The application of a profluorescent nitroxide probe to detect reactive oxygen species derived from combustion generated particulate matter

Queensland University of Technology Discipline of Physics and Chemistry THE APPLICATION OF A PROFLUORESCENT NITROXIDE PROBE TO DETECT REACTIVE OXYGEN SPECIES DERIVED FROM COMBUSTION-GENERATED PARTICULATE MATTER Branka Miljevic A THESIS SUBMITTED TO THE QUEENSLAND UNIVERSITY OF TECHNOLOGY IN FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY September 2010 ABSTRACT Particulate pollution has been widely recognised as an important risk factor to human health In addition to increases in respiratory and cardiovascular morbidity associated with exposure to particulate matter (PM), WHO estimates that urban PM causes 0.8 million premature deaths globally and that 1.5 million people die prematurely from exposure to indoor smoke generated from the combustion of solid fuels Despite the availability of a huge body of research, the underlying toxicological mechanisms by which particles induce adverse health effects are not yet entirely understood Oxidative stress caused by generation of free radicals and related reactive oxygen species (ROS) at the sites of deposition has been proposed as a mechanism for many of the adverse health outcomes associated with exposure to PM In addition to particle-induced generation of ROS in lung tissue cells, several recent studies have shown that particles may also contain ROS As such, they present a direct cause of oxidative stress and related adverse health effects Cellular responses to oxidative stress have been widely investigated using various cell exposure assays However, for a rapid screening of the oxidative potential of PM, less time-consuming and less expensive, cell-free assays are needed The main aim of this research project was to investigate the application of a novel profluorescent nitroxide probe, synthesised at QUT, as a rapid screening assay in assessing the oxidative potential of PM Considering that this was the first time that a profluorescent nitroxide probe was applied in investigating the oxidative stress potential of PM, the proof of concept regarding the detection of PM–derived ROS by using such probes needed to be demonstrated and a sampling methodology needed to be developed Sampling through an impinger containing profluorescent nitroxide solution was chosen as a means of particle collection as it allowed particles to react with the profluorescent nitroxide probe during sampling, avoiding in that way any possible chemical changes resulting from delays between the sampling and the analysis of the PM Among several profluorescent nitroxide probes available at QUT, bis(phenylethynyl)anthracene-nitroxide (BPEAnit) was found to be the most suitable probe, mainly due to relatively long excitation and emission wavelengths (λex= 430 nm; λem= 485 and 513 nm) These wavelengths are i long enough to avoid overlap with the background fluorescence coming from light absorbing compounds which may be present in PM (e.g polycyclic aromatic hydrocarbons and their derivatives) Given that combustion, in general, is one of the major sources of ambient PM, this project aimed at getting an insight into the oxidative stress potential of combustion-generated PM, namely cigarette smoke, diesel exhaust and wood smoke PM During the course of this research project, it was demonstrated that the BPEAnit probe based assay is sufficiently sensitive and robust enough to be applied as a rapid screening test for PM-derived ROS detection Considering that for all three aerosol sources (i.e cigarette smoke, diesel exhaust and wood smoke) the same assay was applied, the results presented in this thesis allow direct comparison of the oxidative potential measured for all three sources of PM In summary, it was found that there was a substantial difference between the amounts of ROS per unit of PM mass (ROS concentration) for particles emitted by different combustion sources For example, particles from cigarette smoke were found to have up to 80 times less ROS per unit of mass than particles produced during logwood combustion For both diesel and wood combustion it has been demonstrated that the type of fuel significantly affects the oxidative potential of the particles emitted Similarly, the operating conditions of the combustion source were also found to affect the oxidative potential of particulate emissions Moreover, this project has demonstrated a strong link between semivolatile (i.e organic) species and ROS and therefore, clearly highlights the importance of semivolatile species in particleinduced toxicity ii KEYWORDS Combustion aerosols, combustion-generated particulate matter, cigarette smoke, wood smoke, diesel exhaust, health aspects of aerosol, health effects of particulate matter, radicals, reactive oxygen species, ROS, oxidative stress, oxidative potential, inflammatory potential, in vitro, profuorescent nitroxides; BPEAnit; fluorescence, impinger, bubbling, collection efficiency iii STATEMENT OF ORIGINAL AUTHORSHIP The work contained in this thesis has not been previously submitted to meet requirements for an award at this or any other higher education institution To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made Signature: Date: iv Table of Contents ABSTRACT i KEYWORDS iii STATEMENT OF ORIGINAL AUTHORSHIP iv LIST OF PUBLICATIONS ix LIST OF TABLES x LIST OF FIGURES x ABBREVIATIONS xv ACKNOWLEDGEMENTS xvii Chapter INTRODUCTION 1.1 Description of scientific problem investigated 1.2 Overall aims of the study 1.3 Specific objectives of the study 1.4 Account of scientific progress linking the scientific papers Chapter LITERATURE REVIEW 2.1 Introduction – particles and health effects 2.1.1 Summary of epidemiological findings 2.1.2 Summary of toxicological findings 10 2.2 Aerosol fundamentals and basic terminology 12 2.3 Combustion-generated PM 15 2.3.1 Cigarette smoke 16 2.3.2 Diesel exhaust PM 17 2.3.3 Wood smoke particles 20 2.4 Particle characteristics relevant for health effects 22 v 2.4.1 Size and surface area 22 2.4.2 Composition 24 2.5 Measurement of oxidative stress capacity of the PM 28 2.5.1 In vitro studies 28 2.5.2 Cell-free assays 30 2.6 Nitroxides 36 2.6.1 Profluorescent nitroxides 37 2.7 An overview of particle sampling approaches for toxicological studies 42 2.8 Summary of research needs 44 2.9 References 46 Chapter 63 ON THE EFFICIENCY OF IMPINGERS WITH FRITTED NOZZLE TIP FOR COLLECTION OF ULTRAFINE PARTICLES 63 Abstract 65 3.1 Introduction 66 3.2 Experimental 67 3.3 Results and discussion 70 3.4 Conclusion 76 3.5 References 77 Chapter 79 THE APPLICATION OF PROFLUORESCENT NITROXIDES TO DETECT REACTIVE OXYGEN SPECIES DERIVED FROM COMBUSTION-GENERATED PARTICULATE MATTER: CIGARETTE SMOKE – A CASE STUDY 79 Abstract 81 4.1 Introduction 82 4.2 Experimental 86 4.2.1 Materials 86 vi 4.2.2 Cigarette smoke sampling 86 4.2.3 Fluorescence measurements 89 4.2.4 Calibration curve 90 4.3 Results 90 4.3.1 Mainstream cigarette smoke - linearity 90 4.3 Sidestream cigarette smoke - sensitivity 92 4.4 Discussion 96 4.5 Conclusion 98 4.6 References 99 4.7 Supplementary Information 103 4.7.1 Reaction of BPEAnit with peroxyl radicals 103 Chapter 105 PARTICLE EMISSIONS, VOLATILITY AND TOXICITY FROM AN ETHANOL FUMIGATED COMPRESSION IGNITION ENGINE 105 Abstract 108 5.1 Introduction 109 5.2 Methodology 111 5.2.1 Engine, fuel and testing specifications 111 5.2.2 Particle measurement methodology 112 5.2.3 Particle volatility methodology 114 5.2.4 ROS concentration measurement – BPEAnit assay 115 5.3 Results 116 5.3.1 Particle size distributions 116 5.3.2 Particle volatility 119 5.3.3 ROS concentration results 122 5.4 Discussion 124 5.5 References 127 5.6 Supporting Information 130 vii Chapter 135 OXIDATIVE POTENTIAL OF LOGWOOD AND PELLET BURNING PARTICLES ASSESSED BY A NOVEL PROFLUORESCENT NITROXIDE PROBE 135 Abstract 138 6.1 Introduction 139 6.2 Experimental 141 6.2.1 Wood burners 141 6.2.2 Sampling setup and instrumentation 142 6.2.3 BPEAnit assay 144 6.3 Results 145 6.3.1 Particle emissions 145 6.3.2 ROS from logwood burning particles 147 6.3.3 Correlation between ROS and organics 148 6.3.4 ROS from pellet burning particles 152 6.4 Discussion 153 6.5 References 157 6.6 Supporting information 160 Chapter 163 CONCLUSIONS 163 7.1 Principal significance of findings 164 7.2 Directions for future research 171 7.3 References 173 viii Naeher, L.P., Brauer, M., Lipsett, M., Zelikoff, J.T., Simpson, C.D., Koenig, J.Q., Smith, K.R., 2007 Woodsmoke health effects: A review Inhalation Toxicology 19, 67-106 Nel, A., 2005 ATMOSPHERE: Enhanced: Air pollution-related illness: Effects of particles Science 308, 804-806 Oser, M., Nussbaumer, T., Schweizer, B., Mohr, M., Figi, R., 2001 Influence on aerosol formation in an automatic wood furnace Proceedings of the International Seminar of IEA Bioenergy Task 32: Aerosols from Biomass Combustion59-64 Pope, C.A., Burnett, R.T., Thun, M.J., Calle, E.E., Krewski, D., Ito, K., Thurston, G.D., 2002 Lung cancer, cardiopulmonary mortality, and long-term exposure to fine particulate air pollution J Am Med Assoc 287, 1132 - 1141 Pou, S., Huang, Y.I., Bhan, A., Bhadti, V.S., Hosmane, R.S., Wu, S.Y., Cao, G.L., Rosen, G.M., 1993 A fluorophore-containing nitroxide as a probe to detect superoxide and hydroxyl radical generated by stimulated neutrophils Analytical Biochemistry 212, 85-90 Rau, J.A., 1989 Composition and Size Distribution of Residential Wood Smoke Particles Aerosol Science and Technology 10, 181 - 192 Schmidl, C., Marr, I.L., Caseiro, A., Kotianová, P., Berner, A., Bauer, H., Kasper-Giebl, A., Puxbaum, H., 2008 Chemical characterisation of fine particle emissions from wood stove combustion of common woods growing in mid-European Alpine regions Atmospheric Environment 42, 126-141 Surawski, N.C., Miljevic, B., Roberts, B.A., Modini, R.L., Situ, R., Brown, R.J., Bottle, S.E., Ristovski, Z.D., 2009 Particle emissions, volatility and toxicity from an ethanol fumigated compression ignition engine Environmental Science & Technology 44, 229-235 Szidat, S., Prevot, A.S.H., Sandradewi, J., Alfarra, M.R., Synal, H.A., Wacker, L., Baltensperger, U., 2007 Dominant impact of residential wood burning on particulate matter in Alpine valleys during winter Geophysical Research Letters 34, 1-6 Tao, F., Gonzalez-Flecha, B., Kobzik, L., 2003 Reactive oxygen species in pulmonary inflammation by ambient particulates Free Radical Biology and Medicine 35, 327-340 Venkatachari, P., Hopke, P.K., Brune, W.H., Ren, X.R., Lesher, R., Mao, J.Q., Mitchel, M., 2007 Characterization of wintertime reactive oxygen species concentrations in Flushing, New York Aerosol Science and Technology 41, 97-111 159 Venkatachari, P., Hopke, P.K., Grover, B.D., Eatough, D.J., 2005 Measurement of particlebound reactive oxygen species in Rubidoux aerosols Journal of Atmospheric Chemistry 50, 49-58 6.6 Supporting information Impinger efficiency: In order to conduct quantitative chemical analysis of the particles collected by the impingers, it is important to know the collection efficiency of the impinger The size dependent collection efficiency for 20 mL of DMSO and flow rate of L min-1 was determined as described previously (Miljevic et al., 2009) The portion of particles that remain in the impinger upon bubbling was calculated by multiplying the size distribution as measured by the SMPS with the impinger collection efficiency curve Scan time of 150 sec was used for the SMPS system and size range was 15 – 750 nm Correction for impinger losses: In order to perform correction for impinger losses, the following procedure was applied: Number size distributions as measured by the SMPS were multiplied by the impinger collection efficiency curve (Figure S1), giving the size distribution of particles that remained in the impinger The resulting number size distributions were converted into mass size distributions (assuming particles to be spherical and having a unit density) The initial mass size distributions (prior to correcting for the impinger efficiency) and the mass size distributions corrected for the impinger efficiency were fitted to a lognormal distribution, giving the total initial mass concentrations and total corrected mass concentrations, respectively Dividing corrected mass concentration with the initial mass concentration, gave the mass impinger efficiency The mass impinger efficiency obtained by this procedure was 59 ± % (the error indicating one standard deviation; = 51%; max = 69%) and it was used to correct the particle mass measured by the TEOM 160 Figure S 6-1 Size dependent collection efficiency of the impinger used in this study when bubbling aerosol through 20 ml of DMSO and at flow rate of L min-1 (adopted from Miljevic et al., 2010 ) Miljevic, B., Fairfull-Smith, K.E., Bottle, S.E., Ristovski, Z.D., 2010 The application of profluorescent nitroxides to detect reactive oxygen species derived from combustiongenerated particulate matter: Cigarette smoke - A case study Atmospheric Environment 44, 2224-2230 Miljevic, B., Modini, R.L., Bottle, S.E., Ristovski, Z.D., 2009 On the efficiency of impingers with fritted nozzle tip for collection of ultrafine particles Atmospheric Environment 43, 1372-1376 161 162 Chapter CONCLUSIONS Particulate pollution has been widely recognised as an important risk factor to human health In addition to increases in respiratory and cardiovascular morbidity associated with exposure to PM, WHO estimates that urban PM causes 0.8 million premature deaths globally and that 1.5 million people die prematurely from exposure to indoor smoke generated from the combustion of solid fuels (World Health Organization, 2002) Despite the fact that specific mechanisms responsible for the negative health impacts of PM are still not entirely clear, oxidative stress caused by generation of ROS at the sites of deposition has been widely accepted as a probable mechanism for many of the adverse health outcomes associated with exposure to PM In addition to the particle-induced generation of ROS, several recent studies have shown that particles may also contain ROS, presenting in that way a direct cause of oxidative stress Therefore, to gain more insight into the nature of the particles relevant for their negative health impact, it is important to evaluate the oxidative stress potential of PM in terms of their inherent ROS and that was the motivation for the work presented in this thesis The basis of this research project was to develop a rapid screening test for assessing the oxidative potential of PM using a novel profluorescent nitroxide probe, synthesised at our university It is important to note that this was the first time that a profluorescent nitroxide probe was applied in investigating the oxidative stress potential of PM Therefore, the main goal of this project was to demonstrate the proof of concept regarding the detection of PM–derived ROS by using a profluorescent nitroxide probe, as well as to develop a sampling methodology for assessing the oxidative stress potential of PM using the same probe Given that combustion, in general, is one of the major sources of ambient PM, this project aimed at getting an insight into the oxidative stress potential of 163 combustion-generated PM, namely cigarette smoke, diesel exhaust and wood smoke PM Liquid impingement (sampling directly into a solution containing a profluorescent nitroxide probe) was chosen as a means of particle collection as it allowed particles to react with the profluorescent nitroxide probe during sampling, avoiding in that way any possible chemical changes resulting from delays between the sampling and the analysis of the PM Among several profluorescent nitroxide probes available at QUT, BPEA-nitroxide (BPEAnit) was selected as the most suitable probe, mainly due to relatively long excitation and emission wavelengths (λex= 430 nm; λem= 485 and 513 nm) The amount of BPEAnit being converted to a fluorescent product(s) was normalised with respect to PM mass providing a measure for ROS concentration During the course of this research project, it was demonstrated that BPEAnit assay is sufficiently sensitive and robust enough to provide a rapid estimate of the oxidative potential of PM Specific findings of the project, as well as their significance are discussed in the following section 7.1 Principal significance of findings As mentioned previously, this research project applied for the first time QUT’s profluorescent nitroxides in an investigation of the oxidative stress potential of PM Therefore, the first step of the project was to design a sampling methodology To collect particles for reaction with the BPEAnit, liquid impingement was thought to be a good sampling approach as it allowed particles to react directly with the profluorescent nitroxide probe in a sampling liquid during sampling and, thus, eliminated any possible chemical changes in PM resulting from delays between the sampling and the analysis of the PM However, impingers are known to have relatively low efficiency for collection of PM smaller than 0.5 µm in diameter, and, therefore, a collection efficiency of the impingers that were going to be used in this project needed to be determined The efficiency of impingers with fritted nozzle tip for collection of ultrafine and near-ultrafine (13-220 nm) particles and factors influencing the collection efficiency were investigated in a paper 164 entitled “On the efficiency of impingers with fritted nozzle tip for collection of ultrafine particles” and the findings of the paper are summarized as follows: • During bubbling, a substantial portion of particles is deposited on the fritted nozzle tip The percentage of particles being lost on the fritted tip increases as the particle diameter decreases and it is dependent on the porosity grade of the fritted tip - particle losses on the impinger with the porosity grade (40–100 µm) are substantially higher than for the impinger with the porosity grade (100–160 µm) • Volume and the type of collection liquid affect the collection efficiency Using 40 mL of either water or heptane resulted in higher collection efficiency than using 20 mL of the same solvent and values for collection efficiency obtained with water were on average 35% higher than the values obtained with heptane • Obtained values for overall collection efficiency were substantially higher (~30–95%) than previously reported, mainly due to the high deposition of particles in the fritted nozzle tip, especially in case of finer porosity frits and smaller particles In other words, removal efficiency is a combination of the filter-like behaviour of the fritted tip and liquid impingement Values for the capture efficiency of the solvent alone ranged from 20 to 45%, depending on the type and the volume of solvent This is higher than 10%, which has been previously reported, indicating that the increased dispersion of airstream into bubbles increases trapping of particles by the liquid As it is easy to generate in a laboratory, cigarette smoke was chosen to be a model combustion-generated aerosol in the evaluation of the probe for applications in PM-derived ROS detection By exposing BPEAnit solution to cigarette smoke it was shown that: • In the case of mainstream cigarette smoke, there is a linear increase in fluorescence intensity with an increasing number of cigarette puffs This 165 pattern was reproducible, although values varied with each individual cigarette This result verifies the proper response of BPEAnit when exposed to equal amounts of aerosol with approximately the same physical and chemical characteristics (freshly generated equal volumes of mainstream cigarette smoke puffs) • When sampling under standard smoking conditions (35 mL in duration of s, repeated every 60 s), an average of 101 nmol ROS per cigarette was obtained, with ~40% of fluorescence increase coming from the gas phase and ~60% coming from the particle phase This value is within the upper range of the previously published results • Much lower concentrations of particles as produced by sidestream cigarette smoke and sampled from a test chamber also resulted in an increase in fluorescence intensity with sampling time In adition, the fluorescence increase was well above the detection limit This demonstrates that the probe is sensitive enough to be potentially successful in determining the levels of ROS in studies where close to ambient concentrations of particles are observed • To the best of our knowledge, this study has provided the first quantitative estimates of the ROS arising from sidestream cigarette smoke It was calculated that the amount of ROS was ~50 nmol per mg of PM; however, this value decreased with ageing of the particles in the chamber Overall, these results demonstrated the proof of concept regarding the detection of PM–derived ROS by using BPEAnit and provided a good basis for employing the new BPEAnit probe for the assessment of the oxidative potential arising from particles generated by other combustion sources The results of this study were presented in a paper entitled “The application of profluorescent nitroxides to detect reactive oxygen species derived from combustion-generated particulate matter: Cigarette smoke – a case study” 166 After evaluation of the probe using cigarette smoke, BPEAnit was applied to assess theoxidative potential of PM generated by a diesel engine using ethanol substitution This study was presented in the paper “Particle emissions, volatility and toxicity from an ethanol fumigated compression ignition engine” The study has found that: • The greatest particulate mass reduction was achieved with ethanol fumigation at full load By using neat diesel at full load only accumulation mode particles (dCMD = 90 nm) were generated, whereas a 40% ethanol substitution (E40) resulted in a nucleation mode occurrence and reduction in the accumulation mode particle concentrations On the other hand, fluorescence intensity of BPEAnit was ~4.5 times higher for E40 than for neat diesel When the amount of BPEAnit being converted to fluorescent product(s) was normalised to the PM mass, the obtained values for ROS concentrations were almost 40 times higher for E40 test than for neat diesel • Size distributions at all other loads (idle, 25% and 50%), for the neat diesel and ethanol tests (E10, E20, E40), were quite similar, i.e nucleation mode particles occurred in each size distribution Accordingly, at a particular load setting, the ROS concentrations for the E10 and E20 tests, relative to E0, did not differ by any more than 20% The exemption was half load, E40 test, which resulted in approximately double the ROS concentration relative to E0 In addition, ROS concentrations for all other tests were 30 – 100 times higher than for neat diesel full load test, where only accumulation mode particles were generated • ROS concentrations exhibit an increase with decreasing engine load An explanation for the mechanism governing the formation of ROS due to ethanol fumigation and its relationship to the formation of a nucleation mode was not possible with the limited data collected in this initial study However, the important finding of this study lies in the association of the increase in fluorescence response of BPEAnit and ROS concentrations with the occurrence of the nucleation 167 mode particles Nucleation mode particles contribute very little to PM mass and are composed mainly of organic species, whereas accumulation mode particles comprise the majority of submicron PM mass and are primarily made of soot This demonstrates that the mass of particulates emitted by an engine may not be the most appropriate metric for assessing the potential health effects of diesel PM In addition, this study shows that operational practice and resulting combustion conditions affect the toxicological potential of particulate emissions The significant increase in potential particle toxicity with ethanol substitution over 20% may provide a substantial barrier for the uptake of fumigation technology using ethanol as a supplementary fuel BPEAnit was also applied to assess the potential of particles produced by an automatic pellet boiler and a traditional logwood stove to cause oxidative stress The results are reported in a paper entitled “Oxidative potential of log wood and pellet burning particles assessed by a novel profluorescent nitroxide probe” and the important findings are summarised as follows: • Particulate emissions produced by a traditional logwood stove induced an increase in fluorescence of BPEAnit, indicating the presence of ROS and other redox active species in these particles, whereas particles produced by the automatic pellet boiler did not induce an increase in BPEAnit fluorescence This indicates that within the detection limit of this method there are no ROS present in these particles and suggests that particulate emissions from pellet boilers have a lower toxicological potential than emissions from logwood stoves • The cold-start phase (beginning of burning) resulted in much higher ROS concentrations than the warm-start logwood burning phase ROS concentrations related to particle emissions from the stable phase of the cold start (4000 ± 260 nmol mg-3) were about times higher than ROS concentrations from start-up phase of cold start (1230 ± 100 nmol mg-3), 168 and about -18 times higher than the ROS concentrations from the warmstart burning (450 ± 250 nmol mg-3 and 220 ± 50 nmol mg-3) phases • The study hypothesize that combustion temperature might play a role in the amount of ROS being detected in the particulate emissions, with higher temperature of the combustion chamber resulting in lower ROS concentrations The hypothesis is supported by a strong negative correlation between ROS concentrations and logwood combustion chamber temperatures and it is proposed to be one of the reasons for the lack of fluorescence increase in case of sampling pellet boiler emissions A similar trend was observed in the study on ethanol substituted diesel emissions, where ROS concentrations were found to decrease as the engine load and subsequently the combustion temperature increased • Another important observation from this study was that sampling logwood burning emissions after passing through a thermodenuder at 250oC resulted in very small or no increase of BPEAnit fluorescence, indicating that the majority of reactive species were semivolatile • Furthermore, the amount of reactive species showed a strong correlation with the amount of particulate organic material This indicates the involvement of semivolatile organics in particle-related toxicity This study has demonstrated the importance of combustion appliance and type fuel (wood) on the oxidative potential of the particles emitted While particle size, mass and number concentration of particles from logwood burning in the traditional logwood stove and pellet burning in the automatic pellet boiler were rather similar, they were quite different in terms of estimating their oxidative potential as measured by the BPEAnit assay Similarly, operational practice in batch burning of logwood and the resultant combustion conditions were shown to affect the oxidative potential of particulate emissions Moreover, this study has demonstrated the importance of semivolatile species in particle-induced toxicity 169 Considering that for all three aerosol sources (i.e cigarette smoke, diesel exhaust and wood smoke) the same assay was applied, the results presented in this thesis allow direct comparison of the oxidative potential measured for all three sources of particles Particles from sidestream cigarette smoke were shown to have 4-9 and 30-80 times less ROS per unit of mass than particles produced during warmstart and cold-start logwood combustion, respectively This finding sheds a new light on logwood smoke particles and draws attention to the importance of expanding the knowledge on the toxicological properties of wood smoke particles Diesel exhaust particles generated under full engine load were found to have similar ROS concentrations as sidestream cigarette smoke particles (~50 nmol mg-1) On the other hand, diesel exhaust particles generated during idling were found to have ROS concentrations similar to those observed for stable phase of cold-start logwood burning The main difference between diesel exhaust particles generated under full engine load and during idling was in the occurrence of the nucleation mode during idling While diesel exhaust PM generated under full engine load consisted only of accumulation mode, the important feature of the size distribution for PM generated during engine idling was a strong nucleation mode occurrence and a reduction in the accumulation mode particle concentration Nucleation mode particles contribute very little to PM mass and are composed mainly of organic (semivolatile) species The significant increase of ROS concentration for particles generated during idling was associated with the semivolatile species present in the nucleation mode In case of logwood smoke we have observed a decrease of the ROS concentration upon removal of the semivolatile species Both studies indicate a strong link between semivolatile species and ROS and therefore the importance of semivolatiles in the oxidative potential of the PM This project shows that there are, as measured by the BPEAnit assay, significant differences between ROS concentrations obtained from particles produced by different sources Current air pollution controls are solely based on ambient particle mass standards, so studies linking adverse health effects with emissions coming from specific sources could provide a body of evidence for 170 adopting a source apportionment-based regulatory approach in air quality management 7.2 Directions for future research As stated earlier, the work presented in this thesis demonstrated a proof of concept regarding the applicability of BPEAnit in detecting of particle-derived ROS and, therefore, estimating the oxidative potential of PM The next step would be to gain an understanding about the specifics of the underlying chemistry leading to a fluorescence generated from BPEAnit when exposed to PM One way to gain insight into specific species involved could be to expose BPEAnit to model ROS compounds and other redox active compounds likely to be found in PM and to then monitor BPEAnit fluorescence and analyse the products generated HPLC and LC-MS (both available at QUT) are powerful analytical tools that could be employed to achieve this For such an analysis it would be interesting to compare products resulting from exposure of BPEAnit to PM generated by different sources In the BPEAnit assay, the overall increase of fluorescence is measured and based on that, the amount of probe that reacted can be calculated A potential source of error when measuring only overall fluorescence increase might arise from the fact that not all fluorescent products will have exactly the same fluorescence quantum yield Variations in the quantum yields of the specific fluorescent products generated after exposing BPEAnit to PM would need to be explored The sampling methodology could also be improved Liquid impingement is good as it allows particles to directly react with the nitroxide during sampling and, therefore, eliminates any delays between the sampling and the analysis of PM In addition, it is a rather simple sampling methodology and from that point of view it provided a convenient methodology to initiate the project However, for particles smaller than 0.5 µm, impingers have relatively low and size dependent collection efficiency, which needs to be taken into account when calculating the mass of particles being collected Another potentially suitable method for particle collection 171 into a liquid involves a combination of a growth of submicron particles in a condensational growth chamber and their subsequent collection using a wetted wall cyclone, which has been found to be highly efficient particle collection method (Orsini et al., 2008) If connected in-line with a flow through spectrofluorimeter, this sampling system would enable a continuous and real-time ROS detection Profluorescent nitroxides are stable and nonreactive towards oxygen which suggests they have a good potential for continuous and long-term particle/ROS sampling During the course of this PhD project, cigarette smoke, diesel exhaust and wood smoke were investigated In future research, the BPEAnit assay could be applied to some other combustion sources (for example, diesel engine fuelled with various biodiesel fuels), as well as to Secondary Organic Aerosol (SOA) SOA make a significant contribution to the total atmospheric burden of fine PM and it is formed by photochemical transformation of volatile organic compounds (VOCs) coming from biogenic (e.g forests, oceans) and anthropogenic sources into less-volatile compounds which can subsequently transfer from the gas phase to a particle phase either through nucleation to form new particles or condensing onto pre-existing particles In addition, combustion aerosols could be exposed to such photochemical conditions in order to stimulate transformations that would happen upon ageing in the atmosphere In this case, the BPEAnit assay could be applied to compare the toxicological potential of freshly generated and aged (photochemically transformed) combustion PM An important finding of this project was a strong correlation between the amount of particulate organic material and the amount of ROS as measured by BPEAnit assay, indicating the involvement of organics in particle-related toxicity In the future research, this could be further explored For that purpose, an aerosol mass spectrometer (AMS) could be used and AMS results and results of the BPEAnit assay could be directly compared This could potentially reveal specific class(es) of compounds contributing to the oxidative potential of PM 172 A thing that was originally anticipated to be investigated during the course of this project and is still yet to be explored is oxidative potential as a function of particle size For that purpose, particles of the same chemical composition throughout the whole size range would be needed (e.g diesel exhaust accumulation mode) as well as a means for particle size selection (e.g electrostatic classifier) BPEAnit assay is an acellular assay and, as such, its ability to predict PM toxicity in vivo (or at least in cells) would need to be assessed To investigate whether BPEAnit assay is biologically meaningful, a comparison of this assay with cell exposure assays should be performed A positive correlation would then allow employment this assay in routine measurements as a rapid screening test for the oxidative potential of PM It would also be interesting to incorporate BPEAnit into lung tissue cells, which would subsequently be exposed to PM Fluorescence from BPEAnit could then be monitored using confocal microscopy and flow cytometry and used as an indicator of oxidative stress induced in the cells upon exposure to PM However, BPEAnit has a very low solubility in water and, as such, would not be a good choice for this type of experiments For that purpose, a new, water soluble probe would need to be designed 7.3 References Orsini, D.A., Rhoads, K., McElhoney, K., Schick, E., Koehler, D ,Hogrefe, O., 2008 A water cyclone to preserve insoluble aerosols in liquid flow - An interface to flow cytometry to detect airborne nucleic acid Aerosol Science and Technology 42, 343-356 World Health Organization, 2002 World Health Report http://www.who.int/whr/2002/en/whr02_en.pdf (accessed February 2010) 173 ... combustion- generated aerosols and it is easy to generate in a laboratory Among several profluorescent nitroxide probes available at QUT, BPEAnitroxide (BPEAnit) was selected as the most suitable... entitled The application of profluorescent nitroxides to detect reactive oxygen species derived from combustion- generated particulate matter: Cigarette smoke – a case study” and has been published... during sampling, avoiding in that way any possible chemical changes resulting from delays between the sampling and the analysis of the PM Among several profluorescent nitroxide probes available at