Online exposure systems for toxicological effects of combustion emissions and nanoparticles

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Dissertations in Forestry and Natural Sciences

DISSERTATIONS | TUUKKA IHANTOLA | ONLINE EXPOSURE SYSTEMS FOR TOXICOLOGICAL EFFECTS OF COMBUSTION... | No 447

TUUKKA IHANTOLA

Online exposure systems for toxicological effects of combustion emissions and

nanoparticles

PUBLICATIONS OF

THE UNIVERSITY OF EASTERN FINLAND

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ONLINE EXPOSURE SYSTEMS FOR

TOXICOLOGICAL EFFECTS OF COMBUSTION

EMISSIONS AND NANOPARTICLES

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Tuukka Ihantola

ONLINE EXPOSURE SYSTEMS FOR

TOXICOLOGICAL EFFECTS OF COMBUSTION EMISSIONS AND NANOPARTICLES

Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences

No 447

University of Eastern Finland Kuopio

2021

Academic dissertation

To be presented by permission of the Faculty of Science and Forestry for public examination in the Auditorium CA101 in the Canthia Building at

the University of Eastern Finland, Kuopio, on December 4, 2021, at 12 o’clock noon

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Punamusta Oy Joensuu, 2021 Editor: Pertti Pasanen

Myynti: Itä-Suomen yliopiston kirjasto ISBN: 978-952-61-4394-1 (nid.) ISBN: 978-952-61-4395-8 (PDF)

ISSNL: 1798-5668 ISSN: 1798-5668 ISSN: 1798-5676 (PDF)

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5 Author’s address: Tuukka Ihantola

University of Eastern Finland

Depart. of Environmental and Biological Sciences P.O. Box 1627

70211 KUOPIO, FINLAND Email: tuukka.ihantola@uef.fi

Supervisors: Associate Professor Pasi Jalava, Ph.D., title of docent University of Eastern Finland

70211 KUOPIO, FINLAND Email: pasi.jalava@uef.fi

Professor emerita Maija-Riitta Hirvonen, Ph.D.

University of Eastern Finland

70211 KUOPIO, FINLAND

Email: maija-riitta.hirvonen@uef.fi

Mikko Happo, Ph.D., title of docent Ramboll Finland Oy

Itsehallintokuja 3 P.O. Box 25

02601 ESPOO, FINLAND

Email: mikko.happo@ramboll.fi

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Reviewers: Espen Mariussen, Ph.D

Norwegian Institute of Public Health Depart. of Air Quality and Noise PO Box 222 Skøyen

0213 Oslo, NORWAY

Email: espen.mariussen@fhi.no

Professor Dario Greco, Ph.D

FHAIVE (Finnish Hub for Development and Validation of Integrated Approaches),

Faculty of Medicine and Health Technology, Tampere University.

33520 Tampere, FINLAND Email: Dario.Greco@tuni.fi

Opponent: Professor Harri Alenius

Institute of Environmental Medicine (IMM), Karolinska Institutet C6,

Systems toxicology, Box 210 171 77, Stockholm, Sweden

Email: harri.alenius@ki.se

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7 Ihantola, Tuukka

Online exposure systems for toxicological effects of combustion emissions and nanoparticles.

Kuopio: University of Eastern Finland, 2021 Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences; 447 ISBN: 978-952-61-4394-1 (print)

ISSNL: 1798-5668 ISSN: 1798-5668

ISBN: 978-952-61-4395-8 (PDF) ISSN: 1798-5676 (PDF)

ABSTRACT

Air pollution is a health hazard that affects every human being. One key source for local air pollution is residential heating by combustion. In the combustion process, several harmful compounds such as polyaromatic hydrocarbons are produced. They can cause several adverse health issues, and to assess the effects of combustion emissions several toxicological endpoints are studied.

Combustion emissions toxicity evaluation can be done on living beings such as mice (in vivo) or cell cultures (in vitro). In in vivo studies, the exposure can be done naturally by inhalation in whole-body exposure or directly applying the studied compounds into lungs in intratracheal instillation. However, there are several problems with in vivo experiments that hinders their use. The most relevant methods are highly resource-intensive, time-consuming, and experiments are not always representative of human exposure. In vitro, on the other hand, can answer these problems, but these tests are often too simple to represent the complex systems of a living being. However, these studies rely on presentative exposure of tested compounds, i.e. to have unchanged tested compounds exposed to cells, which has been the foremost restriction for toxicological studies. In earlier studies, exposure has been done with the submerged method in which the test substance is dissolved in a suitable liquid such as culture medium and added into the cell culture. The problem with the submerged method is that it only allows the study of particulate matter (PM) but not the gaseous compounds. Furthermore, this method does not present PM in its original form as some PM reacts with the solution and

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some agglomerates. Thus, the PM that reacts with cells is different from the PM in the air, but not PM reaches the cells due to dissolving into solution and floating.

Newer exposure methods have been invented that rely on an air-liquid interface in which cells have a culture medium on one side and on the other side air from which the cells are exposed. There are commercial systems available, but these systems generally have low deposition efficiency, especially with nanosize particles.

Thus, better exposure mechanisms are required, and thermophoretic forcing has been introduced to overcome the low deposition efficacy. In thermophoretic forcing, aerosols between two different temperature surfaces are guided towards the lower temperature side.

In this work, the aim was to assess thermophoretic exposure systems suitability for combustion emission toxicological studies. In more specific aims, the toxicity of spruce, pine and brown coal briquette was evaluated using different methods and several toxicological responses such as cytotoxicity, inflammatory response and genotoxicity. Furthermore, complete combustion emissions (particles and gaseous compounds) were compared to filtered emissions (gaseous compounds), and the 1^st hour of a four-hour combustion experiment was compared to the 3^rd hour.

Results from the studies show that the spruce and pine combustion emissions had similar responses in monoculture cells as in mice; however, they had the opposite order of the highest genotoxicity among the tested samples. Interestingly, gaseous compounds caused almost as high an effect as the complete combustion emission (particles and gaseous compounds). Finally, it was shown that the brown coal briquette and spruce had a similar toxic effect in co-cultured cells, even though the emissions were notably different.

In conclusion, the thermophoretic exposure system was proven to work in combustion emission studies and has a much higher deposition efficiency that does not change depending on aerosol size. All fuels caused mild cytotoxicity, a notable increase in genotoxicity and slightly increased inflammatory responses except with monoculture cells exposed to spruce.

National Library of Medicine Classification: WA 754

CAB Thesaurus: combustion; burning; stoves; biomass; fuelwood; Picea abies; Pinus sylvestris; brown coal; air pollution; air pollutants; emissions; exposure; particles;

particulate matter; nanoparticles; aerosols; gases; toxicity; cytotoxicity; genotoxicity;

inflammation; laboratory animals; cell culture

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9

TIIVISTELMÄ

Ilmansaasteet ovat kaikkia ihmisiä koskeva terveysriski. Yksi tärkeimmistä ilmansaasteiden lähteistä on asunnon lämmitys erilaisia polttoaineita polttamalla.

Palamisessa syntyy useita haitallisia yhdisteitä, kuten polyaromaattisia hiilivetyjä, jotka voivat aiheuttaa useita terveysongelmia. Palamispäästöjen vaikutuksien selvittämisessä tutkitaan useita toksikologisia vaikutuksia.

Palamispäästöjen myrkyllisyys voidaan arvioida elävillä eläimillä, kuten hiirillä (in vivo) tai soluviljelmillä (in vitro). In vivo-tutkimuksissa altistaminen voidaan tehdä inhaloimalla tai laittamalla tutkittuja yhdisteitä suoraan keuhkoihin, kuten intratrakeaalisessa annostelussa. In vivo-kokeissa on kuitenkin useita muita ongelmia, jotka vaikeuttavat niiden käyttöä. Tärkeimpinä haittoina ovat kokeiden vaativat resurssit, ne ovat paljon aikaa vieviä ja kokeet eivät aina vastaa ihmisen altistumista. Toisaalta in vitro-kokeet voi vastata näihin ongelmiin, mutta ovat usein liian yksinkertaisia vastaamaan kokonaisen eläimen monimutkaisia järjestelmiä.

Näissä kokeissa kuitenkin suurin este on se ettei solujen altistuminen tapahdu realistisella tavalla toisin sanoen tutkittavan aineen siirtäminen soluun muuttumattomana jolloin testiyhdisteet olisivat todellisessa muodossaan.

Aiemmissa tutkimuksissa altistuminen on tehty upotusmenetelmällä, jossa testiaine liuotetaan sopivaan nesteeseen, kuten elatusaineeseen, ja lisätään soluviljelmään.

Upotusmenetelmän ongelma on se, että sen avulla voidaan tutkia vain hiukkasia (PM) mutta ei kaasumaisia yhdisteitä. Lisäksi upotusmenetelmässä hiukkaset eivät esiinny alkuperäisessä muodossaan, koska osa hiukkasista reagoi nesteen kanssa ja jotkut agglemeroituvat. Tästä johtuen solujen kanssa reagoiva hiukkanen on erilainen kuin se olisi ilmassa. Myöskään kaikki hiukkaset eivät päädy soluhin koska ne liukenevat tai kelluvat nesteessä.

Uudempia ilma-neste-rajapintaan perustuvia altistusmenetelmiä on kehitetty.

Ne perustuvat siihen, että soluilla on elatusaine toisella puolella soluviljelyinsertin kalvoa ja toisella puolella on ilmaa, josta altistus tehdään. Tähän menetelmään perustuen on olemassa muutamia kaupallisia järjestelmiä, mutta näillä järjestelmillä on yleensä alhainen altistusteho varsinkin nanokokoisten hiukkasten kanssa. Tästä johtuen tarvitaan parempia altistusmekanismeja, ja yksi keino ratkaisuksi alhaiselle altistusteholle on termoforeesin hyödyntäminen altistuksissa.

Termoforeettisessa voimassa kahden eri lämpöisen pinnan väliset aerosolit ohjataan alemman lämpötilan puolelle.

Tässä työssä tavoitteena oli arvioida termoforeettisten altistusjärjestelmien soveltuvuutta palamispäästöjen toksikologisiin tutkimuksiin.

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Yksityiskohtaisempana tavoitteena oli arvioida kuusi- ja mäntyhalkojen, sekä ruskohiilibriketin päästöjen toksisuutta käyttäen hyväksi eri toksikologisilla menetelmillä, kuten sytotoksisuus, tulehdusreaktio ja genotoksisuus. Lisäksi vertailtiin koko palamispäästöä (hiukkaset ja kaasumaiset yhdisteet) suodatettuun päästöön (kaasumaiset yhdisteet) ja neljän tunnin palamiskokeen ensimmäistä tuntia kolmanteen tuntiin.

Tämä työ osoitti, että kuusen ja männyn palamispäästöillä oli samanlaiset vasteet yksisolumallissa kuin hiirillä; kuitenkin korkeimman genotoksisuuden järjestys oli päinvastainen. Mielenkiintoisinta oli, että kaasumaiset yhdisteet aiheuttavat melkein yhtä suuren vaikutuksen kuin kokonainen palamispäästö (hiukkaset ja kaasumaiset yhdisteet). Lopuksi työssä osoitettiin, että ruskohiilibriketillä ja kuusella oli samankaltainen toksinen vaikutus kaksoisolumalliin, vaikka päästöissä oli merkittävä ero.

Yhteenvetona voidaan todeta, että termoforeettinen altistusjärjestelmä osoittautui toimivaksi palamispäästötutkimuksissa ja sillä on paljon suurempi altistustehokkuus, joka ei muutu aerosolin koosta riippuen. Kaikki polttoaineet aiheuttivat lievää sytotoksisuutta, merkittävästi lisääntynyttä genotoksisuutta ja hieman lisääntynyttä tulehdusvastetta lukuun ottamatta yksisolumallia, altistettaessa kuusen savulle.

Yleinen suomalainen ontologia: palaminen; poltto; polttopuu; halot; metsäkuusi;

metsämänty; ruskohiili; ilmansaasteet; päästöt; altistuminen; pienhiukkaset;

hiukkaspäästöt; nanohiukkaset; aerosolit; kaasut; myrkyllisyys; tulehdus; koe-eläimet;

soluviljely

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Acknowledgement

In The Return of the King, Frodo said,” it’s over, it’s done”, and so is this thesis project. Now I can thank all those great people that had my back during this journey.

No journey would have been possible without support. I want to thank my financial supporters; the doctoral school funding (Doctoral Programme in Environmental Physics, Health, and Biology) from the University of Eastern Finland, the North Savo Region Fund (Leena and Jouko Tuomisto Fund) of the Finnish cultural foundation and Kuopion seudun Hengityssäätiö.

Every hero needs a mentor, and luckily I had three of them. Pasi, you were the rock that I could rely on. Every time I had something troubling me, a visit to your office was all that was needed to reassure me that everything could be worked out.

Mikko, even though we had a short time at the University, you have taught me a lot during this journey, for which I am grateful. Moreover, and finally, Maija-Riitta, our fearless leader, thank you for pushing me forward and keeping me in line. Without my mentors, this would not be possible and thus, thank you all.

No hero could achieve anything without his fellowship, and luckily I had the best people on my side to make the sanity rolls successful. I´m deeply grateful to all my friends and co-workers in my department and especially in Intola. You all have made my work more fun and enjoyable than I would have ever hoped. Maria, you shared the dark humour that would institutionalize us immediately if being seen by others.

Thank you for the memes and constant support. Teemu, my brother in arms, you had my back and gave me a deep dive in statistics, which I did not ask for but thanks anyway. Finally, Herra Hakkarainen is the best masters student that I have had the pleasure to guide. Let me pass the metaphorical sceptre to you. You will be an amazing scientist, and keep the faith.

Shit, this is going on the second page. Wait, what, can I write here shit? Damn it.

I must include the second page because there are too many to thank and so little space. Why didn’t I copy the acknowledgements from Teemu like I copied all other things. Disclaimer I did not copy anything from the thesis by Teemu, but I used his thesis as a source of inspiration. So yeah, I dodged that bullet, back to the thanks.

The reader must know that no fellowship can achieve any major victory without allies, and I did have excellent groups of people to challenge me. This collaboration in the HICE project made my studies much larger than I initially planned but at the

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same time more fascinating. I have had the pleasure of getting to know the FINE group, especially Mika, Anni, and Olli. You all have shared this journey with me and managed to make it somewhat more understandable. Mika, you helpED me with the exposure system and guidED me into the world of physics of deposition. You made exposures (especially with zinc) more pleasED to do, and I learnED so much about everything from you. Anni, your help has saved me several times, and without your support, I would have had severe difficulties finishing my publications. Finally, Olli, not only has working with you been a pleasure, but we have an acquaintance outside work, which allows us to know each other better. Thank you for all the teaching about particle physics and corrections on my incorrect particle physic texts.

A major part of my data comes from our collaboration with Ralf Zimmermann and his group. I will not name any other person because there is waaaayyy too many of them to be named. This collaboration was memorable and figuratively phrased as a baptism of fire for being a scientist. Furthermore, I would like to present my thanks to collaborators from the Finnish Meteorological Institute and Luxenburg Institute of Health.

Not all allies come from actual work, but from common interest and I luckily had great support to keep my health bar on the positive side. My Oulu friends, we have had many epic meetings, and all of you deserve a whole section, but writing is hard, so only two of you are mentioned as you two had a notable yet not statistically significant role in this journey. Ulla and Kaisa, thank you for several legend… wait for it…dary moments. We have had so much fun every time we meet, and we share the same love for science. However, I still remember my lab note and how you enhanced it, out of nowhere, without any cause. Then there is Jaakko, Thank you for being such a good friend all of these years, and frankly, you were one of the role models I had for being a scientist. We had had some truly memorable times.

I must include my friends from Seinäjoki, the family Ketomäki. I would have had so much more sleep if I did not visit your home every time I was in Seinäjoki, but I would not have my life lengthened by all the laughter and fun time I had with you.

Moreover, to my godchildren (isn’t that an odd word), now your godfather has done something awesome, so you’re welcome. You have the bragging right from now on.

I must include here shortly my good friends Karl Franz, Mannfred and Isabella Von Carstein and Luthor Harkon, to name a few of you. Without you all, I would have had much higher stress levels and sleepless nights. Thank you, CA, for all the good times.

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13 No one can have acknowledgements without thanking his family, and so here it is. Thank you all.

Third page???? Come on, Tuukka, keep your shit together. Can you try to be professional for just this time? Keep it short and straightforward, no special “in your head discussions” but only thank and move on to the next person. This part would have been much shorter without all of this nonsense.

And now the next group of people. Anna, Tatu, Saara and Onni, thanks for the time we lived together and thank you for all the times we had tea in your kitchen.

Moreover, I would like to thank Sirpa, Risto and the rest of the family-in-law. You all made our time in Kuopio too much fun, and it was really hard to move out.

And here I must stop you, reader, as I was joking when I just thanked my family with one word. I am not that cold bastard; hence we will go back to my family and, more specifically, to my brother. Miikka, you held our family together in one of the hardest times of my life as my father passed away in the early parts of my journey, and there are no words to present the gratitude I have for you. Your family has given me strength and the constant reminder that I should get in better shape since my nephews have the energy of the nuclear plant. The following sentences are to my mom, and I will write them so she can understand. Why did I explain myself like that to you? Damn, this is the reason this is so long. Äiti kiitos loppumattomasta tuestasi, innostuksestasi (varsinkin väitöstä ja karonkkaa kohtaan) sekä kaikista rukouksistasi. Ne kaikki oli todellakin tarpeen ja ei haittaa ettet täysin ymmärrä mitä teen sillä minäkään en ymmärrää… tuota noin… tota ei olisi varmaan kirjoittaa tähän. Ööö… missäs se on se poisto-nappi, äh, tällä mennään. Ei se ole niin nöpön nuukaa.

As the grand finale to my wife, thank you for everything. You pushed, pulled, supported, and made me do this. You believed I could do it in times I was ready to give up. You were the extra life I needed to push the final meters, and I am extremely happy that you are on my side. I would not have chosen a better person to share this journey and life. I love you.

Kuopio, December 2021 Tuukka Ihantola

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15 LIST OF ABBREVIATIONS

ALI Air-liquid interface

AhR Aryl hydrocarbon receptor

ADHD Attention deficit hyperactivity disorder A549 Human alveolar epithelial cell line B[a]P Benzo[a]pyrene

BC Black carbon

BEAS-2B Bronchial epithelial cell line

BrC Brown carbon

CO Carbon monoxide

CASP7 Caspase7

COPD Chronic obstructive pulmonary disease COX-2 Cyclooxygenase-2

CYP Cytochrome P450

DEP Diesel exhaust particle GDP Gross domestic product GRO-α Growth related oncogene-α HAC Heterocyclic aromatic compound HBEC Human bronchial epithelial cells H2O2 Hydrogen peroxide

IL-8 Interleukin 8

IARC International Agency for Research on Cancer LDL Low-density lipoprotein

MS Mass spectrometry NPAH Nitrated PAH NO2 Nitrogen dioxide

NO Nitrogen oxide

NOx Nitrogen oxides

Nrf2 Nuclear factor erythroid 2-related factor 2 NFκB Nuclear factor kappaB

OC Organic carbon

OPAH Oxygenated PAH

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O3 Ozone

PN Particle number PM Particulate matter

PBBD Physiologically based biodynamic PBBK Physiologically based biokinetic PAH Polycyclic aromatic hydrocarbons POA Primary organic aerosols

QIVIVE Quantitative in vitro–in vivo extrapolation ROS Reactive oxygen species

RH Relative humidity RNA Ribonucleic acid

SOA Secondary organic aerosol SO2 Sulfur dioxide

THP-1 Human monocytic cell line TP-ALI Thermophoretic ALI

TNF-α Tumour necrosis factor-alpha UFP Ultrafine particles

UV Ultraviolet radiation

VOC Volatile organic compounds VB-ALI Volatile organic compounds β2AR β2-adrenergic receptors

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17 LIST OF ORIGINAL PUBLICATIONS

This thesis is based on data presented in the following articles, referred to by the Roman Numerals I-III.

I Ihalainen M., Jalava P.I., Ihantola T., Kasurinen S., Uski O., Sippula O., Hartikainen A., Hirvonen M.-R., Jokiniemi J. (2019). Design and validation of an air-liquid interface (ALI) exposure device based on thermophoresis. Aerosol Science and Technology, 53(2), 133–145.

II Ihantola T., Di Bucchianico S., Happo M., Ihalainen M., Uski O., Bauer S., Kuuspalo K., Sippula O., Tissari J., Oeder S., Hartikainen A., Rönkkö T.J., Martikainen M.-V., Huttunen K., Vartiainen P., Suhonen H., Kortelainen M., Lamberg H., Leskinen A., Sklorz M., Michalke B., Dilger M., Weiss C., Dittmar G., Beckers J., Irmler M., Buters J., Candeias J., Czech H., Yli-Pirilä P., Abbaszade G., Jakobi G., Orasche J., Schnelle-Kreis J., Kanashova T., Karg E., Streibel T., Passig J., Hakkarainen H., Jokiniemi J., Zimmermann R., Hirvonen M.-R., Jalava P.I.. (2020). Influence of wood species on toxicity of log-wood stove combustion aerosols: A parallel animal and air-liquid interface cell exposure study on spruce and pine smoke. Particle and Fibre Toxicology, 17(1), 27.

III Ihantola T., Hirvonen M.-R., Ihalainen M., Hakkarainen H., Sippula O., Tissari J., Bauer S., Di Bucchianico S., Narges R., Hartikainen A., Leskinen J., Yli-Pirilä P., Miettinen M., Suhonen H., Rönkkö T.J., Kortelainen M., Lamberg H., Czech H., Martens P., Orasche J, Michalke B, Jokiniemi J., Zimmermann R., Jalava P.I., (2021). Genotoxic and inflammatory effects of spruce and brown coal briquettes combustion aerosols on lung cells at the air-liquid interface. Science of the Total Environment 806.

The above publications have been included at the end of this thesis with their copyright holders’ permission.

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AUTHOR’S CONTRIBUTION

I) Author performed cell-related experiments and data analysis of the cell-related results. The in vitro toxicological experiments were planned in co-operation with Pasi Jalava, Oskari Uski, Stefanie Kasurinen and Maija-Riitta Hirvonen. The author also participated with Pasi Jalava and Maija-Riitta Hirvonen in writing the toxicological parts of the manuscript. The author contributed to manuscript revisions, mainly considering the toxicological parts.

II) Author performed Tox-ALI cell experiment, Tox-ALI related endpoint experiments and data analysis of the cell-related results with assistance of Henri Hakkarainen. The author also assisted in the animal result analysis. Planning the in vitro toxicological experiments was done in co-operation with Pasi Jalava, Mikko Happo, Oskari Uski and Maija-Riitta Hirvonen. The author drafted the initial manuscript with Sebastiano Di Bucchianico. All authors contributed to manuscript revision.

III) Author performed UEF-ALI cell experiment, exposures and data analysis of the UEF-ALI related results. Planning of the in vitro toxicological experiments was planned in co-operation with Pasi Jalava and Maija-Riitta Hirvonen. The author drafted the initial manuscript.

All authors contributed to manuscript revision.

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1 LITERATURE REVIEW

Particulate matter (PM) is harmful to human cells, which is clearly seen in several epidemiological studies (1–4). However, while the epidemiological studies present the most relevant public health results; they cannot show which PM components or which type of emissions causes the harmful effects on cells or living organisms. Thus, to investigate PMs’ harmful effects, toxicological studies are required. These studies can be roughly divided into two groups; in vivo and in vitro (5). An in vivo study refers to animal or human experiments showing how responses perform at the systemic level, and an in vitro experiment refers to cell-based models to mimic tissues and microenvironments to satisfy basic research questions (6). Unfortunately, in many cases, in vivo models would increase the cost, complexity of experiments and also have ethical considerations (5).

On the other hand, in vitro models enable the identification of cell-specific activation mechanisms behind the reported outcomes and overcome the problems with in vivo. Yet, in vitro models might be over simple, i.e.

presenting cellular responses of a single cell type or organ, rather than the actual effect on a human being. To overcome this problem quantitative in vitro–in vivo extrapolation (QIVIVE) has been invented and this extrapolation allows the prediction of chemical risk in an ethical manner. QIVIVE is created from in silico methods including modelling of physiologically based biokinetic (PBBK) and physiologically based biodynamic (PBBD), and combined with findings of the compounds distribution, metabolism and excretion (7). QIVIVE can be then used in per endpoint inspection such as cytotoxicity. This extrapolation has mainly been used in drug, pesticide and other chemical risk evaluations and it has been only scarcely used in inhalation toxicity testing (8,9). However, compounds that are present in air pollution are not optimally studied in in vitro models, currently having a problem presenting exposure in a manner corresponding to real-life, which drastically affects the extrapolation to humans.

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Air-liquid interface (ALI) exposure systems have been designed to overcome this problem, but currently available systems have low and variable deposition efficiency on different PM sizes (10–12). Thus, toxicological tests require better methods to be able to show relevant outcomes of tested compounds. Therefore, this literature review focuses on toxicity measurements of air pollution originating from combustion, with different exposure systems.

1.1 Toxicological testing of inhalable compounds

A good toxicological test requires exposure to be as close to the real world as possible, reproducible, and representative of the tested substance (5).

The exposure here contains the PM production, collection and deposition method, which all affect the exposure. Reproducibility generally refers to having similar results from similar conditions, and representativity refers to having the test substance simulating the real-world as closely as possible, i.e. not modified in any way before the deposition. In many studies, the reproducibility and representativity are balanced by the exposure, leading to a compromise between these factors. However, increasing reproducibility could decrease the relevance of the test subject. This imbalance comes from the uniqueness of combustion, which forms unique emissions. To increase reproducibility, the test substance must be the same; for example, collecting the PM from several emissions and using the collected PM in exposures.

However, this approach will decrease the combustion emission representability since test substance react during the collecting or exposure process whereas increasing representability by direct exposure reduces reproducibility since the test substance is not identical in every individual exposures (5).

1.1.1 In vivo studies

In vivo experiments have been the cornerstone of toxicology testing since they allow inspection of the toxic compound’s systemic, pharmacokinetic and pharmacodynamic effects with all the clearance and defence systems of a living organism (13). Moreover, animals can present the acute and chronic

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23 effects of the studied compounds, and the effects on each organ can be studied simultaneously (14). However, in vivo experiments also have issues as they are resource-intensive, time-consuming, and are not always representative of human exposure. In addition, test animals show a notable difference in responses between species, genders and even breeding batches.

Furthermore, animal studies have significant issues when translating findings into humans due to differences in anatomy, physiology, and, concerning air pollution studies, in breathing. These differences were observed in several animal experiments, such as thalidomide, and monoclonal antibody TGN 1412, where animal tests revealed no safety issues but it was later found that these drugs have severe effects on humans (15).

In addition, ethical aspects have to be taken into account. These kinds of problems are the reason for the three Rs’ idea; reduce, replace, and refine to justify animal experiments. Therefore, before any in vivo experiments, the three Rs must be considered, and the ethical committees must approve these considerations. Also, in toxicological experiments, the amount of tested substance used is highly important to estimate the toxicity. In many studies, the dose is hard to estimate due to the exposure method used, e.g.

inhalation studies. Methods such as intratracheal instillation allow more controlled exposure doses but are laborious. In addition, it is a non- physiological method (16). Moreover, intratracheal instillation can study only solid and soluble compounds leaving out the gaseous compounds such as volatile organic compounds (VOCs) or small polycyclic aromatic hydrocarbons (PAHs), thus it does not represent the actual exposure emission but only part of it.

1.1.2 In vitro studies

In vitro models mainly use cells of human origin to study possible cellular effects in human cells, minimising animal studies’ requirements and possibly replacing them altogether. The cell-based toxicological tests are usually done with continuous epithelial cell lines that are either

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immortalized or tumour-originated, but tissue sample cell lines are also used. There are several reasons for the use of continuous cell lines 1) cells require less work than primary cells and require cheaper materials, 2) the results obtained from these cells are easier to compare since cells are derived from one individual, 3) a longer lifespan allows more experiments to be done, 4) they can be used in high-throughput screening (17).

Furthermore, the most widely used cell type in air pollution studies is the immortalized adenocarcinomic human alveolar basal epithelial cells (A549).

Their morphology and functionality, such as surfactant synthesis, microvilli at the apical side or transport properties, are similar to the pulmonary alveolar type II cells in vivo (18–20). However, the downside of using A549 cells in more complex cultures is that the cells do not form tight junctions (21).

Nevertheless, in more complex cell cultures^, airway epithelial cells can differentiate into cells that resemble their original morphology, organization and stratification (17). Other widely used cell lines are tumour cell line Calu- 3 and bronchial epithelial cell lines BEAS-2B and 16HBE14o. BEAS-2B is similar to airway basal epithelial cells, but these cells do not form tight junctions, whereas 16HBE14o has cobblestone morphology, and is more differentiated than BEAS-2B cells.

Primary cells are taken from human tissue, e.g. by biopsies and cultivated.

Using primary cultures allows a more reliable interpretation of their physiological function than immortalized cell lines. However, this direct isolation and cultivating is limited due to cells having a very limited life span.

In addition, these cells are harder to use, and they are much more expensive, resulting in more complex interpretations caused by the differences in their source individuals (17).

There are complex commercially available primary cell models such as MucilAir™ and EpiAirway™ that resembles more in vivo conditions than in vitro. These models have several different cell types in multiple forms, and they present actual disease conditions due to the patient's disease from which they are taken. However, these are more expensive and laborious than simpler models. Moreover, some conditions can cause milder

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25 toxicological responses than in simpler models, and they may require priming with additional compounds such as tumour necrosis factor-alpha (TNF-α) (17,22,23).

Monoculture is the basic model in in vitro testing, and in this model, as the naming suggests, there is only a single type of cell. Therefore, cellular responses from these exposures reveal how this type of cell reacts to exposure with the tested compounds. However, in vivo response to air pollution is not an outcome of a single cell type but an outcome of several cells. Thus, monoculture’s simplicity prevents the cells from presenting a relevant response to toxic exposure (24), and the cells can have different responses in monoculture than in more complex cultures (23,25). An example of this change in response was apparent in a study, in which epithelial cells and macrophages revealed monoculture to hide realistic responses to ozone (O3). Therefore, monoculture is the most straightforward cell model, but it can be made more complex by adding additional cell types.

Increased complexity of cell model allows more realistic outcomes than from monoculture and more natural responses from cells since cells are able to communicate with each other (25,26). Co-cultures are thought to be more sensitive (20,27–29), but due to cellular communication, the cellular response to exposure compounds can, in fact, be less sensitive (30–34).

Interestingly, a recent study showed that the co-culture of A549, human monocytic cell line (THP-1) and human bronchial epithelial cells (HBEC) had a similar trend of response as A549 cells alone but in higher strength (199).

Furthermore, the inflammatory response was even more potent when co- cultures were used, indicating cellular communication’s importance (137).

The cell model can be made more even more complex by using pluripotent stem cells to form organoids. Organoids have the airway epithelium cell types such as goblet cells and ciliated cells, but organoids are difficult to expose directly. Thus, organoids are not currently suitable for air pollution toxicity studies while they have practical use, for example, in drug discovery (13).

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Therefore, the cell model defines the possible endpoints, and by increasing the cell models complexity, the outcome resembles a more in vivo condition. Moreover, the exposure method has increased importance for evaluating the toxicity of PM since it can affect the studied compounds more than the cell model.

1.1.3 Submerged cultures

The most widely used exposure method used in air pollution studies has been the submerged culture method. It is also the easiest to perform. In this method, the cells are grown at the bottom of the culture dish or plate and studied substances such as collected PM are added into culture via culture medium or other suitable liquid. Cells are exposed to non-soluble particles via sedimentation, whereas soluble particles can float into cells (Figure 1).

However, diluted samples can agglomerate and/or react with the diluted solution, thus not presenting the actual morphology of combustion PM (35).

Therefore, there are problems in determining the deposition due to unknown particle changes (36,37). Furthermore, studies using submerged culture conditions generally use much higher concentrations than would generally occur in the real-world. Submerged culture is also an unrealistic method to simulate real-life conditions except when studying pathological conditions such as pulmonary oedema. However, a lower cost and easier setup make this method ideal for larger-scale screening tests or large-scale toxicological experiments.

Furthermore, submerged exposure is mainly used with collected PM without the gaseous compounds, thus showing the effects of only one part of the combustion emissions. Nevertheless, it is highly standardizable, i.e.

reproducible and easily comparable, cheap to perform, and allows studying PM from places where more direct exposure methods would be problematic to set up or perform (5,36).

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27 Figure 1. Submerged method compared to ALI exposure. The submerged method (left) allows study of collected PM but requires PM collection, storage, and dilution before exposure. The ALI method (right) allows direct PM exposure without affecting the PM but requires a specialized exposure system that can affect the cells or perform an unwanted selection of deposited PM.

1.1.4 Air-liquid interface systems

ALI exposure systems were invented to overcome the problems with the submerged method. In ALI, cells are grown on a semipermeable surface on a supporting frame in which cells are exposed to air from the apical side.

The basolateral side of the culture instead is exposed to culture medium.

This structure is called a semipermeable membrane insert, and the semipermeable membranes have several different pore sizes. Pores of 0.4 µm are usually used because this size prevents particle translocation from side to side (14,38). ALI allows direct cell exposure from the apical side, and here epithelial cells can oxygenate and differentiate if possible for the cell type used. Instead, the medium side maintains the cells well-being by supporting nutrition through the semipermeable membrane (13,36,39). ALI, also allow surfactant formation (20). While submerged conditions only allow the use of collected and dissolved particles, the ALI systems can operate

Kuvallinen aloitussivu, kuvan koko 230 x 68mm

Combustio emission

+ Direct exposure for both PM and gaseous compounds + Resembles conditions to those in the lungs - Expensive to perform

- Require complex exposure system - Repeatability is not identical filter

PM solubilization

+ Simplest exposure method + Cheap to perform

+ High repeatability due to identical PM sample + Can be used with high-throughput techniques - Medium can affect study compounds - Only PM and no gaseous compounds - Hard to estimate cell deposition

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closer to realistic conditions in which there are solid and liquid particles present in a complex mixture, including gases. The deposition occurs directly on cells (36,40). Studies have shown that ALI exposure presents a more in vivo situation than the submerged method (13,41).

A549 cells in submerged conditions show few microvilli, but cells cultured on semipermeable membrane but with submerged conditions had much more microvilli. Interestingly, cells cultivated in semipermeable membrane and ALI condition had a smooth surface without any microvilli. Also, A549 cells in ALI were observed to be smaller than in submerged conditions(20).

Reproducibility is weaker when ALI condition is used than in submerged conditions if the test substance is generated during the exposure since combustion emissions vary. In other words, ALI exposure sacrifices reproducibility with the representability of the tested substance. However, it must be pointed out that an increasing number of independent exposures can overcome the decrease in reproducibility. In many cases, multiple individual exposures are hard to achieve due to increased workload and cost, as cellular exposure in ALI conditions is much more expensive than in submerged conditions (42).

There are several kinds of different ALI systems used in individual research laboratories (22,43–46). Currently, two manufacturers provide ALI systems to be purchased, and these systems are the most used ones;

VitroCell^®, Cultex™ (12,47). This literature review will focus on these two systems as these systems have been the most widely used, and thus, there is more information available from these systems. These ALI systems can perform direct cellular exposure to air pollution, and with a nebulizer, liquid substances can be aerosolized for exposures (13). This nebulizer is not the only possible additional system for these exposure systems, and there is additional equipment such as vapour generation and cigarette smoking robots (13). Both systems use inlets that allow airflow with tested substances towards cells and in which gravity is the primary deposition method, but gravity only has high efficacy on larger particles, i.e. >600 nm, with low efficacy on smaller particles. Smaller particles move by Brownian movement, and gravity does not affect them; therefore, diffusion becomes

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29 the deposition mechanism for small particles. Deposition of smaller sizes can be enhanced with high voltage in both systems, and high voltage polarizes the particles so that the electrical attraction pulls the particles towards the electrical field close to the cells (42,48). However, even with this enhancement, the nanoparticles are deposited with relatively low efficiency.

Thermophoretic forcing is a novel deposition method in ALI systems.

Figure 2 presents the thermophoretic forcing in detail, but in summary, thermophoresis or thermodiffusion drives aerosols towards a colder surface instead of the hotter surface due to gaseous compounds movement.

This forcing allows higher efficacy in nanosize aerosols. Furthermore, thermophoresis has no discrimination for aerosols shape and size; thus, the efficacy is the same for any particle (14).

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Figure 2. Different methods to deposit particles onto cells. A) In the submerged method, particles are added into an exposure solution, which is usually a cell medium, and in this solution, the particles are deposited into the cells by sedimentation. B) In thermophoresis, gas molecules move with Brownian motion, and warmer side gas molecules have more movement due to higher temperature than colder gas molecules. This movement difference causes gas molecules to bounce aerosols more frequently from the warmer side than the colder. This imbalanced bouncing of gas molecules causes aerosol to move towards the colder side. C) In exposure systems such as VitroCell^®, particle deposition occurs mainly via gravitation in which particles are directed towards cells by nozzle directed airflow and particles land on the cell surface. D) This deposition can be enhanced by including a high voltage source that attracts charged particles and charges uncharged particles.

1.1.5 On-a-chip model

A newer cell and exposure method is an organ-on-a-chip where the organ/body is mimicked in a microfluid system where cells are usually cultivated in a multichannel chip. Terminology is not defined with the on-a- chip models, but generally, if the chip presents a single organ, it is called organ-on-a-chip, and if there is an organ per chamber in a chip, it is called body-on-a-chip. In the chip, fluids flow from chamber to chamber where

Gas molecule Particle

High temperature

Low temperature

Drag force Thermophoreticforce

Temperature gradient

A) B)

C) D)

Charged particle

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31 cells are present and react with the compounds travelling in the flowing fluid. Chambers can have a specific structure such as semipermeable membranes to better mimic the target organ or part of the organ. In organ- on-a-chip, cells can be cultivated at ALI conditions, but the direct exposures are only under development and not currently implemented (49). In this exposure system, the test substance must be processed similarly to the submerged method. However, it is possible to test systemic effects and observe the effects of cellular communication between different cells and organs. Also, in this system, it is possible to mimic movements such as breathing movements. However, further studies are still needed to determine the possibilities and limitations of this method as it has only recently been introduced in toxicological studies (17).

1.2 Air pollution from combustion emissions

Air pollution has been considered a significant issue for human health after several dramatic historical events such as the London smog, the Donora event, and even the aftermath of the World Trade Center building collapse (50–52). Air pollution was estimated in 2015 to have caused 4.2 million premature deaths. Furthermore, it has been suggested that there will be an increase of 6 to 9 million annual premature deaths by 2060. This increase would cost 1% of global gross domestic product (GDP), approximately 2.6 trillion USD (53). Therefore, air pollution is not only a health issue but also a significant economic issue. Air pollution can be any compound naturally generated or artificial in the atmosphere that can cause harmful effects on humans (54,55). However, in this work, the focus is on small scale combustion, i.e. combustion done in residential properties, which is one of the major sources of combustion derived air pollution.

1.2.1 Characteristics of air pollution

Air pollution is a mixture of particles and gaseous compounds. Aerosols can consist of liquid or solid PM with different sizes, shapes and chemical compounds depending on the source (56). Different PM sizes are divided into groups based on the particles’ aerodynamic diameter (55). These

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groups are referred to as inhalable particles or PM10 (<10 µM), fine or PM2.5

(<2.5 µM) and ultrafine particles (UFPs) or PM0.1 (<100 nm). Larger size groups have a higher mass concentration than smaller, albeit smaller size groups have a higher particle number concentration (PN) (57). Differences are presented in Figure 3. Particle size dictates deposition location and affects uptake, biodistribution, and bioclearance (58). Moreover, air pollution is not only inhaled but also ingested and dermally absorbed.

However, from these mechanisms, less PM is transferred into the bloodstream than from particles in the lungs (55). Combustion generated particles typically have a carbonaceous core and a complex mixture of compounds on the surface.

Earlier studies have focused on the core chemical composition of PM to reveal the source of exposure toxicity (10,11). However, the PM’s surface is the first to react with the cells due to solubility; thus, compounds bound on the surface have the first reactions with biological systems. The mechanisms of toxicity include; cytotoxicity, reactive oxygen species (ROS) production, inflammation, or genotoxicity (5,59). The surface also dictates the PM’s dissolution rate, uptake mechanism and biodistribution. For example, PM might have compounds on the surface recognized by immune defence cells, leading to PM removal (37). However, there is no knowledge currently about the circumstances in which this recognition occurs.

Figure 3. PM differentiation illustrations. Schematic illustration of idealized mass concentrations and sizes of PM in the outdoor air (A) and an illustration of wood combustion with different stages of combustion (B). Adapted from (60,61).

Ignition Flaming Smoldering

A) B)

RelativeMassConcentration

Particle Aerodynamic Diameter (µm)

Diameter (µm) dN/d(logD) (cm-3)

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33 1.2.2 Chemical composition

In this work, the main focus is on combustion emissions from wood fuels, and these fuels generally have a chemical composition of combustible material (50–52% carbon; 40–44% oxygen; 6–6.5% hydrogen; ~0.2% sulfur;

~0.2% nitrogen), water (20-60%) and minerals (calcium, magnesium, phosphorus <0.5%) (62).

In optimal combustion reactions, the gaseous fraction consists by volume almost entirely (99%) of non-toxic gases such as nitrogen, water vapour, carbon dioxide (CO2) and oxygen and the rest of toxic gases such as carbon monoxide (CO), nitrogen oxide (NO) and nitrogen dioxide (NO2). Inorganic gases are not the only ones produced in combustion, but organic compounds such as methanol, ethylene or formaldehyde are also emitted.

However, most organic compounds are from large and complex aliphatic compounds referred to as PAHs, nitrated PAH (NPAH and heterocyclic aromatic compounds (HACs) (59,63–66). There are also other PAH derivates such as oxygenated PAH (O-PAH), and therefore further mentions of PAH includes its derivates for simplicity. Nevertheless, there are generally low levels of heavy metals such as As, Cd, Cr, Cu, Hg, Ni, Pb and Zn in biomass combustion emissions (54).

In incomplete combustion, PAH compounds, PM and several toxic gases such as CO form in higher amounts than in optimal conditions. The main cause for formation of these compounds is the lack of oxygen in the combustion processes (67). Carbonaceous compounds are an important group as they form the core of PM and other organic compounds. Carbon components can be divided into organic carbon (OC), black carbon (BC) and brown carbon (BrC). The OC is abundant in incomplete combustions, but other carbonaceous components also have high levels. These compounds are interesting because they cause toxic responses and affect climate change by absorbing solar radiation, especially BC (68,69).

One method to differentiate compounds from combustion emissions is to consider two groups: organic and inorganic compounds. Organic compounds are an important group of compounds found from combustion

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emissions, and from these compounds, PAH compounds are among the most studied ones (70–73). Several different PAH compounds are emitted in different phases of the combustion process, depending on the combustion quality. PAH compounds containing four or fewer aromatic rings are mostly found from the gas phase. Instead, PAHs containing more than four rings are usually attached to aerosols. Interestingly, three and four-ring PAHs can also be found attached to combustion aerosols such as diesel exhaust particles from engines before EURO 4 standard, and these PAH compounds are usually the most abundant ones attached to diesel exhaust particles (74).

One of the most studied toxic PAH compound, benzo[a]pyrene (B[a]P), is found from combustion emissions, and it has been shown that it can enter into the blood circulation from the aerosols at the alveolar region in an unmetabolized form. PAH compounds are then metabolized inside the body as part of the removal mechanism, but in some PAH such as B[a]P, the intermediate can be more toxic than the unmetabolized form. In the case of high levels of PAH, the intermediates can cause adverse effects in the body (75). PAH compounds are quite a heterogeneous group, even if most of them are considered to be genotoxic. For example, B[a]P cause hypoxia, oxidative stress and DNA damage that it has been used as reference point for other PAHs, whereas dibenzo[def,p]chrysene cause inflammation and DNA damage (76).

From the inorganic compounds, metals are the most interesting concerning toxicity. Metals and metal-oxides may produce ROS, which can trigger antioxidant defence, inflammatory response or cytotoxic effects (5).

ROS is produced in Fenton-type reactions in which hydrogen peroxide (H2O2) reacts with a transition metal such as Fe (77–81). This reaction then produces ROS, an example of one of ROS compound is superoxide O2-, which can in turn react with cellular structures and compounds such as DNA. However, metals can be regenerated and thus, the reaction is cyclic. The oxidative stress level has been mostly connected with levels of Fe, Mn, Cu and Zn from which the Fe and Zn has been found to the most reactive.

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35 1.2.3 Particle aging

Primary organic aerosols (POA) are called organic aerosols straight from the source, and these aerosols are susceptible to atmospheric aging. This atmospheric ageing is a rapid process in which POA has multiple photochemical reactions with oxidants, ultraviolet (UV) or both (82). Both UV and oxidative compounds are found in the atmosphere, and after the rapid ageing process with POA, the POA transforms to a secondary organic aerosol (SOA) (83,84). Thus, humans are generally exposed to SOA instead of POA.

Generally, POA is an inhomogeneous group of compounds, but the further these aerosols travel, the more atmospheric processes they undergo and thus become more homogenous. Moreover, the smaller the aerosol is, the more it changes, and thus SOA consists of larger particles than POA, especially the older it is. Larger particles of SOA are generally below a few hundred nanometers (85). It has also been shown that aged emissions compared to fresh emissions can increase toxic responses such as genotoxicity (86). For example, one major group of compounds, PAHs, partially explain this increase as these compounds can transform in the atmosphere rapidly into oxygenated PAH compounds (OPAH), which are more genotoxic than their original forms (87). However, the longer ageing process will, in the end, transform these OPAHs into other compounds, thus lowering the toxicity of the SOA (88).

1.3 Residential biomass combustion

Biomass combustion, which refers to combustion of wood and other natural substances, is a crucial source for heating and in rural areas for cooking, for example, in China, approximately half of the population uses biomass fuels as the primary energy source (89). In Europe, biomass is used mainly for heating instead of cooking, and it is estimated in 2015 to produce 45% of total PM2.5 emissions (90). Furthermore, the EU aimed to have at least 18% of energy production from renewable sources, and in 2030 renewable sources should cover a minimum of 32% of all energy sources (91). In addition, the number of furnaces in Europe is around 70 million, and most

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of them are outdated or improperly used (92). It has been estimated that aerosols from biomass combustion affect around 3 billion people worldwide, in perspective with tobacco smoking which affects 1 billion, making biomass combustion a significant risk for developing chronic obstructive pulmonary disease (COPD) among other disease conditions (93).

Moreover, several factors contribute to biomass fuel usage, such as it is attainable, easy to use, does not require special systems and is low cost (94–

97). For example, during the economic crisis in 2013-2014, wood usage compared to oil and natural gas increased due to the cheapness of wood fuels (95). Recently, an increasing demand to use a “carbon neutral” energy source has risen, affecting wood fuel usage. Wood fuel is considered carbon- neutral since it absorbs CO2 as much in growing as it releases at burning, i.e.

the more extended the growth period, the more carbon it can store (98,99).

However, if the replacing wood is grown for a shorter period than the earlier woods, then the new wood cannot be considered carbon neutral.

Batch combustion of wood logs has four distinct phases; start-up (ignition), steady (efficient), intermediate (a state where the output is lower than the fireplace’s maximum), and burn-out. In addition to these normal combustion phases, worse conditions also occur because of improper use of small-scale appliances. Improper use includes use of wet or too much wood, and outdated or poorly maintained fireplaces. Due to these improper practices, carcinogens and other toxic compounds are released at much higher levels than in proper combustion (100). This condition is called incomplete combustion, but there are no perfect combustions, and thus incomplete combustion is present in all combustion processes. However, it is mainly a result of three factors that have been neglected or abused; fuel type, combustion technology, and user practices (29,34,38–44); the following sections are focused on these issues. In this work, the focus is on residential combustion or also known as small-scale combustion, which differs significantly from large-scale or medium-scale powerplants (>1 MW) where the combustion conditions are better controlled (i.e. higher temperature) than in small-scale, and emissions are controlled before releasing into the atmosphere (101).

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37 1.3.1 Biomass fuel

Fuel type is the source of the elements that combustion emissions could include. Thus better quality fuel will allow better combustion conditions and cleaner combustion emissions than lower-quality fuel, which might have impurities contributing to worsening combustion emissions (100). The European parliament and council on industrial emissions label biomass as either 1) any vegetable matter originating from forestry or agriculture, 2) waste of forestry, agriculture, food industry if the heat can be recovered, pulp or paper production if heat can be recovered, cork waste or wood waste except when the wood contains halogenated organic compounds or heavy metals due to preservation or coating (102). Biomass fuels have been estimated to represent 30% of global energy demand and are considered the world’s third-largest energy source (96,103). These different fuels have their own positive and negative effects on the produced emissions. Wood logs are the most basic and thus the cheapest form of wood fuel if waste materials are not counted. Generally, wood logs are hardwoods such as oak, maple, birch and beech or softwoods such as pine, spruce and douglas fir.

Wood logs are dried to 16-20% to achieve the best combustion condition as dry wood burns better and hotter than wet wood (100). The shape or size of the fuel can also affect the combustion process as the higher the surface-to- volume increases, the possibility of insufficient air mixture increases. In that case, the PM-related emission factors such as organic compounds and PM1

increase (104). One example of fuel, which has a high surface-to-volume ratio, is pellets that are usually used in highly controlled systems. Pellets are compressed organic matter of wood shavings, wood chips, sawdust, grasses, straws, lumber mill scrap or energy crop waste. In preparation of pellets, the pellet material is first heated to dry the material and break down and melt lignin inside the material, solidifying and holding the pellet together. If there is not enough lignin to hold the pellet together, some flour, for instance, cornstarch, can be added to act as glue. The pellet is then pushed through a pellet mould using high pressure and afterwards cut to a suitable length. Studies have shown that pellets produce less toxic

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combustion emissions than other wood fuels due to their structure, making them the most suitable fuel (100,105,106). The only downside with pellet fuel is that it produces more ash than a log of the same wood. Pellets are most suitable for automatic feeder systems, and they are vulnerable to moisture, which disintegrates pellets structure. Briquettes are similar to pellets but larger and thus also suitable for manually fed systems (100).

1.3.2 Combustion technology

Combustion technology limits the combustion process, controlling how efficiently the fuel can be burned and what emissions will result from the combustion process. There are mainly two different groups of heaters;

primary and secondary heaters. Primary heaters are made to heat the entire residence, and with some models, the residents hot water. These heaters usually have large combustion chambers that heat water (hydronic heater) or air (furnace) guided to warm the entire home. These stoves can perform single-stage or two-stage gasification (Figure 4). A single-stage system has one chamber for combustion and one to two air dampers. The two-stage system, on other hand, has one chamber for combustion and another chamber to combust the off-gases, which were not fully burned in the earlier chamber. However, the single-stage system is usually less effective than the two-stage system and produces more air pollution. These systems are fueled with cordwood, wood chips or pellets and these heaters are controlled using thermostats and are usually automated. Secondary heaters are made to heat a single room or used as a recreational fire. The thermostat does not control these heaters, but the newest models might have automation with secondary combustion. Furthermore, heaters can be coated with catalytic agents such as rare-earth metals allowing volatile gases to be combusted at lower temperatures. Masonry heaters, compared to stoves, are built into the residence, and they can release the heat for a much longer duration as the heat is efficiently stored in the brick mass, which is heated by hot combustion emission (100,107,108). Masonry heaters usually have a mass of 800 to 3000 kg (108). Several studies have shown that newer

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39 heaters produce less toxic emissions and toxicological effects than older ones (109–112).

Figure 4. Schematic of 1-stage and 2-stage heaters. Adapted from (100).

1.3.3 User practices

User practices, in the end, dictate the outcome of any combustion as perfect fuel in an optimal heating appliance can produce the most air pollution due to improper usage and fuel loading (107,113,114). As previously mentioned, fuel has an important role, and unsuitable wood fuel with high moisture levels produces more PAH and other toxic carbon compounds (115–117). Biomass tends to absorb moisture when possible, and therefore biomass fuel storage will require special care. Wet biomass causes a suboptimal combustion process as fuel needs to dry before it can be optimally reacted with oxygen, and this drying process will lower the fuels heating value (100,118). It has been found that biomass with higher moisture levels produces combustion emissions containing more PAH and CO than drier biomass fuels.

Furthermore, improper storage allows the wood to decompose, gradually decreasing the wood density even though the wood’s body stays intact due

To chimney To chimney

Wood loading door

1-stage 2-stage (gasification)

primary air

blower

Online exposure systems for toxicological effects of combustion emissions and nanoparticles

Dissertations in Forestry and Natural Sciences

TUUKKA IHANTOLA

Online exposure systems for toxicological effects of combustion emissions and

nanoparticles

ONLINE EXPOSURE SYSTEMS FOR

TOXICOLOGICAL EFFECTS OF COMBUSTION

EMISSIONS AND NANOPARTICLES

ONLINE EXPOSURE SYSTEMS FOR

TOXICOLOGICAL EFFECTS OF COMBUSTION EMISSIONS AND NANOPARTICLES

Acknowledgement

Table of contents

1 LITERATURE REVIEW

1-stage 2-stage (gasification)