels and exhaust emissions on biological effects of particle extracts: a multivariate statistical analysis of ten diesel fuels.

The emission of diesel exhaust particulates is associated with potentially severe biological effects, e.g., cancer. The aim of the present study was to apply multivariate statistical methods to identify factors that affect the biological potency of these exhausts. Ten diesel fuels were analyzed regarding physical and chemical characteristics. Particulate exhaust emissions were sampled after combustion of these fuels on two makes of heavy duty diesel engines. Particle extracts were chemically analyzed and tested for mutagenicity in the Ames test. Also, the potency of the extracts to competitively inhibit the binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to the Ah receptor was assessed. Relationships between fuel characteristics and biological effects of the extracts were studied, using partial least squares regression (PLS). The most influential chemical fuel parameters included the contents of sulfur, certain polycyclic aromatic compounds (PAC), and naphthenes. Density and flash point were positively correlated with genotoxic potency. Cetane number and upper distillation curve points were negatively correlated with both mutagenicity and Ah receptor affinity. Between 61% and 70% of the biological response data could be explained by the measured chemical and physical factors of the fuels. By PLS modeling of extract data versus the biological response data, 66% of the genotoxicity could be explained, by 41% of the chemical variation. The most important variables, associated with both mutagenicity and Ah receptor affinity, included 1-nitropyrene, particle bound nitrate, indeno[1,2,3-cd]pyrene, and emitted mass of particles. S9-requiring mutagenicity was highly correlated with certain PAC, whereas S9-independent mutagenicity was better correlated with nitrates and 1-nitropyrene. The emission of sulfates also showed a correlation both with the emission of particles and with the biological effects. The results indicate that fuels with biologically less hazardous potentials should have high cetane number and contain less PAC and sulfur. The results also indicate that engine factors affect the formation and emission of nitrated PAC.

Structure elucidation of two tryptophan-derived, high affinity Ah receptor ligands.

BACKGROUND: Environmental contaminants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other structurally related 'environmental hormones', exert their harmful biological effects through the Ah receptor signaling pathway. Several naturally occurring substances also bind to this receptor, but its natural role is still obscure. Tryptophan derivatives of the indolo[3,2-b]carbazole type, earlier suggested by us to be endogenous ligands for the receptor, should be a powerful tool in understanding receptor function. We therefore set out to determine their identity. RESULTS: The two tryptophan-derived Ah receptor ligands have been chemically analyzed and characterized by means of mass spectrometry, 1H NMR and 13C NMR. UV, infra-red and fluorescence spectra were also recorded. All data are in accordance with the two compounds being closely related indolo[3,2-b]carbazole derivatives. Evidence is presented that compound A (MW = 312) is the symmetrical 6,12-diformylindolo[3,2-b]carbazole, and compound B (MW = 284) is the monosubstituted 6-formylindolo[3,2-b]carbazole. CONCLUSIONS: The elucidation of the structures of the two high affinity Ah receptor ligands 6,12-diformylindolo[3,2-b]carbazole and 6-formylindolo[3,2-b]carbazole provides the necessary basis for further mechanistic studies of this important group of compounds, and will help in determining the natural role of the Ah receptor.

Human exposure to urban air pollution.

This study deals with some methods of making human exposure estimates, aimed at describing the human exposure for selected air pollutants in Sweden that are suspected carcinogens. Nitrogen oxides (NOx) have been chosen as an indicator substance for estimating the concentration of the urban plume. Earlier investigations have shown that the traffic in Swedish cities contributes around 85% to the measured NOx concentrations, and that most of the mutagenicity in urban air originates from traffic. The first section of this paper describes measurements in Stockholm of some unregulated light hydrocarbons, such as ethene, ethyne, propane, propene, butane, and isobutane. In addition, measurements of some volatile aromatic hydrocarbons are presented. Simultaneous measurements of carbon monoxide (CO) were made. The ratios between CO and the individual specific compounds were determined by linear regression analysis. By analysis of relationships between CO and NOx, NOx concentrations can be used as a tracer to describe the exposure for these specific compounds. NOx are considered to be a better tracer than CO, because NOx or NO2 values exist for many places over a long time, while CO is measured mostly in streets with high concentrations. At low concentrations, instruments that measure normal CO levels give no detectable signals. Through use of atmospheric dispersion models and models that describe how people live and work in urban areas it has been possible to describe the average exposure to NOx in cities of different sizes. The exposure to NOx for people living in the countryside has also been estimated. In this way, it has been possible to calculate the average exposure dose for NOx for the Swedish population. This figure is 23 micrograms/m3. By use of the relationships between NOx and specific compounds the average dose has been calculated for the following compounds: polyaromatic compounds (PAH); ethene, propene, and butadiene; benzene, toluene, and xylene; formaldehyde and actaldehyde; nickel, chromium (VI), arsenic, and cadmium; asbestos; and silicon.

Exhaust emissions from light- and heavy-duty vehicles: chemical composition, impact of exhaust after treatment, and fuel parameters.

This paper presents results from the characterization of vehicle exhaust that were obtained primarily within the Swedish Urban Air Project, "Tätortsprojektet." Exhaust emissions from both gasoline- and diesel-fueled vehicles have been investigated with respect to regulated pollutants (carbon monoxide [CO], hydrocarbon [HC], nitrogen oxides [NOx], and particulate), unregulated pollutants, and in bioassay tests (Ames test, TCDD receptor affinity tests). Unregulated pollutants present in both the particle- and the semi-volatile phases were characterized. Special interest was focused on the impact of fuel composition on heavy-duty diesel vehicle emissions. It was confirmed that there exists a quantifiable relationship between diesel-fuel variables of the fuel blends, the chemical composition of the emissions, and their biological effects. According to the results from the multivariate analysis, the most important fuel parameters are: polycyclic aromatic hydrocarbons (PAH) content, 90% distillation point, final boiling point, specific heat, aromatic content, density, and sulfur content.

Chemical analysis and biological testing of a polar fraction of ambient air, diesel engine, and gasoline engine particulate extracts.

Extracts of gasoline and diesel vehicle exhaust and ambient air particles were fractionated into five fractions according to polarity on a silica gel column. Two medium polar fractions showing high genotoxic activity in the Ames test were further subfractionated, using normal-phase high-performance liquid chromatography. Chemical analyses were performed by means of gas chromatography combined with mass spectrometry and flame ionization and detection. The crude extracts, fractions, and subfractions were assayed with the Ames test, with and without S9, and the most abundant compounds in the subfractions are reported.

Uptake and metabolism of ethene studied in a smoke-stop experiment.

Knowledge of the relationships between exposure levels and levels of hemoglobin adducts are essential when the latter are to be used for exposure monitoring or risk estimation, the hygienic control being based on measurements of exposure. These ratios are mostly very uncertain, mainly due to difficulties of determining the time-weighted average exposure concentration. A solution to this problem has been suggested involving adduct measurement before and after two consecutive periods of about 1 week, the first with absence from exposure, the second with careful measurement of exposure. This model was tested in two smokers who abstained from smoking for one week. Analysis of inhaled ethene and of adducts from ethylene oxide (EO) to N-terminal valine of hemoglobin are compatible with metabolism of 2% of inhaled ethene to EO and a detoxification rate of 1 h-1 of EO.

Human sperm motility is affected by plasticizers and diesel particle extracts.

In order to test various drugs and possibly hazardous compounds on living cells in vitro a system with human spermatozoa was employed. A population of human spermatozoa was transferred into a defined medium by a swim-up procedure or by separation on a Percoll gradient. Such a population is rather homogenous with respect to motility characteristics and was found to be useful for this purpose. Different modes of response were recorded, indicating various effect mechanisms. Effects of various phthalates used as plastic softeners in the production of medical equipment, and extracts from diesel particulate material were recorded. All these compounds interfered with sperm motility in a dose-response fashion. Immediate effects of phthalates were modest, but upon prolonged exposure effects became more evident. Sperm motility was more affected by diethyl-hexyl and dibutyl phthalates. Significant effects were noted for the different phthalates with regard both to percent motility and to some of the various qualities of motility, such as velocity, linearity and amplitude of the track. Thus, the pattern of response considering the motion variables was not the same with the different phthalates. With regard to the effects on sperm motion di-n-octyl phthalate seemed to be the least toxic, followed by dibutyl phthalate. The initial effects of diesel particulate extracts were moderate and mainly restricted to percent motile sperm but upon exposure for 18 hr the effects became more pronounced for all the movement variables.

Certain photooxidized derivatives of tryptophan bind with very high affinity to the Ah receptor and are likely to be endogenous signal substances.

The purpose of the present study was to determine whether ultraviolet light (UV) irradiation of amino acids produces compounds with affinity for the Ah receptor. Aqueous solutions of L-tryptophan were exposed to radiation from an unfiltered high-pressure mercury lamp. The photoproducts formed were solvent-extracted or concentrated on Sep-Pak C18 cartridges. The concentrated extracts or eluants were treated for their ability to compete with 3H-labeled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Binding was assayed in liver cytosolic preparations from Sprague-Dawley rats using a technique based on hydroxylapatite separation. Photoproducts with receptor affinity were formed in a time-dependent manner. Histidine and tryptamine also gave products upon UV irradiation that competed with TCDD. Commercial tryptophan, at least aged, contained trace amounts of impurities with receptor affinity. Analysis by TLC and high-pressure liquid chromatography of the photo-products of tryptophan showed a minimum of three different binding compounds. Two of the products were studied in greater detail. One of them, showing UV absorbance and yellow fluorescence, gave a molecular ion (M+) of 284 and the other gave M+ 312 but showed little UV absorption and fluorescence. The concentration, based on mass spectrometry quantifications, of the two compounds that displaced more than 50% of TCDD was found to be extremely low, giving Kd values of 0.44 nM (M+ 312) and 0.07 nM (M+ 284). The existence of high affinity receptors for oxidized amino acids is postulated and their possible role in the proliferative cellular responses to TCDD and tryptophan is discussed briefly.

Applicability of a cryogradient technique for the enrichment of PAH from automobile exhausts: demonstration of methodology and evaluation experiments.

A cryogradient system for the enrichment of polycyclic aromatic hydrocarbons from gasoline and diesel powered vehicles is described. The sampling involves particle trapping on a filter followed by gas phase enrichment in three separate condensers. The filter is extracted with dichloromethane (DCM). For the extraction of the condensers three different solvents have been used; cyclohexane, acetone and DCM. The latter has also been used together with three buffers, pH 3, pH 7 and pH 11. Analyses of polynuclear aromatic hydrocarbons were performed by means of glass capillary gas chromatography and mass spectrometry. These analyses of diluted gasoline exhausts show that of the phenanthrene/anthracene, fluoranthene/pyrene and their monoalkylated forms found, between 90 and 30% are present in the gas phase. For diesel emissions, corresponding values are between 50% and 5%, respectively. However, the distribution of PAH between gas phase and particles is dependent on dilution ratio and filter temperatures. The addition of NO2 (approximately 7 ppm) prior to filter sampling involves degradation of cyclopenteno(cd)pyrene (CPedP) and benzo(a)pyrene (BaP) on the particles from diluted gasoline exhausts. This also occurs with BaP on diesel particles sampled under equivalent conditions. Mutagenicity data from these experiments support the theory of formation of direct-acting mutagens, probably due to nitration. Parallel sampling of particles with Teflon-coated and glass fiber filters does not show that components which are reactive to NO2, e.g., CPcdP, are degraded to a lower extent when glass fiber filters are used.

Emission of carcinogenic components with automobile exhausts.

Different sampling methods for mutagenic polynuclear aromatic hydrocarbons (PAH) are described. These methods involve either direct sampling of raw exhausts which prior to filtering are cooled in a condenser, or filter sampling of exhausts diluted in a tunnel. The relevance of gas-phase PAHs of samples from diluted exhausts is discussed; methods used are either adsorbents (XAD-2) or cryogenic condensation. The emission of benzo(a)pyrene and certain other PAHs is reported from vehicles using different fuels (gasoline, diesel, LPG, alcohols) or different emission control systems. The emission of some volatiles, such as benzene, ethylene and alkylnitrites, is also presented from different types of fuels used.