Persistent Organic Pollutant Determination in Killer Whale Scat Samples: Optimization of a Gas Chromatography/Mass Spectrometry Method and Application to Field Samples.

Biologic sample collection in wild cetacean populations is challenging. Most information on toxicant levels is obtained from blubber biopsy samples; however, sample collection is invasive and strictly regulated under permit, thus limiting sample numbers. Methods are needed to monitor toxicant levels that increase temporal and repeat sampling of individuals for population health and recovery models. The objective of this study was to optimize measuring trace levels (parts per billion) of persistent organic pollutants (POPs), namely polychlorinated-biphenyls (PCBs), polybrominated-diphenyl-ethers (PBDEs), dichlorodiphenyltrichloroethanes (DDTs), and hexachlorocyclobenzene, in killer whale scat (fecal) samples. Archival scat samples, initially collected, lyophilized, and extracted with 70 % ethanol for hormone analyses, were used to analyze POP concentrations. The residual pellet was extracted and analyzed using gas chromatography coupled with mass spectrometry. Method detection limits ranged from 11 to 125 ng/g dry weight. The described method is suitable for p,p'-DDE, PCBs-138, 153, 180, and 187, and PBDEs-47 and 100; other POPs were below the limit of detection. We applied this method to 126 scat samples collected from Southern Resident killer whales. Scat samples from 22 adult whales also had known POP concentrations in blubber and demonstrated significant correlations (p < 0.01) between matrices across target analytes. Overall, the scat toxicant measures matched previously reported patterns from blubber samples of decreased levels in reproductive-age females and a decreased p,p'-DDE/∑PCB ratio in J-pod. Measuring toxicants in scat samples provides an unprecedented opportunity to noninvasively evaluate contaminant levels in wild cetacean populations; these data have the prospect to provide meaningful information for vital management decisions.

Susceptibility to quantum dot induced lung inflammation differs widely among the Collaborative Cross founder mouse strains.

Quantum dots (QDs) are engineered semiconductor nanoparticles with unique physicochemical properties that make them potentially useful in clinical, research and industrial settings. However, a growing body of evidence indicates that like other engineered nanomaterials, QDs have the potential to be respiratory hazards, especially in the context of the manufacture of QDs and products containing them, as well as exposures to consumers using these products. The overall goal of this study was to investigate the role of mouse strain in determining susceptibility to QD-induced pulmonary inflammation and toxicity. Male mice from 8 genetically diverse inbred strains (the Collaborative Cross founder strains) were exposed to CdSe-ZnS core-shell QDs stabilized with an amphiphilic polymer. QD treatment resulted in significant increases in the percentage of neutrophils and levels of cytokines present in bronchoalveolar lavage fluid (BALF) obtained from NOD/ShiLtJ and NZO/HlLtJ mice relative to their saline (Sal) treated controls. Cadmium measurements in lung tissue indicated strain-dependent differences in disposition of QDs in the lung. Total glutathione levels in lung tissue were significantly correlated with percent neutrophils in BALF as well as with lung tissue Cd levels. Our findings indicate that QD-induced acute lung inflammation is mouse strain dependent, that it is heritable, and that the choice of mouse strain is an important consideration in planning QD toxicity studies. These data also suggest that formal genetic analyses using additional strains or recombinant inbred strains from these mice could be useful for discovering potential QD-induced inflammation susceptibility loci.

A sensitive LC-MS/MS method for measurement of organophosphorus pesticides and their oxygen analogs in air sampling matrices.

A rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for determination of levels of the organophosphorus (OP) pesticides chlorpyrifos (CPF), azinphos methyl (AZM), and their oxygen analogs chlorpyrifos-oxon (CPF-O) and azinphos methyl-oxon (AZM-O) on common active air sampling matrices. XAD-2 resin and polyurethane foam (PUF) matrices were extracted with acetonitrile containing stable-isotope labeled internal standards (ISTD). Analysis was accomplished in Multiple Reaction Monitoring (MRM) mode, and analytes in unknown samples were identified by retention time (±0.1 min) and qualifier ratio (±30% absolute) as compared to the mean of calibrants. For all compounds, calibration linearity correlation coefficients were ≥0.996. Limits of detection (LOD) ranged from 0.15-1.1 ng/sample for CPF, CPF-O, AZM, and AZM-O on active sampling matrices. Spiked fortification recoveries were 78-113% from XAD-2 active air sampling tubes and 71-108% from PUF active air sampling tubes. Storage stability tests also yielded recoveries ranging from 74-94% after time periods ranging from 2-10 months. The results demonstrate that LC-MS/MS is a sensitive method for determining these compounds from two different matrices at the low concentrations that can result from spray drift and long range transport in non-target areas following agricultural applications. In an inter-laboratory comparison, the limit of quantification (LOQ) for LC-MS/MS was 100 times lower than a typical gas chromatography-mass spectrometry (GC-MS) method.

Transport of a solvent mixture across two glove materials when applied in a paint matrix.

The transport of mixed paint solvents through natural rubber latex (4 mil) and nitrile rubber (5 mil) gloves was evaluated after spray application of the paint formulation directly on the glove surface. Glove materials and thicknesses were those selected by the majority of spray painters in the local automobile repair industry. A flat panel containing glove specimens mounted in multiple permeation cells permitted evaporation of solvents from the applied paint and incorporated a solid sorbent receiving medium for measuring glove membrane transport. The panel was sprayed in a paint booth to simulate use conditions. Charcoal cloth under the glove adsorbed transported solvents, which were quantified by gas chromatography. For each solvent component, results were expressed as mass transported through the glove relative to the mass applied, per unit area, during 30 min after spray application. The paint formulation contained ketones, acetates, and aromatics. Natural rubber latex allowed 6-10 times the transport of solvents relative to nitrile rubber for all eight solvent components: methyl ethyl ketone, toluene, styrene, ethyl benzene, xylene isomers, and 2-heptanone. m-Xylene showed the largest difference in transport between the two glove materials. This solvent also had the highest transport for each material. The results indicate that nitrile rubber gloves offer somewhat greater chemical resistance to all eight solvents studied compared with natural rubber latex gloves, regardless of the chemical properties of the individual solvent components. However, it must be emphasized that neither of the glove materials, in the thicknesses used in this study, provide adequate protection when exposed by direct spray painting. Simulation of realistic spray conditions may offer a source of useful information on the performance of chemical protective gloves because it accounts for solvent evaporation and the effect of paint polymerization after application on glove transport.

Ethanol-induced increase in the metabolic clearance of 1,1,1-trichloroethane in human volunteers.

This study evaluated the effect of moderate doses of ethanol over a short period of time on the toxicokinetics of an organic solvent, 1,1,1-trichloroethane. A group of 10 moderate drinkers were recruited and exposed via inhalation for 2 h to a low concentration of 1,1,1-trichloroethane (175 ppm) on two separate occasions. Subjects were administered ethanol (0.35 g/kg body weight) on each of the 7 days preceding one of the exposures. Blood and urine samples were collected during and following each exposure, with blood analyzed for 1,1,1-trichloroethane and urine analyzed for the metabolites of 1,1,1-trichloroethane: trichloroethanol and trichloroacetic acid. Prior ethanol consumption resulted in a significant increase in apparent metabolic clearance of 1,1,1-trichloroethane (mean increase = 25.4%). The results of this study demonstrate that ethanol consumption over time can affect the rate at which an organic solvent is cleared through metabolism in humans. For chemicals with toxic metabolic products, this inductive effect of ethanol consumption on the rate of biotransformation could be potentially harmful to exposed individuals. Metabolic clearance of compounds with high hepatic extraction may not be affected by enzyme induction as it is likely that these compounds are essentially completely metabolized while passing through the liver.

Phthalate metabolite levels and menopausal hot flashes in midlife women.

During the menopausal transition, a woman's reproductive capacity declines, her hormone milieu changes, and her risk of hot flashes increases. Exposure to phthalates, which can be found in personal care products, can also result in altered reproductive function. Here, we investigated the associations between phthalate metabolite levels and midlife hot flashes. Eligible women (45-54 years of age) provided detailed information on hot flashes history and donated urine samples (n=195). Urinary phthalate metabolite levels were measured by HPLC-MS/MS. A higher total sum of phthalate metabolites commonly found in personal care products was associated with an increased risk of ever experiencing hot flashes (odds ratio (OR)=1.45; 95% confidence interval (CI)=1.07-1.96), hot flashes in the past 30days (OR=1.43; 95%CI=1.04-1.96), and more frequent hot flashes (OR=1.47; 95%CI=1.06-2.05). These data suggest that some phthalate exposures from personal care products are associated with menopausal hot flashes in women.

Urinary polycyclic aromatic hydrocarbon metabolites as biomarkers to woodsmoke exposure - results from a controlled exposure study.

Woodsmoke contains harmful components - such as fine particulate matter (PM2.5) and polycyclic aromatic hydrocarbons (PAHs) - and impacts more than half of the global population. We investigated urinary hydroxylated PAH metabolites (OH-PAHs) as woodsmoke exposure biomarkers in nine non-smoking volunteers experimentally exposed to a wood fire. Individual urine samples were collected from 24-h before to 48-h after the exposure and personal PM2.5 samples were collected during the 2-h woodsmoke exposure. Concentrations of nine OH-PAHs increased by 1.8-7.2 times within 2.3-19.3 h, and returned to baseline approximately 24 h after the exposure. 2-Naphthol (2-NAP) had the largest post-exposure increase and exhibited a clear excretion pattern in all participants. The level of urinary OH-PAHs, except 1-hydroxypyrene (1-PYR), correlated with those of PM2.5, levoglucosan and PAHs in personal PM2.5 samples. This finding suggests that several urinary OH-PAHs, especially 2-NAP, are potential exposure biomarkers to woodsmoke; by contrast, 1-PYR may not be a suitable biomarker. Compared with levoglucosan and methoxyphenols - two other urinary woodsmoke biomarkers that were measured in the same study and reported previously - OH-PAHs might be better biomarkers based on sensitivity, robustness and stability, particularly under suboptimal sampling and storage conditions, like in epidemiological studies carried out in less developed areas.

Evaluation of dynamic headspace with gas chromatography/mass spectrometry for the determination of 1,1,1-trichloroethane, trichloroethanol, and trichloroacetic acid in biological samples.

A sensitive and reproducible method is described for the analysis of trichloroacetic acid in urine and 1,1,1-trichloroethane in blood using dynamic headspace GC/MS. Samples were analyzed using the soil module of a modified purge and trap autosampler to facilitate the use of disposable purging vessels. Coefficients of variation were below 3.5% for both analytes, and response was linear in the range of 0.01-7.0 microg/ml for trichloroacetic acid and 0.9 ng/ml-2.2 microg/ml for 1,1,1-trichloroethane. Attempts at using dynamic headspace for the analysis of trichloroethanol in urine were unsuccessful.

Toluene metabolites as biological indicators of exposure.

The measurement of exhaled and excreted xenobiotics and their metabolites can provide accurate, non-invasive, and time-flexible measurements of internal dose. We analyzed rates of exhaled (2)H(8)-toluene and excreted urinary metabolites from 33 exposures of men to 50 ppm of (2)H(8)-toluene for 2 h at rest. The total dose was distributed as follows: exhaled (2)H(8)-toluene, 13 +/- 6.2%; (2)H(5)-hippuric acid, 75 +/- 6.4%; (2)H(7)-o-cresol, 0.31 +/- 0.22%; (2)H(7)-m-cresol, 0.53 +/- 0.44%; and (2)H(7)-p-cresol, 11 +/- 3.8%. Interindividual variability was assessed using the coefficients of variation for peak exhalation or excretion rates, and fractions of dose excreted: (2)H(8)-toluene, c.v.=60, 47%; (2)H(5)-hippuric acid, 29, 8.6%; (2)H(7)-o-cresol, 80, 73%; (2)H(7)-m-cresol, 37, 83%; and (2)H(7)-p-cresol, 38, 34%. Excretion rates of the cresols were stable over the first 5 h post-exposure, and o-cresol was determined to be the best urinary indicator of exposure, given the lower background levels of this isomer. The hippuric acid/cresol rate ratios for the first 5 h post-exposure could be described by single exponential terms, and thus provided a means for estimating time since exposure for any finite toluene duration/exposure combination.

Determination of levoglucosan in atmospheric fine particulate matter.

A microanalytical method suitable for the quantitative determination of the sugar anhydride levoglucosan in low-volume samples of atmospheric fine particulate matter (PM) has been developed and validated. The method incorporates two sugar anhydrides as quality control standards. The recovery standard sedoheptulosan (2,7-anhydro-beta-D-altro-heptulopyranose) in 20 microL solvent is added onto samples of the atmospheric fine PM and aged for 1 hr before ultrasonic extraction with ethylacetate/ triethylamine. The extract is reduced in volume, an internal standard is added (1,5-anhydro-D-mannitol), and a portion of the extract is derivatized with 10% by volume N-trimethylsilylimidazole. The derivatized extract is analyzed by gas chromatography/mass spectrometry (GC/MS). The recovery of levoglucosan using this procedure was 69 +/- 6% from five filters amended with 2 microg levoglucosan, and the reproducibility of the assay is 9%. The limit of detection is approximately 0.1 microg/mL, which is equivalent to approximately 3.5 ng/m3 for a 10 L/min sampler or approximately 8.7 ng/m3 for a 4 L/min personal sampler (assuming 24-hr integrated samples). We demonstrated that levoglucosan concentrations in collocated samples (expressed as ng/m3) were identical irrespective of whether samples were collected by PM with aerodynamic diameter < or = 2.5 microm or PM with aerodynamic diameter < or = 10 microm impactors. It was also demonstrated that X-ray fluorescence analysis of samples of atmospheric PM, before levoglucosan determinations, did not alter the levels of levoglucosan.

Accuracy of prenatal smoking data from Washington State birth certificates in a population-based sample with cotinine measurements.

PURPOSE: To assess the accuracy of smoking data in contemporary U.S. birth certificates. METHODS: We compared data on prenatal smoking as reported on Washington State birth certificates to cotinine measured in archived newborn screening dried blood spots for 200 infants born in 2007 (100 randomly selected from births to self-reported nonsmokers and 100 born to self-reported smokers). We estimated the sensitivity of the birth certificate data to identify prenatal smokers and the precision with which self-identified third trimester smokers report smoking levels. RESULTS: Infants born to two (2%) mothers who reported they did not smoke during the pregnancy had whole blood cotinine concentrations consistent with active smoking by the mother (sensitivity 85%). Sensitivity of the birth certificate to identify reported smokers who continued to smoke throughout pregnancy was similar (89%). Among self-identified third trimester smokers whose infants' specimens were collected shortly after delivery, Spearman rho between infant cotinine and maternal-reported cigarettes/day in the third trimester was 0.54. CONCLUSIONS: Birth certificates may represent a viable option for assessing prenatal smoking status, and possibly smoking cessation and level among smokers, in epidemiologic studies sufficiently powered to overcome a moderate amount of exposure measurement error.

Occurrence of trivalent monomethyl arsenic and other urinary arsenic species in a highly exposed juvenile population in Bangladesh.

Following reports of high cytotoxicity and mutagenicity of monomethyl arsonous acid (MMA(III)) and early reports of urinary MMA(III) in arsenic-exposed individuals, MMA(III) has often been included in population studies. Use of urinary MMA(III) as an indicator of exposure and/or health risk is challenged by inconsistent results from field studies and stability studies, which indicate potential artifacts. We measured urinary arsenic species in children chronically exposed to arsenic in drinking water, using collection, storage, and analysis methods shown to conserve MMA(III). MMA(III) was easily oxidized in sample storage and processing, but recoveries of 80% or better in spiked urine samples were achieved. Attempts to preserve the distribution of MMA between trivalent and pentavalent forms using complexing agents were unsuccessful and MMA(III) spiked into treated urine samples actually showed lower stability than in untreated samples. In 643 urine samples from a highly exposed population from the Matlab district in Bangladesh stored for 3-6 months at ≤-70 °C, MMA(III) was detected in 41 samples, with an estimated median value of 0.3 µg/l, and levels of MMA(III) above 1 µg/l in only two samples. The low urinary concentrations in highly exposed individuals and known difficulties in preserving sample oxidation state indicate that urinary MMA(III) is not suitable for use as an epidemiological biomarker.

Unexpected results in a randomized dietary trial to reduce phthalate and bisphenol A exposures.

Diet is a primary source of exposure for high-molecular-weight phthalates and bisphenol A (BPA), but little is known about the efficacy of various interventions to reduce exposures. We conducted a randomized trial with 10 families to test the efficacy of a 5-day complete dietary replacement (Arm 1; n=21) versus written recommendations to reduce phthalate and BPA exposures (Arm 2; n=19). We measured phthalate and BPA concentrations in urine samples at baseline, intervention, and post-intervention periods. We used Wilcoxon paired signed-rank tests to assess change in concentrations across time and multi-level mixed effects regression models to assess differences between Arms 1 and 2. Urinary di(2-ethylhexyl) phthalate (DEHP) metabolite concentrations increased unexpectedly from a median of 283.7 nmol/g at baseline to 7027.5 nmol/g during the intervention (P<0.0001) among Arm 1 participants, and no significant changes were observed for Arm 2 participants. We observed a statistically significant increase in total BPA concentration between baseline and intervention periods in Arm 1 but no significant changes in Arm 2. Arm 1 food ingredient testing for DEHP revealed concentrations of 21,400 ng/g in ground coriander and 673 ng/g in milk. Food contamination with DEHP led to unexpected increases in urinary phthalate concentrations in a trial intended to minimize exposure. In the absence of regulation to reduce phthalate and BPA concentrations in food production, it may be difficult to develop effective interventions that are feasible in the general population. An estimate of DEHP daily intake for children in the dietary replacement Arm was above the US Environmental Protection Agency oral reference dose and the European Food Safety Authority's tolerable daily intake, suggesting that food contamination can be a major source of DEHP exposure.

Identification and quantification of 1-nitropyrene metabolites in human urine as a proposed biomarker for exposure to diesel exhaust.

1-nitropyrene (1-NP) is one of the most abundant nitrated polycyclic aromatic hydrocarbons (NPAHs) in diesel exhaust particulate matter (DEP) and is a main contributor of direct-acting mutagenicity in DEP. Therefore, the metabolites of 1-NP are expected to be a biomarker for assessment of exposure to DEP. In this study, a highly specific and sensitive analytical method using liquid chromatography with tandem mass spectrometry (LC-MS/MS) was developed to determine urinary 1-NP metabolites. After enzymatic hydrolysis of the conjugated metabolites, the analytes were selectively extracted from the urine matrix with blue rayon. The eluate from the rayon was further purified on an acidic alumina cartridge. Hydroxy-N-acetyl- 1-aminopyrenes (6- and 8-OHNAAP) and hydroxy-1-nitropyrenes (3-, 6-, and 8-OHNP) in human urine were identified by their retention times and MS/MS spectra and quantified by using deuterated internal standards. 1-NP metabolites were quantified in urine from all healthy, nonoccupationally exposed subjects. 6-OHNAAP, 8-OHNAAP, 6-OHNP, and 8-OHNP (means of 117, 109, 203, and 137 pmol/mol creatinine, respectively) were the most abundant isomers in human urine. This report is the first to demonstrate the presence of OHNAAPs and OHNPs in human urine, in agreement with previous in vivo and in vitro studies that predicted that these metabolites should be excreted into human urine. This method for determining urinary 1-NP metabolites should be useful for the surveillance of exposure to NPAHs and DEP and will facilitate the study of cancer risk associated with these exposures.

Evaluation of urinary methoxyphenols as biomarkers of woodsmoke exposure.

Urinary methoxyphenols have been proposed as biomarkers for woodsmoke exposure, but the relationship between exposure and urinary methoxyphenol concentrations has not been characterized. We collected personal particulate matter2.5 and urine samples from 9 adults experimentally exposed to smoke from an open wood fire to characterize this relationship. Personal exposures (PM2.5 mean 1500 microg/ m3) varied 3.5-fold. Twenty-two methoxyphenols, levoglucosan, and 17 polynuclear hydrocarbons were quantified by gas chromatography/mass spectrometry assays for personal filter samples and urine samples. Most methoxyphenols had measurable preexposure levels. Propylguaiacol, syringol, methylsyringol, ethylsyringol, and propylsyringol had peak urinary concentrations after the woodsmoke exposure. Eight subjects had peak urinary elimination of methoxyphenols within 6 h (t1/2 3-5 h), whereas one had delayed elimination. Several metrics for urinary excretion were evaluated. Analyte concentration was greatly affected by diuresis. Excretion rate and analyte concentrations normalized by creatinine gave a clearer signal and were equivalent in predictive ability. Twelve-hour average creatinine-normalized concentrations of each of the 5 methoxyphenols gave a Pearson correlation > or = 0.8 with their particle-phase concentration. The sum of urinary concentrations for the 5 methoxyphenols versus levoglucosan on personal filters gave a regression coefficient of 0.75. This sum versus PM2.5 gave a regression coefficient of 0.79. The intercept of this regression suggests that the threshold for detection of an acute exposure event would be approximately 760 microg/m3 particulate matter from woodsmoke. The signal-to-noise (12-h postexposure average/preexposure average) ranged from 1.1 to 8 for the 5 methoxyphenols. Analysis of multiple compounds provided assurance that elevations were not artifactual due to food or other products.

Determination of methoxyphenols in ambient atmospheric particulate matter: tracers for wood combustion.

Combustion of wood and other biomass fuels produces source-specific organic compounds arising from pyrolysis of lignin, including substantial amounts of 4-substituted methoxylated phenolic compounds (methoxyphenols). These compounds have been used as atmospheric markers to determine the contribution of wood smoke to ambient atmospheric fine particulate matter (PM). However, reliable quantification of methoxyphenols represents an analytical challenge because these compounds are polar, semi-volatile, and somewhat reactive. We reportherein an improved gas chromatographic-mass spectrometric (GC/MS) method for the sensitive and reliable determination of methoxyphenols in low-volume ambient PM samples. Deuterated standard compounds are added to the environmental samples prior to extraction to determine analyte recoveries in each sample. Analytical figures of merit for the assay, as applied to ambient PM2.5 and PM10 samples are as follows: recovery = 63-100%; precision = 2-6%; analytical limit of detection (S/N 2) = 0.002 microg/mL; limit of quantitation = 0.07-0.45 ng/m3 (assuming a 14 m3 sample). The improved method was applied to ambient PM samples collected between 1999 and 2000 in Seattle, WA. Particle-bound methoxyphenol concentrations in the range <0.1 to 22 ng/m3 were observed and the methoxyphenols were present almost exclusively in the fine (PM2.5) size fraction. We also demonstrated that XRF analysis of samples of atmospheric PM collected on Teflon filters significantly reduced the levels of methoxyphenols measured in the PM samples in subsequent assay of the same filters. Therefore, XRF analysis of filters, commonly undertaken to obtain trace element concentrations for use in source apportionment analyses, would preclude the subsequent analysis of those filters for methoxyphenols and other similarly semivolatile or reactive organic chemicals.