PM2.5 Filter Extraction Methods: Implications for

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Ecotoxicology and Human Environmental Health 2.5

PM Filter Extraction Methods: Implications for Chemical and Toxicological Analyses Courtney Roper, Lisandra Santiago Delgado, Damien Barrett, Staci L. Massey Simonich, and Robert L. Tanguay Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b04308 • Publication Date (Web): 03 Dec 2018 Downloaded from http://pubs.acs.org on December 4, 2018

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Environmental Science & Technology

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PM2.5 Filter Extraction Methods: Implications for Chemical and Toxicological Analyses

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Courtney Roper,1 Lisandra Santiago Delgado,1,2 Damien Barrett,3 Staci L. Massey Simonich,1,2 Robert L. Tanguay1*

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Department of Chemistry, Oregon State University, Corvallis, OR 97331

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Department of Microbiology, Oregon State University, Corvallis, OR 97331

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* Corresponding author. Email: [email protected]

Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR, 97331

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ACS Paragon Plus Environment


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Abstract

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Toxicology research into the global public health burden of fine particulate matter (PM2.5)

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exposures frequently requires extraction of PM2.5 from filters. A standardized method for these

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extractions does not exist, leading to inaccurate inter-laboratory comparisons. It is largely

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unknown how different filter extraction methods might impact the composition and bioactivity of

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the resulting samples. We characterized the variation in these metrics by using equal portions of

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a single PM2.5 filter, with each portion undergoing a different extraction method. Significant

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differences were observed between extraction methods for concentrations of elements and

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polycyclic aromatic hydrocarbons (PAHs) of the PM2.5 tested following its preparation for

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biological response studies. Importantly, the chemical profiles differed from those observed

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when using standard protocols for chemical characterization of the ambient sample,

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demonstrating that extraction can alter both chemical component amounts and species profiles

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of the extracts. The impact of these chemical differences on sensitive endpoints of zebrafish

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development was investigated. Significant differences in the percent incidence and timing of

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mortality were associated with PM2.5 extraction method. This research highlights the importance

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of and rationale for considering extraction method when making inter-laboratory comparisons of

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PM2.5 toxicology research.

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Table of Contents (TOC) Art

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Introduction

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Toxicology research is essential to better understand the public health burden from fine

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particulate matter (PM2.5) exposures which are associated with systemic health effects.1-3 In

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addition to exploring the full range of PM2.5 hazard potential, the use of an appropriate whole

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animal model can also identify toxic constituents and the molecular mechanisms underlying the

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associated systemic health effects.4-7 PM2.5 collected on filters can address the global variability

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in PM2.5 from a toxicological perspective, broadening the knowledge previously gained from

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fixed location testing and limited sample number.8, 9 Use of spatially, temporally, and seasonally

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variable samples provides additional information on the toxic potential of PM2.5.

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Research groups currently use various filter extraction methods to prepare samples for these

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investigations,10-14 creating a potential toxicity bias from the extraction method, rather than from

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the actual PM2.5-sample composition. The use of varying filter extraction procedures also

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complicates inter-laboratory comparisons and hence formation of a robust consensus of PM2.5

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exposure hazard. Variability in extraction methods can misrepresent the toxic responses to

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specific PM2.5 samples because of the potential loss of a key toxic driver(s) during the extraction

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process.15 Few studies have compared PM2.5 filter extraction procedures but they indicate

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substantial differences between the actual and observed chemical components of PM2.5 post

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filter extraction.16, 17 Not surprisingly, these differences are associated with similar discrepancies

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in sample oxidative potential18 and bioactivity.19 This previous research explored a single

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biological system and only compared two extraction methods, highlighting a clear knowledge

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gap in filter extraction impacts on chemical and toxicological analyses.

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Recently, the zebrafish (Danio rerio) was utilized as an in vivo surrogate to evaluate particulate

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matter-induced toxicity.20-22 The developing zebrafish is highly sensitive to chemical perturbation.

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Advantages include embryo transparency, rapid external development (3 – 5 days post

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fertilization for most endpoints), and amenability to molecular and genetic techniques.23-25 These


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advantages enable rapid screening of PM2.5 samples with biological activity measurements

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spanning from overt toxicity (malformations and mortality), to subtle but important effects on

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behavior.20, 26 Thus far, PM2.5 extraction method-bioactivity studies have not been reported in

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zebrafish.

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We performed multiple extraction methods on portions of the same PM2.5 filter to determine the

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associated impacts on chemical recovery and bioactivity. Use of a single filter sample allowed

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for interpretation of data independent of physical and chemical properties that would vary from

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different collections of PM2.5. From this we hypothesized that different filter extraction

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procedures on the same PM2.5 sample will impact the chemical and biological response data of

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these samples, introducing a methods bias. This research will guide selection of an extraction

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method that is best suited for bioactivity assessments using PM2.5 samples chemically

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representative of ambient samples.

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Materials and Methods

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Chemicals

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PAHs and isotopically labeled standards information, including abbreviations and vendors, is

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provided in the Supporting Information (Table S1). Solvents including: methanol (MetOH),

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hexane, ethyl acetate (EA), acetonitrile (ACN), acetone (Ace), and dichloromethane (DCM); all

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optima grade were purchased from Thermo Fisher Scientific (Santa Clara, CA). Toluene,

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dimethylsulfoxide (DMSO), and N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide

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(MTBSTFA) were purchased from Sigma-Aldrich (Milwaukee, WI).

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PM2.5 Samples

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Samples were donated by Keith Bein and collected in the winter in downtown Sacramento, CA

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from January 15-24, 2011 on PTFE-coated filters.16 The filter was cut into six equal portions for

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subsequent extraction. Blank PTFE-coated filters (Pallflex fiberfilm, 37 mm) that did not undergo

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PM2.5 collection were extracted to serve as methods controls.



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Extraction Methods

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Six different extraction methods were tested on ambient PM2.5 filters and blank control filters.

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The extraction process consisted of removal of particles from the filter piece, concentration of

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removed extracts, and reconstitution in DMSO (Table 1). Each extraction method is detailed

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below.

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1) Water: The filter was sonicated in a waterbath sonicator (40 kHz, Bransonic) in 15 mL tubes

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with 6 mL of water. After sonication, the filter piece was removed and rinsed with water to

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remove any residual particles remaining on the filter. The sample was then concentrated via

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freeze drying and the dry PM2.5 was reconstituted in DMSO.

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2) Methanol: The filter piece was sonicated and rinsed as described in method 1 but in methanol

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instead of water. The sample was concentrated by N2 stream and then reconstituted in DMSO.

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3) DCM: The filter piece was sonicated and rinsed as described in method 1 but in DCM instead

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of water. The sample was concentrated by N2 stream and then reconstituted in DMSO.

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4) DMSO: The filter piece was sonicated and rinsed as described in method 1 but in DMSO

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instead of water. The sample was solvent exchanged to ethyl acetate (EA), concentrated by N2

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stream, and then reconstituted in DMSO.

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5) Single Vial: A single vial method was created in an effort to reduce sample loss,

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consumables, and sample process time. In this method, a single filter piece was placed into a

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1.5 mL centrifuge tube and the same volume used for DMSO reconstitution in all other methods

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was used. The DMSO and filter were sonicated for 60 min in a waterbath sonicator, as occurred

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with the other sonication extraction methods.

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6) Pressurized Liquid Extraction (PLE): A filter piece was placed in a 33 mL cell (Dionex

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Accelerated Solvent Extractor 350) that underwent two cycles of pressurized liquid extraction: 1)

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DCM followed by 2) EA (1500 psi, 100 °C, 1 cycle, 240s purge). The sample was concentrated

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by N2 stream and then reconstituted in DMSO.


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All dry PM2.5 was reconstituted in an equal volume of DMSO, except for the single vial method

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which already contained the appropriate volume to result in a final concentration of 200 µg/mL.

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The soluble fraction from DMSO extraction was collected as previously described and selected

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for this research as it replicated the findings of the whole particle suspension, the insoluble

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fraction was not tested as it was previously shown to have negligible bioactivity compared to the

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soluble fraction for particulate matter samples.20 The soluble fractions of PM2.5 and blank filter

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extracts resulting from the six different methods were split for chemical and biological testing.

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Ambient sample characterization

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An additional portion of the ambient PM2.5 filter used for all extraction method testing was used

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to determine the chemical constituents present on the filter following standard operating

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procedures (SOP) for chemical characterization, without the additional preparation steps

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required for toxicological research. PM2.5 was removed from the filter via pressurized liquid

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extraction followed by sample clean-up as previously described for PAH analyses.27 For

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characterization of elements, a portion of the filter was sonicated in water for 60 min via water

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bath sonication (60 Hz). This extraction method has previously been shown to produce similar

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extraction efficiencies to liquid-liquid extraction methods with particulate matter samples.28 This

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sample which underwent SOP characterization steps, without toxicology preparation steps

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(concentration and reconstitution), will be referred to as the “Ambient SOP Sample”.

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Table 1. Description of Different PM2.5 Extraction Methods 1: Water

2: MeOH

3: DCM

4: DMSO

5: Single Vial

Removal Sonication Sonication Sonication Sonication Sonication in Water in MeOH in DCM in DMSO in DMSO Concentration

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Reconstitution

Freeze Drying

N2

N2

DMSO

DMSO

DMSO

Solvent exchange N2 DMSO

Chemical Characterization


6: PLE Pressurized Liquid Extraction (DCM:EA)

N/A

N2

N/A

DMSO


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PAHs: Aliquots of the DMSO soluble fraction of PM2.5 and blank filter extracts were solvent

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exchanged to hexane via a TurboVap evaporation system (N2 gas, 30 °C) followed by solid

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phase extraction (SPE) cleanup (SI). Samples were then solvent exchanged to EA and

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concentrated to 300 µL under a stream of N2. Samples were spiked with isotopically labeled

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internal standards, hydroxy-PAH analysis was performed with an aliquot of the concentrated

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sample that was derivatized following addition of internal standards (SI). Organic compounds,

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specifically parent/methyl PAHs (n=19), and nitro- (n=22), oxy- (n=23), hydroxy- (n=36), and

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high molecular weight (MW ≥ 302, HMW, n=14) PAHs, were quantitatively measured using

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Agilent 6890 gas chromatography (GC) coupled with an Agilent 5973N mass spectrometer

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(MS). Selected ion monitoring (SIM) was utilized with spectral data analysis performed with

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ChemStation software (V. E.02.02.1431, Agilent Technologies). Commercially available

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standards were used for all measured compounds (abbreviations and vendors available in S1)

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and all samples and controls were run in triplicate.

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Elements: Aliquots of the DMSO soluble fraction of PM2.5 and blank filter extracts were added to

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ultrapure water, resulting in a 0.1 % DMSO concentration. Elements (n=14), were quantitatively

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measured using an Agilent 5110 inductively coupled plasma optical emission spectrometry

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(ICP-OES) system in axial view mode at the Central Analytical Laboratory at Oregon State

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University. Commercially available standards were utilized for all measured compounds and all

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samples and controls were run in triplicate.

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Developmental Toxicity Screening

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Zebrafish Husbandry: Standard procedures for fish care followed at Sinnhuber Aquatic

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Research Laboratory (SARL) were utilized with adult fish for a wildtype (Tropical 5D) that were

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maintained at 28±1 °C on a recirculating system, with a 14 h light/10 h dark cycle.29 Embryos

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were collected from group spawns of adult zebrafish30 and enzymatically dechorionated at 4

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hours post fertilization (hpf).31 Embryos were then mechanically distributed into individual wells


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of a 96-well plate that contained 90 µl of embryo medium31, 32 and the soluble fraction of PM2.5 or

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vehicle (DMSO)/blank filter controls in embryo medium (10 µl) were added at 6 hpf. Final

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concentrations in all wells were 1 % DMSO in embryo medium. All experiments were conducted

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with fertilized embryos according to Oregon State University Animal Care and Use Protocols.

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Developmental Toxicity Screen: Following embryo exposure at 6 hpf, the 96-well plates were

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sealed with Parafilm to prevent evaporation, wrapped in aluminum foil to prevent

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photodegradation, and placed on an orbital shaker at 235 rpm overnight to ensure gentle mixing

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after the exposure; plates were stored at 28 °C throughout the experiment.29 Developmental

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toxicity was assessed at 24 and 120 hpf in all treatments and controls (n=32 embryos/group).

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Mortality and morphological outcomes (n=22 endpoints) were visually assessed using a

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dissecting microscope as previously described.25

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Statistical Analysis

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For chemical characterization data, histograms and statistical significance calculations (one- or

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two-way analysis of variance (ANOVA) tests and pairwise multiple comparison procedures

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(Hom-Sidak method) with significance set at p

PM2.5 Filter Extraction Methods: Implications for

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