ArticlePDF Available

Abstract and Figures

Ship engine emissions are important with regard to lung and cardiovascular diseases especially in coastal regions worldwide. Known cellular responses to combustion particles include oxidative stress and inflammatory signalling. To provide a molecular link between the chemical and physical characteristics of ship emission particles and the cellular responses they elicit and to identify potentially harmful fractions in shipping emission aerosols. Through an air-liquid interface exposure system, we exposed human lung cells under realistic in vitro conditions to exhaust fumes from a ship engine running on either common heavy fuel oil (HFO) or cleaner-burning diesel fuel (DF). Advanced chemical analyses of the exhaust aerosols were combined with transcriptional, proteomic and metabolomic profiling including isotope labelling methods to characterise the lung cell responses. The HFO emissions contained high concentrations of toxic compounds such as metals and polycyclic aromatic hydrocarbon, and were higher in particle mass. These compounds were lower in DF emissions, which in turn had higher concentrations of elemental carbon ("soot"). Common cellular reactions included cellular stress responses and endocytosis. Reactions to HFO emissions were dominated by oxidative stress and inflammatory responses, whereas DF emissions induced generally a broader biological response than HFO emissions and affected essential cellular pathways such as energy metabolism, protein synthesis, and chromatin modification. Despite a lower content of known toxic compounds, combustion particles from the clean shipping fuel DF influenced several essential pathways of lung cell metabolism more strongly than particles from the unrefined fuel HFO. This might be attributable to a higher soot content in DF. Thus the role of diesel soot, which is a known carcinogen in acute air pollution-induced health effects should be further investigated. For the use of HFO and DF we recommend a reduction of carbonaceous soot in the ship emissions by implementation of filtration devices.
Content may be subject to copyright.
RESEARCH ARTICLE
Particulate Matter from Both Heavy Fuel Oil
and Diesel Fuel Shipping Emissions Show
Strong Biological Effects on Human Lung
Cells at Realistic and Comparable In Vitro
Exposure Conditions
Sebastian Oeder
1,2,3
, Tamara Kanashova
1,4
, Olli Sippula
1,5
, Sean C. Sapcariu
1,6
,
Thorsten Streibel
1,7,8
, Jose Manuel Arteaga-Salas
1,8
, Johannes Passig
1,7
,
Marco Dilger
1,9,10
, Hanns-Rudolf Paur
1,9
, Christoph Schlager
1,9
, Sonja Mülhopt
1,9
,
Silvia Diabaté
1,10
, Carsten Weiss
1,10
, Benjamin Stengel
1,11
, Rom Rabe
1,11
,
Horst Harndorf
1,11
, Tiina Torvela
5
, Jorma K. Jokiniemi
1,5,12
, Maija-Riitta Hirvonen
1,5,13
,
Carsten Schmidt-Weber
2
, Claudia Traidl-Hoffmann
3,14
, Kelly A. BéruBé
1,15
, Anna
J. Wlodarczyk
1,15
, Zoë Prytherch
1,15
, Bernhard Michalke
16
, Tobias Krebs
1,17
, André S.
H. Prévôt
18
, Michael Kelbg
1,19
, Josef Tiggesbäumker
1,19
, Erwin Karg
8
, Gert Jakobi
8
,
Sorana Scholtes
1,8
, Jürgen Schnelle-Kreis
8
, Jutta Lintelmann
8
, Georg Matuschek
8
,
Martin Sklorz
7
, Sophie Klingbeil
1,7
, Jürgen Orasche
8
, Patrick Richthammer
1,8
,
Laarnie Müller
8
, Michael Elsasser
8
, Ahmed Reda
8
, Thomas Gröger
8
, Benedikt Weggler
1,8
,
Theo Schwemer
7
, Hendryk Czech
7
, Christopher P. Rüger
7
, Gülcin Abbaszade
8
,
Christian Radischat
1,7
, Karsten Hiller
1,6
, Jeroen T. M. Buters
1,2,3
, Gunnar Dittmar
1,4
,
Ralf Zimmermann
1,7,8
*
1 HICEHelmholtz Virtual Institute of Complex Molecular Systems in Environmental HealthAerosols and
Health, www.hice-vi.eu, Neuherberg, Rostock, Munich, Karlsruhe, Berlin, Waldkirch, Germany, Kuopio,
Finland, Cardiff, UK, Esch-Belval, Luxembourg, 2 Center of Allergy and Environment (ZAUM), Helmholtz
Zentrum München and Technische Universität München, Member of the German Center for Lung Research
(DZL), Munich, Germany, 3 CK-CARE, Christine Kühne Center for Allergy Research and Education, Davos,
Switzerland, 4 Mass Spectrometry Core Unit, Max Delbrück Center for Molecular Medicine Berlin-Buch,
Germany, 5 University of Eastern Finland, Department of Environmental Science, P.O. Box 1627, FI-70211
Kuopio, Finland, 6 Luxembourg Centre for Systems Biomedicine, University of Luxembourg, L-4362 Esch-
Belval, Luxembourg, 7 Joint Mass Spectrometry Centre, Chair of Analytical Chemistry, Institute of
Chemistry, University Rostock, Rostock, Germany, 8 Joint Mass Spectrometry Centre, CMA
Comprehensive Molecular Analytics, Helmholtz Zentrum München, Neuherberg, Germany, 9 Institute for
Technical Chemistry (ITC), Karlsruhe Institute of Technology, Campus North, Karlsruhe, Germany,
10 Institute of Toxicology and Genetics (ITG), Karlsruhe Institute of Technology, Campus North, Karlsruhe,
Germany, 11 Chair of Piston Machines and Internal Combustion Engines, University Rostock, Rostock,
Germany, 12 VTT Technical Research Centre of Finland, P.O. Box 1000, FI-02044 VTT, Espoo, Finland,
13 National Institute for Health and Welfare, Department of Environmental Health, P.O. Box 95, FI-70701,
Kuopio, Finland, 14 Institute of environmental medicine, UNIKA-T, Technische Universität, Munich,
Germany, 15 Lung and Particle Research Group, School of Biosciences, Cardiff University, Cardiff, Wales,
United Kingdom, 16 Research Unit Analytical BioGeoChemistry, Helmholtz Zentrum MünchenGerman
Research Center for Environmental Health GmbH, Neuherberg, Germany, 17 Vitrocell GmbH, Waldkirch,
Germany, 18 Laboratory of Atmospheric Chemistry, Paul Scherrer Institute (PSI), Villigen, Switzerland,
19 Institute of Physics, University Rostock, Rostock, Germany
These authors contributed equally to this work.
These authors also contributed equally to the manuscript.
* ralf.zimmermann@helmholtz-muenchen.de
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 1/17
OPEN ACCESS
Citation: Oeder S, Kanashova T, Sippula O,
Sapcariu SC, Streibel T, Arteaga-Salas JM, et al.
(2015) Particulate Matter from Both Heavy Fuel Oil
and Diesel Fuel Shipping Emissions Show Strong
Biological Effects on Human Lung Cells at Realistic
and Comparable In Vitro Exposure Conditions. PLoS
ONE 10(6): e0126536. doi:10.1371/journal.
pone.0126536
Academic Editor: Shama Ahmad, University of
Alabama at Birmingham, UNITED STATES
Received: October 14, 2014
Accepted: April 2, 2015
Published: June 3, 2015
Copyright: © 2015 Oeder et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Additionally, transcriptomics data are available from
Gene Expression Omnibus (accession number
GSE63962). Proteomics data are available from
ProteomicsDB (ID: PRDB004215).
Funding: HICE partners received funding from the
Impulse and Networking Funds (INF) of the
Helmholtz Association (HGF), Berlin, Germany. The
support of HICE by the Helmholtz Zentrum München
Abstract
Background
Ship engine emissions are important with regard to lung and cardiovascular diseases espe-
cially in coastal regions worldwide. Known cellular responses to combustion particles in-
clude oxidative stress and inflammatory signalling.
Objectives
To provide a molecular link between the chemical and physical characteristics of ship emis-
sion particles and the cellular responses they elicit and to identify potentially harmful frac-
tions in shipping emission aerosols.
Methods
Through an air-liquid interface exposure system, we exposed human lung cells under realis-
tic in vitro conditions to exhaust fumes from a ship engine running on either common heavy
fuel oil (HFO) or cleaner-burning diesel fuel (DF). Advanced chemical analyses of the ex-
haust aerosols were combined with transcriptional, proteomic and metabolomic profiling in-
cluding isotope labelling methods to characterise the lung cell responses.
Results
The HFO emissions contained high concentrations of toxic compounds such as metals and
polycyclic aromatic hydrocarbon, and were higher in particle mass. These compounds were
lower in DF emissions, which in turn had higher concentrations of elemental carbon (soot).
Common cellular reactions included cellular stress responses and endocytosis. Reactions
to HFO emissions were dominated by oxidative stress and inflammatory responses, where-
as DF emissions induced generally a broader biological response than HFO emissions and
affected essential cellular pathways such as energy metabolism, protein synth esis, and
chromatin modification.
Conclusions
Despite a lower content of known toxic compounds, combustion particles from the clean
shipping fuel DF influenced several essential pathways of lung cell metabolism more
strongly than particles from the unrefined fuel HFO. This might be attributable to a higher
soot content in DF. Thus the role of diesel soot, which is a known carcinogen in acute air
pollution-induced health effects should be further investigated. For the use of HFO and DF
we recommend a reduction of carbonaceous soot in the ship emissions by implementation
of filtration devices.
Introduction
Epidemiological studies provide compelling evidence that pollution by airborne particulate
matter (PM) derived from fossil fuel combustion is an important cause of morbidity and
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 2/17
and University of Rostock is gratefully acknowledged.
Sebastian Oeder also received funding from CK-
CARE Teilbereich A. Sean Sapcariu and Karsten
Hiller acknowledge financial support from the Fonds
National de la Recherche (FNR), specifically the
ATTRACT program Metabolomics Junior Group.
Funding from the Academy of Finland (Grant No:
258315 & 259946), Saastamoinen foundation and the
strategic funding of the University of Eastern Finland
for project sustainable bioenergy, climate change
and health is acknowledged. Funding from the
German Science Foundation (DFG ZI 764/5-1, ZI
764/3-1, INST 264/56-1 and 264/77-1) helped to
achieve the presented results. We also thank SNSF
and DFG for funding for the DACH project WOOSHI.
Vitrocell GmbH provided support in the form of a
salary for author T. Krebs, but did not have any
additional role in the study design, data collection and
analysis, decision to publish, or preparation of the
manuscript. The specific roles of the authors are
articulated in the author contributions section.
Competing Interests: Tobias Krebs is an employee
of Vitrocell GmbH, Tübingen, Germany. This does not
alter the authors' adherence to PLOS ONE policies
on sharing data and materials.
premature death [1, 2]. Chronic PM exposure can induce short-term (e.g., cardiovascular dis-
eases or asthma) and long-term health effects, most notably cancer. Diesel automobile emis-
sions were recently classified as human carcinogens by the Intern ational Agency for Research
on Cancer [3].
Diesel ship emissions substantially contribute to worldwide anthropogenic PM levels, which
account for up to 50% of the PM-related air pollution in certain coastal areas, rivers and ports
[47]. Epidemiological studies attribute up to 60,000 annual deaths from lung and cardiovascu-
lar disease [ 8] to ship engine PM. A variety of new regulations will soon be implemente d to en-
sure cleaner ship emissions [ 911]. Low-grade heavy fuel oils (HFOs) contain high levels of
sulphur, toxic polycyclic aromatic hydrocarbons (PAHs) and transition metals. Current regula-
tions target HFO use by limiting their sulphur content. In this context, the maximum sulphur
content in shipping fuel is internationally regulated by the International Maritime Organisa-
tion (IMO) at 3.5%; in most European and US coastal areas, the maximum allowed sulphur
content is 1% (Sulphur Emission Control Areas, SECA) [12, 13]. Furthermore, in 2015, a 0.1%
sulphur fuel limit will be implemented in the Baltic and North Sea SECAs [14]. To comply
with these new sulphur limits, highly refined distillate fuels are necessary (diesel fuel, DF, or
marine gas oil, MGO). Currently, MGO is the most used distillate fuel for marine shipping and
contains up to 1% sulphur. In 2011, 170 million tons of HFO and 43 million tons of MGO and
DF were consumed by ship diesel engines worldwide [15, 16]. This volume corresponds to ap-
proximately 21% of global fuel consumption [17].
The biological and health effects of land-based diesel engine emissions have been extensively
studied using submersed cell cultures subjected to collected diesel exhaust particles [18, 19].
This submersed cell culture approach neglects the effect of airborne particle exposure, which
can result in low sensitivity in measuring biological effects [20]. An alternative is the air-liquid
interface (ALI) cell exposure technology. Current systems are technically mature enough to en-
able reproducible, direct, on-site exposure of lung cell culture to emission aerosols under realis-
tic dilution, flow and humidity conditions [21]. Multiple ALI-exposure studies using car diesel
engines [2225] highlight the improved sensitivity of ALI systems compared with submerged
toxicological test systems that use collected diesel exhaust particles (DEP) [20].
Up to now three main causes for PM-induced health effects have been identified: genotoxi-
city, inflammation and oxidative stress; other mechanisms have also been described [19]. Thus
far, all information on diesel PM has been inferred from research on car emissions. However,
diesel emissions from ships differ greatly from car or truck diesel emissions due to the fuel
composition (HFO) and combustion characteristics of ship engines [26]. Thus, the practice
currently used to estimate the health impacts of ship diesel emissions based on analogous car
or truck emissions [8, 12, 27] is problematic. The high levels of toxic compounds [6, 28] suggest
that HFO emissions produce more detrimental acute and chronic toxic effects than car or
truck diesel emissions.
This study targets the biological effects of airborne PM from both diesel and HFO ship
emissions based on their chemical compositions. The joint analysis of the biological multi-
omics data with the comprehensive aerosol analysis results provides an extensive overview of
affected biological mechanisms and pathways and further identifies potentially harmful frac-
tions of the shipping aerosols.
Results and Discussion
Experimental setup
The experimental setup is illustrated in Fig 1 (details in S1 and S2 Figs and in S1 Text). Briefly,
we operated a four-stroke, one-cylinder common rail research ship diesel engine (80 kW)
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 3/17
using either HFO (HFO 180) containing 1.6% sulphur or DF containing less than 0.001% sul-
phur and 3.2% plant oil methyl ester in compliance with the 2014 IMO-SECA-legislation (DIN
EN590, see S1 Fig for the engine and fuel properties), which represents the common dual-fuel
use in commercial shipping [10, 29]. The engine was operated according to the test cycle ISO
81784 E2 for ship diesel engines with a balance between harbour-manoeuvring and cruising
engine-loads (Fig 2). The combustion aerosol was cooled and diluted with sterile air. Chemical
and physical properties of the HFO and DF aerosol were comprehensively characterised using
state-of-the-art, on-line and real-time techniques as well as off-line filter sample analyses. Re-
sults are summarised in Fig 2 (for details, see SI). In parallel with aerosol characterisation, con-
fluent layers of two human epithelial lung cell lines (the human lung alveolar cancer cell line
A549, purchased from the American Type Culture Collection, ATCC CCL-185; http://www.
lgcstandards-atcc.org/Products/All/CCL-185.aspx, and human SV40-immortalised bronchial
epithelial cells BEAS-2B, purchased from ATCC, CRL-9609; http://www.lgcstandards-atcc.org/
Fig 1. Experimental set-up and global omics analyses. (A) An 80 KW common-rail-ship diesel engine was operated with heavy fuel oil (HFO) or refined
diesel fuel (DF). The exhaust aerosols were diluted and cooled with clean air. On-line real-time mass spectrometry, particle-sizing, sensor IR-spectrometry
and other techniques were used to characterise the chemical composition and physical properties of the particles and gas phase. Filter sampling of the
particulate matter (PM) was performed to further characterise the PM composition. Lung cells were synchronously exposed at the air-liquid-interface (ALI) to
aerosol or particle-filtered aerosol as a reference. The cellular responses were characterised in triplicate at the transcriptome (BEAS-2B), proteome and
metabolome (A549) levels with stable isotope labelling (SILAC and
13
C
6
-glucose). (B) Heatmap showing the global regulation of the transcriptome, proteome
and metabolome.
doi:10.1371/journal.pone.0126536.g001
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 4/17
Products/All/CRL-9609.aspx)) [30] were exposed to the diluted engine exhaust for 4 h at the
ALI (Fig 1). Epithelial lung cells have direct contact to inhaled aerosol particles and gases and
were therefore used as a model of aerosol inhalation. The cell lines A549 and BEAS-2B have
been widely used for testing particles and gases at the air-liquid-interface [3136]. The BEAS-
2B cells are considered to better resemble the situation in human lung tissue while require-
ments for the cultiva tion of the cancer derived cell line A549 are better suited for labeling with
the L-D
4
-Lysine isotope maker for the quantitative proteomics. The transcriptomics methodol-
ogy is not based on metabolic labelling and thus well suited for the analysis of BEAS-2B cells.
The quantitative comparative proteomics approach requires the labelling of the cells with
D4-Lysine. However the BEAS-2B cells require specialized media and coating of the plates,
which is currently incompatible with the metabol ic labelling. Therefore simultaneuos SILAC-
based proteomic and metabolic analysis was performed with the establis hed A549 cell model.
In summary the cells were analysed using transcriptome (BEAS-B), SILAC-proteome (A549),
metabolome and metabolic flux measurements (A549) as well as cytotoxicity tests (A549). The
omics data are stored in Gene Expression Omnibus (GSE63962) and Proteomics DB
Fig 2. Chemical and physical aerosol characterisation. (A) The ship diesel engine was operated for 4 h in
accordance with the IMO-test cycle. (B) Approximately 28 ng/cm
2
and 56 ng/cm
2
were delivered to the cells
from DF and HFO, respectively, with different size distributions. The HFO predominantly contained particles
<50 nm, and the DF predominantly contained particles >200 nm, both in mass and number. (C) Number of
chemical species in the EA particles. (D) Transmission electron microscope (TEM) images and energy-
dispersive X-ray (EDX) spectra of DF-EA and HFO-EA; heavy elements (black speckles, arrow); and
contributions of the elements V, P, Fe and Ni in the HFO particles using EDX (* = grid-material). (E)
Exemplary EA concentrations (right) and concentration ratios (left) for particulate matter-bound species. For
all experiments, n = 3.
doi:10.1371/journal.pone.0126536.g002
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 5/17
(PRDB004215), respectively. All experiments were performed in triplicate (3 independent ex-
posures) and referenced to filtered aerosol (for normalising the effects induced by the gas
phase) because particles and particle-related chemicals play an important role in the health rel-
evance of diesel exhaust [ 37] and are therefor in the focus of this study.
The first phase of the experiment was used to find the optimal dose for the large-scale analy-
sis. Cells in the setup were exposed to different concentrations of aerosols. The reaction of the
cells was moni tored using the Alamar Blue viability test. Due to the higher particle concentra-
tion in HFO- exhaust (see belo w) a dilution of 1:100 was required to achieve a non-impaired
cell status while for DF-exhaust a lower dilution of 1:40 was possible without any viability im-
pairment (i.e., a no acute toxicity exhaust dilution; S3A Fig). By applying the different dilution
ratios of 1:40 (DF) and 1:100 (HFO) for the exhaust gases for no acute toxicity at 4 h exposure,
a similar deposition dose (deposited particle mass per confluent cell culture surface area, see
below) was achieved. Based on a gravimetric filter analysis of PM 2.5 and assuming a size-inde-
pendent, constant deposition probability of 1,5% after Comouth et al. [38], the accumulated
particle mass deposited on the lung cell monolayer surface area was roughly estimated as
28 ± 1.5 (DF) and 56 ± 0.7 ng/cm
2
(HFO) per 4 h exposure duration (see S3C Fig) with the var-
iance of the mass measurement expressed by the standard deviation of the filter samples. A
more elaborated model taking into account the particle size distribution from an electric low
pressure impactor (ELPI) and a size dependent deposition probability after Comouth et al.
[38], which was determined using previous measurements from ALI exposure systems , predicts
15.7 (DF) and 41.5 ng/cm
2
(HFO) per 4 h exposure. Even for improved deposition approxima-
tion model, the estimated uncertainties, however, are rather high (about a factor of 2). There-
fore the deposition dose in both cases can be considered being approximately equal for DF and
HFO. We decided to perform the exposure for omics measureme nts at these dilutions, in order
to compare th e specific molecular biological effect strength at an about equal deposition dose.
Note that in the following all aerosol parameters are reported considering the specific emis-
sion-aerosol dilution factors (i.e. the exposure aerosol, EA, as delivered to the cells).
Chemical and physical analysis
Consistent with previous studies [29], only small concentration differences of gaseous com-
pounds were found in the emissions of the ship engine using the two fuels. An exception was
SO
2
(4 mg/m
3
), which was below toxicity threshold after dilution in the HFO-EA. In addition,
the EA concentrations of the further potential ly toxic gases NO, NO
2
and CO were below 16.3,
0.4 and 7 ppm, respectively. These values are below the reported toxicity thresholds for the air-
liquid interface [39, 40] and even below the general NIOHS lifetime workplace 8-hr exposure
limit values of 25, 1 and 35 ppm, respectively [41].
The concentration of particles with an aerodynamic diameter larger than 200 nm was higher
for the DF-EA (particle number and mass concentration), whereas nanoparticles smaller than
50 nm were approximately 100-fold more abundant for the HFO-EA (see the size distributions
in Fig 2). However, note that the mass contribution of these nanoparticles is very small. TEM
images of the particles show that the smaller HFO particles (Fig 2) contained high levels of
amorphous organic material around carbonaceous fractal cores with metal inclusions. The
DF-EA particle analysis reveals a different picture (Fig 2), in which the particles appear larger
and are mostly composed of pure carbonaceous aggregates with spherical soot cores ~ 20
30 nm). A layered graphite-like car bon structure became visible at a higher TEM magnification
(S3 Fig). Based on the size-dependent deposition function described by Comouth et al.[38](S3
Fig) and the low specific density of the observed fractal soot aggregates in DF-EA (Fig 2), the
deposited mass for the DF-EA cell exposure experiments is slightly lower than the above
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 6/17
estimates. The particles deposited from the HFO-EA were of a higher dose in mass and number
compared to the DF-EA exposure.
Energy-dispersive X-ray spectroscopy (EDX, Fig 2) on TEM showed large differences be-
tween HFO-EA and DF-EA particles with regard to the abundance of heavy elements. High in-
tensities of elements such as vanadium, nickel, sulphur and iron were detected in the HFO
particles, whereas the DF particles primarily contained carbon and oxygen in the EDX spec-
trum. Fig 2 shows an overview of the differences in the inorganic and organic chemical compo-
sition (Fig 2) as well as the absolute concentrations of the respective substances in the DF- and
HFO-exposure aerosol particles (Fig 2 and S4 Fig). Almost all of the measured components, ex-
cept elemental carbon and black carbon, were more abundant in HFO-EA compared with
DF-EA, despite a 2.5-fold higher dilution for HFO-EA.
On-line aerosol mass spectrometry and off-line analyses showed considerably higher mass
concentrations of particle-bound organic material and much more complex organic material
in the HFO-EA (S1 Table). High-resolution mass spectrometry (ESI-FTICR-MS) revealed
3631 different polar organic compounds in the HFO particles compared with only 321 in the
DF particles (Fig 2); 244 compounds were common to both fuel types. The quantification of ar-
omatic and aliphatic compounds (S4 Fig ) revealed that higher molecular weight components
were more abundant in the HFO particles (green text in Fig 2), such as the higher molecular
weight carcinogenic PAH benzo[a]pyrene (Fig 2 and S4 Fig). The sum of PAH toxicity equiva-
lency factor s (Fig 2), which ranks different toxic PAHs weighted by their concentration and rel-
ative toxicity, was more than 10-fold higher in HFO-PM compared with DF-PM (Fig 2). The
only component over-represented in the DF-PM was the elemental carbon fraction (EC) and
the corresponding optically measured black carbon factor (BC; Fig 2).
Summarising the chemical and physical characterisations, particles emitted from ship en-
gines differ in concentration, size distribution, morphological appearance and chemical com-
position depending on whether DF or HFO is used. The DF particles in the inhalable size
region were dominated by elemental carbon-rich soot-aggregate particles [29], whereas the
HFO particles were smaller (nanoparticles) and rich in organic material, including known or-
ganic air toxicants (PAHs and their derivatives) and reactive transition metals such as V, Ni, Fe
and Zn (S4 Fig). However, it shall be noted that also DF-PM contains organic compounds in
relatively high concentrations. The HFO-PM just contains much higher concentrations (Fig 2).
Exposure and deposition dose
We exposed human lung cells for 4 h to concentrations which are corresponding to occupation-
al exposure scenarios or 10 times the concentration of an ambient high concentration scenario
(EA ~ 390 μg DF PM2.5/m
2
and ~760 μg HFO PM2.5/m
2
). This concentration corresponds to
an ALI mass deposition dose of about 28 and 56 ng PM/cm
2
for DF and HFO respectively.
These doses can be related to the human respiratory tract using the specific deposition effi-
ciency for different lung regions. From the measured size distribution and an estimated effective
particle density based on the mass-mobility-relation for aggregated diesel particles (between 1.1
and about 0.1 g cm
-3
, derived from [42, 43]), a deposition simulation was performed using a re-
cently updated model [44, 45] for the tracheobronchial lung region. A 4 h exposure of a human
being to the EA concentrations used in our experiments would result to a tracheobronchial de-
position of about 1.5 and 5 ng PM/cm
2
for DF and HFO, respectively. Thus the deposited mass
in an ALI experiment corresponds to about 3 days (DF) or 2 days (HFO) exposure time for an
exposed person (note that for an equal dilution of 1:100 in both EAs the actual deposited tra-
cheobronchial dose for DF would correspond to a 7.5 days exposure of a person). However, one
should keep in mind that the size distribution may change quickly in the ambient atmosphere
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 7/17
and in the airways, e.g. by coagulation or water condensation, causing additional uncertainty
thereby. The similarity between the size dependent deposition curve [38] for the ALI-system
and for the lung deposition curve [44] suggests a good transferability of the results, in particular
for the tracheobronchial region. In conclusion, the deposited mass concentration of at an equal
dilution of DF PM mass would be about ¼ of the deposited HFO PM. This however, only holds
true for directly emitted aerosols. In the atmosphere the more polar, sulphate containing HFO
emission particle will quickly grow considerably by water condensation, while the hydrophobic
DF particles size distribution will remain stable for longer time [46, 47]. Therefore, emission
size distributions might, to some extent, equalise soon.
Biological analysis
To relate the extensive chemical and physical characterisation of the exhaust aerosols to biolog-
ical effects, the HFO and DF emission particles were directly deposited on human lung cells
using ALI exposure technology. Transcriptome, proteome, metabolome and metabolic flux
analyses were performed, which yielded parallel and relative quantification of 42205 different
transcripts, 6192 proteins and 400 metabolic molecules. To reduce variability, the proteins and
metabolites were extracted from the same cell material (A549) that was previously metabolical-
ly labelled using D
4
-lysine (SILAC proteomics) and
13
C
6
-glucose (metabolic flux analysis). Ri-
bonucleic acid (RNA) was isolated from BEAS-2B cells exposed through the same ALI
exposure system and was used for the transcriptome analyses [20].
The transcriptome, proteome and metabolome analyses revealed widespread changes in the
cellular system upon exposure to both HFO and DF aerosol particles. Surprisingly, more gene
expression levels were regulated in the DF-particle-exposed cells (i.e., the response was more
widespread compared with the HFO-particle-treated cells on all omic-levels; p<0.001, Figs 1
and 3 and S5 Fig). The most significantly regulated genes, proteins and metabolites also dif-
fered between the DF and HFO (S6 Fig), which shows that the response to emissions of each
type of fuel differed quantitatively and qualitatively in both human lung cell lines. A higher reg-
ulation alone only proofs a stronger biological reaction onto the deposited PM at the given ex-
posure conditions (i.e. 4 h exposure at a deposition dose below measurable cytotoxicity) and
does not necessarily indicate a higher toxicity or risk of disease.
Further conclusions can be drawn from a specific biological pathway analysis. Pro-inflamma-
tory signaling, chemical response (such as xenobiotic metabolism) and oxidative stress pathways
were indicated by the regulated genes (Fig 3 and S6 Fig)[19]. The HFO particles specifically in-
duced the transcription of primary and secondary inflammation markers (IL-8, IL-6 and IL-1),
and both fuel types affected the cytokines CSF3, CXCL1, and CXCL2. Considering xenobiotic
metabolism, CYP1A1 (PAH metabolism) was induced by exposure to HFO particles (which cor-
responds to the higher PAH concentrations in HFO PM), whereas the DF particles affected
other cytochromes (CYP3A4 and CYP17A1) and the carbosulphotransferase CHST6 (Fig 3).
In addition to these, in the context of aerosol exposure well-known pathways [19, 48], we
searched for other cellular responses undergoing modulation. A meta-analysis combining the
proteome and transcriptome data was performed to examine the significant enrichment of
gene ontology (GO) terms. The results indicate that the HFO and DF particle effects were dis-
tinct (Fig 3 and, in more detail, S7 Fig). Particles from both fuels induced effects on cell motili-
ty, th e cellular stress response, the response to organic chemicals, proliferation and cell death
(Fig 3 and S7 Fig). Genes and proteins associated with vesicle transport pathways were en-
riched, which might be connected to the endocytosis of diesel particulate matter.
The pathways specifically regulated by DF particle exposure included the general translation
pathway (Fig 3, S7 Fig and S2 Table). The translational elongation, RNA-processing and
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 8/17
ribosome translation pathways were down-regulated, whereas the pathways that affect chroma-
tin organisation and modification were up-regulated. The down-regulated pathways included
histone acetylation, which may result in DF particle-induced epigenetic changes. Other path-
ways modulated by DF particles were involved in processes such as cell junction organisation
and cell adhesion. Pathways such as the energy metabolism, cell junction and cell adhesion
were clearly affected in both cell lines when assessed using transcriptomics and proteomics but
differed in the direction of regulation (Table 1, S2 Table and Fig 3), which indicates a time-de-
layed reaction in the cell. Exposure to DF particles induced mitochondria-associated genes and
proteins, which indicates that mitochondrial stress was induced, whereas the HFO particles did
not yield this response.
Pathways specifically regulated by the HFO particles include the homeostasis, oxidative
stress and inflammatory response pathways, whereas the metabolic and biosynthetic processes
were slightly down-regulated (Fig 3 and S2 Table).
Fig 3. Effects of shipping particles on lung cells. The net effects from the particles were referenced against the gaseous phase of the emissions. (A)
Number of the regulated components in the transcriptome shows more genes regulated by the DF than the HFO particles (in BEAS-2B cells). Similar results
were observed for the proteome (B) and metabolome (C) (in A549 cells). (D) Meta-analyses for the transcriptome and proteome using the combined Gene
Ontology (GO) term analysis of the 10% most regulated transcripts and proteins. Individual GO terms are listed in S2 Table; the hierarchical pathways are
indicated on the right. (E) Gene regulation of Wiki-pathway bioactivation; (F) gene regulation of Wiki-pathway inflammation; g, secreted metabolites; and h,
metabolic flux measurements using
13
C-labelled glucose. For all experiments, n = 3.
doi:10.1371/journal.pone.0126536.g003
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 9/17
Interestingly, the proteomics data reveal a direct induction of cell-cell interaction remodel-
ling, whereas the transcriptomics data show a down-regulation of simila r GO terms. This find-
ing can be explained by assuming an immediate response of the proteome e.g. by stabilizing
the already synthesized proteins, while the transcriptome shows the shut-down of the system
in a time-de layed response. Although 40% of the observed protein regulation can be explained
by the changes in mRNA abundance, most of the changes indicate other modes of regulation.
Protein can be degraded in direct response to PM exposure, and translation or transcription
may be too slow to change the protein concentrations after 4 h of exposure [49].
The metabolome analyses supported the finding that biosynthetic and protein synthesis
processes were down-regulated in the DF particle-treated cells. ATP-binding cassette trans-
porters, which are involved in actively transporting biomolecules across membranes, were also
affected (S2 Table). Further information supporting the inhibition of biosynthetic activities in-
cludes the negatively affected metabolites secreted by the cells (Fig 3). The pathways affected
by HFO particle exposure include glycolysis and pyrimidine metabolism. Glycolysis is a path-
way that is typically altered during inflammation and is generally increased in cells under in-
flammatory conditions [50].
Glucose flux into lactic acid through glycolysis was significantly reduced (p<0.05) in cells
treated with DF particles (Fig 3 and S9 Fig). Mammalian cells oxidise glucose and glutamine in
the TCA cycle to produce NADH/H
+
, which is re-oxid ised in the respiratory chain to produce
ATP. DF exposure strongly decreases the levels of relative glucose oxidation in the TCA cycle
compared with HFO, as reflected by the significantly lower levels of labelled citric acid
(p<0.001; ratio data: Fig 3). Simultaneously, we observed an increase in gl ucose-derived carbon
flux into glycine (Fig 3); enhanced glycine metabolism has previousl y been associated with
tumourigenesis in lung cancer [51]. These observations suggest a lower ATP production and,
hence, lower available energy compared with HFO. Increased carbon flux into glycine is
Table 1. Summary of the main HFO- and DF-particle exposure effects.
Effect HFO DF
Pro-inammatory signaling " -
Oxidative stress " -
Cell homeostasis " -
Response to chemicals "#"
Cellular stress response ""
Motility ""
Endocytosis ""
Cellular signalling MAPK, TGF beta, PDGF, EGF, GPCR ID, kinase cascade
Energy metabolism - #"
x
Protein synthesis - #
Protein degradation - "
RNA metabolism - #
Chromatin modications - "
Cell junction and adhesion - #"*
The arrows indicate the direction of regulation for cellular functions derived from the most statistically
signicant enriched Gene Ontology terms from the transcriptome, proteome, and metabolome (details in S2
Table).
x
BEAS-2B up, A549 down
* BEAS-2B down, A549 up
doi:10.1371/journal.pone.0126536.t001
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 10 / 17
directly linked to the increased transformation of hydroxymethyl groups through one-carbon
metabolism. The latter is essential for DNA synthesis and repair.
Conclusions
We assessed human lung cell resp onses to ship exhaust particles. A unique combination of ex-
tensive chemical and physical aerosol characterization and multiple omics analysis was used to
generate a broad overview on cellular mechanisms affected by shipping particles and to identify
possibly harmful constituents of two types of ship exhaust aerosols. While not providing a clas-
sical toxicological risk assessment, which would require the testing of multiple doses and time-
points, this study rather gives a comprehensive picture on the cellular responses to ship exhaust
particles after short-term exposure, which should be used as starting point for more mechanis-
tic studies. Although the HFO particles deposited in the ALI system were about equal in mass,
higher in number and contained a large excess of toxic compounds, DF particle exposure in-
duced a broader biological reaction in the human lung cells (BEAS-2B and A549) on all investi-
gated "omic" levels. As discussed, a stronge r affected cell metabolism is not an adverse effect
per se, but it holds a higher risk of disturbance of normal cell functions. Within known path-
ways, such as pro-inflammatory signaling, oxidative stress and xenobiotic metabolism, the lev-
els of certain well-known indicators (e.g., IL-1/6/8 and CYP1A1) surged followin g HFO
particle exposure. In contrast, DF particles strongly affected basic cellular functions (energy
and protein metabolism) and mechanisms little yet known to be affected by aerosol treatment,
such as mRNA proc essing and chromatin modification.
The obtained results also suggest formulating specific hypotheses and are motivating further
experiments to proof or disproof those. In this context the role of freshly formed elemental
carbon, EC fractions and the influence of organic compounds on the biological activity should
be investigated. The relatively large EC fractio n in DF exhaust is one of the prominent differ-
ences between the two particle types. The chemical and physical surface propertie s of freshly
formed EC fractions might be of relevance here. Laboratory experiments using e.g. combustion
aerosol standard generator (CAST,[52]), which allows to generate fresh combustion particles
with adjustable EC/OC ratios, are currently under preparation.
There is no doubt that the carcinogenic emissions from HFO-operated vessels need to be
minimized and HFOs should be replaced by refined modern DF (at least if no flue gas cleaning
systems are installed). HFO emissions contain among other constituents high concentrations
of toxic metals (V, Ni etc.) and polycyclic aromatic hydrocarbons. However, also emission of
diesel engines operated with refined DF, are known to be toxic and carcinogenic, although the
toxicant concentrations are much lower [8] than in HFO emissions. Consequently the imple-
mentation of emission reduction measures for land-based diesel engines started decades ago
(e.g. with sulfur-reduced fuels) [18] and current efforts are directed towards the reduction of
particle emissions from diesel automobiles. Due to the substantial contribution of ship emis-
sions to global pollution, ship emissions are the next logical target for improving air quality
worldwide, particularly in coastal regions and harbour cities. In this contex t our findings on
the biological effects of HFO and DF ship diesel emissions can contribute to the current debate
about the reduction measures to be implemented for shipping. The results from this study pro-
vide the information that at comparable lung deposition doses the acute biological activity of
particles of ship emissions from DF fuelled ships is not less relevant than the activity of HFO
emission particles. This supports the suggestion that a general reduction of the PM emissions
(not the SO
2
emission) from shipping in harbours and the vicinity of the coast should be imple-
mented for both, HFO- and DF-opera ted ships. Efficient particle filter technology (e.g., electro-
static precipitation or bag-filtration) is available. From a regulatory perspective, the next step
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 11 / 17
should be the introduction of legal emissions limits for respirable PM (e.g. PM 2.5, in [mg/m
3
])
from ship emissions [29].
Supporting Information
S1 Fig. Sampli ng setup. (A) Simplified scheme of the sampling and measurement setup.
DR = dilution ratio, TC = temperature control, T = temperature measurement, P = pressure
meter. (B) Detailed setup of the used sampling train with porous tube and ejector diluter units.
(C) Properties of the used diesel fuel (DF) and heavy fuel oil (HFO). Most noticeable are the
high viscosity and high sulfur content of HFO compared with distillate fuels like EN 590 diesel.
(D) Experimental engine parameters. The engine is a single cylinder engine with common rail
injection system representing state of the art medium speed marine diesel engines. The dual
fuel system allows operation with both distillate and residual fuels.
(EPS)
S2 Fig. Air-Liquid-Interface (ALI) exposure. HICE exposure system: the left part shows the
data acquisition and control unit for the mass flow controllers, humidity and temperature. The
exposure unit in the right part contains three Vitrocell modules and is thermostated to 37°C.
Each module has six positions for cell exposure to either complete or filtered aerosol for gas
phase referencing. The flow through each of the exposure positions is individually controlle d
by a mass flow controller (lower left) Cell exposure: the aerosol passes through the aerosol inlet
and is streaming directly over the cell cultures.
(EPS)
S3 Fig. Particle dosing and morphology. (A) Cell viability at DF and HFO aerosol particle
dose. A549 cells were exposed for 4h to 1:40 diluted DF or 1:100 diluted HFO. Directly after ex-
posure, cell viability was mea sured by reduction of Alamar Blue and compared to cells exposed
to the filtered aerosols. Reported are the means relative to filtered aerosol ± SD from 3 (HFO)
or 2 (DF) independent experiments. As requested for the further omics study, the viability is
not impaired by the DF or HFO particle exposure. (B) Size dependent deposited dose of DF
and HFO particles (left ordinate) as well as deposition probability (W, right ordinate) calculat-
ed according to Comouth et al. (1) for a size dependent density profile. (C) Mass dose of DF
and HFO particles deposited per cell area. Data are estimated from gravimetric filter samples
(case 1, 2) and from electrical low pressure impactor (ELPI) size distributions (case 3, 4). Cal-
culations are performed assuming a constant deposition probability of W = 1.5% for all particle
diameters (case 1, 2). For comparison, calculations are performed additionally using the size
dependent probability Wρ(D) based on Comouth et al. (31) and a particle density based on a
mass-mobility relationship for DF and HFO (case 3, 4). In all cases the deposited PM dose is
about a factor 2 higher for the HFO case. d-g, TEM images of diesel fuel exposure aerosol parti-
cles. The typical soot agglomerate structure (D,E) and the layered graphitic structure (F,G) is
typical for rather pure, elemental carbon containing soot. (H-L), TEM images of heavy fuel oil
exposure aerosol particles. The often much smaller particles consist of heavier elements (black
speckles) and tarry substance (crusted appearance). The HFO-EA soot particles have a more
amorphous structure than the diesel fuel soot (J).
(EPS)
S4 Fig. Compounds in particulate matter. (A) Exemplary sum-parameters and compound-
class data for exposure aer osol (EA) particu late matter for HFO-EA and DF-EA. Particular
abundance and statistic parameters ratios (a), absolute concentrations (b) and statistic param-
eters on the sample complexity (c) reveal a substantial complexity of the organic-chemical
composition of the particulate matter.
1
EC/OC coupled to SPI,
2
EC/OC coupled to REMPI,
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 12 / 17
3
EC/OC-analysis (thermal-optical method),
4
AMS,
5
Filter weighing.
6
Aethalometer,
7
Compre-
hensive two-dimensional gas chromatography/Time-of-flight mass spectrometry,
8
Fourier-
Transform Ion Cyclotron Resonance Mass Spectrometry with atmospheric chemical ioniza-
tion,
9
Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry with electrospray ioni-
zation,
10
Thermal desorption/direct derivatization gas chromatography/Mass spectrometry,
11
GC/MS. (B) Elemental analysis of the particulate matter. Exemplary concentrations-ratios
(HFO-EA- over DF-EA-particles) of elements (left). Absolute concentrations of the species in
the HFO-EA- (red bars) and DF-EA-particles (blue bars) are also shown (right). Method:
ICP-AES. (C) Exemplary concentration-ratios (HFO-EA- over DF-EA-particles) of polycyclic
aromatic hydrocarbons (PAH) (left). Absolute concentrations of the species in the HFO-EA-
(red bars) and DF-EA-particles (blue bars) are also shown (right). The larger the PAH-struc-
ture, the stronger is the prevalence of the compound in the HFO-EA-particles. Methods:
1
Thermal desorption/derivatization gas chromatography/Mass spectrometry,
2
Gas chromatog-
raphy/mass spectrometry,
3
Liquid chromatography/Tandem mass spectrometry. (D) Exempla-
ry concentration-ratios (HFO-EA- over DF-EA-particles) of aliphatic hydrocarbons (left).
Absolute concentrations of the species in the HFO-EA- (red bars) and DF-EA-particles (blue
bars) are also shown (right). The same behaviour as in the PAH compound class is observed:
The larger the aliphatic-structure, the stronger is the prevalence of the compound in the
HFO-EA-particles.
(EPS)
S5 Fig. DF regulates more transcripts, proteins and metabolites than HFO. (A-C) Compari-
son of regula tion magnitude and regulation significance (obtained with a two-tailed t-Students
t-test on the replicate measurements). Mean of log2 fold change aerosol/filtered is plotted vs.
-log10 p-value of comp lete datasets of transcriptome in BEAS-2B cells (A), proteome (B) and
metabolome (C) in A549 cells for DF and HFO. (D-F), Comparison of regulation magnitude
and abundance of regulated transcripts, proteins or metabolites. Mean of log
2
fold change aero-
sol/filtered is plotted vs. mean of log
10
fold intensity of complete datasets of transcriptome (D),
proteome (E) and metabolome (F) for DF and HFO.
(EPS)
S6 Fig. Cellular responses to DF and HFO differ qualitatively. (A-C) Distinct patterns of reg-
ulation of DF and HFO. Hierarchical clustering of highest regulated entities of each omic ap-
proach: transcriptomics (A) (BEAS-2B), proteomics (B) and metabolomics (C) (A549). (D,E)
Pathways known to be affected by diesel particle exposure. Transcriptome pathway analysis
was performed using 1.5-fold regulated genes. Typical PM-influenced pathways were selected
and according gene regulation were clustered hierarchically. Apoptosis (D, pro- and anti-apo-
ptotic genes), Oxidative stress (E).
(EPS)
S7 Fig. Meta-analysis of gene ontology-terms in the proteomi c and transcriptomic mea-
surement of DF and HFO particle-treated samples. Significantly regulated proteins in A549
cells were determined using 10% of lowest and 10% of highest log2 fold change in the ratio
Aerosol/Gas and a cut-off of log10(p-value) >1 for 3 replicates. According to the high identi-
fication number, significantly regulated transcripts in BEAS-2B cells were determined using
5% of lowest and 5% of highest log2 fold change of Aeroso l/Gas and a cut-off oflog10(p-
value) >1 for 3 replicates. GO term analysis was performed using David Tool. The p-values of
GO-terms were z-transformed, hierarchically clustered, and plotted as a heat map.
(EPS)
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 13 / 17
S8 Fig. DF- and HFO-particles disrupt lung epithelial integrity. (A) Histopathology of
HFO-/DF-particle treated NHBE cells. Light microscopy histological analysis of sections of the
NHBE cultures treated with PBS (control), and (B) HFO, (C) DF and (D) CB120 at a dose
150 μg/cm
2
for 24 h. Hematoxylin and eosin staining, scale bar = 50 μm. (E) TEM micrographs
of HFO- and (F) DF-particle treated NHBE cells. Ribosome agglomeration in cells of the
NHBE cultures after 24 h incubation at a dose 150 μg/cm
2
; n = 5. Scale bar = 2 μm.
(EPS)
S9 Fig. Secreted metabolites and metabolomic flux analysis. Met abolism of U-
13
C-Glucose
through central carbon metabolism in A549 cells. Reduced model of central carbon metabo-
lism, with labeled atom transition marked for selected metabolit es. Red circles =
13
C labeled
carbon; Blue circles =
13
C labeled carbon from Malic Enzyme activity; White circles =
12
C unla-
beled carbon. Selected Secreted Metabolite Ratios. Selected metabolites were measured through
GC/MS analysis of cellular medium post exposure. Values shown are the ratios of unfiltered
treatments to filtered treatments for each fuel type during three replicates. Metabolic flux mea-
surements based on
13
C-labeled glucose. Filtered and unfiltered aerosol samples were
analyzed separately.
(EPS)
S10 Fig. Exemplary light microscopic image of a confluent A459 cell layer. 4x10
5
A549 cells
were seeded into a 24mm trans-well insert. After 24h and just before ALI-exposure, confluence
was checked by light microscopy.
(TIF)
S1 Table. Chemical Analytics of Ship Exhaust Particles.
(XLSX)
S2 Table. Biological Responses to Ship Exhaust Particles.
(XLSX)
S1 Text. Mater ials and Methods.
(DOCX)
Acknowledgments
The technical efforts of Anita Wüst, Evelyn Hübner, Renate Effner, Jenny Ghelfi, Thekla
Cordes, and Christian Jäger are greatly appreciated. We thank Patrick Beaudette for carefully
reading the manuscript.
Author Contributions
Conceived and designed the experiments: SO T. Kanashova OS SCS T. Streibel JP MD HRP
SM SD CW HH JKJ MRH KAB MK EK GJ MS JO LM ME AR TG CR KH JB GD RZ. Per-
formed the experiments: SO T. Kanashova OS SCS JP MD CS BS RR TT AJW ZP BM AP MK
EK GJ JL GM MS JO PR LM ME AR BW T. Schwemer HC CPR GA CR. Analyzed the data:
SO T. Kanashova OS SCS T. Streibel JMAS JP MD SD CW BS RR TT AJW ZP BM AP MK JT
EK GJ JSK JL GM MS SK JO PR LM ME AR TG BW T. Schwemer HC CPR GA CR KH JB GD
RZ. Contributed reagents/materials/analysis tools: SO T. Kanashova OS SCS T. Streibel JMAS
JP MD HRP CS SM SD CW BS RR HH TT JKJ MRH CSW CTH KAB AJW ZP BM T. Krebs
AP MK JT EK GJ SS JSK JL GM MS SK JO PR LM ME AR TG BW T. Schwemer HC CPR GA
CR KH JB GD RZ. Wrote the paper: SO T. Kanashova OS SCS T. Streibel JMAS JP MD SM BS
KAB AJW ZP JT EK GJ SS JSK JL GM MS SK JO LM ME AR TG BW T. Schwemer HC CPR
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 14 / 17
GA CR KH JB GD RZ. Initially conceived and designed the study: T. Streibel HRP CW HH JKJ
MRH KAB T. Krebs JT EK JSK JL KM MS TG KH JB GD RZ.
References
1. Pope CA 3rd, Dockery DW. Air pollution and life expectancy in China and beyond. Proc Natl Acad Sci
USA. 2013; 110(32):128612. doi: 10.1073/pnas.1310925110 PMID: 23847200
2. Chen Y, Ebenstein A, Greenstone M, Li H. Evidence on the impact of sustained exposure to air pollu-
tion on life expectancy from China's Huai River policy. Proc Natl Acad Sci USA. 2013; 110(32):12936
41. doi: 10.1073/pnas.1300018110 PMID: 23836630
3. Benbrahim-Tallaa L, Baan RA, Grosse Y, Lauby-Secretan B, El Ghissassi F, Bouvard V, et al. Carcino-
genicity of diesel-engine and gasoline-engine exhausts and some nitroarenes. Lancet oncol. 2012; 13
(7):6634. PMID: 22946126
4. Dalsoren SB, Eide MS, Endresen O, Mjelde A, Gravir G, Isaksen ISA. Update on emissions and envi-
ronmental impacts from the international fleet of ships: the contribution from major ship types and ports.
Atmos Chem Phys. 2009; 9:217194.
5. Matthias V, Bewersdorff I, Aulinger A, Quante M. The contribution of ship emissions to air pollution in
the North Sea regions. Environ pollut. 2010; 158(6):224150. doi: 10.1016/j.envpol.2010.02.013 PMID:
20226578
6. Ault AP, Moore MJ, Furutani H, Prather KA. Impact of emissions from the Los Angeles port region on
San Diego air quality during regional transport events. Environ Sci Technol. 2009; 43(10):35006.
PMID: 19544846
7. Poplawski K, Setton E, McEwen B, Hrebenyk D, Graham M, Keller P. Estimation and assesment of
cruise ship emissions in Victoria, BC, Canada. Atmos Environ. 2011; 45:82433.
8. Corbett JJ, Winebrake JJ, Green EH, Kasibhatla P, Eyring V, Lauer A. Mortality from ship emissions: a
global assessment. Environ Sci Technol. 2007; 41(24):85128. PMID: 18200887
9. EPA. Diesel Boats and Ships. Available: http://www.epa.gov/otaq/marine.htm. Accessed March 2014)
2014.
10. Lack DA, Cappa CD, Langridge J, Bahreini R, Buffaloe G, Brock C, et al. Impact of fuel quality regula-
tion and speed reductions on shipping emissions: implications for climate and air quality. Environ Sci
Technol. 2011; 45(20):905260. doi: 10.1021/es2013424 PMID: 21910443
11. Blatcher DJ, Eames I. Compliance of Royal Naval ships with nitrogen oxide emissions legislation. Ma-
rine pollution bulletin. 2013; 74:108. doi: 10.1016/j.marpolbul.2013.07.010 PMID: 23906471
12. Winebrake JJ, Corbett JJ, Green EH, Lauer A, Eyring V. Mitigating the health impacts of pollution from
oceangoing shipping: an assessment of low-sulfur fuel mandates. Environ Sci Technol. 2009; 43
(13):477682. PMID: 19673264
13. Borrell Fontelles J, Straw J. Directive 2005/33/EC of the Europen Parliament and the council. OJEU.
2005; L191/59(22.7.2005).
14. Khan MY, Giordano M, Gutierrez J, Welch WA, Asa-Awuku A, Miller JW, et al. Benefits of two mitigation
strategies for container vessels: cleaner engines and cleaner fuels. Environ Sci Technol. 2012; 46
(9):504956. doi: 10.1021/es2043646 PMID: 22468877
15. Gaetjens. http://smm-hamburg.com/fileadmin/img/content/programme/downloads/programmpunkte_
de/491_7351_gaetjens.pdf 2012.
16.
Eyring V, Isaksen ISA, Berntsen T, Collins WJ, Corbett JJ, Endresen O, et al. Transport impacts on at-
mosphere and climate: Shipping. Atmospheric Environment. 2010; 44(37):473571. doi: 10.1016/j.
atmosenv.2009.04.059
17. Eyring V, Köhler HW, van Aardenne J, Lauer A. Emissions from international shipping. J Geophys Tes.
2005; 110:D17305.
18. Lloyd AC, Cackette TA. Diesel engines: environmental impact and control. J Air & Waste Manag
Assoc. 2001; 51(6):80947.
19. Schwarze PE, Totlandsdal AI, Lag M, Refsnes M, Holme JA, Ovrevik J. Inflammation-related effects of
diesel engine exhaust particles: studies on lung cells in vitro. BioMed research international. 2013;
(685142: ):113. doi: 10.1155/2013/685142 PMID: 23509760
20. Holder AL, Lucas D, Goth-Goldstein R, Koshland CP. Cellular response to diesel exhaust particles
strongly depends on the exposure method. Toxicol Sci: an official journal of the Society of Toxicology.
2008; 103(1):10815. doi: 10.1093/toxsci/kfn014
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 15 / 17
21. Paur H-R, Cassee F, Teeguarden J, Fissan H, Diabate S, Aufderheide M, et al. In-vitro cell exposure
studies for the assessment of nanoparticle toxicity in the lungA dialog between aerosol science and
biology. J Aerosol Sci. 2011; 42(10):66892. doi: 10.1016/j.jaerosci.2011.06.005
22. Tsukue N, Okumura H, Ito T, Sugiyama G, Nakajima T. Toxicological evaluation of diesel emissions on
A549 cells. Toxicol in vitro. 2010; 24(2):3639. doi: 10.1016/j.tiv.2009.11.004 PMID: 19900534
23. Cooney DJ, Hickey AJ. Cellular response to the deposition of diesel exhaust particle aerosols onto
human lung cells grown at the air-liquid interface by inertial impaction. Toxicol in vitro. 2011; 25
(8):195365. doi: 10.1016/j.tiv.2011.06.019 PMID: 21756993
24. Oostingh GJ, Papaioannou E, Chasapidis L, Akritidis T, Konstandopoulos AG, Duschl A. Development
of an on-line exposure system to determine freshly produced diesel engine emission-induced cellular
effects. Toxicol in vitro. 2013; 27(6):174652. doi: 10.1016/j.tiv.2013.04.016 PMID: 23684770
25. Kooter IM, Alblas MJ, Jedynska AD, Steenhof M, Houtzager MM, Ras M. Alveolar epithelial cells
(A549) exposed at the air-liquid interface to diesel exhaust: First study in TNO's powertrain test center.
Toxicol in vitro. 2013; 27(8):23429. doi: 10.1016/j.tiv.2013.10.007 PMID: 24161370
26. Adam TW, Chirico R, Clairotte M, Elsasser M, Manfredi U, Martini G, et al. Application of modern online
instrumentation for chemical analysis of gas and particulate phases of exhaust at the European Com-
mission heavy-duty vehicle emission laboratory. Anal Chem. 2011; 83(1):6776. Epub 2010/12/04. doi:
10.1021/ac101859u PMID: 21126058
27. Mueller D, Uibel S, Takemura M, Klingelhoefer D, Groneberg DA. Ships, ports and particulate air pollu-
tionan analysis of recent studies. J Occup Med Toxicol. 2011; 6:31. doi: 10.1186/1745-6673-6-31
PMID: 22141925
28. Cooper J. Exhaust emissions from ships at berth. Atmos Environ. 2003; 37:381730.
29. Winnes H, Fridell E. Particle emissions from ships: dependence on fuel type. J Air & Waste Manag
Assoc. 2009; 59(12):13918.
30. Reddel RR, Ke Y, Gerwin BI, McMenamin MG, Lechner JF, Su RT, et al. Transformation of human
bronchial epithelial cells by infection with SV40 or adenovirus-12 SV40 hybrid virus, or transfection via
strontium phosphate coprecipitation with a plasmid containing SV40 early region genes. Cancer Res.
1988; 48(7):19049. PMID: 2450641
31. Kooter IM, Alblas MJ, Jedynska AD, Steenhof M, Houtzager MM, van Ras M. Alveolar epithelial cells
(A549) exposed at the air-liquid interface to diesel exhaust: First study in TNO's powertrain test center.
Toxicol in vitro. 2013; 27(8):23429. doi: 10.1016/j.tiv.2013.10.007 PMID: 24161370
32. Steinritz D, Mohle N, Pohl C, Papritz M, Stenger B, Schmidt A, et al. Use of the Cultex(R) Radial Flow
System as an in vitro exposure method to assess acute pulmonary toxicity of fine dusts and nanoparti-
cles with special focus on the intra- and inter-laboratory reproducibility. Chemico-biol Int. 2013; 206
(3):47990. doi: 10.1016/j.cbi.2013.05.001 PMID: 23669118
33. Herzog F, Clift MJ, Piccapietra F, Behra R, Schmid O, Petri-Fink A, et al. Exposure of silver-nanoparti-
cles and silver-ions to lung cells in vitro at the air-liquid interface. Part Fibre Toxicol. 2013; 10:11. doi:
10.1186/1743-8977-10-11
PMID: 23557437
34. Persoz C, Achard S, Momas I, Seta N. Inflammatory response modulation of airway epithelial cells ex-
posed to formaldehyde. Toxicol Lett. 2012; 211(2):15963. doi: 10.1016/j.toxlet.2012.03.799 PMID:
22484645
35. Baber O, Jang M, Barber D, Powers K. Amorphous silica coatings on magnetic nanoparticles enhance
stability and reduce toxicity to in vitro BEAS-2B cells. Inhal Toxicol 2011; 23(9):53243. doi: 10.3109/
08958378.2011.592869 PMID: 21819260
36. Diabate S, Mulhopt S, Paur HR, Krug HF. The response of a co-culture lung model to fine and ultrafine
particles of incinerator fly ash at the air-liquid interface. Atla-Altern Lab Anim. 2008; 36(3):28598.
PMID: 18662093
37. Patel MM, Chillrud SN, Deepti KC, Ross JM, Kinney PL. Traffic-related air pollutants and exhaled mark-
ers of airway inflammation and oxidative stress in New York City adolescents. Environ Res. 2013;
121:718. doi: 10.1016/j.envres.2012.10.012 PMID: 23177171
38. Comouth A, Saathoff H, Naumann K-H, Muelhopt S, Paur H-R, Leisner T. Modelling and measurement
of particle deposition for cell exposure at the airliquid interface. J Aerosol Sci. 2013; 63(0):10314.
doi: 10.1016/j.jaerosci.2013.04.009
39. Karthikeyan S, Thomson EM, Kumarathasan P, Guenette J, Rosenblatt D, Chan T, et al. Nitrogen diox-
ide and ultrafine particles dominate the biological effects of inhaled diesel exhaust treated by a cata-
lyzed diesel particulate filter. Toxicol Sci. 2013; 135(2):43750. doi: 10.1093/toxsci/kft162 PMID:
23897985
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 16 / 17
40. Ghio AJ, Dailey LA, Soukup JM, Stonehuerner J, Richards JH, Devlin RB. Growth of human bronchial
epithelial cells at an air-liquid interface alters the response to particle exposure. Part Fibre Toxicol.
2013; 10(1):25. doi: 10.1186/1743-8977-10-25
41. Dept. of Health and Human Services. NIOHS pocket guide to chemical hazards. Barsan M, editor. Cin-
cinnati Ohio: NIOHS publications; 2007.
42. Pagels J, Khalizov AF, McMurry PH, Zhang RY. Processing of Soot by Controlled Sulphuric Acid and
Water Condensation: Mass and Mobility Relationship. Aerosol Sci Technol. 2009; 43(7):62940. doi:
10.1080/02786820902810685
43. Park K, Cao F, Kittelson DB, McMurry PH. Relationship between particle mass and mobility for diesel
exhaust particles. Environ Sci Technol. 2003; 37(3):57783. doi: 10.1021/es025960v PMID: 12630475
44. Ferron GA, Upadhyay S, Zimmermann R, Karg E. Model of the Deposition of Aerosol Particles in the
Respiratory Tract of the Rat. II. Hygroscopic Particle Deposition. J Aerosol Med Pulm Drug Deliv. 2013;
26(2):10119. doi: 10.1089/jamp.2011.0965 PMID: 23550602
45. Karg E, Ferron GA. The hygroscopic particle lung deposition model Neuherberg / Munich: Helmholtz
Zentrum München; 2014 [cited 2014]. Available: http://www.helmholtz-muenchen.de/en/neu-cma/
research/facilities/lung-deposition-model/index.html.
46. Wehner B, Birmili W, Gnauk T, Wiedensohler A. Particle number size distributions in a street canyon
and their transformation into the urban-air background: measurements and a simple model study.
Atmos Environ. 2002; 36(13):221523.
47. Vignati E, Berkowicz R, Palmgren F, Lyck E, Hummelshoj P. Transformation of size distributions of
emitted particles in streets. Sci Total Environ. 1999; 235(13):3749. PMID: 10535125
48. Oeder S, Jorres RA, Weichenmeier I, Pusch G, Schober W, Pfab F, et al. Airborne indoor particles from
schools are more toxic than outdoor particles. Am J Respir Cell Mol Biol. 2012; 47(5):57582. doi: 10.
1165/rcmb.2012-0139OC PMID: 22904196
49. Schwanhausser B, Busse D, Li N, Dittmar G, Schuchhardt J, Wolf J, et al. Global quantification of mam-
malian gene expression control. Nature. 2011; 473(7347):33742. doi: 10.1038/nature10098 PMID:
21593866
50. Palsson-McDermott EM, O'Neill LA. The Warburg effect then and now: from cancer to inflammatory dis-
eases. BioEssays. 2013; 35(11):96573. doi: 10.1002/bies.201300084 PMID: 24115022
51. Zhang WC, Shyh-Chang N, Yang H, Rai A, Umashankar S, Ma S, et al. Glycine decarboxylase activity
drives non-small cell lung cancer tumor-initiating cells and tumorigenesis. Cell. 2012; 148(12):259
72. doi: 10.1016/j.cell.2011.11.050 PMID: 22424234
52. Mueller L, Jakobi G, Orasche J, Karg E, Sklorz M, Abbaszade G, et al. Online determination of polycy-
clic aromatic hydrocarbon formation from a flame soot generator. Anal Bioanal Chem. 2015. doi: 10.
1007/s00216-015-8549-x
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 17 / 17

Supplementary resource (1)

... When investigating the effects of environmental exposure on biological systems, the current trend is to work at the air-liquid interface and the use of human material. Air-liquid interface exposure allows inclusion of (combustion) gases, which were shown to be equally biologically active as diesel particles alone (Oeder et al., 2015). The most prominent allergic sensitizations in northern Europe are against pollen, mainly birch and grass pollen (Burbach et al., 2009;Smith et al., 2014). ...
... We therefore developed a Pollen Sedimentation Chamber (pollen-ALI), which allows exposure of cells at the air-liquid interface to an environmentally valid low dose whole pollen (Candeias et al., 2021). The co-exposure to fresh complete diesel exhaust gases at the air-liquid interface was achieved in this study using a VITROCELL® exposure station (Ihantola et al., 2020;Oeder et al., 2015;Sapcariu et al., 2016). ...
... The culture of the immortalized human bronchial epithelial cell line BEAS-2B (ATCC® CRL-9609™) was performed as in Oeder et al. (2015). For all exposures, 0.4 × 10 6 cells were cultured on transferable 24 mm Transwells® inserts with a 0.4 μm pore polyester membrane (Corning, Tewksbury, USA). ...
Full-text available
Article
Pollen related allergic diseases have been increasing for decades. The reasons for this increase are unknown, but environmental pollution like diesel exhaust seem to play a role. While previous studies explored the effects of pollen extracts, we studied here for the first time priming effects of diesel exhaust on native pollen exposure using a novel experimental setup. Methods Human bronchial epithelial BEAS-2B cells were exposed to native birch pollen (real life intact pollen, not pollen extracts) at the air-liquid interface (pollen-ALI). BEAS-2B cells were also pre-exposed in a diesel-ALI to diesel CAST for 2 h (a model for diesel exhaust) and then to pollen in the pollen-ALI 24 h later. Effects were analysed by genome wide transcriptome analysis after 2 h 25 min, 6 h 50 min and 24 h. Selected genes were confirmed by qRT-PCR. Results Bronchial epithelial cells exposed to native pollen showed the highest transcriptomic changes after about 24 h. About 3157 genes were significantly up- or down-regulated for all time points combined. After pre-exposure to diesel exhaust the maximum reaction to pollen had shifted to about 2.5 h after exposure, plus the reaction to pollen was desensitised as only 560 genes were differentially regulated. Only 97 genes were affected synergistically. Of these, enrichment analysis showed that genes involved in immune and inflammatory response were involved. Conclusion Diesel exhaust seems to prime cells to react more rapidly to native pollen exposure, especially inflammation related genes, a factor known to facilitate the development of allergic sensitization. The marker genes here detected could guide studies in humans when investigating whether modern and outdoor diesel exhaust exposure is still detrimental for the development of allergic disease.
... Many hazardous components derived from anthropogenic activities are concentrated mainly on fine particles (Hien et al., 2007;Hu et al., 2014;Li et al., 2015) and particles smaller than 2.5 μm (PM 2.5 ) are of particular concern because they are able to penetrate deep into the lungs. PM soluble components have been shown to be released within minutes in simulated lung interstitial environment solution (Palleschi et al., 2018) and to affect the energy metabolism, protein synthesis, and chromatin modification of human cells (Oeder et al., 2015). Trace metals in PM are known to have negative health effects (Okuda et al., 2008;Qi et al., 2016), such as impacts on human attention, executive function, mental flexibility and cognitive efficiency (Jiang et al., 2014;Rafiee et al., 2020;Soetrisno and Delgado-Saborit, 2020), lung damage (Palleschi et al., 2018) and cancer (IARC, 2014;Wang et al., 2015;Galon-Negru et al., 2019). ...
... The study of Ho et al. (2019) applied a combination of bottom-up and top-down methods for calculating atmospheric emissions in HCM and found that the seaports contributed up to 5% and 15% of total TSP (total suspended particulate matter) and SO 2 emissions, respectively (Ho et al., 2019). Thus the combustion of HFO by shipping emits not only V and Ni but also various pollutants including PM, inorganic and organic compounds and affects air quality in HCM (Becagli et al., 2012;Oeder et al., 2015). ...
Full-text available
Article
Hanoi and Ho Chi Minh City (HCM), the most populous cities in Vietnam, have received increasing global attention because of their poor air pollution status. As part of the recent UK-Vietnam 2-Cities project, the concentrations of trace metals in fine particulate matter have been characterized. 24-hour samples of PM2 were collected at 2 sites in Hanoi and 3 sites in HCM during two 4-week periods in September/October 2018 and March 2019. The soluble fraction of 15 trace metal(oid)s (Fe, Al, Mn, Ti, Zn, V, Cu, Ni, Co, Cd, Pb, Th, Cr, As, and Sb) bound to PM2 were analyzed by ICP-MS. The results show that Zn was the most abundant soluble metal in PM2 in both cities, with very large numbers of road vehicles (e.g. tyre wear) likely contributing in both cities and non-ferrous metal production being a substantial additional source in Hanoi. Fe and Al, derived from crustal sources, were the dominant metals after Zn. Most trace metals concentrations in Hanoi were higher than in HCM, especially toxic metals such as Pb, Cd, Cr and As. V and Ni were the only two metals having higher concentrations in HCM than in Hanoi, likely due to shipping emissions (combustion of heavy fuel oil) that strongly affect the air quality in HCM. Coal-power plants and non-ferrous metal production are likely to be the major sources of trace metals in Hanoi. Health risk assessment shows that a high carcinogenic risk exists for inhalation exposure of soluble trace metals bound to PM2 in both cities.
... However, it is still largely unknown which PM properties-such as size, mass, shape, surface properties, or chemical composition-induce biological responses (Burkholder et al. 2017;Park et al. 2018;Wyzga and Rohr 2015). Furthermore, our knowledge is mostly limited to the effects of collected airborne particles under submerged exposure conditions rather than the direct deposition and interaction of aerosols with cell cultures (Ihantola et al. 2020;Oeder et al. 2015). Submerged exposures do not reflect the physiological condition of airway barriers because inhalation occurs by aerosol deposition onto airway epithelial cells that form, together with, for example, endothelial cells and basal membranes, a tissue layer enabling gas exchanges between the lungs and the blood circulation (Barosova et al. 2021). ...
... The tested aerosol was guided through a size-selective impactor that excluded particles with size fractions >PM 10 , before entering the inlet of the ALI exposure system and the main reactor. The ALI exposure systems were operated with different settings compared with previous publications (Mülhopt et al. 2016;Oeder et al. 2015). The total aerosol flow (inlet) was reduced to 5 L=min owing to the limited aerosol flow through the PAM reactor. ...
Full-text available
Article
Background: Secondary organic aerosols (SOAs) formed from anthropogenic or biogenic gaseous precursors in the atmosphere substantially contribute to the ambient fine particulate matter [PM ≤2.5μm in aerodynamic diameter (PM2.5)] burden, which has been associated with adverse human health effects. However, there is only limited evidence on their differential toxicological impact. Objectives: We aimed to discriminate toxicological effects of aerosols generated by atmospheric aging on combustion soot particles (SPs) of gaseous biogenic (β-pinene) or anthropogenic (naphthalene) precursors in two different lung cell models exposed at the air-liquid interface (ALI). Methods: Mono- or cocultures of lung epithelial cells (A549) and endothelial cells (EA.hy926) were exposed at the ALI for 4 h to different aerosol concentrations of a photochemically aged mixture of primary combustion SP and β-pinene (SOAβPIN-SP) or naphthalene (SOANAP-SP). The internally mixed soot/SOA particles were comprehensively characterized in terms of their physical and chemical properties. We conducted toxicity tests to determine cytotoxicity, intracellular oxidative stress, primary and secondary genotoxicity, as well as inflammatory and angiogenic effects. Results: We observed considerable toxicity-related outcomes in cells treated with either SOA type. Greater adverse effects were measured for SOANAP-SP compared with SOAβPIN-SP in both cell models, whereas the nano-sized soot cores alone showed only minor effects. At the functional level, we found that SOANAP-SP augmented the secretion of malondialdehyde and interleukin-8 and may have induced the activation of endothelial cells in the coculture system. This activation was confirmed by comet assay, suggesting secondary genotoxicity and greater angiogenic potential. Chemical characterization of PM revealed distinct qualitative differences in the composition of the two secondary aerosol types. Discussion: In this study using A549 and EA.hy926 cells exposed at ALI, SOA compounds had greater toxicity than primary SPs. Photochemical aging of naphthalene was associated with the formation of more oxidized, more aromatic SOAs with a higher oxidative potential and toxicity compared with β-pinene. Thus, we conclude that the influence of atmospheric chemistry on the chemical PM composition plays a crucial role for the adverse health outcome of emissions. https://doi.org/10.1289/EHP9413.
... Compared to MGO, HFO emissions exhibit a particular toxic mixture with higher concentrations of particulate matter (PM), polycyclic aromatic hydrocarbons (PAH), heavy alkanes and metals, such as nickel and vanadium [8][9][10][11][12]. However, combustion of the cleaner distillate fuel MGO emits higher levels of soot, which is a known carcinogen, and induces strong toxicological effects [13]. Furthermore, most organic species in the aerosol are transferred directly from the fuel as unburnt material, especially at lower power operations [14]. ...
Article
In the context of the global transportation of goods, shipping emissions account for a significant proportion of air pollution. Indeed, a focus has been made over recent years on this primary emission source, leading to several regulations with respect to the chemical composition of shipping fuels. However, these regulations mainly concern the fuel sulfur content (FSC) and do not consider other compound classes such as polycyclic aromatic hydrocarbons (PAHs) or metal-containing aromatics, i.e., petroporphyrins, known to be present in bunker fuels. Petroporphyrins are tetrapyrrole-based metal complexes derived from the transformation of chlorophylls through geological time scales. In contrast to PAHs, their fate in the combustion process and effects on environmental health are widely unknown. In this study, we present electron-transfer ionization in matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (ET-MALDI FTICR MS) for the characterization of vanadyl and nickel porphyrins in shipping feed fuels and primary particulate matter emissions. For the first time, these petroporphyrins could successfully be described in the heavy fuel oil (HFO) feeds but also in the particles emitted by the combustion of the respective fuel on a molecular level. Three main alkylated series of porphyrins were observed; these series can be qualified by their double bond equivalent, i.e., 17, 18, and 20, and correspond to various core structures. Our results highlight the molecular fate of the petroporphyrins through combustion and show that a significant amount of petroporphyrins is released unburned or partially dealkylated to the atmosphere. Furthermore, our results suggest that a higher amount of petroporphyrins might be released in harbors than in open sea, due to a less efficient combustion at lower engine load. This last observation motivates a future specific study on porphyrins’ health and environmental effects.
... Relevant equipment not only effectively collects submicron fine particles but also has a low resistance, a long service life, and low energy consumption and is thus suited to the filtration of aerosols [11][12][13]. This technology is widely used to purify cooking oil fume and oily aerosol produced by machining processes, ship engines, and diesel-powered vehicles [14][15][16][17]. In the electrostatically enhanced fibrous filter system, there is air ionization around the electrode to produce O3 because of the application of a high voltage, so the ozone concentration should be monitored in case of causing secondary pollution [18][19][20][21]. ...
Full-text available
Article
The synergistic effect of electrostatically enhanced fibrous filtration originates from the charging characteristics of aerosol particles and electret fibers in an electric field. Two electrostatically enhanced fibrous filter systems are designed in this study to investigate the mechanism of the effects of the charging characteristics of oily aerosol on the filtration efficiency. We investigate the charging characteristics and their effects on the filtration efficiency of dioctyl-phthalate (DOP) aerosol particles of various sizes by setting different filter systems and electric field intensities. The experimental results show that the charge of DOP particles increases with the strength of the electric field, and the average charge increases with the particle size. The maximum charge of DOP particles reaches 4760 eC/P, and the filtration efficiency of the coupled system improves when DOP particles are amply charged. For 0.25 μm DOP particles as the most penetrating particle size, the system had good long-term stability, and the filtration efficiency is approximately 72% higher than that of the fiber acting alone. Meanwhile, the problem of oily aerosol deposition reducing the electret filtration efficiency is solved, providing a basis for long-term filtration and oily aerosol purification by electret fiber.
... PM 2.5 is generated by natural sources (primary particles) or formed in the atmosphere through chemical reactions (secondary particles). These fine particles, as well as organic chemical aerosols, are emitted by both natural events (dust storms [149], forest fires [150]), volcanos [151] as well as from man-made (anthropogenic) sources (biomass burning, fossil fuel combustion, consumer products [152,153], cigarette smoke). Outside of particulate matter, Bruinen de Bruin et al. studied urban inhalation of benzene, formaldehyde and acetaldehyde in the European Union and reported that legal thresholds of these highly toxic compounds were exceeded for an appreciable part of the population leading to significant health side effects [47]. ...
Full-text available
Article
Environmental risk factors, including noise, air pollution, chemical agents, ultraviolet radiation (UVR) and mental stress have a considerable impact on human health. Oxidative stress and inflammation are key players in molecular pathomechanisms of environmental pollution and risk factors. In this review, we delineate the impact of environmental risk factors and the protective actions of the nuclear factor erythroid 2-related factor 2 (NRF2) in connection to oxidative stress and inflammation. We focus on well-established studies that demonstrate the protective actions of NRF2 and its downstream pathways against different environmental stressors. State-of-the-art mechanistic considerations on NRF2 signaling are discussed in detail, e.g. classical concepts like KEAP1 oxidation/electrophilic modification, NRF2 ubiquitination and degradation. Specific focus is also laid on NRF2-dependent heme oxygenase-1 induction with detailed presentation of the protective down-stream pathways of heme oxygenase-1, including interaction with BACH1 system. The significant impact of all environmental stressors on the circadian rhythm and the interactions of NRF2 with the circadian clock will also be considered here. A broad range of NRF2 activators is discussed in relation to environmental stressor-induced health side effects, thereby suggesting promising new mitigation strategies (e.g. by nutraceuticals) to fight the negative effects of the environment on our health.
Article
The impact of ship emission reductions can be maximised by considering climate, health and environmental effects simultaneously and using solutions fitting into existing marine engines and infrastructure. Several options available enable selecting optimum solutions for different ships, routes and regions. Carbon-neutral fuels, including low-carbon and carbon-negative fuels, from biogenic or non-biogenic origin (biomass, waste, renewable hydrogen) could resemble current marine fuels (diesel-type, methane and methanol). The carbon-neutrality of fuels depends on their Well-to-Wake (WtW) emissions of greenhouse gases (GHG) including carbon dioxide (CO2), methane (CH4), and nitrous oxide emissions (N2O). Additionally, non-gaseous black carbon (BC) emissions have high global warming potential (GWP). Exhaust emissions which are harmful to health or the environment need to be equally removed using emission control achieved by fuel, engine or exhaust aftertreatment technologies. Harmful emission species include nitrogen oxides (NOx), sulphur oxides (SOx), ammonia (NH3), formaldehyde, particle mass (PM) and number emissions (PN). Particles may carry polyaromatic hydrocarbons (PAHs) and heavy metals, which cause serious adverse health issues. Carbon-neutral fuels are typically sulphur-free enabling negligible SOx emissions and efficient exhaust aftertreatment technologies, such as particle filtration. The combinations of carbon-neutral drop-in fuels and efficient emission control technologies would enable (near-)zero-emission shipping and these could be adaptable in the short- to mid-term. Substantial savings in external costs on society caused by ship emissions give arguments for regulations, policies and investments needed to support this development.
Article
The comprehensive chemical description of air pollution is a prerequisite for understanding atmospheric transformation processes and effects on climate and environmental health. In this study, a prototype vacuum photoionization Orbitrap mass spectrometer was evaluated for field-suitability by an online on-site investigation of emissions from a ship diesel engine. Despite remote measurements in a challenging environment, the mass spectrometric performance could fully be exploited. Due to the high resolution and mass accuracy in combination with resonance-enhanced multiphoton ionization, the aromatic hydrocarbon profile could selectively and sensitively be analyzed. Limitations from commonly deployed time-of-flight platforms could be overcome, allowing to unraveling the oxygen- and sulfur-containing compounds. Scan-by-scan evaluation of the online data revealed no shift in exact m/z, assignment statistics with root mean square error (RMSE) below 0.2 ppm, continuous high-resolution capabilities, and good isotopic profile matches. Emissions from three different feed fuels were investigated, namely, diesel, heavy fuel oil (HFO), and very low sulfur fuel oil (VLSFO). Regulations mainly concern the fuel sulfur content, and thus, exhaust gas treatment or new emerging fuels, such as the cycle-oil-based VLSFO, can legally be applied. Unfortunately, despite lower CHS-class emissions, a substantial amount of PAHs is emitted by the VLSFO with higher aromaticity compared to the HFO. Hence, legislative measures might need to take further chemical criteria into account.
Article
The emissions of marine diesel engines have gained both global and regional attentions because of their impact on human health and climate change. To reduce ship emissions, the International Maritime Organization capped the fuel sulfur content of marine fuels. Consequently, either low-sulfur fuels or additional exhaust gas cleaning devices for the reduction in sulfur dioxide (SO2) emissions became mandatory. Although a wet scrubber reduces the amount of SO2 significantly, there is still a need to consider the reduction in particle emissions directly. We present data on the particle removal efficiency of a scrubber regarding particle number and mass concentration with different marine fuel types, marine gas oil, and two heavy fuel oils (HFOs). An open-loop sulfur scrubber was installed in the exhaust line of a marine diesel test engine. Fine particulate matter was comprehensively characterized in terms of its physical and chemical properties. The wet scrubber led up to a 40% reduction in particle number, whereas a reduction in particle mass emissions was not generally determined. We observed a shift in the size distribution by the scrubber to larger particle diameters when the engine was operated on conventional HFOs. The reduction in particle number concentrations and shift in particle size were caused by the coagulation of soot particles and formation/growing of sulfur-containing particles. Combining the scrubber with a wet electrostatic precipitator as an additional abatement system showed a reduction in particle number and mass emission factors by >98%. Therefore, the application of a wet scrubber for the after-treatment of marine fuel oil combustion will reduce SO2 emissions, but it does not substantially affect the number and mass concentration of respirable particulate matters. To reduce particle emission, the scrubber should be combined with additional abatement systems.
Article
Several studies indicate that short-sea shipping is an important source of air pollution for coastal areas and port cities. This paper reports results of a non-reactive particles dispersion model and a new set of experiments implemented for the Channel of Procida (Italy), an area with a high signal-to-noise ratio, due to intense marine traffic and low background pollution. The model successfully predicts particle number concentrations but underestimates PM2.5 data. Model and experiments show that thanks to the EU policies on marine fuels, the Channel of Procida already has good air quality levels. Besides, the paper demonstrates that fostering the use of LNG or methanol or the application of an exhaust-gas-cleaning-system may allow reducing particles emissions well above 90%. The reliability of control strategies and the benefits for the population suggest that the introduction of regulations on particles emissions for ships can be a realistic option for the future environmental policy agenda.
Full-text available
Article
We studied the impact of a catalyzed diesel particulate filter (DPF) on the toxicity of diesel exhaust. Rats inhaled exhaust from a Cummins ISM heavy-duty diesel engine, with and without DPF after-treatment, or HEPA-filtered air for 4h, on one day (single exposure) and 3 days (repeated exposures). Biological effects were assessed after 2h (single exposure) and 20h (single and repeated exposures) recovery in clean air. Concentrations of pollutants were: 1) untreated exhaust (-DPF), NO, 43ppm; NO2, 4ppm; CO, 6ppm; hydrocarbons, 11ppm; particles, 3.2x10(5)/cm(3), 60-70nm mode, 269μg/m(3); 2) treated exhaust (+DPF), NO, 20ppm; NO2, 16ppm; CO, 1ppm; hydrocarbons, 3ppm; and particles, 4.4x10(5)/cm(3), 7-8nm mode, 2μg/m(3). Single exposures to diesel exhaust (-DPF) resulted in increased neutrophils, total protein and the cytokines, GRO/KC, MIP1-α, and MCP-1 in lung lavage fluid, as well as increased gene expression of IL-6, PTGS2, MT2A, TNF-α, iNOS, GSTA1, HO-1, SOD2, ET-1 and ECE-1 in the lung, and ET-1 in the heart. Ratio of bigET-1 to ET-1 peptide increased in plasma in conjunction with a decrease in eNOS gene expression in the lungs after exposure to diesel exhaust, suggesting endothelial dysfunction. Rather than reducing toxicity, +DPF exhaust resulted in heightened injury and inflammation, consistent with the 4-fold increase in NO2 concentration. The ratio of bigET-1 to ET-1 was similarly elevated after -DPF and +DPF exhaust exposures. Endothelial dysfunction thus appeared related to particle number deposited, rather than particle mass or NO2 concentration. The potential benefits of particulate matter reduction using a catalyzed DPF may be confounded by increase in NO2 emission and release of reactive ultrafine particles.
Full-text available
Article
This paper's findings suggest that an arbitrary Chinese policy that greatly increases total suspended particulates (TSPs) air pollution is causing the 500 million residents of Northern China to lose more than 2.5 billion life years of life expectancy. The quasi-experimental empirical approach is based on China's Huai River policy, which provided free winter heating via the provision of coal for boilers in cities north of the Huai River but denied heat to the south. Using a regression discontinuity design based on distance from the Huai River, we find that ambient concentrations of TSPs are about 184 μg/m(3) [95% confidence interval (CI): 61, 307] or 55% higher in the north. Further, the results indicate that life expectancies are about 5.5 y (95% CI: 0.8, 10.2) lower in the north owing to an increased incidence of cardiorespiratory mortality. More generally, the analysis suggests that long-term exposure to an additional 100 μg/m(3) of TSPs is associated with a reduction in life expectancy at birth of about 3.0 y (95% CI: 0.4, 5.6).
Full-text available
Article
We tested the hypothesis that normal human bronchial epithelial (NHBE) cells 1) grown submerged in media and 2) allowed to differentiate at air-liquid interface (ALI) demonstrate disparities in the response to particle exposure. Following exposure of submerged NHBE cells to ambient air pollution particle collected in Chapel Hill, NC, RNA for IL-8, IL-6, heme oxygenase 1 (HOX1) and cyclooxygenase 2 (COX2) increased. The same cells allowed to differentiate over 3, 10, and 21 days at ALI demonstrated no such changes following particle exposure. Similarly, BEAS-2B cells grown submerged in media demonstrated a significant increase in IL-8 and HOX1 RNA after exposure to NIST 1648 particle relative to the same cells exposed after growth at ALI. Subsequently, it was not possible to attribute the observed decreases in the response of NHBE cells to differentiation alone since BEAS-2B cells, which do not differentiate, showed similar changes when grown at ALI. With no exposure to particles, differentiation of NHBE cells at ALI over 3 to 21 days demonstrated significant decrements in baseline levels of RNA for the same proteins (i.e. IL-8, IL-6, HOX1, and COX2). With no exposure to particles, BEAS-2B cells grown at ALI showed comparable changes in RNA for IL-8 and HOX1. After the same particle exposure, NHBE cells grown at ALI on a transwell in 95% N2-5% CO2 and exposed to NIST 1648 particle demonstrated significantly greater changes in IL-8 and HOX1 relative to cells grown in 95% air-5% CO2. We conclude that growth of NHBE cells at ALI is associated with a diminished biological effect following particle exposure relative to cells submerged in media. This decreased response showed an association with increased oxygen availability.
Article
In this study, we produced a class of diffusion flame soot particles with varying chemical and physical properties by using the mini-Combustion Aerosol STandard (CAST) and applying varying oxidant gas flow rates under constant propane, quenching, and dilution gas supply. We varied the soot properties by using the following fuel-to-air equivalence ratios (Φ): 1.13, 1.09, 1.04, 1.00, 0.96, and 0.89. Within this Φ range, we observed drastic changes in the physical and chemical properties of the soot. Oxidant-rich flames (Φ < 1) were characterized by larger particle size, lower particle number concentration, higher black carbon (BC) concentration, lower brown carbon BrC.[BC](-1) than fuel-rich flames (Φ > 1). To investigate the polycyclic aromatic hydrocarbons (PAH) formation online, we developed a new method for quantification by using the one (13)C-containing doubly charged PAH ion in a high-resolution time-of-flight aerosol mass spectrometry (HR-ToF-AMS). The time-resolved concentration showed that the larger PAHs prevailed in the fuel-rich flames and diminished in the oxidant-rich flames. By comparison with the offline in situ derivatization-thermal-desorption gas-chromatography time-of-flight mass spectrometry (IDTD-GC-ToF-MS), we found that the concentration by using the HR-ToF-AMS was underestimated, especially for lower mass PAHs (C14-C18) in the fuel-rich flames possibly due to size limitation and degradation of semi-volatile species under high vacuum and desorption temperature in the latter. For oxidant-rich flames, the large PAHs (C20 and C22) were detected in the HR-ToF-AMS while it was not possible in IDTD-GC-ToF-MS due to matrix effect. The PAH formation was discussed based on the combination of our results and with respect to Φ settings.
Article
Air-liquid interface (ALI) exposures enable in vitro testing of mixtures of gases and particles such as diesel exhaust (DE). The main objective of this study was to investigate the feasibility of exposing human lung epithelial cells at the ALI to complete DE generated by a heavy-duty truck in the state-of-the-art TNO powertrain test centre. A549 cells were exposed at the air-liquid interface to DE generated by a heavy-duty Euro III truck for 1.5 hours. The truck was tested at a speed of ∼70 km·h(-1) to simulate free-flowing traffic on a motorway. Twenty-four hours after exposure, cells were analysed for markers of oxidative stress (GSH and HO-1), cytotoxicity (LDH) and Alamar Blue assay) and inflammation (IL-8). DE exposure resulted in an increased oxidative stress response (significantly increased HO-1 levels and significantly reduced GSH/GSSH ratio), and a decreased cell viability (significantly decreased Alamar Blue levels and slightly increased LDH levels). However, the pro-inflammatory response seemed to decrease (decrease in IL-8). The results presented here demonstrate that we are able to successfully expose A549 cells at ALI to complete DE generated by a heavy-duty truck in TNO's powertrain test centre and show oxidative stress and cytotoxicity responses due to DE exposure.
Article
Inflammatory immune cells, when activated, display much the same metabolic profile as a glycolytic tumor cell. This involves a shift in metabolism away from oxidative phosphorylation towards aerobic glycolysis, a phenomenon known as the Warburg effect. The result of this change in macrophages is to rapidly provide ATP and metabolic intermediates for the biosynthesis of immune and inflammatory proteins. In addition, a rise in certain tricarboxylic acid cycle intermediates occurs notably in citrate for lipid biosynthesis, and succinate, which activates the transcription factor Hypoxia-inducible factor. In this review we take a look at the emerging evidence for a role for the Warburg effect in the immune and inflammatory responses. The reprogramming of metabolic pathways in macrophages, dendritic cells, and T cells could have relevance in the pathogenesis of inflammatory and metabolic diseases and might provide novel therapeutic strategies.
Article
Nitrogen oxide (NOx) emissions from marine diesel engines pose a hazard to human health and the environment. From 2021, demanding emissions limits are expected to be applied to sea areas that the Royal Navy (RN) accesses. We analyze how these future constraints affect the choice of NOx abatement systems for RN ships, which are subject to more design constraints than civilian ships. A weighted matrix approach is used to facilitate a quantitative assessment. For most warships to be built soon after 2021 Lean Nitrogen Traps (LNT) in conjunction with Exhaust Gas Recirculation (EGR) represents a relatively achievable option with fewer drawbacks than other system types. Urea-selective catalytic reduction is likely to be most appropriate for ships that are built to civilian standards. The future technologies that are at an early stage of development are discussed.
Article
Characterization of the effects of cruise ship emissions on local air quality is scarce. Our objective was to investigate community level concentrations of fine particulate matter (PM2.5), nitrogen dioxide (NO2) and sulphur dioxide (SO2) associated with cruise ships in James Bay, Victoria, British Columbia (BC), Canada. Data obtained over four years (2005–2008) at the nearest air quality network site located 3.5km from the study area, a CALPUFF modeling exercise (2007), and continuous measurements taken in the James Bay community over a three-month period during the 2009 cruise ship season were examined. Concentrations of PM2.5 and nitrogen oxide (NO) were elevated on weekends with ships present with winds from the direction of the terminal to the monitoring station. SO2 displayed the greatest impact from the presence of cruise ships in the area. Network data showed peaks in hourly SO2 when ships were in port during all years. The CALPUFF modeling analysis found predicted 24-hour SO2 levels to exceed World Health Organization (WHO) guidelines of 20μgm−3 for approximately 3% of 24-hour periods, with a maximum 24-hour concentration in the community of 41μgm−3; however, the CALPUFF model underestimated concentrations when predicted and measured concentrations were compared at the network site. Continuous monitoring at the location in the community predicted to experience highest SO2 concentrations measured a maximum 24-hour concentration of 122μgm−3 and 16% of 24-hour periods were above the WHO standard. The 10-minute concentrations of SO2 reached up to 599μgm−3 and exceeded the WHO 10-minute SO2 guideline (500μgm−3) for 0.03% of 10-minute periods. No exceedences of BC Provincial or Canadian guidelines or standards were observed.
Article
We developed a fluid dynamic model to predict the size and material dependent particle deposition efficiencies for a commercial available exposure chamber. Validated by measurements with SiO2 and polystyrene particle standards between 29 nm and 2μm we obtained a parameterization of the particle deposition efficiency for a specific set of practically relevant flow, pressure and temperature conditions that can be used for accurate dose calculations at these conditions. Furthermore, the model predicts that a significant impact on the deposition efficiency due to the fractal-like structure of nanoparticle agglomerates is expected for cluster sizes beyond 200 nm mobility equivalent diameter. For the commercially available gravitation–diffusion based exposure chamber investigated here at one specific flow rate, the results indicate that deposition rates of particles smaller than 100 nm are too low in order to deposit mass doses that are equivalent to typical cytotoxic LOAELs determined in submerged experiments within reasonable times.