A Simple Method to Determine Cytotoxicity of Water-Soluble Organic

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Energy and the Environment

A simple method to determine cytotoxicity of water-soluble organic compounds and solid particles from biomass combustion in lung cells in vitro Peter Zotter, Stéphane Richard, Marcel Egli, Barbara Rothen-Rutishauser, and Thomas Nussbaumer Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b03101 • Publication Date (Web): 01 Mar 2019 Downloaded from http://pubs.acs.org on March 2, 2019

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Environmental Science & Technology

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A simple method to determine cytotoxicity of water-

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soluble organic compounds and solid particles from

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biomass combustion in lung cells in vitro

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Peter Zotter*,†, Stéphane Richard‡, Marcel Egli‡, Barbara Rothen-Rutishauser#, Thomas

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Nussbaumer*,†

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†Lucerne

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Architecture, Bioenergy Research Group, Technikumstrasse 21, 6048 Horw,

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Switzerland

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‡Lucerne

University of Applied Sciences and Arts, School of Engineering and

University of Applied Sciences and Arts, School of Engineering and

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Architecture, Institute of Medical Engineering, Seestrasse 41, 6052 Hergiswil,

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Switzerland

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#Adolphe

1700 Fribourg, Switzerland

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Merkle Institute, University of Fribourg, Ch. des Verdiers 4,

Keywords: aerosols, emissions, cytotoxicity, wood combustion, WSOC

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TOC art:

ACS Paragon Plus Environment

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ABSTRACT

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Adverse health effects of condensable organic compounds (COC) and potential

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secondary organic aerosols from wood combustion emissions are difficult to determine.

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Hence, available information is usually limited to a small number of specific applications.

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Therefore, we introduced a simple, fast, and economic method where water-soluble

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COC (WSCOC) and WSCOC together with water-soluble primary solid particles

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(WSpSP) from wood combustion were sampled and subsequently exposed to cultured

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human lung cells. Comparing the cell viability of H187 human epithelial lung cells from

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five combustion devices, operated at different combustion conditions, no or only a minor

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cytotoxicity of WSCOC is found for stationary conditions in a grate boiler, a log wood


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boiler and a pellet boiler. All combustion conditions in a log wood stove and

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unfavourable conditions in the other devices induce, however, significant cytotoxicity

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(median lethal concentration LC50 5–17 mg/l). Furthermore, a significant correlation

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between CO and cytotoxicity was found (R2~0.8) suggesting that the simply measurable

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gas phase compound CO can be used as a first indicator for the potential harmfulness

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of wood combustion emissions. Samples containing WSCOC plus WSpSP show no

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additional cytotoxicity compared to samples with COC only, indicating that WSCOC

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exhibit much higher cytotoxicity than WSpSP.

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INTRODUCTION

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Wood and other biomass fuels are an important renewable energy source and their use

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is expected to significantly increase within the next years to substitute fossil fuels1, 2.

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Biomass combustion, however, significantly contributes to ambient air pollution,

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especially with respect to inhalable particulate matter 3-5, which can induce adverse

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health effects including respiratory and cardiovascular diseases, cancer, and increased


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mortality as shown in many in-vitro, in-vivo and epidemiological studies. In-vitro cellular

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effects include cytotoxicity, inflammatory responses, oxidative stress, and genotoxicity

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e.g. DNA and chromosome damage 6-12. Consequently, there is a trade-off between air

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pollution control and the propagation of wood as a renewable energy source.

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With respect to air pollutant emissions, different types of combustion devices and

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combustion modes are related to distinct emission patterns which need to be

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distinguished. In particular, primary particles from wood combustion can be classified

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into three different types, namely inorganic particles (mostly salts, dominant at near-

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complete combustion), soot (formed at high temperatures and lack of oxygen), and

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organic compounds (released during incomplete combustion at lower temperature e.g.

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due to high excess of combustion air) 13. Wood combustion emission also contain

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volatile organic compounds (VOC) which can be transformed to the aerosol phase via

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cooling and/or dilution and/or photochemical reactions. Consequently, the amount of

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organic compounds in the particle phase depends on the combustion conditions, the

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measurement position (directly at the stack or in the atmosphere) and method (no

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dilution, ejector dilution, dilution tunnel). Measurements in the atmosphere usually

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distinguish between primary organic aerosol (POA) and secondary organic aerosol

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(SOA), formed via photochemical reactions of VOCs14-17. In emission measurements,

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when the hot flue gas is characterised, the terms condensable PM and condensable

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organic compounds (COC) are used. In the hot flue gas, COC are mainly in the gas

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phase but when entering the atmosphere they can contribute to POA. The amount and

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composition of COC also depends on the measurement method (flue gas quenching

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and gas into liquid sampling as applied here, dilution tunnel) which aim to assess

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potential POA formation due to cooling and dilution of the emissions leaving the stack

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into the atmosphere.

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In automated wood combustion, near-complete combustion (throughout the manuscript

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denoted as stationary condition) with generally low gas and particle phase emissions

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can be achieved and hence salts dominate the particle fraction. In manual wood

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combustion devices incomplete combustion occurs, where soot and/or COC can be the

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dominant fraction of particulate matter released to the atmosphere. However, COC and


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soot can also be emitted from automated appliances during start-up and phases of

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inappropriate operation. For the three basic particle types, a previous investigation,

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where natural wood fuels were used, revealed the highest cytotoxicity and

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carcinogenicity in Chinese hamster V79 cells for COC followed by soot, while salt

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particles showed the lowest impact18. Other studies also showed that old or manual

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combustion devices with higher organic and soot emissions induce higher cytotoxicity

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and carcinogenicity in mouse RAW264.7 macrophages and human BEAS-2B bronchial

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epithelial cells compared to new or automated devices 19-22. The importance of organic

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compounds was also confirmed from the analysis of organic extracts from filter samples

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collected during wood combustion experiments which showed comparable or higher

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release of the pro-inflammatory markers TNF-α and IL-8 in the monocytic THP-1 cells

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compared to washed solid particles23.

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Since emission limits on particulate matter from stationary combustion processes as

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described e.g. in the New Source Performance Standards of the U.S. Environmental

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Protection Agency24, the German emission control legislation BImSchV25 or the Swiss


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Ordinance on Air Pollution Control26 apply only for the hot flue gas, COC and potential

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SOA are not regularly measured and rarely investigated. In addition, often only health

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effects of water-insoluble organic compounds (WIOC, e.g., polycyclic aromatic

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hydrocarbons) are assessed. Consequently, there is an interest in further information to

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address the knowledge gap between pollutant emissions directly at the source,

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differences in health effects between WISC and water-soluble organic compounds

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(WSOC) and their relevance to ambient air and finally to human health. Experimental

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methods to investigate the influences of wood combustion emissions in the stack on

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possible health effects applied so far are, however, often complex and costly. Hence,

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experiments on adverse effects from wood combustion emissions are typically restricted

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to a small number of specific applications. Furthermore, the inter-comparability between

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different health studies is often limited due to the different applied methods and the

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specific experimental conditions. In addition, different cell lines, biological endpoints

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and/or methods (i.e., in-vitro, in-vivo and epidemiology) might show different results23, 27.


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Therefore, the aim of the present investigation was to develop a simple and economic

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method for the collection and subsequent in-vitro lung epithelial cell cytotoxicity analysis

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of WSCOC and possibly also water-soluble primary solid particles (WSpSP) from wood

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combustion. This method is then applied to compare possible cellular effects of various

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combustion devices operated under different conditions. With such results less health

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relevant technologies could be identified and target oriented air pollution control

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strategies and incentive policies for clean technologies could be developed to minimize

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the environmental impact of wood combustion.

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MATERIALS AND METHODS

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COC and SP sampling. In order to keep the sampling method for the investigation of

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in-vitro cellular effects of COC and primary solid particles (SP) from wood combustion

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emissions simple, the standard sampling method US-EPA method 5H 28 was applied in

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a slightly adapted way. In this method, hot flue gas is isokinetically sampled through a

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heated stainless-steel tube to a heated filter (referred to as filter 1) followed by a series


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of three impingers (borosilicate glass, first two filled with water) placed in a water bath

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cooled to 5 °C and an unheated filter (referred to as filter 2). SP are collected on filter 1.

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Condensable PM (mainly COC, salts as well as water and to a minor extent, metals) is

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formed due to the rapid cooling of the flue gas. WSCOC are collected in the impinge

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fillings, whereas condensable WIOC (WICOC) as well as droplets leaving the impingers

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due to the sampling air stream are trapped on filter 2 (Figure 1). Consequently, in our

124

study WSCOC denote organic compounds that are water-soluble and condense at a

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temperature of 5°C. Filter 1 also corresponds to the VDI 2066 method and several other

126

international standards for the gravimetric determination of total primary SP in the hot

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flue gas 29-33.


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Figure 1. Schematic of the two parallel sampling lines used to collect WSCOC

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(sampling method 1) and WSCOC plus WSpSP (sampling method 2).

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In order to investigate the cellular effects of both, WSCOC and WSpSP in flue gases

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from wood combustion, two sampling lines were set up in parallel which are referred to

133

as sampling method 1 and sampling method 2, respectively (Figure 1). Sampling

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method 1 corresponds to US-EPA 5H and to VDI 2066, whereas in sampling method 2

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the US-EPA 5H method was slightly adapted by omitting filter 1 upstream of the

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impingers and consequently in addition to WSCOC, also WSpSP were precipitated into

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the impingers. Another modification of the US-EPA 5H method was carried out by using

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cell growth media without serum as impinger filling instead of water. After the


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combustion experiments, cells were exposed to the liquids generated with sampling

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method 1 and sampling method 2.

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Experimental setup. The experiments consisted of two parts with sample generation in

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the combustion laboratory and subsequent in-vitro cell analyses in the biomedical lab.

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During the sample generation, detailed characterization of gas and particle phase

145

emissions was conducted as displayed in Figure S1 in the supporting information (SI).

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In the hot flue gas gas-phase species O2, CO, NO, VOC, methane (CH4) and non-

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methane VOC (NMVOC) were measured. CO2 in the flue gas was calculated from the

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measured O2 and CO at standard conditions on dry basis and assuming ideal gas

149

conditions 34. Primary SP mass was determined gravimetrically with isokinetic sampling

150

according to the VDI 2066 standard 29 on filter 1. All experiments were carried out with a

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constant chimney draft of 12 Pa controlled by a flue gas ventilator. More details on the

152

experimental setup can be found in the SI.

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Sample preparation and experimental procedures. Before every experiment, the gas

155

analysers were calibrated using standard reference gases and the glass impingers and

156

connection tubes were sterilized. Filters for the gravimetric SP determination were

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conditioned for 6 h in a desiccator and weighted before and after sampling. For most of

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the experiments 170 ml sampling liquid (80 ml in bottle 1 and 90 ml in bottle 2) was

159

used. The sampling liquids, sterile water and the cell growth medium are stored in a

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fridge at 5 °C and impingers were filled with these liquids and put into the cooled water

161

bath (5 °C). Nozzles for the extraction of flue gas for the primary SP and COC sampling

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were adjusted to assure an isokinetic sampling with 5–15 l/min.

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Test runs were carried out for every investigated combustion device in order to

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determine the sampling durations needed for the collection of a sufficient amount of

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total organic carbon (TOC) within the sampling liquids inducing significant cell viability.

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Sampling durations varied between 5 min and 4 h with shorter sampling durations for

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devices and conditions with high NMVOC emissions and/or short phases (Table 1).

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More details on defining the sampling durations and phases for the different combustion

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devices can be found in the SI.


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After sampling, the impinger fillings were stored in sterile 50 ml conical sterile

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centrifuge tubes in a fridge at 5 °C until cell analysis. TOC was measured by thermal

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oxidation followed by infrared detection according to the reference method DIN EN

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1484-H3 performed by Bachema AG, a laboratory for chemical and microbiological

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analyses of environmental samples.

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To further investigate cytotoxic effects of primary SP, for some experiments (n = 7) SP

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collected on filter 1 (upstream of impingers in sampling method 1) was water extracted

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and cells were exposed to these extracts in the same way as the other samples. Water

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extraction was carried out according to Daellenbach et al.35 (for more details see SI).

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Combustion devices. To develop and characterize the sampling methods, initial test-

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series were performed using a commercial pellet boiler which enabled different

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combustion conditions at high reproducibility13. In addition to an appropriate operation

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with optimum air excess (denoted as λopt) and boiler settings according to the

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manufacturer, modifications of the settings were applied to create non-ideal conditions


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with high excess of combustion air (λ++, resulting in high CO and NMVOC emissions)

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and with lack of combustion air (λ-, resulting in high CO and soot emissions).

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After defining and characterizing the method with the pellet boiler, four additional

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combustion devices with 14 different combustion conditions were investigated (Table 1).

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More details can be found in the SI. In the log wood stove the ignition from the bottom

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represents a traditional method which, however, results in higher cold start emissions

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compared to ignition from the top 36. All measurements with the moving grate boiler

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were conducted with the electrostatic precipitator (ESP) switched off representing raw

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gas conditions. Consequently, primary SP emissions in real-life conditions with regular

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ESP operation are significantly lower. The combustion conditions with high excess and

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with lack of combustion air in the pellet and log wood boiler were investigated since

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these conditions exhibit distinct emissions (COC and soot). In real life operation, these

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conditions occur infrequently within new automated devices and only during severe mal-

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operation.

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Table 1. Combustion devices and conditions investigated in this study. In all devices natural wood fuels were used.

Combust -ion device

Pellet boiler

Log wood stove

Moving grate boiler

Nominal heat output

Combustion technique##

15 kW

two-stage, updraft, automated continuous

Wood

6 kW

one-stage, updraft, batch manual

Beech wood logs (33 cm length)

two-stage, updraft, automated continuous

Forestry wood chips (mainly beech)

150

kW**

Log wood boiler

30 kW

two-stage, downdraft, batch manual

Pellet stove

6 kW

two-stage, updraft,

Wood used

pellets*

Moisture content on dry basis

Combustion conditions

Sampling duration

7.5%

Optimum/best (λopt#) Lack of combustion air (λ-) High excess of combustion air (λ++)

2 h 30–4 h 10min 50 min–1 h 50min 10–35 min

16% and 23.5%

Cold start (bottom ignition) Flaming Warm start Dry and wet wood*

10–30 min 10–15 min 5–10 min

30%

Full-load Part-load (30% of full-load) Going to standby from 30%

1 h 30min– 1 h 45min 2 h 10min– 3 h 10min ~15 min

Beech wood logs (50 cm length)

13%

Cold start Stationary Lack of O2 (λ-)

10–30 min ~1 h 30 min ~10 min

Wood pellets*

7.5%

Part-load (3 kW)

~1 h


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automated continuous 201

*ISO

17225-2-A1 certified

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**Prototype,

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#λ

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##In

down-scale from a 450 kW commercial boiler

is the air-fuel equivalence ratio or excess air ratio describing the overall oxygen availability of a combustion process 37 the pellet stove, the pellet boiler and the log wood boiler combustion air supply is regulated by a flue gas ventilator.

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For the pellet and log wood boiler primary and secondary combustion air is additionally regulated via valves automatically.

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In the moving grate boiler, primary and secondary air supply is automatically regulated via primary and secondary air

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ventilators.

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Cell cultures. H187 (CRL-5804TM) human epithelial lung cells were obtained from

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American Type Culture Collection (ATCC; LGC Standards, Molsheim Cedex, France)

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and cultured in Roswell Park Memorial Institute medium 1640 (RPMI, Thermo Fisher

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Scientific, A10491) containing 10% fetal bovine serum (Brunschwig, CVFSVF00-01).

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The cultures were maintained in a fully humidified atmosphere, at 37 °C and 5% CO2

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and the cells were passaged every two days and are used for experiments only before

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the 15th passage. For the experiments H187 cells were seeded at a density of

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15000 cells/mL on 6-well plates (3 ml per well).

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Cell exposure. H187 cells were incubated for 24 hours with medium containing water

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or RPMI which was exposed to wood combustion flue gases. When RPMI was exposed

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to combustion gases, cells were incubated with “exposed RPMI” with concentrations

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from 0% to 90%, the 10% remaining corresponding to the addition of fetal bovine serum

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(FBS). When water was exposed to combustion gases, cells were incubated with

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“exposed water” from 0% to 80%, the 20% remaining corresponding to the addition of

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FBS and 10 times RPMI medium. Cells were incubated within 24 hours after exposure


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of cell growth media to combustion gases. For every experiment negative controls (cell

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cultures prepared with fresh medium and incubated at the same time as exposed

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samples) were analysed as well.

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Determination of cytotoxicity. Cytotoxicity in this study was assessed by determining

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cell viability after 24 h exposure Propidium Iodide (PI) (detailed staining protocol can be

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found in the SI). The samples were than analysed with a flow cytometer (FACS, ©BD

233

Sciences). No interference of the FACS signal with SP contained within the cell cultures

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is found which was already shown in 22 and can also be seen in Figure S2, where the

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gating of cells is displayed also. Determination of cytotoxicity is also evaluated using

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Triton X (Figure S3 in the SI).

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Data Treatment. Cell viability results in this manuscript are related to the amount of

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TOC in mg/l measured in the sampling liquids and the volume of flue gas which the

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sampling liquids were exposed to (Vflue gas in m3). To derive dose response curves cell

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viability was analysed for different concentrations (initial sample diluted, see sections


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above) and the corresponding TOC and Vflue gas values were determined with the same

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dilution factors. The cell viability results from multiple experiments from the same

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combustion condition (n = 2–7, Table 2) were grouped into TOC and Vflue gas bins from

245

which the averages and standard deviations (error bars in Figures 3-4) were calculated.

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Bin margins for each combustion condition were assigned individually.

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For a better comparison of the cytotoxicity induced by the different investigated

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combustion devices and conditions the LC50 (lethal concentration where 50% of all cells

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are found dead) was used. The LC50 was determined by linearly fitting (Least Squares

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Method) the two data points closest above and below 50% cell viability, with axes

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scaled logarithmically in x and linearly in y. It was shown that LC50 determination using

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other methods (e.g. Probit) is more accurate 38, however, any differences in LC50 due to

253

applying different fitting methods is expected to be lower than the uncertainties induced

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by the limited number of different concentrations per sample (n = 4–6) and the use of

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average dose-response curves for each combustion condition with standard deviations

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for the cell viability of up to 35%.

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Results

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Method characterisation

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Several tests were performed to evaluate the applied method including the method for

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cell viability determination, investigation of the influence of sterility and blanks, type of

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sampling liquid, and sample storage duration. As seen from Figures 2 to 4, a clear dose

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response curve with decreasing cell viability with increasing TOC amount or amount of

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sampled flue gas is observed showing that the applied method can be applied

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successfully applied. Testing blanks and non-sterile sampling equipment did not show

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significant effects on the cell viability indicating that possible contaminations influencing

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the cell analysis due to the procedures in the combustion laboratory can be excluded

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(Figure S4 in the supporting information (SI)). Furthermore, the effect of the duration

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between WSCOC sampling and cell exposure was investigated (Figure S5). Cell

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viability is comparable for different storage durations up to 31 h. However, after two

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weeks a significant decrease in cell viability is found compared to a sample storage of


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only 12 h. Consequently, cell exposure was always carried out the day after WSCOC

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sampling, which is equivalent to storage durations of 15–20 h. Comparing the use of

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sterile water and cell growth medium RPMI used as sampling liquid, no difference in cell

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viability between both sampling liquids in both sampling methods is found (Figure 2).

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This shows that the composition of the RPMI is not altered during sampling thereby

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negatively affecting the cell cultures. Furthermore, this indicates that the collection

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efficiencies of primary SP and WSCOC for sterile water and RPMI are comparable and

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that both liquids can be used to sample wood combustion flue gases with subsequent

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exposure to cells. However, since cell growth media already contain several organic

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and inorganic compounds, in all other experiments sterile water is used to allow a more

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detailed chemical analysis, e.g. TOC, of the samples.

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Figure 2. Cell viability as a function of the sampled flue gas volume (at standard

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conditions (0°C, 1 bar) and are normalized to a reference oxygen content of 13 %) for

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samples generated with sterilized water (solid lines) and the cell growth medium RPMI

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(dashed lines). Experiments were carried out using the pellet boiler operated at λ++

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conditions and applying parallel sampling with methods 1 and 2. Tests with sterile water

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and RPMI were carried out consecutively on the same day. Sample-to-sample

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differences in cell viability due to variable emissions are expected to be low due to the

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stable combustion conditions in the pellet boiler (the average differences in NMVOC

294

and SP emissions between all test runs were 4% and 26%, respectively).

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Emissions. A comparison of selected emissions of the investigated combustion

297

devices and conditions using natural wood fuels is displayed in Table 2 and Figure S6.

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The highest emissions occur in the log wood stove during cold start with ignition from

299

the bottom and reload conditions as well as during cold start and λ- operation in the log

300

wood boiler (highest concentrations of 320 mg/mn3 for SP, 25000 mg/mn3 for CO and

301

2000 mg/mn3 for NMVOC). Also, the other unfavorable conditions (transient phase from

302

part-load to standby in the grate boiler, λ++ and λ- operation in the pellet boiler, as well

303

as part-load operation in the pellet stove) result in high NMVOC (111 mg/mn3), and

304

especially high CO emissions (3000 mg/mn3). Moreover, the use of wet wood in the log

305

wood stove results in approximately two times higher CO and NMVOC emissions,

306

whereas no difference is evident for primary SP. During stationary operation in all

307

devices, the emissions are much lower, i.e. for organic compounds even by more than

308

one order of magnitude (7-850 mg/mn3 and 0.1-50 mg/mn3 for CO and NMVOC,

309

respectively). This highlights the importance of an appropriate operation of combustion

310

devices as shown previously 36, 39. Furthermore, COC concentration (sum of TOC in the

311

impingers and the mass determined on the filter after the impingers in sampling


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method 1) can exceed the one of primary SP measured in the hot flue gas which was

313

also found in other investigations 40, 41. In addition, strong correlations between TOC

314

determined in the sampling liquid and NMVOC (R2 = 0.82) as well as between NMVOC

315

and CO (R2 = 0.80) are found (Figure S7 in the SI) which was also observed in previous

316

studies 42, 43.

317

318

Cytotoxicity of WSCOC. To determine the cytotoxicity of WSCOC without primary SP,

319

samples are generated by the sampling method 1 and the cell viability is related to the

320

amount of TOC determined in the sampling liquids. As seen in Figure 3, no decrease in

321

cell viability is observed during part- and full-load in the moving grate boiler and the log

322

wood boiler during stationary flaming conditions, even when the sampling liquids are

323

exposed to flue gases for up to 4 h. Also, for optimum combustion in the pellet boiler

324

only a small decrease in cell viability is evident (LC50 of 415 mg/l). For the unfavorable

325

combustion conditions in the pellet boiler (λ- and λ++), in the log wood boiler (cold start

326

and λ- operation), in the moving grate boiler (going to standby from part-load), in the


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pellet stove (part-load operation), as well as for all combustion conditions in the log

328

wood stove, cell viability decreases significantly with increasing TOC amount within the

329

sampling solutions (LC50 of 5–17 mg/l, Table 2). These differences in LC50 are relevant,

330

however, do not span orders of magnitude and further indicates that a certain mass of

331

organic compounds emitted from the different combustion devices and conditions, when

332

operated with natural wood fuels, will exhibit a similar cytotoxicity.

333

334

Figure 3. Cell viability after 24h exposure to the samples relative to the negative

335

controls of all samples containing only WSCOC (sampling method 1) as function of

336

TOC. The grey shaded area denotes the average ± standard deviation of the λ- and λ++


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conditions in the pellet boiler, cold start and λ- operation in the log wood boiler, going to

338

standby from part-load in the moving grate boiler, part-load operation in the pellet stove

339

and all combustion conditions in the log wood stove. The individual dose response

340

curves for all combustion devices and conditions can be found in Figure S8 in the SI.


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Table 2. LC50 of WSCOC (sampling method 1) relative to the negative controls and corresponding average NMVOC and

342

CO emissions and emission factors.

Combustio n device

Pellet boiler

Log wood stove

Moving

Combustio n conditions

# of repeated λ experiment s

opt

4

1.56

0.98

10.9

-

5

1.28

9.4

104.4

813

++

7

3.36

112

1,245

Cold start

2

15.49

2,035

Warm start 4

4.53

Warm start 2 wet wood Flaming

LC50 TOC

LC50 Vflue gas

LC50 burnt wood#

[mn3/l]

[g/l]

NMVOC

CO

[mg/mn3] [mg/kg]#

[mg/mn3]

[mg/kg]#

[mg/l]

133

1,489

415.17

663.8

*

7.41*

9,070

10.88*

2.50*

223.8

3,035

33,884

17.44

0.31

27.7

21,051

5,980

61,862

6.47

0.08

7.9

412

4,262

3,760

38,898

5.09

0.11

10.3

8.81

1,019

9,902

7,092

68,912

11.86

0.14

13.9

3

4.25

51.7

535

849

8,783

7.89

1.05

101.0

Flaming wet wood

2

4.41

84.9

825

1,295

12,586

5.68

0.12

12.7

Full-load

2

2.12

0.04

0.4

7.1

65

-**

-**

-**


27


grate boiler

Log wood boiler Pellet stove

Part-load

2

2.08

1.3

12.2

194

1,790

-**

-**

-**

Going to standby

2

1.43

327

3,020

4,963

45,810

8.02

0.08

8.4

Cold start

4

4.94

674

7,154

3,645

38,705

7.38

0.14

13.2

opt

2

1.46

4.4

46.3

32.6

346

-**

-**

-**

-

4

1.05

1,036

11,001

24,879

264,201

8.94

0.05

5.0

Part-load

3

19.76

241

2,689

4,181

46,668

5.43

0.52

343

*LC

344

**Decrease

345

#V

346

factors in mg per kg wood using the conversion factor of 12 mn3/kg for dry wood

347

moisture content.

50

Page 28 of 56

46.6

extrapolated using the slope between the three points with lowest cell viability.

flue gas

in cell viability was < 10% even though sampling durations were between 1.5 h and 4 h.

can be converted to the mass of burnt wood and NMVOC and CO emissions can therefore be converted to emission 44

which is here corrected to the actual

348


28

Page 29 of 56


349

If the cell viability is referred to the flue gas volume at standard conditions (which is

350

proportional to 1 kg of burnt wood or 1 MJ of heating value in the fuel) to which the

351

samplings liquids were exposed to, differences of two orders of magnitude are evident

352

(Figure 4). These differences are much more pronounced than for the comparison of the

353

cell viability based on TOC, which is a consequence of the significantly different

354

emission factors of organic compounds between the investigated combustion devices

355

and conditions. The highest cytotoxicity is found for the combustion with lack of

356

combustion air in the log wood boiler. There, only 5.0 g wood which releases 0.05 m3

357

flue gas during combustion are needed per liter of sampling liquid to induce LC50. For all

358

other unfavorable conditions, in the log wood stove (cold start, re-fill and operation with

359

wet wood), the log wood boiler (cold start) and the grate boiler (going to standby from

360

part-load), the induced cytotoxicity is similar (LC50 between 0.08 m3/l and 0.14 m3/l

361

corresponding to 7.9–13.9 g/l burnt wood, Table 2). Flue gas from stationary conditions

362

in the grate, log wood and pellet boilers shows no or only a minor effect on the cell

363

viability even for long sampling durations and large amounts of Vflue gas.


29


Page 30 of 56

364

When the LC50 of Vflue gas of the different combustion devices and conditions is compared

365

to the corresponding NMVOC and CO emissions a clear correlation is evident (R2 = 0.84

366

and R2 = 0.76, respectively, Figure 5). This indicates that the cytotoxicity of wood

367

combustion flue gases (excluding burn out conditions where NMVOC emissions are

368

usually low but CO emissions high) could be approximated with a power function from

369

emissions which are regularly monitored and for which emission limit values exist.

370

371

372

Figure 4. Cell viability after 24h exposure relative to the negative controls of all samples

373

containing only WSCOC (sampling method 1) as function of the sampled flue gas


30

Page 31 of 56


374

volume at standard conditions (0°C, 1 bar) and normalized to a reference oxygen

375

content of 13 %.

376

377

378

Figure 5. Scatterplots of LC50 (relative to the negative controls) of WSCOC (sampling

379

method 1) vs. NMVOC (left panel) and LC50 of WSCOC vs. CO (right panel) at standard

380

conditions (0°C, 1 bar) and normalized to a reference oxygen content of 13 %. A linear

381

fit (Least Square Method) omitting the intercept was applied on the log normal

382

transformed values.


31


Page 32 of 56

383

384

Cytotoxicity of primary water-soluble SP. To determine the cytotoxicity of primary water-

385

soluble SP (WSpSP) parallel sampling is performed with sampling method 1 and

386

sampling method 2 where, in addition to WSCOC, also WSpSP are precipitated.

387

However, results for eight different combustion conditions reveal no significant

388

differences between the cell viability of WSCOC only and the cell viability of WSCOC

389

and WSpSP (Figure S9). This indicates that for the investigated concentrations in the

390

sampling liquids, no additional cytotoxic effect of WSpSP compared to WSCOC alone is

391

detected. It should be noted that water-insoluble particles are only precipitated to a

392

minor extent into the sampling solutions. Another reason could be that on average in

393

this study, in samples containing WSCOC and WSpSP (sampling method 2) only ~10 %

394

more TOC is detected than in samples with WSCOC alone (sampling method 1). Since

395

the negative controls already contained ~10 % of dead cells the method applied here is

396

most probably not sensitive enough to detect a small additional cytotoxic effect of

397

WSpSP.


32

Page 33 of 56


398

To further investigate the cytotoxicity of WSpSP, cells were exposed to water extracts

399

containing solid SP from filter 1. Also in this analysis (Figure S10) no cytotoxicity of

400

WSpSP is detected. The maximum TOC concentration for the investigated primary SP

401

water extracts is 33 mg/l. On the contrary, LC50 for WSCOC only for the same combustion

402

devices and conditions (except for λopt in the pellet boiler) is already reached for TOC

403

concentrations of 6–19 mg/l.

404

405

Discussion

406

Advantages and limitations of the applied method

407

This study shows that adverse effects on cells from wood combustion emissions can

408

be investigated with a relatively simple experimental setup which allows analysing a larger

409

number of samples. The applied method is based on standard sampling procedures

410

(i.e.US-EPA 5H) which can be deployed relatively easy and could be included in type

411

testing procedures of combustion devise., It can also be applied in the field in relatively

412

harsh environmental conditions, and not only in controlled laboratory conditions.


33


Page 34 of 56

413

Furthermore, the method could also be applied after flue gas dilution, e.g. a dilution

414

tunnel. However then, most probably, longer sampling times and/or more sensitive

415

biological endpoints are necessary, thereby adding complexity to the simple method.

416

Moreover, it should be noted that particle and VOC characteristics using flue gas dilution

417

systems are different, i.e. due to dilution and cooling a larger fraction of SP are expected

418

compared to sampling hot flue gas directly at the stack. Another advantage of the method

419

is, that directly cell grow medium for WSCOC and WSpSP sampling can be used, when

420

no further chemical analysis of the sampling liquids is necessary, further simplifies all

421

necessary steps in the cell exposure. In many studies investigating in-vitro health effects

422

SP is first sampled on filters and then extracted with solvents. This involves mostly organic

423

solvents which are very toxic to the cells and consequently, several steps are necessary

424

before cells can be exposed to the extracted SP making such methods time consuming

425

and thereby limiting the number of samples which can be analysed. Also, the use of a

426

simple biological endpoint supports the aim to keep the entire method fast, simple and

427

economic.

428


34

Page 35 of 56


429

One limitation of the applied method is that the used sampling liquids, i.e. water and

430

cell growth medium, are polar solvents and consequently mainly WSCOCs and WSpSP

431

compounds can be investigated with our method. Also, only WSCOC precipitated into

432

the impinger fillings, excluding WIOC collected on the filter after the impingers, were

433

analysed. Salts exhibit only minor cell effects compared to COC and organic primary

434

SP18. Heavy metal emissions are of minor importance for natural wood fuels as used

435

here 39, 45, 46 and, therefore were not considered. Consequently, cytotoxicity in this study

436

is mainly related to TOC content of WSCOC which condense at 5°C. This includes not

437

only water-soluble condensed semi-volatile but also some volatile organic compounds

438

when the boiling point is considered for discrimination of volatility classes (semi-volatile

439

organic compounds: 240-260°C to 380-400°C, volatile organic compounds: 50-100°C to

440

240-260°C and very volatile organic compounds: 50 mg/l) as well as possible different cytotoxic responses due to SP extracted

481

with water or organic solvents need to be investigated.

482

483


38

Page 39 of 56

484

485

486

487


ASSOCIATED CONTENT

Supporting Information. The Supporting Information is available free of charge on the ACS Publications website.

PDF document containing more details on 1) the experimental setup and

488

instrumentation, 2) sample preparation and experimental procedures, 3) investigated

489

combustion devices, 4) determination of cytotoxicity including the detailed PI staining

490

protocol, 5) additional result figures from the method characterisation, emissions from

491

the combustion devices and cytotoxicity.

492 493

AUTHOR INFORMATION

494

Corresponding Authors

495

*Peter

496

*Thomas

497

Notes

Zotter: Phone: +41 41 349 38 34; E-mail: [email protected] Nussbaumer: Phone: +41 41 349 35 19; E-mail: [email protected]


39


498

Page 40 of 56

The authors declare no competing financial interest.

499

500

ACKNOWLEDGMENTS

501

This work was funded by the Swiss Federal Office for the Environment, the Swiss

502

National Science Foundation (NRP 70) and it is part of the Swiss Competence Center for

503

Energy Research SCCER BIOSWEET of the Swiss Innovation Agency Innosuisse. The

504

experiments were supported by Attika Feuer AG, SCHMID AG energy solutions, Sigmatic

505

AG, and Tiba AG.


40

Page 41 of 56


506

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