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Beskæftigelsesudvalget 2018-19 (2. samling)
BEU Alm.del Bilag 2
Offentligt

Airport emission particles: Exposure characterization and toxicity following intratracheal

instillation in mice

--Manuscript Draft--

Manuscript Number:

Full Title:

Article Type:

Funding Information:

Abstract:

Background

Little is known about the exposure levels and adverse health effects of occupational

exposure to airplane emissions. Diesel exhaust particles are classified as carcinogenic

to humans and jet engines produce potentially similar soot particles. Here, we

evaluated the potential occupational exposure risk by analyzing particles from a non-

commercial airfield and from the apron of a commercial airport. Toxicity of the collected

particles was evaluated alongside NIST standard reference diesel exhaust particles

(NIST2975) in terms of acute phase response, pulmonary inflammation, and

genotoxicity after single intratracheal instillation in mice.

Results

Particle exposure levels were up to 1 mg/m3 at the non-commercial airfield. Particulate

matter from the non-commercial airfield air consisted of primary and aggregated soot

particles, whereas commercial airport sampling resulted in a more heterogeneous

mixture of organic compounds including salt, pollen and soot, reflecting the complex

occupational exposure at an apron. The particle contents of poly aromatic

hydrocarbons and metals were similar to the content in NIST2975. Mice were exposed

to doses 6, 18 and 54 µg alongside carbon black (Printex 90) and NIST2975 and

euthanized after 1, 28 or 90 days. Dose-dependent increases in total number of cells,

neutrophils, and eosinophils in bronchoalveolar lavage fluid were observed on day 1

post-exposure for all particles. Lymphocytes were increased for all four particle types

on 28 days post-exposure as well as for neutrophil influx for jet engine particles and

carbon black nanoparticles. Increased Saa3 mRNA levels in lung tissue and increased

SAA3 protein levels in plasma were observed on day 1 post-exposure. Increased

levels of DNA strand breaks in bronchoalveolar lavage cells and liver tissue were

observed for both particles, at single dose levels across doses and time points.

Conclusions

Pulmonary exposure of mice to particles collected at two airports induced acute phase

response, inflammation, and genotoxicity similarly as standard diesel exhaust particles

and carbon black nanoparticles, suggesting similar physicochemical properties and

toxicity of jet engine particles and diesel exhaust particles. Given this resemblance as

well as the dose-response relationship between diesel exhaust exposure and lung

cancer, occupational exposure to jet engine emissions at the two airports should be

minimized.

Corresponding Author:

Ulla Vogel

National Research Centre for the Working Environment

Copenhagen, DENMARK

[email protected];[email protected];[email protected]

PFTX-D-18-00186R2

Airport emission particles: Exposure characterization and toxicity following intratracheal

instillation in mice

Research

Particle and Fibre Toxicology

Corresponding Author E-Mail:

Corresponding Author Secondary

Information:

Corresponding Author's Institution:

Corresponding Author's Secondary

Institution:

First Author:

First Author Secondary Information:

National Research Centre for the Working Environment

Katja Maria Bendtsen, DVM, PhD

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Order of Authors:

Katja Maria Bendtsen, DVM, PhD

Anders Brostrøm

Antti Joonas Koivisto

Ismo Koponen

Trine Berthing

Nicolas Bertram

Kirsten Inga Kling

Miikka Dal Maso

Oskari Kangasniemi

Mikko Poikkimäki

Katrin Loeschner

Per Axel Clausen

Henrik Wolff

Keld Alstrup Jensen

Anne Thoustrup Saber

Ulla Vogel

Order of Authors Secondary Information:

Response to Reviewers:

Dear Roel Schins

Thank you for the opportunity to submit a second revision of our manuscript "Airport

emission particles: Exposure characterization and toxicity following intratracheal

instillation in mice". Enclosed, please find a revision manuscript for your consideration.

We have addressed the comment by reviewer 2 (please see the point-by-point

response below), and thank the reviewer for pointing out the mistake.

We hope that you will be able to accept the revised manuscript, and that the

manuscript will be published as soon as possible.

Kind regards Katja Bendtsen and Ulla Vogel

Reviewer reports:

Reviewer #1: The authors have addressed the comments of the reviewer. The paper is

significantly improved and especially Table 1 is very informative as the auhtors have

not only described the samples but also have given an overview of the measurements.

Response: thank you very much!

Reviewer #2: Two Comments on revised manuscript:

1.In my first review I had criticized the calculation of the mouse minute ventilation used

by the authors (Line 451 of the original submission) as being way too low, and the

authors had disagreed (With all due respect…..) . As author I would have responded

more strongly, e.g. "The reviewer is dead wrong, reminding us of the Latin saying: Si

tacuisses…". Embarrassing for me; of course, the authors are right, and I (Günter

Oberdörster) apologize for seemingly not being able to multiply 0.18 by 255.

That still leaves the issue of Dose - Rate to be considered, but this is ok since the

study deals just with hazard characterization and not with risk characterization.

RE: thank you for this comment!

2. In their response describing the mouse intratracheal instillation procedure the

authors state that right after the instillation "….the mouse was carefully shaken head-

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down twice……to ensure confinement of the instilled material in the lungs and

spreading downwards the alveoli." A head-down position would just do the opposite?

Please explain.

RE: We thank the very alert reviewer for noting this mistake! We have deleted the word

'head-down'. As the reviewer correctly noted, the whole purpose of shaking is to make

sure that the particles are delivered deeply into the lungs.

Additional Information:

Question

Response

<b>Is this study a clinical

trial?</b><hr><i>A clinical trial is defined

by the Word Health Organisation as 'any

research study that prospectively assigns

human participants or groups of humans

to one or more health-related

interventions to evaluate the effects on

health outcomes'.</i>

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Manuscript

Click here to view linked References

Click here to access/download;Manuscript;Airport REVISED

version R2.docx

Airport emission particles: Exposure characterization and toxicity following intratracheal

instillation in mice

Katja Maria Bendtsen

, Anders Brostrøm

1,2

, Antti Joonas Koivisto

, Ismo Koponen

1,3

, Trine

Berthing

, Nicolas Bertram

, Kirsten Inga Kling

, Miikka Dal Maso

, Oskari Kangasniemi

, Mikko

Poikkimäki

, Katrin Loeschner

, Per Axel Clausen

, Henrik Wolff

, Keld Alstrup Jensen

, Anne

Thoustrup Saber

, Ulla Vogel

1,7*

*Corresponding author: [email protected]

National Research Centre for the Working Environment, Nanotoxicology, Lersø Parkallé 105,

DK-2100 Copenhagen, Denmark

Technical University of Denmark, National Centre for Nano Fabrication and Characterization,

Fysikvej, Building 307, DK-2800 Kgs Lyngby

FORCE Technology, Park Allé 345, 2605 Brøndby, Denmark

Tampere University of Technology, Aerosol Physics, Laboratory of Physics, Faculty of Natural

Sciences, PO Box 527, FI-33101 Tampere, Finland

Technical University of Denmark, National Food Institute, Research Group for Nano-Bio Science,

Kemitorvet 201, DK-2800 Kgs. Lyngby

Finnish Institute of Occupational Health, P.O. Box 40, FI-00032 Työterveyslaitos, Finland

Technical University of Denmark, Department of Health Technology, DK-2800 Kgs. Lyngby,

Denmark

Email adresses:

Katja Maria Bendtsen: [email protected]

Anders Brostrøm:

[email protected];

[email protected]

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Antti Joonas Koivisto: [email protected]

Ismo Koponen: [email protected]

Trine Berthing: [email protected]

Nicolas Bertram: [email protected]

Kirsten Inga Kling

[email protected]

Miikka Dal Maso: [email protected]

Oskari Kangasniemi: [email protected]

Mikko Poikkimäki: [email protected]

Katrin Loeschner: [email protected]

Per Axel Clausen: [email protected]

Henrik Wolff: [email protected]

Keld Alstrup Jensen: [email protected]

Anne Thoustrup Saber: [email protected]

Ulla Vogel: [email protected]

Abstract

Background

Little is known about the exposure levels and adverse health effects of occupational

exposure to airplane emissions. Diesel exhaust particles are classified as carcinogenic to humans

and jet engines produce potentially similar soot particles. Here, we evaluated the potential

occupational exposure risk by analyzing particles from a non-commercial airfield and from the

apron of a commercial airport. Toxicity of the collected particles was evaluated alongside NIST

standard reference diesel exhaust particles (NIST2975) in terms of acute phase response, pulmonary

inflammation, and genotoxicity after single intratracheal instillation in mice.

BEU, Alm.del - 2018-19 (2. samling) - Bilag 2: Orientering om videnskabelig artikel om flyemmissioner, fra beskæftigelsesministeren

Results

Particle exposure levels were up to 1 mg/m

at the non-commercial airfield. Particulate

matter from the non-commercial airfield air consisted of primary and aggregated soot particles,

whereas commercial airport sampling resulted in a more heterogeneous mixture of organic

compounds including salt, pollen and soot, reflecting the complex occupational exposure at an

apron. The particle contents of poly aromatic hydrocarbons and metals were similar to the content

in NIST2975. Mice were exposed to doses 6, 18 and 54 µg alongside carbon black (Printex 90) and

NIST2975 and euthanized after 1, 28 or 90 days. Dose-dependent increases in total number of cells,

neutrophils, and eosinophils in bronchoalveolar lavage fluid were observed on day 1 post-exposure

for all particles. Lymphocytes were increased for all four particle types on 28 days post-exposure as

well as for neutrophil influx for jet engine particles and carbon black nanoparticles. Increased

Saa3

mRNA levels in lung tissue and increased SAA3 protein levels in plasma were observed on day 1

post-exposure. Increased levels of DNA strand breaks in bronchoalveolar lavage cells and liver

tissue were observed for both particles, at single dose levels across doses and time points.

Conclusions

Pulmonary exposure of mice to particles collected at two airports induced acute phase

response, inflammation, and genotoxicity similarly as standard diesel exhaust particles and carbon

black nanoparticles, suggesting similar physicochemical properties and toxicity of jet engine

particles and diesel exhaust particles. Given this resemblance as well as the dose-response

relationship between diesel exhaust exposure and lung cancer, occupational exposure to jet engine

emissions at the two airports should be minimized.

Keywords

Airport - Nanoparticles - Diesel - Diesel engine exhaust - Pulmonary exposure

–

Exposure risk

–

Jet

engine exhaust

–

Instillation

–

Occupational exposure

Background

Airport personnel are at risk of complex occupational exposures originating from many sources,

including combustion particles from jet engines and diesel-fueled handling vehicles. Exposure to

ultrafine particles (UFP, diameter

≤100

nm) from combustion exhaust has consistently been

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associated with a wide range of health risks [1, 2]. Diesel engine exhaust and diesel exhaust

particles, which are a major component of ultrafine particles (UFP) in urban aerosols, have been

classified as carcinogenic to humans (group 1) by the International Agency for Research on Cancer

(IARC) [3] and cause lung cancer and systemic inflammation and inflammatory responses in the

airways [4].

There is increasing awareness of the potential health risk due to occupational fuel combustion

exposures at airports and studies of airport personnel health and exposure are accumulating. A large

cohort study following 69,175 workers at Copenhagen Airport from 1990-2012 included data such

as lifestyle characteristics, work tasks, and air pollution. By linkage to health registers this cohort

will be monitored for incidence of cardiovascular diseases, cancer, and pulmonary diseases [5]. An

Italian study reported DNA aberrations in airport staff (sister chromatid exchange and total

structural chromosomal changes in lymphocytes and exfoliated buccal cells) with increased tail

moment in the comet assay compared to unexposed controls [6]. Evaluation of airport workers in

Turkey [7] and at an American aircraft equipment military station [8] also showed a significant

increase in the frequency of sister chromatid exchange in the exposed workers. Recently, it was

shown that two hours of normal breathing in a high-concentration airport-particle zone downwind

of Los Angeles airport increased the acute systemic inflammatory cytokine IL-6 of non-smoking

adults with asthma [9]. However, studies assessing the potential health hazards of jet engine

particles without confounding life style factors are limited. A study of the jet fuel JP-8, where mice

were exposed to vapor and aerosol exposure, reported potential effects on lung surfactant [10].

Studies of the hazard potential of environmental exposures benefit from inclusion of well-

characterized control particles or standard reference materials (SRM) because this allows

comparison of the studied exposures with exposures to particles of well-known toxicity. Diesel

exhaust particles have been extensively studied animal studies and in humans [11, 12, 13, 14] and

are therefore suitable as bench mark particles. The standard reference material SRM 2975

(forwardly referred to as NIST2975) from the National Institute of Standards and Technology

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(NIST, Gaithersburg, MD, USA) is a sample of diesel exhaust particles collected from an industrial

fork lift [15] which contains low levels of polycyclic aromatic hydrocarbons (PAH). The NIST

SRM 1650b (forwardly referred to as NIST1650) is diesel particles collected from a heavy-duty

diesel truck engine and contains more PAH compared to NIST2975. The pigment carbon black has

been classified as possibly carcinogenic to humans [3]. Carbon black Printex 90 (CB) is black

pigment used in printing ink and consists of carbon nanoparticles with very low levels of

contaminants. We previously showed that intratracheal instillation with NIST1650 and CB induces

pulmonary acute phase response, neutrophil influx, and genotoxicity [16, 17, 18, 19, 20, 21, 22].

Genotoxicity was observed even at very low doses of CB [23]. The potential similarity of jet engine

exhaust particles with diesel exhaust particles and carbon nanoparticles, such as CB, warrants a

hazard risk assessment of jet engine exhaust particles.

The purpose of the current study was to assess the pulmonary toxicity of airplane emissions in mice

and to compare this with reference particles of known toxicity. We characterized the exposure at a

commercial airport and at a non-commercial airfield and characterized the physical/chemical

properties of collected particles from both locations. Finally, we assessed the acute phase response,

inflammation, and genotoxicity following pulmonary exposure to these two different samples of

airplane emissions at three different dose levels and three different time points in mice (Table 1

gives an overview of the data and relevant figures). Standard reference materials with known

toxicity, namely diesel particle NIST2975 and carbon black Printex90 (CB) nanoparticles as well as

available data on NIST1650 [23] were included in the study for comparison.

Table 1. Overview of samples

Particle type Measurement

Exposure

characterization

Instruments/Method

1 ELPI

4 DISCminis

1 NanoScan

1 OPC

Micro INertial Impactor

(MINI)

Micro INertial Impactor

(MINI)

Electrostatic

precipitator

Relevant figures

Figure 1:

Table 2:

Additional File S1 A:

Additional File S1 B:

Exposure characterization

Exposures and doses

Position of instruments Jet

engine test facility

Non-

commercial

airfield particles

(JEP)

Background

characterization

Emission

characterization

Particle collection for

physical and chemical

characterization and

mouse instillations

Results not shown

Additional File S1 C:

Table 3:

Table 4:

Description of impacted

aerosols and TEM images

PAH contents

Metal contents

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JEP particles

suspended in

instillation vehicle

TEM (dropcast)

Table 5:

Additional File S1 D:

Figure 2:

Additional File S1E:

Figure 1:

Additional File S1 A:

Additional File S1 C:

Table 3:

Table 4:

Exposure

characterization

Emission

characterization

Particle collection for

physical and chemical

characterization and

mouse instillations

CAP particles

suspended in

instillation vehicle

4 DISCminis

1 NanoScan

1 OPC

Micro INertial Impactor

(MINI)

Electrostatic

precipitator

Size distribution

DLS figures

SEM images

Elemental composition by

EDS analysis

Exposure characterization

Position of instruments

Description of impacted

aerosols and TEM images

PAH contents

Metal contents

Commercial

airport particles

(CAP)

TEM, dropcast

Table 5:

Additional File S1 D:

Figure 2:

Additional File S1 E:

Figure 3:

Table 6:

Figure 4:

Figure 5:

Additional File S2 A:

Lung pathology

Cellular composition in

the lungs

Mouse

instillations of

JEP and CAP

Serum Amyloid A

levels in tissues

DNA damage

Histology

Broncho-alveolar

lavage (BAL)

mRNA expression

DNA strand breaks

(Comet Assay)

Figure 6:

Additional File S2 B:

Figure 7:

Additional File S2C:

Size distribution

DLS figures

SEM images

Elemental composition by

EDS analysis

Histopathology of lung

sections

BAL fluid cell composition

Dose-response relationship

of instilled particles

Neutrophil influx

Scatter plots of cellular influx

Eosinophil influx

SAA day 1

SAA day 28 and 90

Tail Length

% DNA in Tail and data table

Results

Aerosols

Particle exposure characterization at a non-commercial airfield

Two full cycles representative of a normal workflow of Plane Leaving (PL), Plane Arriving (PA)

and refueling by a Fuel Truck (FT) were recorded in a jet shelter using both stationary and portable

devices (see Additional File S1 A for outline). During the main combustion events of PL and PA,

the instruments reached their upper detection limits of 10

(DiSCmini) and 10

(ELPI)

particles/cm

. Importantly, this included the breathing zone monitor of the airfield personnel.

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Overall, but especially in main peaks, the ELPI detected mainly particles under 500 nm (Fig. 1A).

The number size distributions during PL, PA, and FT suggested that the prevalent particle sizes

were probably below the detection limit of the ELPI, suggesting that the jet engine combustion

particles are below 10 nm in aerodynamic diameter (Fig. 1B). This was similar to the particle

number and size distributions in measurements of jet engine exhaust conducted in a jet engine test

facility (see Additional File S1 B). In the size-resolved mass distributions for PL, PA, and FT, there

was a mode around 150-200 nm and the remaining mass was allocated with larger particle sizes up

to the detection limit of 10 µm (Fig. 1B). The particle concentrations measured by the four

DiSCmini devices followed the same event-specific trends with a slightly lower background signal

for the personal monitor. The two events of PA (large peak) and FT arrival (subsequent shoulder)

were not fully discernable and have therefore been combined into a single event in the analysis. The

event-related air concentrations and the corresponding predicted lung deposition are shown in Table

Table 2. Average exposures and doses of jetfighter personnel at a non-commercial airfield

Event

PM4

Particles Mass

[min] ×10

[µg

×10

[µg

[×10

[µg]/

[cm

-3

]

-3

]

[min

HA,

TB,

AL,

min

-1

] HA,

TB,

AL,

Event

]

n[%] n[%] n[%]

m[%] m[%] m[%]

15.1

7.7 1086

537

15 21.2 27.2 51.6

18.7 84.6

4.7 10.7

2.26

280

PA+FT

21.3 2.67

410

228

5.4 21.7 27.7 50.7

7 83.6

4.9 11.5

1.15

150

PM4

170 1.22

194

2.4 21.4 27.4 51.3

3.5 85.8

4.6

9.6

4.12

600

Average exposures and doses during Plane Leaving (PL), Plane Arrival and fueling the plane (PA+FT combined), and over one

flight cycle (t

PM4

). From left to right: average event time (t) in minutes, average particle number concentration (n), mass

concentration (m) and mass fraction smaller than 4 µm (m

PM4

), inhaled number dose per minute (DR

), predicted fraction of

particles deposited in extra-thoracic (HA), tracheo-bronchial (TB) and alveolar (AL) lung regions, inhaled mass dose per minute

(DRm), predicted fraction of mass deposited in extra-thoracic (HA), tracheo-bronchial (TB) and alveolar (AL) lung regions, total

particles per event and total mass per event.

Collected samples of jet engine particles (JEP) from a non-commercial airfield

One JEP impactor sample was acquired when no jetfighters were running and another sample was

collected near a running jet fighter in taxi, each with an electron microscopy (EM) grid installed on

all three stages. The low number density observed on the grids from the background sample even

after 60 seconds of sampling suggested that the background aerosol contained very few particles

(results not shown), and therefore could be ignored when analyzing the take-off sample, which was

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collected for 5 seconds. The EM grids from the first and second stage of the take-off sample were

densely populated with highly agglomerated soot particles ranging from approximately 500 nm to

tens of micrometers in equivalent circular diameter (ECD). The primary soot particles were in the

order of 10 to 30 nm and displayed a typical soot structure with fringes of graphene like flakes (see

Additional File S1 C for detailed description and EM images). Due to the high particle loadings on

the grids, it was not possible to determine whether the large soot agglomerates were a result of co-

deposition during sampling, or whether they were airborne as agglomerates.

Particle exposure characterization at a commercial airport

The average DiSCmini geometric mean particle concentration and lung deposited surface area

(LDSA) were 2.2×10

-3

(Geometric Standard Deviation (GSD) 3.6) and 24.1 cm

-3

(GSD 2.6)

over the measurement period, respectively . High GSD was caused by high variation in

concentration levels (Fig. 1C). According to the NanoScan, the particles were mainly below 300 nm

in diameter and distributed in two modes with geometric mean diameters of <20 nm and

approximately 140 nm. The measured respirable mass concentrations were all below detection

limits, which corresponded to concentration levels of <66 µg/m

when an aircraft engine was

running close by, <18.6 µg/m

when there was no engines running in close vicinity, and <14 µg/m

when sampled over the measurement day from 10:27 am to 3:00 pm.

Collected samples of commercial airport particles (CAP)

A single CAP impactor sample was collected for 30 seconds at the apron of the commercial airport

(see Additional File S1 A for placement). The first stage contained many micrometer-sized particles

ranging between 1 and 50 µm. The particles were mainly dominated by rectangular or square salt

crystals and a few micrometer-sized particles, which appeared to be pollen. The second stage

contained only very few particles, which were in the size range between 500 nm and 1 µm in ECD.

The last stage of the impactor displayed an area covering approximately 12 grid squares, which was

densely populated with particles. Particle sizes varied from approximately 1 µm to a few nm in

ECD. Soot particles were found in three different states: as free, individual agglomerates, as well as

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agglomerated to other particles (e.g. larger particles, salts, and others) and associated with or

captured in droplets (see Additional File S1 C for detailed description and EM images).

Consequently, the aerosol at the non-commercial airfield appeared to be mainly aggregates of nano-

sized carbon particles (soot), whereas the aerosol at the apron of the commercial airport appeared

much more complex dominated by agglomerated soot particles, salt crystals, and low volatile

compounds.

Physicochemical characterization of particles for mouse instillation

From electrostatic precipitator (ESP) sampling [24, 25, 26, 27, 28, 29] at the jet shelter during a

time span of approximately 15 hours, 11.7 mg of JEP were collected and at the commercial airport

during 4 hours and 40 minutes, 12.3 mg particles of CAP were collected.

Contents of polycyclic aromatic hydrocarbons (PAH)

Analysis of the content of polycyclic aromatic hydrocarbons (PAH), showed

∑PAH

concentrations

(sum of 16 PAH (Table 2), ND=0) of 0.081 mg/g in CAP and 0.05 mg/g in JEP, respectively,

including contents of benzo(a)pyrene (Table 3). The PAH profiles of JEP and CAP were roughly

similar. For comparison, NIST1650 and NIST2975 contained 0.22 and 0.086 mg/g, respectively, of

the same PAHs.

Table 3. Content of 16 PAH in airport-collected particles.

PAH

Naphthalene

Acenaphthylene

Acenaphthene

Fluorene

Phenanthrene

Anthracene

Fluoranthene

Pyrene

Benz(a)anthracene

Chrysene

Benzo(b)fluoranthene +

Benzo(k)fluoranthene

Benzo(a)pyrene*

Dibenz(a.h)anthracene

CAP mg/g particles

0.009(0.0009)

0.001(0.00007)

0.008(0.0005)

0.008(0.00007)

0.04(0.0007)

0.01(0.0009)

0.005(0.0004)

JEP mg/g particles

0.01(0.002)

0.001(0.0002)

0.001(0.00008)

0.001

0.001(0.00008)

0.007(0.00007)

0.02

0.009(0.0004)

NIST1650B

(mg/g)

NIST2975

(mg/g)

0.007(0.0004)

0.001(0.00004)

0.0002(0.00002)

0.001(0.00004)

0.07(0.004)

0.008(0.0004)

0.05(0.001)

0.04(0.001)

0.006(0.0004)

0.01(0.0006)

0.009(0.0009)

0.001(0.0001)

0.0004(0.00008)

0.004(0.0001)

0.0005(0.00003)

0.003(0.0002)

0.02(0,0003)

0.00005(0.000002)

0.03(0.0005)

0.002(0.0002)

0.001(0.00004)

0.006(0.0001)

0.01(0.003)

0.0008(0.00004)

0.0005(0.00005)

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Ideno(1.2.3-cd)pyrene

0.004(0.0002)

0.002(0.0001)

Benzo(g.h.i)perylene

0.006(0.0003)

0.002(0.00009)

∑PAH

0.081

0.05

0.22

0.086

PAH was measured by GC-MS and listed as blank corrected mean values (N=2) with standard deviation in parenthesis. The PAH

were extracted with cyclohexane from the two water suspensions of each particle used for the instillation in mice. ND=Not Detected.

The highest concentrations given in the Certificate of Analysis measured by several different methods and the associated expanded

uncertainty given in parenthesis.

For NIST2975 the value is for Dibenz[a,h + a,c]anthracene

Metal contents

Semi-quantitative analysis of elemental contents by inductive coupled plasma mass spectrometry

(ICP-MS) detected metals in both JEP and CAP, including lead, cobalt, nickel, arsenic, cadmium

and mercury (Table 4). The metal content profiles for JEP, CAP, and NIST2975 were generally

similar, but the CAP sample had the overall highest metal contents. Noteworthy, CAP contained

more than three times higher concentrations of Mg, Al, Cu, Zn, Sr and Pb than JEP and NIST2975.

NIST2975 contained more Zn than JEP. No metal content was detected in CB.

Table 4. Extracted elements from analysis of 4 mg of jet engine particles (JEP ) and

particles from a commercial airport (CAP).

Ref. NIST2975

Ref. CB

JEP

CAP

NIST 2975

1/ND

3/ND

950

8,655

291/281

ND /ND

3,057

9,735

203/0

134

2,788

200

1,147

7,433

100

146

125

5,386

249

14,884

31,897

427

103

658

5/1

90/102

11/11

814/743

7/8

55/65

24/5

13,926/17,003

1/2

ND /2

8/1

4/ND

97/105

1 /ND

498/-

0/-

13/3

-/1

2/1

3/3

0.0±0.0

0.0±13

0.1±0.1

0.5±0.7

0.9±0.6

16±4

<10

<0.4

<0.2

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1/1

Elemental concentrations are shown in units of µg/g particle (ND = not detectable). Blank concentrations

were subtracted. NIST2975 and CB were analyzed in duplicates (separated by slash).

Reference values

from Ball et al. (2000) [30] (the study only analyzed Co, Cu, Fe, Ni, V, and Zn). Note that we extracted for

significantly longer time (several days vs. overnight) and with 25% nitric acid instead of 0.1 M phosphate

buffer.

Reference values from the MAK‐Collection for Occupational Health and Safety (written

communication of unpublished data of Degussa) [31].

Particle size distribution in dispersion

All particles were dispersed in Nanopure water and sonicated to obtain stable dispersions [32]. The

hydrodynamic number size distribution and intensity were measured by Dynamic Light Scattering

(DLS) for particle concentrations of 3.24 mg/ml, 1.08 mg/ml, 0.36 mg/ml and 0.12 mg/ml,

corresponding to 162, 54, 18 and 6 µg particulate matter in 50 µL instillation volume per mouse.

The average hydrodynamic particle zeta-size (Z

ave

) varied from 136-269 nm for CAP and from 143-

196 nm for JEP, depending on concentration (Table 5). CB and NIST2975 formed uniform

agglomerates of 50-60 nm, whereas JEP and CAP appeared more heterogeneous with particles in

the Z

ave

size range of 50-60 nm as well as larger aggregates resulting in poor poly dispersivity

indices (Table 5 and Additional File S1 D, fig. S1D).

Table 5. Size distribution in dispersion for collected airport particles, NIST2975 and carbon black Printex90

(CB).

Dose

6 µg

18 µg

54 µg

162 µg

ave

(d.nm)

CAP

JEP

NIST2975

136.04

143.50

N/A

PdI

0.57

0.42

N/A

ave

(d.nm)

168.67

142.68

126.40

PdI

0.54

0.35

0.15

ave

(d.nm)

269.00

196.03

138.52

PdI

0.57

0.45

0.23

ave

(d.nm)

N/A

136.62

PdI

N/A

0.22

N/A

148.74

0.28

N/A

All particles were dispersed in Nanopure water. Z-Average (intensity based harmonic mean) relates to particle sizes and

Polydispersity Index (PdI) relates to the distribution. N/A: Not applicable (doses not included in the study)

Electron microscopic analysis of dispersed particles used for mouse instillation

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In EM images, JEP appeared homogenous with small and larger

aggregates and/or agglomerates

primary soot particles (Fig. 2A-C). A few organic structures, likely pollen, were also observed

alongside large titanium particles (Fig. 2D and Additional File S1 E (1)), likely originating from the

titanium probe used for sonication. The estimated size of smaller particles forming larger JEP

aggregates and/or agglomerates

was approx. 45 nm. CAP appeared to be a more heterogenous

mixture of particles (Fig. 2F-H) that also contained large plant fibers and collapsed pollen grains

(Fig. 2I) along with smaller aggregates and/or agglomerates up to approx. 45 nm and silicates. In

correspondence with results from the metal analysis, the EDS showed a heterogenic mixture of

different metals and compounds, including silicon, titanium, iron, copper, magnesium, and zinc

(Additional File S1 E (2)). The agglomerated soot particles, pollen and other organic elements of

both JEP and CAP were decorated with silver (Ag) nanoparticles (Fig. 2E+J), which likely

originates from the ESP silver plates. NIST2975 particles appeared as smooth-looking large carbon

aggregates and/or agglomerates mixed with smaller fragments and clear metal reflections,

consisting of mainly titanium. Silicon, iron and sulfur were also abundant. The large aggregates

and/or agglomerates consisted of smaller similar-appearing particles or aggregates and/or

agglomerates, of approx. 45 nm (Additional File S1 D (3)).

In summary, both JEP and CAP dispersions consisted of small-sized aggregated carbon particles,

similar to standard diesel particles in size, shape, and chemical composition as measured by EDS.

The JEP particles in suspension appeared homogenous compared to the CAP suspension and

appeared to consist mainly of jet engine exhaust, whereas CAP suspension was more representative

of the complex occupational exposure at the apron of the commercial airport.

Pulmonary particle deposition and histopathology of exposed C57BL/6 mice

Female C57BL/6 mice were exposed to JEP, CAP, NIST2975, and CB by single intratracheal

instillation at different dose levels and followed for 1, 28, or 90 days.

Histopathological evaluation was performed on samples from mice exposed to 54µg JEP, 54µg

CAP, and 162µg NIST2975 on day 28 and day 90. The tissue samples showed heterogeneity

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between animals. Particles were not readily apparent in mice instilled with JEP particles and no

significant histological changes were detected on day 28 and 90 (Fig. 3A+B).

In mice instilled with CAP, some particles were visible in macrophages (Fig. 3C) and on one

occasion in a granuloma. Pronounced eosinophil infiltration and eosinophil vasculitis was observed

on day 28, characterized by infiltrates in the perivascular region and smooth muscle hyperplasia

(Fig. 3D+E). In the portal areas of the liver, eosinophilia was seen, most pronounced in mice

exposed to CAP (not shown). This was also present in some JEP-instilled mice and in some control

mice as well. Kidney and spleen were unaffected by exposure. NIST2975 lung sections had visible

particles and particle-loaded macrophages (Fig. 3F-H), along with modest inflammation-related

changes.

In summary, histological lung sections from day 28 and 90 post-exposure to airport particles

showed small remnants of particles, likely due to clearance and relocation, and the pronounced

degree of eosinophilic cell infiltrates especially in the CAP-instilled mice reflected the

heterogenetic nature of CAP including pollen and plant fibers, which are associated with

eosinophilic responses.

BAL fluid cell composition

BAL fluid cellular content was evaluated by total cell count and composition of inflammatory cell

subsets (Table 6, Additional File S2 A).

Table 6. BAL fluid cell composition on day 1, 28 and 90 post-exposure.

Day 1

Vehicle control

CB 54 µg

CAP 6 µg

CAP 18 µg

CAP 54 µg

JEP 6 µg

JEP 18 µg

JEP 54 µg

NIST2975 18 µg

Total cell count

56.43±6.42

144.80±16.23(****)

53.32±9.87

82.22±11.96

147.50±10.64(****)

(¤¤¤¤)(’’’)

66.37±21.58

91.02±9.67

160.50±17.40(****)

(¤¤¤¤)(’’’)

47.92±7.36

Neutrophils

2.84±0.89

100.11±11.85(****)

6.43±0.88

36.72±10.00(***)(¤)

101.09±11.07(****)

(¤¤¤¤)(’’’)

6.29±3.00

25.89±8.57(*)

110.88±14.66(****)

(¤¤¤¤)(’’’)

1.67±0.46

Macrophages

46.58±6.00

28.52±3.05

42.62±8.90

38.28±2.94

38.79±5.78

43.83±7.92

57.40±3.43(xx)

37.40±6.94

42.05±7.23

Eosinophils

1.16±0.79

9.96±2.58(***)

0.30±0.07

1.40±0.42

1.85±0.58(*)

(’’)

10.01±9.69

1.58±0.63

4.27±1.75(*)

(’’)

0.17±0.09

Lymphocytes

0.84±0.47

1.43±0.43

0.04±0.04

0.33±0.15

0.67±0.30

1.46±1.26

1.01±0.38

3.31±1.05(¤)

(’’’)

0.19±0.12

Epithelial

cells

5.00±1.59

3.85±0.78

3.94±1.65

5.49±1.60

5.10±1.24

4.77±1.40

5.15±1.40

4.65±0.97

2.68±0.51

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NIST2975 54 µg

NIST2975 162 µg

61.50±9.22

191.33±11.98(****)

87.55±7.706

72.238±8.993

177.375±16.756

Total cell count

51.10±3.89

75.63±13.40

50.20±7.92

60.23±8.02

48.85±9.20

51.47±11.08

61.75±7.01

46.43±8.56

60.67±7.71

50.37±6.07

81.85±8.40(*)

61.01±3.13

58.26±7.56

83.94±10.64

Total cell count

54.03±5.14

86.93±8.78(**)

62.75±4.30

50.90±7.07

48.42±7.09

25.57±5.82(*)

148.46±9.74(****)

10.94±2.78

12.07±5.46

120.54±11.80

Neutrophils

0.10±0.05

1.68±0.75(*)

1.19±0.84

1.05±0.68

0.10±0.07

0.13±0.08

0.27±0.12

0.68±0.38(*)

0.27±0.19

0.21±0.18

2.14±0.92(**)

0.44±0.22

0.18±0.07

1.86±0.83

Neutrophils

0.45±0.16

2.07±0.49(**)

0.92±0.33

0.92±0.47

0.46±0.18

31.28±3.61

32.55±4.13

62.91±4.77

50.56±3.23

48.92±5.75

Macrophages

47.09±3.89

66.83±12.12

42.50±5.05

46.83±2.83

39.18±6.33

49.07±10.53

63.01±4.84

40.18±7.73

54.98±7.61

43.35±6.67

70.53±7.43

46.46±2.98

45.83±5.68

61.86±6.29

Macrophages

45.23±4.39

73.33±6.72

56.68±4.52

42.69±5.78

45.11±6.94

1.05±0.41

5.07±2.26

0.36±0.13

1.03±0.98

1.23±0.49

Eosinophils

0.16±0.08

0.04±0.04

2.82±2.64

7.90±5.98

3.90±2.76

0.02±0.02

0.23±0.17

0.14±0.07

0.09±0.06

0.08±0.05

0.30±0.30

0.11±0.05

1.33±0.78

0.28±0.12

Eosinophils

0.98±0.91

0.09±0.09

0.10±0.10

0.14±0.07

0.00±0.00

0.93±0.27

1.77±0.59

0.09±0.09

0.10±0.07

0.21±0.15

Lymphocytes

0.19±0.08

3.20±0.77(*)

1.03±0.73

1.43±0.80

2.42±1.15(*)

0.26±0.09

0.59±0.25

0.87±0.37(*)

0.47±0.11

0.45±0.27

4.12±1.93(**)

0.60±0.12

1.27±0.54

4.01±1.25

Lymphocytes

3.57±3.39

4.70±1.79

1.30±0.82

1.74±1.40

0.38±0.21

3.48±0.75

4.78±0.93

NIST1650

18 µg

NIST1650

54 µg

NIST1650

162 µg

Day 28

Vehicle control

CB 54 µg

CAP 6 µg

CAP 18 µg

CAP 54 µg

JEP 6 µg

JEP 18 µg

JEP 54 µg

NIST2975 18 µg

NIST2975 54 µg

NIST2975 162 µg

NIST1650

18 µg

NIST1650

54 µg

NIST1650

162 µg

Day 90

Vehicle control

CB 54 µg

CAP 54 µg

JEP 54 µg

NIST2975 162 µg

Epithelial

cells

3.55±0.75

3.88±0.55

2.66±0.88

3.02±0.65

3.25±0.90

2.00±0.64

2.94±0.72

4.56±1.37

4.87±0.88

6.27±1.88

4.75±0.43

Epithelial

cells

3.80±0.74

6.75±1.58

3.74±0.39

5.42±1.51

2.47±0.44

NIST1650 data was included for comparison and obtained from a previously published study (Kyjovska et. al. Mutagenesis 2015)

P-value summary: (*)

–

(****) = p<0.05 - p < 0.0001 increase compared to

vehicle control,

(x)

–

(xxxx) = p<0.05 - p < 0.0001 increase

compared to

CB 54 µg,

(¤)

–

(¤¤¤¤) = p<0.05 - p < 0.0001 increase compared to

NIST2975

of same dose, (‘) – (‘’’’) = p<0.05

- p <

0.0001 increase compared to

NIST1650

of same dose. Data are shown as Mean ± SEM (x 10

BAL= broncho-alveloar lavage, CAP=commercial airport particles, JEP=jet engine particles, CB=carbon black Printex 90

BAL cells on day 1 post-exposure

On day 1 post-exposure, dose-response relationships were observed for JEP, CAP, and NIST2975

for total cell count, neutrophils, eosinophils, and lymphocytes. Significant linear trends were

verified for the observed dose-response relationships for neutrophils and total cell numbers (not

shown) with R-square values between 0.76 and 0.95 (Fig. 4).

Exposure to JEP and CAP at 18 and 54 µg, resulted in significantly increased neutrophil influx,

compared to vehicle control (JEP 18 µg: p=0.0215, JEP 54 µg: p<0.0001, CAP 18 µg: p=0.0008;

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CAP 54 µg: p=0.0001) (Fig. 5A). In addition, at 54 µg, JEP- and CAP-exposure induced

significant eosinophil influx, compared to vehicle control (JEP: p=0.0158, CAP 54 µg: p=0.0205)

(Additional File S2A (4)). By exclusion of statistically determined outliers (see In vivo data

statistics), this difference was further increased (JEP: p = 0.0011, CAP: p = 0.001) with an addition

of significance for 18 µg as well (JEP: p = 0.0422, CAP: p = 0.0139). By removal of outliers in

lymphocyte counts, there was an additional significant difference between JEP at 54 µg and vehicle

control (p = 0.0004) (see Additional File S2 A (1)). However, the results were qualitatively similar

with and without outliers.

Exposure to 54 µg CB significantly increased total cells (p<0.0001), neutrophils (p<0.0001) and

eosinophils (p=0.0002) compared to vehicle controls. NIST2975 instilled mice had significantly

increased cell numbers compared to vehicle for neutrophils at 54 µg (p=0.0299) and at 162 µg

(p<0.0001) (Fig. 5A). It was apparent that CB 54 µg, NIST 162 µg, and the two airport-collected

particles JEP and CAP at 54 µg induced similar responses when compared to vehicle control for

most of the assessed cell types, and that JEP and CAP responses were increased when compared to

same mass dose of NIST2975 and NIST1650b (Table 6). There was the expected dose-response

relationships between total deposited surface area for CB (182 m

/g for CB [33]), NIST2975 (91

/g) [15], NIST1650 (108 m

/g) [15] and neutrophil influx (Additional File S2A (4)).

BAL cells on day 28 post-exposure

On day 28 post-exposure, there was still a significant increase in neutrophil numbers compared to

vehicle controls for JEP at 54 µg (Fig. 5B), and a significantly increased number of lymphocytes for

both JEP and CAP at 54 µg dose level (JEP: p = 0.0328, CAP: p = 0.0223). Total cell count for

NIST2975 162 µg were still significantly increased compared to vehicle control (p = 0.0153) (Table

6). Neutrophil counts for CB and NIST2975 at 162 µg were still significantly increased compared

to vehicle control (CB: p = 0.446; NIST2975: p = 0.0068) (Fig. 5B). In addition, there was a

significant increase for lymphocytes (CB: p = 0.0228; NIST2975 162 µg: p = 0.0023) (Table 6). By

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removing statistically determined outliers, this difference was increased (CB: p = 0.0001;

NIST2975: p = 0.0031).

BAL cell on day 90 post-exposure

Mice from the highest dose groups were followed until day 90 post-exposure, and there was still

increased total cell counts (p = 0.0022) and neutrophils (0.0045) for CB, compared to vehicle

control mice (Fig. 5C, Table 6, and Additional File S2 A (3)).

In summary, both JEP and CAP particles induced high pulmonary inflammatory responses on day 1

post-exposure, similar or higher compared to same mass dose of NIST control particles and CB. On

day 28, there was still active inflammation in mice exposed to JEP and CB, and CB still induced

increased neutrophil influx on day 90.

Serum amyloid A

Serum amyloid (Saa) 3 (Saa3) mRNA in lung tissue and

Saa1

mRNA levels in liver tissue were

used as biomarkers of pulmonary [34] and hepatic [35] acute phase response, respectively. SAA3

protein was measured in plasma as biomarker of systemic acute phase response [35].

Saa

expression in lung and liver was measured on day 1, 28 and 90 post-exposure, and SAA3 in plasma

on day 1 and on day 28 for the highest particle doses.

Exposure to JEP, CAP and NIST2975 resulted in significant dose-dependent increases in

Saa3

mRNA levels in lung tissue compared to vehicle control mice on day 1 (CAP 18 µg: p=0.0151,

CAP 54 µg: p=<0.0001, JEP 54 µg: p=0.0038, NIST2975 54 µg: p=0.0008, NIST2975 162 µg:

p<0.0001) (Fig. 6A+B). CB induced a 447-fold

Saa3

mRNA level increase (p<0.0001) (Fig. 6), in

agreement with previous findings [36]. On day 90,

Saa3

mRNA levels in the CB-exposed group

were still increased compared to control (day 90: p=0.0192) (Additional File S2 B). On day 1, liver

Saa1

mRNA levels were significantly increased for JEP of 54 µg, compared to control (p=0.0415;

12-fold increase) and for NIST2975 of 162 µg (p=0.0025, 22-fold increase) (Fig. 6C and D). On

day 1 post-exposure, plasma SAA3 was increased for JEP 54 µg (p=0.0305) and for NIST2975 at

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162 µg (p=0.0205) (Fig. 6E). No significant differences were found for

Saa1

mRNA in liver tissue

or for SAA3 plasma protein level on day 28 (see Additional File S2 B).

Thus, JEP and CAP exposure induced dose-dependent pulmonary acute phase response on day 1

post-exposure that was paralleled by a systemic circulation of SAA3 protein for JEP. The acute

phase response had returned to baseline levels on 28 days post-exposure for JEP, CAP, and

NIST2975.

DNA damage

Genotoxicity was evaluated as DNA strand breaks in the comet assay, using comet tail length and

% tail DNA in BAL derived cells, lung cells and liver cells. Increased levels of DNA strand breaks

were occasionally observed across particles types, dose and time points, but no dose-response

relationships was observed (Fig. 7 and Additional File S2 C).

DNA damage on day 1 post-exposure

On day 1 post-exposure, increased DNA damage levels were observed for JEP and NIST 2975 at 18

µg as compared to vehicle control (JEP: p = 0.0132, NIST2975: p = 0.0304) for tail length in BAL

cells (Fig. 7A and Additional File S2 C).

DNA damage on day 28 post-exposure

On day 28, tail length and % tail DNA (see Additional File S2 C) in liver cells were increased

compared to vehicle control for CAP 6 µg (% tail DNA: p = 0.0151; tail length: p = 0.0214)

(Additional File S2 C and Fig. 7B).

DNA damage on day 90 post-exposure

On day 90, there were no significant differences compared to vehicle controls (Fig. 7C and

Additional File S2 C).

In summary, increased levels of DNA strand breaks were observed in single dose groups on day 1

and 28 post-exposure, with a pattern of most DNA damage in BAL cells for JEP and in liver cells

for CAP.

Discussion

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In this study, mice were exposed to particles collected at two different airport facilities and

compared to standard diesel particle NIST2975 and to published data on NIST1650. With ESP

collection, 11.7 mg of JEP were collected during a time span of approximately 15 hours and 12.3

mg particles of CAP were collected at the commercial airport during 4 hours and 40 minutes. JEP

and CAP both contained metals and PAH. Total PAH content was similar to the declared content of

NIST2975 and substantially lower than for NIST1650. The metal contents in the CAP and JEP were

considerably higher than for NIST2975.

The sizes, shapes and structures of the primary soot particles found predominantly in JEP and also

in CAP samples were very similar to those found in NIST2975 and to particles from previous

studies [37]. Thus, they likely have comparable surface area and physicochemical properties.

Inflammation

After intratracheal instillation in mice, both JEP and CAP particles produced highly increased influx

of inflammatory cells in BAL fluid on day 1 post-exposure, similar or higher compared to same

mass dose of NIST control particles and CB. On day 28, there was still influx of inflammatory cells

in BAL fluid in mice exposed to JEP and CB. Only CB still induced increased cellular responses on

day 90. We used water as vehicle for intratracheal instillation to ensure least amount of vehicle-

induced artefacts [32]. The inflammatory profile on day 1 post-exposure could potentially be partly

attributed to lipopolysaccharides (LPS) from the air and environment, however, the inflammation

was still present on day 28 post-exposure, which would not be expected from acute inflammation

mediated by organic material. As an example of the pulmonary response to organic material,

inflammation induced by pulmonary exposure to bulk cellulose was observed 1 day post-exposure,

but not 28 days post-exposure [38]. The histopathological evaluation of lung tissue showed limited

JEP and CAP-inflammatory changes 28 and 90 days post-exposure.

We did not collect sufficient material to determine BET surface area, and therefore, we could not

compare the inflammatory response induced by JEP and CAP with standard diesel particles and

CB-induced inflammation when normalized to surface area. However, we observed strong mass

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dose-dependency. The cytological changes in BAL fluid induced by CAP and JEP were remarkably

similar. Assuming that the combustion particles indeed have a diameter of 10 nm as our data

suggested, then the specific surface area of JEP and CAP would be at least similar to that of CB,

which has a diameter of 14 nm and BET of 182 m

/g [33]. The BET of NIST1650 and 2975 are 108

/g and 91 m

/g, respectively. We found dose-response relationships between total deposited

surface area for CB, NIST2975, NIST1650 and neutrophil influx. Thus, the observed stronger

inflammatory response, as determined by BAL, induced by JEP and CAP compared to NIST2975

would be consistent with the expected larger specific surface area of the smaller jet engine

combustion particles.

Acute phase response

Saa3

mRNA levels were used as biomarker of pulmonary acute phase response [34]. Particle-

induced dose-dependent pulmonary acute phase response was observed in parallel with the

neutrophil influx as previously reported for CB and NIST1650b [23, 34]. The hepatic acute phase

response evaluated with

Saa1

mRNA levels was much smaller than the pulmonary acute phase

response, as previously seen for NIST 2975 and CB [36, 39]. Systemic SAA3 levels were also

increased by JEP exposure at 54 µg, and by NIST2975 at the three fold higher dose 162 µg. SAA is

causally related to increased plaque progression [40] and SAA stimulates the formation of

macrophages into foam cells [41]. Increased levels of acute phase proteins SAA and C-reactive

protein (CRP) are associated with increased risk of cardiovascular disease in prospective

epidemiological studies [42]. Furthermore, inhalation of ZnO nanoparticles increased systemic

levels of CRP and SAA in human volunteers in a dose-dependent manner [43].

Genotoxicity

Increased levels of DNA strand breaks were observed with the Comet assay at single dose levels

across doses and post-exposure time points, with a pattern of most DNA damage in BAL cells for

JEP and in liver cells for CAP. BAL cells are not relevant cell types in relation to lung cancer, but

may be more homogeneously exposed to particles following IT exposure as compared to epithelial

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cells, even though we have previously documented that IT exposure result in exposure of all lung

lobes [2, 44]. The observed levels of DNA damage were overall low, but at the same level as for the

NIST diesel particles and CB [23]. We have previously validated our comet assay set up for in vivo

samples using chemical-induced DNA damage and found strong dose-response relationships in all

assessed tissues [45]. We have previously assessed DNA damage in BAL cells, lung and liver tissue

of mice after pulmonary exposure to many different nanomaterials [23, 25, 44, 46, 47, 48, 49, 50].

As previously discussed [23], we observe the same lack of dose-response relationship in the three

tissues in the majority of our studies. Instead of dose-response relationship, we generally observe

that particle exposure at all dose levels increases the level of DNA strand breaks with 50-100%, an

increase that will only be statistically significant in some cases depending on the variation in the

assay. The lack of dose-response relationship may indicate a maximal rate of particle-induced DNA

strand breaks was achieved already at low doses. This, in turn, could indicate that particle-induced

DNA strand breaks in the lung are formed by a mechanism that is fundamentally different from

chemically-induced DNA damage [23]. CAP exposure induced DNA strand breaks in liver tissue,

as previously observed for CB [36, 46]. We have recently shown the genotoxicity in liver following

pulmonary exposure to CB is likely caused by direct genotoxicity caused by surface-dependent

reactive-oxygen-species (ROS) generation of translocated particles [51]. Translocation from lung to

systemic circulation is very size-dependent, and consistent with this, the primary airport-collected

combustion particles were small (10-30 nm in diameter).

Metals and PAH

Both CAP and JEP contained toxic metals including lead, cobalt, nickel, arsenic, cadmium and

mercury, measured with ICP-MS. The content of Ag in JEP and CAP was likely attributed to

contaminations from the ESP silver plates. Our analysis of the reference particles NIST2975 and

CB were in overall agreement with the literature [24]. The discrepancy between the current study

and previously published values for NIST2975 [24, 25] may be caused by longer extraction times

and the use of 25% nitric acid instead of phosphate buffer.

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In our study, the

∑PAH

concentration was 0.081 mg/g in CAP and 0.05 mg/g in JEP, respectively.

In comparison, CB was previously shown to contain 0.000074 mg/g PAH [52], NIST2975 contains

0.086 mg/g and NIST1650b contains 0.22 mg/g of the 16 PAH according to NIST [15, 23].

However, based on two-year inhalation studies in rats, it was previously concluded that the

carcinogenic effect of diesel exhaust particles cannot be explained by the content of carcinogenic

PAH alone [12, 53]. Likewise, inhalation of carbon black nanoparticles was just as carcinogenic as

diesel exhaust in a two year inhalation study in rats, suggesting that the carbon core of the particles

contributes significantly to the carcinogenic effect of diesel particles [14]. In vitro, NIST1650 and

Printex 90 carbon black nanoparticles had similar mutagenic potential in the murine fibroblast cell

line FE-1 [52, 54]. Thus, even though CAP and JEP have similar PAH content as NIST2975, the

carbon particle core is likely an important driver of pulmonary toxicity as previously observed for

diesel particles and carbon black nanoparticles.

Histology and doses

JEP and CAP appeared different on EM images. CAP induced a higher eosinophil response

compared to JEP, reflecting the complex mixture of the commercial airport air with pollen and plant

fibers, compared to the more homogenous jet engine sample. Histological examination of lung and

liver tissue revealed eosinophilic pulmonary vasculitis in CAP-exposed mice, likely reflecting the

exposure to pollen grains, which can be associated with allergic response. This type of

histopathology was previously reported in association with asthma models in mice [55]. To the bset

of our knowledge this has previously not been reported in association with particle exposures. The

samples for histology were collected on day 28 and 90, and generally very few particle

agglomerates were observed in 54 µg JEP- and CAP-exposed mice, in contrast to mice exposed to

the 3-fold higher dose of 164 µg NIST2975 reference particles. The smallest retained dose seemed

to be in JEP-exposed mice, where in most cases no material could be detected. This could be due to

clearance of particles from lungs and liver before day 28, or because the JEP de-agglomerated in the

lung and single JEP were too small for detection by conventional microscopy.

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Distribution and human risk

Environmental ESP particle collection, extraction, dispersion, and instillation are all experimental

procedures that may modify the final deposited material in mice lungs as compared to occupational

inhalation exposure. The impactor EM images represent the mixed ambient air contents, but are not

necessarily a representative sample of aerosol contents over time, as the impactor efficiency varies

with particle size and only sampled over a short time period. The ESP collection method seems to

have contributed with additional silver (Ag) to the CAP and JEP suspensions instilled in the mice,

which were not present in the reference particles. However, the silver mass content was very low.

Titanium nanoparticles were also detected, likely originating from the sonication probe. The vehicle

control was also sonicated to account for this bias. High amounts of sea salt crystals were apparent

in the impactor sampling of CAP, reflecting close proximity to the sea. This might result in higher

particle CAP aerosol measurements. These salt crystals were absent in EM images of particles in

suspension, since the salt dissolves in the water used as vehicle. JEP appeared to have low

background levels, based on the low number densities on the impactor grids representing

background exposure. JEP impactor samples were in turn dominated by soot particles, representing

collection in the proximity of a running jet engine during taxi.

Occupational exposure tracking of JEP showed that the main combustion events of the jetfighter

(plane leaving and plane arriving) resulted in high exposure levels, including in the breathing zone

monitor of the airfield personnel. The average exposures and doses of one full cycle of 170 minutes

were measured to yield at least 4.12 x10

particles, where 9.6% were predicted to deposit in the

alveolar region of the lung. A comparison of all the DiSCmini event peaks (including breathing

zone) suggested that the shelter room air volume is continuously mixed and that the actual

geometrical measuring point is of less importance. Both the turbofan taking in large quantities of air

and the airflow exiting the jet engine nozzle are sufficient to drive the jet shelter ventilation. There

was a larger variation in DiSCmini signals in later stages of the second jetfighter occupational

cycle, which can be attributed to local activity in the sampling volume and to instrument drift after

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extensive measuring time. In the current study, event-dependent air concentrations of up to 1000

µg/m

were measured. Based on the size distribution data in the exposure measurements and

assuming 1.8 L/h ventilation for mice [56], the estimated alveolar deposited dose for a mouse at

1000 µg/m

for an 8-hour workday would be:

(1000 µg/m

) x (8 h) x (1.8 L/h) x 0.096 = 1.38 µg deposited material/8 h workday.

The mice were instilled with the collected particles at doses 6, 18 and 54 µg. We therefore estimate

that the lowest dose of 6 µg and the highest dose of 54 µg JEP and CAP in this study equals to 4

and 39 workdays, respectively.

The physicochemical characterization of JEP suggests that JEP are comparable to the standard

diesel particles and carbon black Printex 90 (CB in this study). The inflammatory and genotoxic

responses following pulmonary exposure to JEP were similar to standard diesel particles and CB.

The biological response following pulmonary exposure to CAP was very similar to JEP even

though CAP appeared more heterogeneous on EM images. This was seen as pronounced

eosinophilic cell infiltrates in CAP-instilled mice, reflecting the contents of organic material

including pollen and plant fibers, which are associated with eosinophilic responses.

In a recent meta-analysis of the association between occupational exposure to diesel exhaust and

lung cancer, it was estimated that occupational exposure to 1 µg/m

diesel exhaust particles

measured as elemental carbon would induce 17 excess lung cancer cases per 10,000 exposed

humans [11]. This warrants continuous research in reduction of particle emissions and diesel engine

refinements, to ensure more efficient combustion to reduce particles both in diesel-origin emissions

and in jet engines. Given the results in this study and further resemblance between JEP and diesel

exhaust particles as well as the dose-response relationship between diesel exhaust exposure and

lung cancer, the observed occupational exposure to jet engine emissions at the two airfields should

be minimized.

Conclusions

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In conclusion, we collected particulate matter from the ambient air at two different airport facilities,

a non-commercial airfield (JEP) and a commercial airport (CAP). The physicochemical

characterization showed that JEP were primarily agglomerated carbon nanoparticles with levels of

metals and PAH comparable to those found in the standard diesel particles NIST2975 and

NIST1650. CAP was more heterogeneous and contained large organic particles, agglomerated

carbon nanoparticles and condensed volatile organic compounds and was representative of the

complex occupational exposure on the apron of a commercial airport. Pulmonary exposure to JEP

and CAP induced acute phase responses as well as time and dose-dependent cytological changes in

BAL cell composition, which were similar to the responses observed for NIST2975 and CB, and to

previously published results for NIST1650. JEP, CAP and NIST2975 induced increased levels of

DNA strand breaks across doses and time points. Our study suggests that jet engine particles have

similar physicochemical properties and toxicity as diesel exhaust particles.

Methods

Particle collection, characterization and preparation

See Table 1 for an overview of measurements and instruments.

Non-commercial airfield particle exposure measurements

Sampling stations were placed in a jet shelter of 4721 m

(see Additional File S1 A) to measure the

airborne particle concentrations in the near field, far field and in the breathing zone of the flight

personnel. In order to track occupational exposure, two full cycles representative of a normal

workflow were observed of Plane Leaving (PL), Plane Arriving (PA) and refueling by a Fuel Truck

(FT).

Measurement strategy

Real-time particle monitoring was performed with an Electrical Low Pressure Impactor (ELPI,

Dekati model ELPI+, Dekati Ltd., Tampere, Finland) and four DiSCmini (Matter Aerosol AG,

Wohlen, Switzerland) deployed at several locations

–

ELPI at position 1 and DiSCmini at positions

2-4 and P (personal breathing zone) (Additional File S1 A). DiSCmini is a compact and portable

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instrument that measures particle number concentration, mean particle size and lung-deposited

surface area (LDSA) [57]. LDSA is correspond to lung deposited surface area of particles in size

range of ca. 20 to 400 nm for males during light exercise [58]. This method has high uncertainties,

which are discussed in details by Koivisto et al. [59]. ELPI collects and classifies particles in a

cascade impactor system according to aerodynamic mobility [60]. By combining these two

instruments airborne particles with diameters from approx. 6 nm to 10 µm can be characterized with

a detection emphasis on nanoparticles (DiSCmini optimum range is 10-700 nm) and particle

concentrations up to 10

particles/cm

for DiSCmini and up to 10

particles/cm

for the ELPI.

Particle number to mass conversion

Particle number size distributions measured by the ELPI were converted to mass distributions by

assuming that particles effective density is equal to nonvolatile effective particle density measured

from a CFM56-5B4/2P turbine engine [61]. The size dependent relation given by Johnson et al. [55]

for CFM56-5B4/2P turbine engine is

��

.9 × ��

��

−

[kg m

-3

]

The respirable mass distribution (mPM4) was calculated by multiplying the particle mass size

distribution by the simplified respirable fraction penetration efficiency according to Hinds [62].

Calculating deposited dose of inhaled particles

Particle deposition rates were calculated from particle concentrations measured by the ELPI.

Particle concentrations were multiplied with the simplified ICRP [63] human respiratory tract

deposition probabilities for the upper airways, the tracheobronchial region, and the alveolar region

[62]. The respiratory minute volume was assumed to be 25 L/min, which corresponds to the typical

respiration rate of a 70 kg male during light exercise (dose rates are described in detail elsewhere

[64]).

Commercial airport measurements

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Particle concentrations were measured using four DiSCminis and a NanoScan (TSI NanoScan

model 3091, TSI Inc., Shoreview, MN, USA) for particles from 10 to 420 nm in 60 second intervals

(Additional File S1 A).

Impactor collection

Aerosol samples were collected at a non-commercial airfield and commercial airport using a three

stage cascade impactor, referred to as the Micro INertial Impactor or MINI [65]. A diaphragm gas

pump model NMP 830 (KNF Neuberger, Germany) was used to generate the flow through the

MINI, resulting in a flow rate of 0.76 L/min. At ambient conditions this gives theoretical cut-off

diameters of 1.36, 0.59, and 0.055 μm

[66]. Each stage of the MINI can be equipped with TEM

grids, allowing particle collection directly onto microscope-suited surfaces. Here the stages were

equipped with 400 mesh nickel TEM grids coated with a 10 nm Formvar substrate with 1 nm

carbon deposited on top (Electron Microscopy Sciences, USA). Nickel grids were chosen as they

are magnetic, thereby allowing them to be held in place with weak magnets, which were inserted

into the impactor stages from the bottom. This ensured minimal movement of the grids during

sampling.

Particle collection for physical and chemical characterization and mouse instillations

Respirable dust (PM4; particles below 4 µm in diameter, see definition from the European

Committee for Standardization [67]) was collected using three sampling cyclones (BGI Model

GK2.69, BGI Inc., Waltham, MA, USA) at volume flow of 4.2 L/min on 37 mm PTFE filters with a

0.8 μm pore size (Millipore, Billerica, MA, USA). The collections were 1) with a running jet

engine, and 2) when there was no jet engines on in close vicinity, and 3) sampled over the

measurement day. Particles for suspensions were collected by a commercial electrostatic

precipitator (ESP) without using a prefilter, originally characterized by Sharma et al. [24], and

previously used for sampling in a range of particle exposure studies [25, 26]. The collected particles

were freeze dried for further processing.

Electron Microscopy

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The particles were visualized and characterized by electron microscopy, both from direct impactor

collection and in suspension following ESP collection.

The impactor samples were analyzed with a Nova NanoSEM 600 (FEI, The Netherlands), equipped

with an OPTIMUS TKD detector (Bruker, Germany), functioning as a scanning transmission

electron microscopy (STEM) detector. The SEM was operated in high vacuum mode with

acceleration voltages of 10-20 keV, a probe current of 12 nA, and at magnifications varying

between 5k and 40k, corresponding to resolutions of 15 to 2 nm/pixel respectively. The Esprit

software (Bruker, Germany) was used for automated analysis of the samples, where an imaging

pattern was defined to cover an entire square of the TEM grid. The square chosen for analysis was

situated directly under the impactor orifice and therefore displayed a high particle number density.

Once the imaging routine is setup the software automatically acquire the images, segments them

using a mean adaptive threshold technique, and performs subsequent energy dispersive x-ray (EDS)

analysis on recognized particles larger than a given size criteria. For these samples the minimum

particle size accepted for EDS analysis was set to 200 nm, as smaller particles were found to give

limited x-ray counts. Exposure times for the EDS analysis was set to 30 seconds. Particles touching

the image borders were discarded, as well as particles with equivalent circular diameters (ECD)

smaller than 50 nm. The size criteria were necessary to minimize the number of misclassified

substrate artefacts, which sometimes occurred during the automated analysis.

The impactor samples from the lowest stage were also analyzed at higher magnification using a

Tecnai T20 G2 (FEI, Netherlands) TEM microscope. The TEM was operated in high vacuum mode,

at an acceleration voltage of 200 keV, and with a probe current of 38 nA. In the TEM resolutions up

to 0.02 nm/pixel were achieved, allowing visualization of the onion like structure of collected soot

particles. In order to determine primary particle sizes of agglomerates the TEM images were

analyzed manually with the open source image analysis program ImageJ (https://imagej.net/Citing).

Particles in suspension were analyzed by field emission scanning electron microscopy SEM-EDX

(ULTRA-55, Carl Zeiss NTS GmbH, Oberkochen, Germany) equipped with an energy dispersive

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X-ray spectroscopy system (Oxford X-Max 50 mm2, Oxford Instruments, Oxfordshire, UK). The

particles were filtered onto Nucleopore Membranes with a hole size of 0.1 µm and hereafter carbon-

coated by carbon thread evaporation. SEM images were acquired at magnifications between 100-

50.000X and high tension at 5 and 20 kV. Detectors used were SE2, InLens and RBSD for options

of visualizing surface topology, high resolution details, or material contrast. Identification of

elemental composition identification was carried out with x-ray spectra acquired at 20 kV with a

live time of 30 seconds.

Positive control particles

Carbon black Printex90 used as benchmark particle with previously well-characterized properties

[16, 52, 68] was provided by Evonik Degussa GmbH (Frankfurt, Germany). Benchmark diesel

particle SRM 2975 (referred to as NIST2975) was obtained from the National Institute of Standards

and Technology (Gaithersburg, MD, USA). The certificate of analysis is available at

http://www.nist.gov.

Dynamic Light Scattering

Particles were dispersed in nanopure water. Hydrodynamic size distributions in particle-suspensions

were analyzed by Dynamic Light Scattering (DLS), on a Malvern Zetasizer Nano ZS (Malvern

Instruments Ltd., UK). The distributions were determined directly in the instillation solutions in 1

ml polystyrene cuvettes at 25° C. Six repeated measurements on the same sample were carried out

and averaged. For the calculation of hydrodynamic size, the refractive (R

) and absorption indices

) of carbon black Printex90 of 2.020 and 2000 were applied for all particles, with standard

optical and viscosity properties for H

PAH contents

PAH contents were evaluated by GC-MS and extracted with cyclohexane from the Nanopure water

suspensions of each particle [69].

Metal contents

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Sample preparation:

As it was not possible to transfer the amount of 4 mg airport particle matter

from the collection flasks to vials for microwave-assisted acid digestion, a volume of 1 mL of 25 %

(v/v) nitric acid was directly added to the flasks for acid extraction. Additionally, NIST2975 and

CB were included in the analysis. For the preparation of these samples, approximately 1 mg of

material were weighed into 13 mL polypropylene tubes (Sarstedt, Nümbrecht, Germany) and 1 mL

of 25 % (v/v) nitric acid added. All samples very gently agitated for 30 min to assure the dispersion

of the particles. Afterwards, the flasks and tubes were transferred to a shaker (Stuart Scientific SF1)

and agitated at 600 oscillations per min for 30 min. After incubation for approximately 72 hours at

room temperature without agitation, the samples were placed in the shaker for another 24 hours and

finally transferred with 6 mL of ultrapure water into polypropylene tubes. An empty flask (same

type as used to collect the airport particles) and polypropylene tubes (as used for NIST2975 and

CB) were treated in the same way as the samples to obtain suitable blank solutions.

Analysis:

Before analysis, the samples were centrifuged for 5 min at 4500 x g (Heraeus Multifuge

X3 FR, Thermo Scientific), because no complete digestion of the particles was achieved. A volume

of 5 mL of the supernatant was transferred to a new polypropylene tube and 0.05 mL of 100 ng/mL

rhodium (Rh) solution added as internal standard. The samples were further diluted 5- or 100-fold

with 5 % nitric acid. A triple quadrupole inductive coupled plasma mass spectrometer (ICP-MS)

(Agilent 8900 ICP-QQQ, Santa Clara, USA) equipped with a MicroMist borosilicate glass

concentric nebulizer and a Scott type double-pass water-cooled spray chamber was run in no gas

(Cd, Hg, Pb, Bi, U) or helium (remaining elements) mode with 0.1 - 3 s integration time per mass.

The following plasma parameters were used: 1550 W RF power, 15 L min

−1

plasma gas, 0.9 L

min

−1

auxiliary gas and 0.99 L min

−1

nebulizer gas. The cell gas flow in helium mode was 5 mL

min

−1

. The auto sampler (SPS4, Agilent Technologies) introduced the samples into the ICP-MS

with a sample uptake time of 30 s (0.5 rps) and a stabilization time of 30 s (0.1 rps). Quantification

was performed based on external calibration (multi-element standards of 5, 10, 25, 50 and 100 µg L

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; for mercury 0.5, 1.0, 2.5, 5.0 and 10 µg L

-1

) with internal standardization (1 µg L

-1

Rh). As

quality control, a mixture of 1 µg L

-1

Li, Ba, Bi, V and As was analyzed.

Mice

A total of 212 female C57BL/6Tac mice 7 weeks old at arrival (BW at instillation: 19±1.1) were

used in this study. The mice were group-housed in standard cages with 6-8 mice with ad libitum

access to tap water and Altromin 1324 rodent diet, and provided with saw dust bedding, mouse

house, wooden chew blocks and Enviro Dri nesting material. The mice were kept at 21±1°C and

50±10% humidity and a 12 hour light-dark circle.

Study design

After one week of acclimatization, mice were exposed to a single dose of collected particles of

either 6 µg, 18 µg or 54 µg per mouse by intratracheal instillation (6-8 mice per dose per particle

exposure) in three different exposure series. For each euthanization date, all vehicle control mice

were pooled together into one control group: e.g. for day 90 exposures there were six different

euthanization dates, hence there were in total 12 vehicle control mice. Across doses and time

points, 52 mice were used for JEP, 51 mice for CAP, 50 mice for NIST2975, 18 mice for CB, and

41 vehicle control mice. On day 28 and day 90, five of these mice per treatment were used

separately for histology (no histology was performed on CB instilled mice).

Instillation procedure

JEP, CAP, NIST2975, and CB were prepared as previously described [23]. Briefly, particles were

suspended in Nanopure Diamond Water and sonicated for 16 minutes using a Branson Sonifier S-

450D (Branson Ultrasonics Corp, Danbury, CT, USA). The suspensions were diluted and the

dilutions were re-sonicated for 2 minutes. Nanopure Diamond Water was prepared similarly as

vehicle. All solutions were freshly prepared and instilled within 1 hour.

Instillation procedure was carried out essentially as described by others [70]. Intratracheal

instillation procedure: A syringe was prepared with correct instillation dose in 50 µl vehicle located

at the top and 200 µl air located after the instillation volume, to ensure maximum delivery into the

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lung. One cage of mice was simultaneously placed in an anesthesia box, and induced with 4%

isoflurane and subsequently maintained at 2.5 % isoflurane. In preparation for instillation, one

mouse at a time was fixated by the front teeth in a customized fixation bracket on a 40‐degree

sloped platform with back support. A diode light was placed at the larynx visualizing the breathing

pattern. With a blunt non-harmful forceps the tongue was grabbed and pressed towards the lower

jaw by a small spatula in the opposite hand, to expose the pharynx. The trachea was then intubated

using a 24‐gauge BD Insyte catheter (Ref: 381212, Becton Dickinson, Brøndby, Denmark) with a

shortened needle. Upon placement of the catheter, the spatula holding the pharynx was removed. To

ensure correct location of the catheter, a small but highly sensitive pressure transducer was placed at

the top of the catheter (developed by our laboratory in collaboration with John Frederiksen (FFE/P,

Copenhagen, Denmark). When the catheter was correctly placed, this was indicated by a clicking

sound triggered by the pressure variation as air was inhaled and exhaled, and the mouse was

instilled. The catheter and syringe was removed, and the mouse was carefully shaken

head-down

twice, fully cupped and secured in one hand, to ensure confinement of the instilled material in the

lungs and spreading downwards towards the alveoli. The mouse was then returned to its home cage,

placed on a heating plate, to ensure optimal recovery from anesthesia. The entire procedure took <1

minute per mouse. The mice were weighed afterwards. The mice were all observed and evaluated

for signs of discomfort immediately after anesthetic seponation, and evaluated frequently until

euthanization, by visual inspections and body weight monitoring. Humane endpoints were weight

loss of maximum 20%, clear signs of discomfort such as ruffled fur, isolation, facial pain

expression, and changed respiration.

Organ harvest and preparation

For bronchoalveolar lavage (BAL), the mice were anesthetized with 25 mg/ml tiletamin and 25

mg/ml zolazepam (Zoletil™

Vet. 250 mg, Virbac), xylaxin (Rompun™ Vet. 20 mg/ml, Bayer), and

fentanyl 50 mg/ml in sterile saline. The lungs were flushed twice with 1 ml sterile saline per flush to

obtain BAL fluid. BAL fluid was kept on ice and centrifuged at 400 G at 4 °C for 10 minutes within

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1 hour. The supernatant was allocated into smaller lots, snap-frozen and stored at -80 °C for further

processing. The BAL cell pellet was further processed for automated total cell count

(NucleoCounter NC-200TM,

Chemometec, Denmark) following manufacturer’s protocol, and

manual differential count of inflammatory cell subsets, or further processed for Comet assay. The

still sedated mice were euthanized by heart-puncture and blood was collected in EDTA tubes and

plasma was stored at -80 °C. Lung and liver tissue were harvested for extraction of RNA, mRNA

expression, genotoxicity determination by comet assay, and histopathology for which kidney and

spleen were harvested as well. BAL samples and samples for Comet assay were prepared and

analyzed as previously described [23, 46]. Saa mRNA (Taq-Man Reverse Transcriptation Reagent

Kit and RTqPCR on ViiA

7, ThermoFischer Scientific, Denmark) and SAA3 plasma protein

(Mouse SAA-3 ELISA, EZMSAA3-12K, Merck Millipore, Denmark; Epoch

microplate

spectrophotometer, BioTek, Winooski, USA) were prepared and measured according to

manufacturer’s protocols

and lung and liver tissue was prepared and dyed for histopathological

examination, as previously described [46].

In vivo data statistics

Statistical analysis was performed in GraphPad Prism (GraphPad Prism, version 7.03 for Windows,

GraphPad Software, La Jolla California USA, www.graphpad.com). Data was assessed for

normality, variation and outliers by inspection of scatter plots and by statistical evaluation (Brown-

Forsythe F-test for variance and ROUT for outliers, provided by GraphPad Prism). Serum Amyloid

A data was log2 transformed to achieve equal variance and normalization. Due to abundance in

values equal to zero, log transformations were not applicable for BAL data. Due to sample sizes,

outliers were included and depicted on figures; however, data was analyzed with and without

outliers and reported in-text if deviant. Data following the Gaussian distribution and equal variance

assumptions was analyzed by one-way

ANOVA followed by Dunnett’s

(comparison to control

group)

or Sidak’s

multiple comparison test (pre-selected column pairs). Nonparametric data was

analyzed by Kruskal-Wallis followed by

Dunn’s

multiple comparisons test. The following

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comparisons were made: 1) Exposure groups compared to vehicle control group (reported as

asterisks on figures) 2) Exposure groups compared to CB benchmark particle exposure group

(reported in data tables) 3) Exposure groups compared to standard diesel particle exposure groups

(NIST2975 and published data on NIST1650) (reported in data tables). Increasing dose-response

effects were confirmed with test for linear trend, where the

alerting R

(referred to as R

in the text)

is the fraction of the variance between group means that is accounted for by the linear trend

(Altman/Sheskin, provided by GraphPad Prism).

List of abbreviations

BAL

Broncho-Alveolar Lavage

CAP

Commercial Airport Particles

Carbon Black

DLS

Dynamic Light Scattering

ECD

Equivalent Circular Diameter

ELPI

Electrical Low Pressure

Impactor

ESP

Electrostatic Precipitator

Fuel Truck

GC-MS

Gas Chromatography-Mass

Spectrometry

ICP-MS

Inductive Coupled Plasma Mass Spectrometry

JEP

Jet Engine Particles

NIST

National Institute of Standards and Technology

Plane Arriving

PAH

Polycyclic

Aromatic Hydrocarbons

Plane Leaving

SAA

Serum Amyloid A

SEM

Scanning Electron

Microscopy

SRM

Standard Reference Material

TEM

Transmission Electron Microscopy

UFP

Ultra-Fine Particles

Declarations

Ethics approval and consent to participate

The study was carried out in agreement with Directive 2010/63/EU of the European Parliament and

of the Council of 22 September 2010 on the protection of mice used for scientific purposes, and the

Danish Animal Experimentation Act (LBK 474 15/05/2014). The study was approved by The

Animal Experiments Inspectorate under The Ministry of Environment and Food of Denmark

(License: 2010/561-1779) and the local Animal Welfare Committee responsible for ensuring the

implementation of 3R policy at the National Research Center for the Working Environment.

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Consent for publication

Not applicable

Availability of data and material

The datasets used and/or analyzed during the current study are available from the corresponding

author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by Danish Centre for Nanosafety II financed on the Financial Law in

Denmark.

Authors' contributions

Idea and study design: UBV and ATS. Electron Microscopy: ABB. Light Microscopy: TB and HW.

Metal contents: KL and JJS. PAH contents: PAC. Particle collection and exposure characterization:

IK, KA, NB, JK, MDM, OK, MP. In vivo data: KMB, ATS and UBV. Interpretation of data: KMB,

AAB, JK, UBV. KMB drafted the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Excellent technical assistance from Anne-Karin Asp, Yasmin Akhtar, Noor Irmam, Anne Abildtrup,

Eva Terrida, Michael Guldbrandsen, Signe Hjortkjær Nielsen, Ulla Tegner, Vivi Kofoed-Sørensen,

Birgitte Koch Herbst, and Sauli Savukoski is gratefully acknowledged. The authors wish to thank

the non-commercial airfield and the commercial airport for letting us in. KL thanks Agilent for

providing the Agilent 8900 ICP-QQQ instrument. KB thanks Hans Erik Magnus Wisaeus from the

Danish Technological Institute (www.dti.dk) for expert assistance with electron microscopy.

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Figure legends

Fig. 1

Particle concentrations measured inside a jetfighter shelter at a non-commercial airfield (A and B)

and at a non-commercial airport (C) (see also Additional File S1 A). A: Total particle number

concentrations (a) and particle number size distribution time series (b) inside the shelter measured

during jetfighter leaving the shelter (PL), arriving at the shelter (PA), and fuel truck (FT) fueling the

plane. The vertical solid and dashed black lines show when the jet engine is started or fuel truck

arrives to the shelter and when the engine is switched off or fuel truck leaves the shelter. Horizontal

thick black line shows the averaging period to calculate exposure and dose levels presented in Table

2. Particle sampling time for one flight cycle (tPM4) for mass fraction smaller than 4 µm (mPM4)

gravimetric analysis is shown with gray vertical bar. B: Average particle number (a) and mass (b)

size distributions. C: Total particle number concentrations measured at a commercial airport (CAP).

The inserted sub-figure shows the average particle size distribution measured by the NanoScan

during the measurement period.

Fig. 2

Scanning electron micrographs of collected particles dispersed in water. A+F: Overview of

dispersed particles showing difference in homogeneity between JEP and CAP. B+G: Detail of

agglomerates consisting of smaller particles. C+H: Detail of primary soot particles in agglomerates.

D+I: Details of collapsed pollen grains and plant fiber. E+J: Details of silver particles covering

agglomerates and plant fragments.

Fig. 3

Histopathology of the lung on 28 and 90 days following exposure to 54 µg particles collected at a

non-commercial airfield (JEP) and at the apron of a commercial airport (CAP). The sections were

stained with HE. Control: Section of lung from a control mouse instilled with water only. A and B:

Particles were not readily apparent in mice instilled with JEP and no significant pathological

changes were found on day 28 or 90. C: In mice instilled with CAP, some particles were visible in

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macrophages. D and E: Pronounced eosinophil infiltration and eosinophil vasculitis was observed

on day 28 and 90, characterized by infiltrates in the perivascular region and smooth muscle

hyperplasia. F-H. Day 28 and 90. Lung sections of mice exposed to 162 µg NIST2975 had visible

particles along with particle-loaded macrophages, along with modest inflammation-related changes.

Fig. 4

Illustration of dose-response linearity between instilled doses of airport-collected particles and

NIST2975 and NIST1650 and neutrophil influx in BAL. Increasing dose-response effects were

confirmed with test for linear trend, where the

alerting R2

(referred to as R2) is the fraction of the

variance between group means that is accounted for by the linear trend (Altman/Sheskin, provided

by GraphPad Prism). Data for NIST1650 was obtained from a previously published study [19].

Significant linear trends were verified for total cell numbers (not shown) and neutrophils in BAL

fluid, with R2 between 0.76 and 0.95.

Fig. 5

Neutrophil influx in BAL fluid on day 1, 28, and 90 following exposure to jet engine particles

(JEP), commercial airport particles (CAP), and reference particles NIST2975, NIST1650, and

Carbon black Printex90 (CB) (Tukey plots, +: mean, line: median, diamonds: outliers). Mice were

exposed to 6, 18, and 54 µg of JEP and CAP, to 54 µg of CB, and to 18, 54, and 162 µg of NIST

particles with 6 mice in each group. Data for NIST1650 was obtained from a previously published

study [19].

Fig. 6

mRNA levels of

Saa3

in lung,

Saa1

liver, and SAA3 plasma protein on day 1 (scatter plots, mean +

SEM).

Saa3

mRNA in lung tissue and

Saa1

mRNA in liver tissue were used as biomarkers of

pulmonary and hepatic acute phase response, following exposure to particles collected at the apron

of a commercial airport and in a jet shelter at a non-commercial airfield. SAA3 protein was

measured in plasma.

Saa

in lung and liver was measured on day 1, 28 and 90 post-exposure, and

SAA3 on day 1 and on day 28 for highest particle doses.

Fig. 7

DNA strands break levels evaluated by tail length in the Comet assay on day 1, 28, and 90

following exposure to jet engine particles (JEP), commercial airport particles (CAP), and reference

particles NIST2975, NIST1650, and carbon black Printex90 (CB) (scatter plots, mean + SEM).

Mice were exposed to 6, 18, and 54 µg of JEP and CAP, to 54 µg of CB, and to 18, 54, and 162 µg

of NIST particles. Data for NIST1650 was obtained from a previously published study [19].

Additional File S1 (pdf)

A. Illustrations of measurement strategies (figure)

B. Jet engine test facility measurement (figure and text)

C. Aerosols characterized by EM of impactor samples (text and images)

D. Dynamic Light Scattering (text and figure)

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E. EDS analysis (images)

Additional File S2 (pdf)

A. Scatter plots of BAL fluid cells on day 1, 28, and 90 post-instillation, eosinophil influx, and BET

area vs neutrophil influx (figures)

B. Saa3 in lung and liver on day 28 and 90 (figure)

C. % DNA in comet tail and DNA strand breaks (figure and table)

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Additional File S1

Supplementary Material

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Additional File S2

Supplementary Material

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