Research article Aerosol characterisation including oxidative potential as a proxy of health impact: a case of a residential site in a highly industrialised area Miroslav Josipovic *1a, Catherine Leal-Liousse 2a, Belinda Crobeddu 3a, Armelle Baeza-Squiban 3b, C. Keitumetse Segakweng 1b, Corinne Galy-Lacaux 2b, J. Paul Beukes 1c, Pieter G. van Zyl 1d, Gerhard Fourie4 1Unit for Environmental Sciences and Management, North-West University, Potchefstroom Campus, South Africa a) Micky.Josipovic@nwu.ac.za b) 16516966@nwu.ac.za c) Paul.Beukes@nwu.ac.za d) Pieter.VanZyl@nwu.ac.za 2Laboratoire d’Aérologie, Université Paul Sabatier, Toulouse III, France a) cathy.leal-liousse@aero.obs-mip.fr b) corinne.galy-lacaux@aero.obs-mip.fr 3Unit of Functional and Adaptive Biology, UMR-CNRS 8251, Laboratory of Molecular and Cellular Responses to Xenobiotics, University Paris Diderot, Sorbonne Paris Cité, France a) belinda.crobeddu@gmail.com b) baeza@univ-paris-diderot.fr 4EnviroNgaka (Pty) Ltd., Brits, South Africa, gerhard.fourie@environgaka.co.za Received: 4 February 2019 - Reviewed: 20 March 2019 - Accepted: 25 August 2019 https://doi.org/10.17159/caj/2019/29/2.7517 Abstract This study aimed to characterise aerosols sampled in the vicinity of a major industrialised area, i.e. the Vaal Triangle. It included the determination of oxidative potential as a predictive indicator of particle toxicity. Aerosol samples were collated through the cascade filtering during an eight-month period (12 h over three days in one week). Three size fractions were analysed for organic carbon (OC), black carbon (BC) and oxidative potential (OP), while ionic content was presented as monthly and seasonal concentrations. The continuous measurement of black carbon by an optical attenuation instrument was collated concurrently with cascade filtering. The carbonaceous content was low compared to the ionic one. Within the carbonaceous concentrations, the organic carbon was higher than concentrations of black carbon in both seasons in the ultra-fine fraction; the opposite was the case for the fine fraction, while the coarse fraction concentrations of organic carbon in the dry season had higher concentrations than black carbon in the wet season and organic carbon in the wet season. The OP tended to increase as the size was decreasing for wet season aerosols, whereas, for the dry season, the highest OP was exerted by the fine fraction. The ultrafine fraction was the one showing the most contrasting OP between the two seasons. Continuous monitoring indicated that the higher BC concentrations were recorded in the dry/winter part of the year, with the daily pattern of concentrations being typically bimodal, having both the morning and evening peaks in both seasons. Within the ionic content, the dominance of sulphate, nitrate and ammonium was evident. Multiple linear correlations were performed between all determined compounds. Strong correlations of carboxylic acids with other organic compounds were revealed. These acids point to emissions of VOC, both anthropogenic and biogenic. Since they were equally present in both seasons, a mixture of sources was responsible, both present in the wider area and throughout the year. Keywords carbon, ions, acellular assays, cascade impactors, aethalometer Introduction mixtures to which humans are usually exposed lead to adverse impacts on human (and similarly to animal) health that range Context and problem from acute to chronic effects. Epidemiological studies and Pollution is now recognised to be a huge environmental threat, animal model data state that primarily affected systems are the with air pollution being a leading cause of premature human cardiovascular and the respiratory system (Kampa and Castanas, death (Landrigan et al., 2018). The different air pollutants, their 2007). In terms of the health effects of the particulate matter doses and time of exposure and in particular the pollutant (PM), there has been substantial progress in the evaluation of CLEAN AIR JOURNAL 1 © 2019. The Author(s). Published under a Volume 29, No 2, 2019 Creative Commons Attribution Licence. Research article: Aerosol characterisation including oxidative potential as a proxy of health impact Page 2 of 15 PM health effects at different time-scales of exposure and in the is produced by coal-fired power stations. A substantial fraction exploration of the concentration-response function (Pope and of liquid fuel is also distilled from coal as well as from natural Dockery, 2006). gas. The main axes of South Africa’s economy remain the large mining and associated metallurgical industries (e.g. Beukes Inhalation toxicology research has demonstrated that the et al., 2010). Domestic biomass and fossil fuel combustion for generation of reactive oxygen species (ROS) and the associated space heating and cooking is also widely practised, especially induction of oxidative stress in target cells may represent a key in informal settlements that are found around most towns and pathway by which ambient fine and ultrafine (sub-micrometre) cities (e.g. Kimemia et al., 2010; Vakkari et al., 2013; Butt et al., size particles cause adverse health effects. Particle surface 2016; Makonese et al., 2016). reactivity, metals and redox cycling organic compounds are properties shown to be involved in ROS generation. All of the afore-mentioned has led to increased environmental concerns with atmospheric pollution being a major worry. New Air quality (AQ) regulations in most countries rely on PM volumetric industrial installations in South Africa are being equipped with mass without taking into consideration PM composition. Since cleaner technologies. However, emissions of sulphur dioxide PM is a complex mixture including metallic, inorganic and organic (SO2), oxides of nitrogen (NOx), black carbon (BC) and carbon compounds, with differing proportions depending on the sources dioxide (CO2) are predicted to increase. of emissions and ambient atmospheric conditions, alternative metrics such as the oxidative potential (OP) can and have been These emissions, combined with a potential change in biomass proposed to predict PM toxicity (Boogaard et al., 2012). burning due to global warming, and the associated dryer climate in certain parts of southern Africa, can significantly influence the Oxidative properties of aerosol are considered to be important regional and global climate (Boko et al., 2007). Climate change attributes to explain many of the aerosol biological/health may also enhance migration to already densely populated urban effects (Borm et al., 2007). As such, aerosol OP constitutes a areas, and potentially increase environmental concerns. unifying factor explaining its pathological activity and has been considered as an additional metric to determine aerosol health Overall, the atmospheric PM in this region originates from a impact (Donaldson et al., 1996; Borm et al., 2007). Li et al. (2003) mixture of natural and anthropogenic emission sources (such indicated that the ultra-fine mode of ambient aerosols collected as industry, domestic burning, biomass burning, transport), from Los Angeles (USA) had a higher intrinsic oxidative capacity and is spatially and temporally variable (Tiitta et al., 2014). On than the fine and coarse aerosol modes. the southern African sub-continent, number concentrations and optical properties of sub-micrometre aerosol particles have To date, not many health impact studies have been undertaken in been investigated intensively during the SAFARI 1992 and 2000 Africa. The POLCA project (“POLlution des Capitales Africaines”) measurement campaigns (Swap et al., 2003; Ross et al., 2003; in western Africa was a pioneering study that addressed the Eck et al., 2003). Recently, more studies have been published physicochemical characterisation of aerosol pollution in two on the aerosol measurements regionally giving more insight west African capitals (Bamako, Mali and Dakar, Senegal). These into the sub-micrometre number-size distribution, formation of cities experience high population growth. They are impacted by secondary particles, and trends and loadings of non-refractive a variety of emission sources, dominated by traffic and domestic species (Hirsikko et al., 2012; Vakkari et al., 2013; Tiitta et al., burning, and strongly subjected to contrasted meteorological 2014; Sundström et al., 2015). The aerosol organic compounds conditions (Doumbia et al., 2012). were studied (Booyens et al., 2015), BC modelled results were compared with the regional BC (continuous) measurements (Kuik The sampling campaigns in these West African capitals were et al., 2015), and impacts of aerosols from residential activities performed during the dry season, and the influence of the Saharan were also modelled (Butt et al., 2016). dust events and biomass burning was recognised in addition to prevailing anthropological pollution sources, mainly traffic and This study focused on characterising aerosol pollution impacting domestic burning (Assamoi and Liousse, 2010). Val et al. (2013) a residential suburb amid a major South African industrialised demonstrated that the finest size fractions of these aerosols area, i.e. the Vaal Triangle. In addition to the carbonaceous induced a pro-inflammatory response in human bronchial cells, and ionic content, the oxidative potential of the aerosols was indicating that aerosol pollution in West African cities may have determined to provide a predictive indicator of their toxicity. a strong impact on its population health. These cities have high ambient aerosol concentrations originating from anthropogenic The particles from 0.25 to over 2.5 µm in aerodynamic diameters sources known to contribute to health effects (Doumbia et al., were collected through three cascade impactor devices running 2012; Liousse and Galy-Lacaux, 2010; Cassee et al., 2013). in parallel at the Vaal Park Primary School, in Sasolburg. They were sampled for 12 hours in each of three sampling days during South Africa has one of the largest economies in Africa and remains the last week of every month from March 2012 to October 2012 the largest industrialised regional energy producer. South Africa to encompass the three local weather seasons: entire autumn, is on a continuing trend of increasing fossil fuel consumption entire winter and early to mid-spring. The thermal analytical and demand for electricity. Most of the electricity in South Africa method was used to determine the black carbon (BC) and organic CLEAN AIR JOURNAL 2 © 2019. The Author(s). Published under a Volume 29, No 2, 2019 Creative Commons Attribution Licence. Research article: Aerosol characterisation including oxidative potential as a proxy of health impact Page 3 of 15 carbon (OC) content (Cachier et al., 1989), whereas (water soluble) ionic compounds were determined by ion chromatography and inductively coupled-plasma-mass spectroscopy. Three different acellular assays were applied to characterise the oxidative potential. A determination of trace metals content was out of the scope for this study. Site selection The study area is colloquially called the Vaal Triangle, after the three towns forming each of its three spatial angles (Sasolburg, Vanderbijlpark and Vereeniging). The Vaal Triangle area is where a large part of the South African petrochemical and other chemical industries is located. In addition, other large point sources, including a coal-fired power station (without SOx and NOx scrubbing) and several large (iron) metallurgical smelters, are also within this area. This area, together with the southern Figure 1: Location of the Vaalpark Primary School sampling site within the section of Gauteng was proclaimed a national air pollution Vaal Triangle area (Source: Google Earth with own markings). area for intervention in terms of the South African National Environmental Management: Air Quality Act (Government Gazette Republic of South Africa, 2007) termed the “Vaal Triangle Priority Area” (constituting effectively more intensely monitored air-shed air quality (AQ) management area). The Vaal Triangle is also home to a number of formal (towns and townships) and informal settlements (slums), which mainly use coal, paraffin and wood as fuel sources. This, in turn, impacts directly on the health and well- being of the people residing in these communities. Other sources of concern contributing to the pollution emission mixtures within the area include transport-related emissions, biomass burning (domestic/anthropogenic and wild/natural), water treatment works and landfill areas, agricultural activities and various other fugitive sources. This is a strong indication that air quality in this area often exceeds or is close to exceeding National Ambient Air Quality Standards for criteria pollutants. The study sampling site was set at the grounds of a primary Figure 2: The wind speeds and directions near the site over the sampling school in the target area (Vaalpark Primary School). The sampling periods (source: SASOL wind data). site’s latitude is 26˚46’ 2” south, and the longitude is 27˚51’ 12” east (Figure 1). The sampling site was chosen for its proximity to precipitation cycle strongly affects pollutant concentrations, the Sasolburg industrial complex. This was done to determine i.e. enhanced wet scavenging (wet deposition/wash-out) of the level of aerosol pollution possibly received by the population pollutants during the wet season, while pollution levels are working, visiting and residing in close proximity to the major increased during the dry season through the occurrence of large- petrochemical complex (Sasol Industries) and metallurgical scale biomass burning and wind-blown dust emissions. industries (in Vanderbijlpark and Vereeniging municipalities) (Figure 1). An important attribute of the regional climate is protracted periods of cold mainly night temperatures with often sub-zero Regional weather and climate temperatures, which contribute to lower relative humidity in the Geo-morphologically, the sampling site is located on the South ambient air as well as drying moisture content from vegetation. African Highveld (an elevated plateau of 1400 m a.s.l. to 1700 m The lower temperatures also coincide with increased use of a.s.l.). Laakso et al. (2012) recently gave a concise description coal, paraffin and wood as fuels in low-income residential areas of the weather conditions over the South African Highveld. The (formal and informal). This contributes to the overall ambient description in this paper is, therefore, limited. pollution levels and subsequently to the increase in aerosol concentrations. During the winter and early spring months, Over the South African Highveld, literally all precipitation falls multiple inversion layers form regularly at various altitudes. during the wet season (from mid-October to end of April), with Such layered atmospheric structure significantly reduces almost no precipitation during the dry season (May to mid- vertical mixing, impacting significantly on the residence time October). The exact duration of the dry and wet seasons can differ of atmospheric pollutant concentrations. The region is also between years, especially if the onset of rains is late. This distinct dominated by anti-cyclonic circulation, especially during the CLEAN AIR JOURNAL 3 © 2019. The Author(s). Published under a Volume 29, No 2, 2019 Creative Commons Attribution Licence. Research article: Aerosol characterisation including oxidative potential as a proxy of health impact Page 4 of 15 winter months (June-Aug). Such re-circulation can trap pollutants African Weather Service (SAWS) precipitation information for a number of weeks (Garstang et al., 1996; Tyson and Preston- because the measurements at the Vaalpark site did not Whyte, 2000; Freiman and Piketh, 2002, Mafusire et al., 2016). include any rain gauge iii. a nearby Sasol company AQ monitoring station (Vaal Eco- The more frequent and highest wind speeds are observed Park) acquisition of the wind speed and wind directions during the end of the dry season (August, September and part of data. October), towards the beginning of the wet season (mid- to end- October) (Figure 2). From this, a decision was taken to keep the April filter samples within the wet season dataset, while the May data/filters were During the wet season, besides higher precipitation, higher allocated to the dry season. September’s filters were included temperatures and relative humidity with frontal and convective in the dry season set, while October’s filters were included in air mass movements enable horizontal and vertical mixing of the the wet season. There were several days with rain at the end of atmosphere over the area, and across the entire region, allowing October (when our aerosol samplings were taken). Although better dispersion and dilution of pollutants. October could fit in either season, the data were kept in the “wet season” data grouping. Methods Analytical methods Carbonaceous content analysis by a thermal method Aerosol filter (impactor) sampling Black carbon (BC) and total carbon (TC) contents were The filter collection of aerosol was performed by means of three determined from quartz filters with a thermal method developed cascade impactors (5-stage Sioutas-type impactor) at the flowrate by Cachier et al. (1989). Two similar aliquots of the same filter of 9 l.min-1, running in parallel for 12 h a day over three days (8am- were separately analysed. One portion was directly examined for 8pm), resulting in 36 h of sampling per month. Each cascade total carbon content (TC). The other portion was first submitted impactor (Sioutas) was mounted with four 25 mm diameter to a pre-combustion step (2 h at 340°C under pure oxygen) to filters, and one 37 mm filter (one Sioutas with polycarbonate eliminate OC and then analysed for BC content. Organic carbon nuclepore filters with 1 µm porosity). A second (Sioutas) impactor, (OC) concentrations were calculated as the difference between equipped with quartz filters (QMA, Whatman), was dedicated to TC and BC. carbonaceous aerosol measurements (for black carbon – BC and organic carbon – OC determination). A third cascade impactor ran Major ions (ion chromatographer) analysis with Teflon filters (Zefluor, Pall Corporation®) for water-soluble This method was used to determine water-soluble compounds. ions and trace element analysis. Aerodynamic particle diameters Teflon filters are weighted on a METTLER MC21S electronic collated by the cascade impactors were in the following cut-off microbalance with a sensitivity of 1 µg and measure the precision size ranges (marked as M5 - M1 in Table 1): <0.25 µm, 0.25-0.50 of ± 5 µg, to determine the aerosol mass loadings before analysis. µm, 0.50-1 µm, 1-2.5 µm, and ≥2.5 µm. For the data analysis, the In order to determine the water-soluble components (WSOC), half results were presented in three particle size fractions, which were of the samples are extracted in 15 ml of Milli-Q water (resistivity combined out of five sizes: ultrafine particle, UF [<0.25 µm], fine, ≈ 18.2 MΩ) by ultrasonic stirring for 15 minutes. Three ion F [>0.25<1 µm], and coarse C [>1µm and >2.5 µm]. For oxidative chromatographs, DX-100, ICS 1000 and DX 500 of Dionex were potential measurements, particles from the same season were equipped respectively with an anion exchange column IonPac pooled. AS40, a cation exchange column IonPac AS50 and a carbonate exchange column IonPac AS50, which are used for separating the Aethalometer (BC) measurement ions. A combination of 1.8 mM Na2CO3 and 1.7 mM NaHCO3 is used Black carbon (BC) equivalent concentrations were also measured as the anion eluent, while a 20 mM solution of methane sulphonic by using an optical attenuation method applied in a seven wave- acid (CH3SO2OH) is used as the cation eluent, and 100 % water length aethalometer (Magee Scientific®, model AE-42) ranging de-ionised is used for eluting carbonate ion. WSOCs obtained from 370 to 950 nm wavelengths. Just the periods towards the were Cl-, SO 2-, NO -, CO 2-4 3 3 , K +, Na+, NH + ²+ ²+4 , Mg and Ca expressed end of June 2012 and at the beginning of July 2012 were excluded in ppb. The limits of detection were less than 10 ppb for anions due to temporary instrument stoppage, which left some periods and cations. with data gaps. Aerosol oxidative potential (OP) analysis Meteorological data The three size fractions presented above were reconstituted from An essential task in this study was to correctly allocate collated sampled nuclepore membrane filters. Recovery of the particles data into two main weather seasons with similar prevailing was achieved as previously described by Ramgolam et al. (2009). climatic conditions, i.e. wet and dry season, as to better categorise Briefly, polycarbonate membranes were sonicated (Ultrasonic the entire dataset. This, we based on the following: Processor, Bioblock Scientific), 3 × 5 sec at 60 Watt in 15mM i. prevailing climatic patterns (Tyson and Preston-Whyte, HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-sulfonic acid) 2000), (Life Technologies, Gibco®). Blank filters were prepared in the ii. the NOAA’s ARL data for the sampling week and the South same way and were used as a control in the experiments. Particle CLEAN AIR JOURNAL 4 © 2019. The Author(s). Published under a Volume 29, No 2, 2019 Creative Commons Attribution Licence. Research article: Aerosol characterisation including oxidative potential as a proxy of health impact Page 5 of 15 suspensions were stored at -20°C (to prevent degradation and bacterial development) until use, and were again sonicated (3 × 10 sec) just before dilution in the culture media for acellular assays. Three different acellular assays were performed to characterise the OP: • the scission of plasmid DNA; • the consumption of dithiothreitol (DTT), a reducing agent, and • the depletion of antioxidants (AO): ascorbic acid (AA), uric acid (UA) and glutathione (GSH) in synthetic lung surfactant, according to the methods specified in Crobeddu et al. (2017). Figure 3: Vaalpark aerosol concentrations (µg.m-3) over the wet season Plasmid assay sampling period for all main sizes (UF, F, C) in comparison with total It served as a determination of the ability of PM to induce DNA suspended particulates (TSP), mean ± standard error of measurement breaks. As such, it is a good indicator of PM ability to generate free (SEM). radicals reactive enough to break DNA. The DNA used was a plasmid (φX174) of 5386 bp. It is super- helicoidal except where there is a simple strand DNA break leading to a relaxed form. These forms can be separated by gel electrophoresis and quantified to evaluate their respective proportion. Plasmid (290 ng) was incubated with 100 µg/ml particles in the presence of hydrogen peroxide (0.4 mM) in 15 mM Hepes buffer for 4 hours at 37°C under gentle agitation (Rotolab 3D). After incubation, samples were electrophoresed on a 1 % agarose gel at 50 V for 5 hours in a 1X Tris-Borate-EDTA buffer (Euromedex). At the end of the migration, DNA bands were visualised using ethidium bromide (2 µg/ml) by soaking for 10 min. The Figure 4: Vaalpark aerosol concentrations (µg.m-3) over the dry season fluorescence is visualised on UV trans-illuminator and the sampling period for all main sizes (UF, F, C) in comparison with total suspended particulates (TSP), (mean ± SEM). intensity of the fluorescence of the super-helicoidal and relaxed forms was quantified using “Image J” software in order to express the results as a percentage (%) of relaxed form. The products were monitored spectrophotometrically at 243 nm (AA); 280 nm (UA) and 210 nm (GSH; GSSG) and quantified by Anti-oxidant depletion assay integration of the peak absorbance area. It served as a determination of the ability of PM to deplete anti-oxidants in a synthetic lung lining fluid. Three specific DTT (dithiothreitol) consumption assay anti-oxidants used were: ascorbic acid (AA), uric acid (UA) and The redox-active compounds catalyse the reduction of oxygen to glutathione (GSH). superoxide by DTT, which is oxidised to disulphide (Kumagai et al., 2002). PM of each size fraction at 25, 50 and 100 µg/ml were incubated for 4 h at 37°C with AA, UA and GSH, under agitation. At the end PM of each size-fraction at 25, 50 and 100 µg/ml was incubated of every incubation, each sample was centrifuged in 0.22 µm with 200 µM of DTT for 1 h at 37°C. After incubation, samples spins (Costar®, Fisher Scientific) (30 sec at 12 000 g) to eliminate were centrifuged for 15 min at 3500 g at 4°C to eliminate PM. 5 the nanoparticle. AA, UA, GSH and the oxidised GSH form (GSSG) mM of DTNB was added to the supernatant. The absorbance was were analysed by means of reversed-phase HPLC (Shimadzu analysed at 405 nm (micro-plate reader Multiskan-EX, Thermo HPLC system interfaced with the “LabSolution” software). Scientific). Samples were injected onto a C18 column (length 250 mm; internal diameter 4.6 mm; particle size 5 µm) at 40°C. The mobile For all the valid samples, the final results’ blank sample values phase composition for the gradient system was 25 mM sodium were taken into account at the time of calculation. phosphate monobasic; 0.5 mM octane sulphonic acid (pH 2.7) for mobile phase A; and 100 % acetonitrile for mobile phase B. The gradient programme was 0-5min 0 % B; 5-10 min 0-12 % B; the initial conditions (100 % A) were then maintained for 10 min. CLEAN AIR JOURNAL 5 © 2019. The Author(s). Published under a Volume 29, No 2, 2019 Creative Commons Attribution Licence. Research article: Aerosol characterisation including oxidative potential as a proxy of health impact Page 6 of 15 Figure 6: Concentrations over three main sizes over the entire sampling period (mean ± SEM). Considering the concentrations averaged over the main sizes across the two main seasons, the following trend is evident: overall, the coarse fraction was recorded highest and Ultra-Fine 2nd highest concentrations over both seasons (Figure 6). The fine fractions were lower in both seasons. The concentrations of BC, OC and TC for the three size-fractions and across the two main seasons were determined. Whatever the season, the highest carbonaceous concentrations were observed in the UF fraction, followed in C fraction and then in F fraction (Table 1). Both the organic carbon (OC) and black carbon (BC) Figure 5: Back trajectories (HYSPLIT 6-hourly, arriving heights at 200 m) for the June (upper figure) and July (lower figure) sampling periods (after ARL, had higher concentrations in the dry season compared to the wet GDAC, NOAA). season for all fractions. Results Table 1: Carbonaceous content mean concentrations; BC, OC and TC in the wet season Aerosol filter concentrations, its Wet M5 M3+ M4 M1+M2 carbonaceous ratios and seasonal season Total (µg.m-3) UF F C carbonaceous trends Considering the distribution of the aerosol filter concentrations BC 0.210 0.105 0.230 0.545 over each main season, it was evident that the concentrations OC 0.352 0.056 0.183 0.591 were comparable between the months of the wet season (Figure 3). TC 0.562 0.161 0.413 Dry M5 M3+ M4 M1+M2 During two dry season months (June and July), concentrations season Total carbonaceous decreased ‘unseasonably’, which constituted the anomaly for this (µg.m-3) UF F C region (dry and cold temperature, low ambient relative humidity, BC 0.350 0.158 0.236 0.745 increased residential burning as well as the start of occasional wildfires/biomass burning) (Figure 4). OC 0.546 0.111 0.433 1.09 TC 0.897 0.269 0.668 For these two anomalous months (June and July), air mass movement and origin were investigated. The wind speeds during In general, the concentrations of OC were higher than those of BC, these sampling periods increased and the back trajectory analysis whatever the season, except for the fine fraction. for the sampling period indicated that the air masses were overpassing from the S-SW (June) and NW-W (July) directions The following ratios, i.e. OC/BC, BC/TPM (total particulate matter) (Figure 5) (ARL, GDAC, NOAA, 2013). The prevailing air mass and TC/TPM concentration ratios were considered for each originated from cleaner and essentially background regions. season and were higher in the dry season (except for the UF OC/ This influenced the aerosol concentrations at the sampling site. BC ratio). The highest ratios of OC/BC, BC/TPM and TC/TPM were Therefore, these questionable concentrations could be attributed found for the ultra-fine fraction (UF), followed by the coarse (C) to the intrusions of cleaner air masses that changed the prevailing and fine (F) fraction during the wet season (Table 2). For the dry ambient air concentrations (Figure 5). season, the OC/BC ratio was the highest. The lowest ratios were found for each BC/TPM and TC/TPM in the wet season (Table 2). CLEAN AIR JOURNAL 6 © 2019. The Author(s). Published under a Volume 29, No 2, 2019 Creative Commons Attribution Licence. Research article: Aerosol characterisation including oxidative potential as a proxy of health impact Page 7 of 15 Table 2: Main carbonaceous ratios over three major mean size fractions Inter-comparison of aethalometer BC with filter determined BC for the wet and dry season content Wet season UF F C The comparison of BC results from filter analysis and ratios aethalometer data obtained for the same days is shown in Figure OC/BC 1.678 0.526 0.795 8. Both sets of data followed a similar trend during the entire sampling period, gradually increasing in concentrations from BC/TPM 0.008 0.004 0.008 the late spring towards winter and higher levels over winter as TC/TPM 0.021 0.006 0.015 expected. Although there were substantial differences between Dry season their absolute values, it needs to be taken into account that the ratios UF F C filter sampling ran 12 h a day while the aethalometer measured 24 h. As elaborated earlier, the June and July ‘dip’ in the trend OC/BC ratios 1.559 0.701 1.836 level is explained by the cleaner air mass overpasses (HYSPLIT BC/TPM 0.013 0.006 0.009 modelled back trajectories, ARL, NCAR, NOAA, 2013). This is also TC/TPM 0.034 0.01 0.025 evident when both datasets for the same period were plotted alongside one another (Figure 8). Aethalometer measured BC trends Since this instrument ran continuously, the diurnal trend for the equivalent BC was determined using the mean BC results obtained from the absorptions near infrared (880 nm) wavelength (Hansen et al., 1984). The results are presented as two main South African Highveld seasons (Figure 7). Figure 8: Comparison of filter sampling BC with aethalometer BC trends over the same three days in each sampled month. Note that the aethalometer values were adjusted 30% lower to account for scattering and other reasons as per Hansen et al. (1984). Impactor concentrations were unchanged (mean ± SEM). Figure 7: Diurnal aethalometer BC concentration trends. Wet season sampling (upper graph) and dry season sampling period (lower graph) (mean ± one STD). The observed bimodal diurnal distribution is typical of the BC pollution reported (and PM2.5 and PM10 trends) for South Africa’s low-cost residential settlements over the SA Highveld region (Hersey et al., 2015; Venter et al., 2012). However, the morning peaks of the Vaalpark aethalometer BC data appear higher than the evening peaks for both seasons. As expected, the dry period Figure 9: Ionic concentrations over the wet season sampling periods had higher concentrations overall. However, the evening peak (mean ± SEM). was lower than the morning peak. The mid-day to mid-afternoon diurnal concentration levels were low. Should there be much Ionic aerosol content industrial pollution reaching/impacting the sampling site, these The content of important non-carbonaceous compounds was concentrations would be higher after the break of nocturnal and also determined. To a certain extent, the ionic concentrations development of convective boundary layer, ~3-4h after sunrise reflect similar trends to the carbonaceous measurements. (Korhonen et al., 2014; Gierens et al., 2018) We considered the ionic concentrations over the two main sampling season periods and also per species (Figure 9). CLEAN AIR JOURNAL © 2019. The Author(s). Published under a Volume 29, No 2, 2019 7 Creative Commons Attribution Licence. Research article: Aerosol characterisation including oxidative potential as a proxy of health impact Page 8 of 15 Considering the dry season months, the concentrations in the May, August and September sampling periods followed the well- known trends of increased concentrations of S and N compounds. However, two mid-dry season sampling months (June and July) were at much lower levels (Figure 10). Figure 12: Aerosol composition concentrations’ relative fraction in the dry season (%). (7 %) and nitrate (14 %) increased. This points to an increased influence of the biomass and residential fossil fuel burning and Figure 10: Ionic composition concentrations over the dry season sampling not necessarily a decrease in industrial emissions (Figure 12). periods (mean ± SEM). The same reason for the lower concentration levels was It is also essential to look at the ratios between the major ions and applicable, as in case of carbonaceous content concentrations: the BC and OC with regard to the total particulate matter (TPM). the sampling site weather played a crucial role – having been influenced by the cleaner air masses arriving from the regional The higher sulphate ratios within TPM and higher sulphate background. This background has neither much industrial and over BC ratios indicate the prevalence of industrial combustion residential burning, nor wildfires. Similarly, the emissions from emissions, while increased BC/TPM ratios indicate the prevalence traffic in the regional background are also limited. of domestic and biomass burning combustion emissions. The lack of higher sulphate ratios in the dry season can be explained Relative contributions of aerosol components by the fact that most of the production (petrochemical and Considering the wet season, it is evident that the sulphate, metallurgical industries) stacks are designed to be high, over ammonium and nitrate were dominant species (Figure 11). ~200 m (Beukes et al., 2012, Held et al., 1996), so the emissions are Black carbon and organic carbon appear almost equally (~3 released aloft the inversion capping layer in the colder and drier %). However, the majority of the aerosol content was sulphate, part of the year. The reduced moisture content also impedes the and this fact points to the prevailing anthropogenic origin of the formation of sulphate (and nitrate), leaving the residential and aerosol sampled at the sampling site. Together with nitrate and traffic pollution content higher, likely emanating from within, as ammonium, these compounds represent more than two-thirds of well as farther from the concerned area (Figure 13 a-d). the filter-sampled aerosol content (Figure 11). Figure 11: Aerosol composition concentrations’ relative fraction in the wet Figure 13: Ratios between aerosol major compounds over the main season (%). sampling periods: a) OC/BC, b) sulphate/BC, c) BC/TPM d) sulphate/TPM. The dry season had just slightly different aerosol composition The linear regression as the coefficient of correlation (Pearson), concentrations. The fractions of sulphate (45 %) and ammonium between each of ions, was calculated, and the output (R) has (14 %) were lower, while the relative fractions of BC (5 %), OC been indicated as a matrix. Certain high correlations (in dark red) CLEAN AIR JOURNAL © 2019. The Author(s). Published under a Volume 29, No 2, 2019 8 Creative Commons Attribution Licence. Research article: Aerosol characterisation including oxidative potential as a proxy of health impact Page 9 of 15 Another multiple-linear regression with RMSE was modelled for all the main ions taking OC as the dependent variable while others were left as an independent. This MLR output indicated that the acetic ions and BC are negatively associated, while Na+, Ca2+ and formic ions are positively associated with OC. Modelled showed that to accurately predict OC (i.e. the change in RMSE reaches a minimum), Na+, Ca2+, Acetic, Formic and BC were the most important (Figure 16). Aerosol oxidative potential Figure 14: Linear regression correlation (R values) matrix among all ionic species and carbonaceous fractions (OC and BC). Plasmid assay results To induce DNA breaks, the production of free radicals, such as and some low to anti-correlations (in yellow, green and blue) hydroxyl radical (OH•), is needed. Adding H2O2 (reactive oxygen have been revealed (Figure 14). species) to the incubation mixture allows the Fenton reaction to take place, if there is the presence of transition metals in the The correlation calculations did not indicate any unexpected aerosol. All samples increased the production of DNA damages association. To gain more insight into the species correlations, as measured by the relaxation of the plasmid DNA by comparison a multiple-linear regression (MLR) with root mean square error with the control (T) and H2O2 (reactive oxygen species) alone, (RMSE) output was modelled using Matlab® for all the main ions, suggesting the presence of transition metals. The effects of taking BC as the dependent variable, while others were left as an aerosol from the wet season were increasing as the PM size was independent. decreasing. For the dry season, the F fraction is the most reactive, whereas C and UF exhibited a similar effect (Figure 17). The model output (MLR) also indicated that the Mg2+, Cl- and oxalic were negatively associated and NH +4 and formic positively with BC. The model showed that to accurately predict BC (i.e. the change in RMSE reaches a minimum), NH +4 , Mg 2+, Formic, Cl- and oxalic were the most important and the OC was less significant (Figure 15). Figure 15: Ionic MLR modelled, where BC was dependant variable and all other ions independent. Figure 17: Intrinsic oxidative capacity characterised by plasmid scission assay (%) of relaxed plasmid circular form relative to super-helicoidal form, for the coarse (C), fine (F) and ultra-fine (UF) size fractions of wet (W) and dry (D) season used at 100 µg/mL: n=2, T=control (mean ± SEM).. Anti-oxidant depletion assay results All samples induced an ascorbic acid (AA) and glutathione (GSH) depletion (Figure 18 top and middle) with concomitant production of oxidised glutathione (GSSG) (Figure 18 bottom). There was no depletion of uric acid (UA) for either season or different dilutions (therefore not shown). For wet season samples, there is a trend of more AA and GSH depletion as the aerosol fraction size is Figure 16: Ionic MLR modelled in the instance where OC was dependent decreasing. For the dry season aerosols, we observed a stronger variable and all other ions independent. effect of the fine fraction compared to the other size fractions. The coarse fraction depleted AA and GSH only at the highest concentrations tested. CLEAN AIR JOURNAL 9 © 2019. The Author(s). Published under a Volume 29, No 2, 2019 Creative Commons Attribution Licence. Research article: Aerosol characterisation including oxidative potential as a proxy of health impact Page 10 of 15 and anti-oxidant depletion assays, we observed that as the size is decreasing, the DTT depletion was more pronounced and significantly different for the ultrafine fraction compared to the fine and coarse fractions. The DTT depletion was generally more pronounced with PM from the wet season considering the same size fraction. For the dry season, the higher effect was observed for the fine fraction and is significant at the highest concentration, whereas the other size fractions exhibited similar oxidative potential (OP) (Figure 19). Figure 19: Intrinsic oxidative capacity characterized by DTT depletion for the coarse (C), fine (F) and ultra-fine (UF) size fractions of wet and dry season at different concentrations (µg/ml). n=3 independent experiments with three replicates, different from wet samples of the same size and dose, different from other sizes whatever the season, different from other fractions of the same season (values are mean ± SEM). The three OP assays consistently showed that for aerosols sampled in the wet season, the OP tends to increase as the size is decreasing. By contrast, for the dry season, the size-fraction exhibiting the highest OP is the fine mode. The UF fraction showed the most contrasting OP between the two seasons. Discussion To a certain extent, the gravimetric mass of the filter samples was low, and we attribute this to the type of the environment where the sampling was taken, i.e. an affluent, fully electrified residential neighbourhood, as well as to the prevailing weather patterns effectively upwind from the nearby pollution sources. However, the aerosols still contained representative mixtures of chemical compounds, such as sulphate, nitrate and ammonium, strongly indicating the prevalence of the anthropogenic sources. The carbonaceous content of both organic and black carbon was not high, and they are mostly similar in content in both seasons, although an evident higher organic content in the dry season indicates the typical influence of the biomass burning season, Figure 18: Intrinsic oxidative capacity characterised by the anti-oxidant nd assay for the coarse (C), fine (F) and ultra-fine (UF) size fractions of wet which is prevalent in the 2 part of the dry season (from mid- and dry season at different concentrations. Ascorbic acid depletion (top winter to mid-spring). graph); reduced glutathione (GSH) depletion (middle graph); and oxidised glutathione (GSSG) production in a synthetic lung lining fluid (bottom graph), n=2 independent experiments with three replicates, values are The diurnal pattern seen from the continuous data indicates mean ± SEM. bimodal peaks, with the morning peak being higher than the evening peak. The plausible explanation is that the traffic into and DTT consumption assay results around the area and some residential fossil fuel burning increase All samples induced a significant dose-dependent DTT depletion concentrations in the morning. While the evening peak is not as (Figure 19). For wet season samples, as seen for plasmid assay pronounced, it is indicative of reduced emissions in comparison CLEAN AIR JOURNAL 10 © 2019. The Author(s). Published under a Volume 29, No 2, 2019 Creative Commons Attribution Licence. Research article: Aerosol characterisation including oxidative potential as a proxy of health impact Page 11 of 15 to the morning peaks (reduced traffic and reduced residential high correlations (R2=0.87 in dry and R2=0.99 in wet season). This burning). This is understandable for the sampling site, which is in indicates that the oxalic acid would be strongly correlated to fire a residential electrified area and although a number of electrified origin, both domestic uses as well as biomass burning seasonal households in SA use fossil fuels for heating, this activity is less fires. pronounced than in low-income settlements (Hersey et al., 2015; Venter et al., 2012). The US Department of Health and Human Services (1992) indicated that oxalic acid aids a range of detrimental pulmonary A question was posed why the concentrations of major ionic impacts. Acetic acid is a strong eye, skin and mucous membrane compounds (being characteristic of industrial combustion irritant. Any prolonged skin contact with glacial acetic acid may emissions) in March and October were similar compared with result in tissue destruction. the months in the dry season (May, September). We attribute this to the regular existence of a more turbulent boundary layer Although formic acid shares most of the chemical properties (with higher solar radiation and fewer inversions and horizontal of other carboxylic acids, it has low toxicity. However, the stratification) allowing entrainment/fumigation of the pollution concentrated formic acid is corrosive to the skin (Reutemann and from the industrial sources nearby. The months of March and Kieczka, 2002). Such characteristics hint that the carboxylic acids October fit this description, although April concentrations do play a role in OP of collated aerosol. were reduced, and the April period meteorology had played a determining role. Interestingly, to the best of our knowledge, this study was the first to compare the OP of PM according to the size fraction in South It is important to point to the presence of some carboxylic acids Africa. It revealed that the fine fraction exhibited a constant OP (oxalic, formic, acetic) found among particulate organic matter. whatever the season. By contrast, the ultrafine fraction exhibited However, similar relative content of these carboxylic acids in both the highest OP in the wet season compared to the other size seasons indicates that there are a number of sources/precursors fraction, whereas it was equal to the coarse fraction in the dry of these species during both seasons. Since the main precursors to season. These data suggest the role of the particle composition carboxylic acids are VOC (from both anthropogenic and biogenic on the ultrafine OP that would warrant deeper chemical sources), their presence throughout the year is expected. The characterisation. We can speculate that metals are involved degradation of anthropogenic hydrocarbons photo-chemically, in OP, since the DNA scission can only occur through hydroxyl as well as oxidation of isoprene and monoterpene emissions, radical production that is known to be mediated by transition leads to the formation of such acids (Herrmann et al., 2005; Lim metals (Crobeddu et al. 2017). et al., 2005). Among the identified carboxylic acids, oxalic acid is the most abundant compound in both seasons. Oxalic acid is The acidity of the particles due to the significant content in oxalic frequently observed as one of the most abundant single organic and formic acid could favour the bioavailability of metals. Size- compounds in tropospheric particles. Sources of oxalic acid in wise, UF and F fractions of aerosol in both seasons are capable of the atmosphere are numerous. Biomass burning (Yamasoe et al., evident OP when exposed to in-vitro acellular human cell proxies. 2000) and vehicular exhaust (Kawamura and Kaplan, 1987) are two primary emission sources for oxalic acid. Conclusions Aerosol OP was characterised on particle size fractions collected Atmospheric oxidation processes provide secondary mechanisms in a heavily industrialised area at a residential suburb through for oxalic acid formation from both anthropogenic and biogenic a set of three different assays. By detecting ROS generated by volatile organic compound emissions (e.g. Kerminen et al., 2000; different particle characteristics, OP can be seen as an integrative Warneck, 2003). indicator of aerosol exposure enabling the prediction of the aerosol effects on human/animal health within the South African In the studied environment, there are both types of sources context. In this study, in the wet season, the fine (F) and ultra- present throughout the year as well as seasonally. While the fine (UF) fractions were proven to exhibit stronger OP compared anthropogenic emissions from industries are more or less to the coarse (C) fraction. In addition, the UF fraction had higher constant, plus emission transport from out of the area, the OP in the wet season compared to the dry season when elevated domestic biomass burning is not specific to the case study area, pollution levels are usually recorded on the SA Highveld due although there are a number of low-income dwellings in its to its climate and land topography. The investigated air mass vicinity that rely on biomass burning for cooking and heating. movement point out that cleaner (background) air mass influx In addition, in the winter months, intentional and spontaneous dominated large periods of the sampling campaign, and that the biomass burning in the form of veld (grassland and savannah) sampling site is upwind of major pollution sources in the area, fires is a substantial source of pollutant emissions. such as industrial, and low-cost and slum dwellings. However, should more particulates have been sampled in the dry season, A significant correlation (coefficient of determination) was this could have exerted different effects. Nevertheless, the found between potassium (K) as a well-known fire tracer (Gao chemical analysis confirmed the predominance of anthropogenic et al., 2003; Andreae et al., 1998) with oxalic acid. Correlations compounds present in collated aerosol – sulphate, nitrate and between potassium (K) and oxalic acid per seasons resulted in ammonium. CLEAN AIR JOURNAL 11 © 2019. The Author(s). Published under a Volume 29, No 2, 2019 Creative Commons Attribution Licence. Research article: Aerosol characterisation including oxidative potential as a proxy of health impact Page 12 of 15 The role of combustion from transport and residential burning is Boko, M. I., Niang, A., Nyong, C., Vogel, A., Githeko, M., Medany, also evident through the continuous BC sampling having typical B., Osman-Elasha, R., Tabo, Yanda, P. Africa, in. Climate Change bimodal peaks and a much stronger peak in inversion capped the 2007: impacts, adaptation and vulnerability. The contribution cold and dry season. Furthermore, inevitably, we could pick up of working group II to the fourth assessment report of the evidence of biomass burning, first in BC/OC content and ratios, as intergovernmental panel on climate change, edited by Parry, M. well as in the presence of the organic acids, which are generally L., Canziani, O. F., Palutikof, J. P., van der Linden, P. J., and Hanson, secondary pollutants resulting from oxidation of VOC. The organic C. E. Cambridge University Press, Cambridge UK, 433-467. acids emanate from both anthropogenic and biogenic sources such as biomass burning, transport emission as well as industrial Boogaard, H., Janssen, N.A.H., Fischer, P.H., Kos, G.P.A., Weijers, production and effluents such as carboxylic acids. E.P., Cassee, F.R., van der Zee, S.C., de Hartog, J.J., Brunekreef, B., Hoek, G., 2012. Contrasts in oxidative potential and other Overall, the sampled aerosols represent the majority of the particulate matter characteristics collected near major streets pollution sources being present well in the atmosphere of the and background locations. Environ Health Perspect. 120, 185- wider area and are not mainly and only generated by the industrial 191. https://doi.org/10.1289/ehp.1103667 emission despite being so spatially close to major chemical and metallurgical industries. Residential use of fossil and biomass Booyens, W., Van Zyl, P.G., Beukes, J.P., Ruiz-Jimenez, J., Kopperi, burning with seasonal veld fires contributes to emissions of M., Riekkola, M., Josipovic, M., Venter, A.D., Jaars, K., Laakso, L. solid particulate matter in the area. In terms of overall health 2015. Size-resolved characterisation of organic compounds in effect, by considering the OP assays, we showed significant atmospheric aerosols collected at Welgegund, South Africa. J oxidative properties for the finer sizes and in the more turbulent Atmos. Chem. 72, 43-64. https://doi.org/10.1007/s10874-015- atmosphere, although the main chemical contributors to OP 9304-6 could not be determined. Combined aerosol characterisations, including OP with experimental biological studies, with relevant Borm, P.J.A, Kelly, F., Künzli, N., Schins, R.P.F., Donaldson, K., target cells in vitro (and ex vivo), are necessary to decipher the link 2007. Oxidant generation by particulate matter: from biologically between chemical composition, OP and cellular oxidative stress effective dose to a promising, novel metric. Occup. Environ Med; within the context of South African aerosols. Such in vitro studies 64, 73-74. https://doi.org/10.1136/oem.2006.029090 would allow establishing the predictivity of OP as an exposure indicator to be used in epidemiological studies. Butt E. W., Rap, A., Schmidt, A., Scott, C. E., Pringle, K. J., Reddington, C. L., Richards, N. A. D., Woodhouse, M. T., Ramirez- Villegas, J., Yang, H. 2016. The impact of residential combustion Acknowledgements emissions on atmospheric aerosol, human health, and climate. We acknowledge the French GDRI for making possible the Atmos. Chem. Phys., 16, 873-905. https://doi.org/10.5194/acp-16- collaboration between the French and SA institutions as well as 873-2016 the NRF, its SA counterpart, in this collaboration. We also kindly thank the SAWS for the provision of rainfall data and SASOL Ltd Cachier H, Brémond MP, Buat-Ménard P: 1989. Determination of for the provision of wind data. atmospheric soot carbon with a simple thermal method. Tellus B Chem. Phys. Meteorol., 41(B):379-390. https://doi.org/10.3402/ tellusb.v41i3.15095 References Air Resources Laboratory, Gridded Meteorological Data Archives, Cassee F.R., Héroux M.E., Gerlofs-Nijland M.E., Kelly F.J. 2013. 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