Swarup China

Bingbing Wang

Johannes Weis

Luciana Rizzo

Joel Brito

Glauber G Cirino

Libor Kovarik

Paulo Artaxo

Mary K Gilles

Alexander Laskin

Rupturing of Biological Spores As a Source of Secondary Particles in Amazonia

Airborne biological particles, such as fungal spores and pollen, are ubiquitous in the Earth's atmosphere and may influence the atmospheric environment and climate, impacting air quality, cloud formation, and the Earth's radiation budget. The atmospheric transformations of airborne biological spores at elevated relative humidity remain poorly understood and their climatic role is uncertain. Using an environmental scanning electron microscope (ESEM), we observed rupturing of Amazonian fungal spores and subsequent release of submicrometer size fragments after exposure to high humidity. We find that fungal fragments contain elements of inorganic salts (e.g., Na and Cl). They are hygroscopic in nature with a growth factor up to 2.3 at 96% relative humidity, thus they may potentially influence cloud formation. Due to their hygroscopic growth, light scattering cross sections of the fragments are enhanced by up to a factor of 10. Furthermore, rupturing of fungal spores at high humidity may explain the bursting events of new particle formation in Amazonia.

■ INTRODUCTION

Aerosolized biological particles significantly influence the biosphere, atmosphere, and public health. [START_REF] Despreś | Primary biological aerosol particles in the atmosphere: a review[END_REF][START_REF] Elbert | Contribution of fungi to primary biogenic aerosols in the atmosphere: wet and dry discharged spores, carbohydrates, and inorganic ions[END_REF][START_REF] Grote | Abortive pollen germination: a mechanism of allergen release in birch, alder, and hazel revealed by immunogold electron microscopy[END_REF][START_REF] Taylor | Release of allergens as respirable aerosols: a link between grass pollen and asthma[END_REF] Biological particles impact cloud dynamics and hydrological cycles by forming clouds and ice crystals. [START_REF] Despreś | Primary biological aerosol particles in the atmosphere: a review[END_REF][START_REF] Murray | The relevance of nanoscale biological fragments for ice nucleation in clouds[END_REF][START_REF] Poschl | Rainforest aerosols as biogenic nuclei of clouds and precipitation in the Amazon[END_REF][START_REF] Steiner | Pollen as atmospheric cloud condensation nuclei[END_REF] They influence the Earth's energy budget by scattering and absorbing solar radiation. [START_REF] Despreś | Primary biological aerosol particles in the atmosphere: a review[END_REF][START_REF] Guyon | Sources of optically active aerosol particles over the Amazon forest[END_REF] Primary biological particles, emitted directly from the biosphere, are pollen, fungal spores, bacteria, and fragments of plants and living organisms. High discrepancies exist in estimation of primary biological particle emissions, [START_REF] Despreś | Primary biological aerosol particles in the atmosphere: a review[END_REF][START_REF] Jaenicke | Abundance of Cellular Material and Proteins in the Atmosphere[END_REF]10 ranging between 56 and 1000 Tg yr -1 . Aircraft and balloon measurements suggest that they can be transported to high altitudes and over long distances. [START_REF] Elbert | Contribution of fungi to primary biogenic aerosols in the atmosphere: wet and dry discharged spores, carbohydrates, and inorganic ions[END_REF][START_REF] Prospero | Interhemispheric transport of viable fungi and bacteria from Africa to the Caribbean with soil dust[END_REF][START_REF] Wainwright | Microorganisms cultured from stratospheric air samples obtained at 41 km[END_REF][START_REF] Meier | Airplane collections of sugar-beet pollen[END_REF] The physical dimensions of atmospheric biological particles span several orders of magnitude with diameters ranging from nanometers (e.g., viruses) to hundreds of micrometers (e.g., pollen grains, plant debris). Fungal spores and their fragments are one of the most abundant classes of biological particles in various environments. [START_REF] Elbert | Contribution of fungi to primary biogenic aerosols in the atmosphere: wet and dry discharged spores, carbohydrates, and inorganic ions[END_REF][START_REF] Bauer | Significant contributions of fungal spores to the organic carbon and to the aerosol mass balance of the urban atmospheric aerosol[END_REF][START_REF] Bowers | Seasonal Variability in Bacterial and Fungal Diversity of the Near-Surface Atmosphere[END_REF][START_REF] Crawford | Temporal and spatial variation of indoor and outdoor airborne fungal spores, pollen, and (1→ 3)-β-d-glucan[END_REF] In tropical areas, such as the Amazon basin, primary biological particles contribute up to 80% of coarse mode (2-3 μm) particle mass concentration. [START_REF] Elbert | Contribution of fungi to primary biogenic aerosols in the atmosphere: wet and dry discharged spores, carbohydrates, and inorganic ions[END_REF][START_REF] Poschl | Rainforest aerosols as biogenic nuclei of clouds and precipitation in the Amazon[END_REF][START_REF] Huffman | Size distributions and temporal variations of biological aerosol particles in the Amazon rainforest characterized by microscopy and real-time UV-APS fluorescence techniques during AMAZE-08[END_REF] Even at a high altitude site in North America biological particles contribute an average of 40% of the particulate organic carbon mass. [START_REF] Wiedinmyer | The contribution of biological particles to observed particulate organic carbon at a remote high altitude site[END_REF] The global average loading and emission rates of fungal spores are ∼1 μg m -3 and ∼50 Tg yr -1 , respectively. [START_REF] Elbert | Contribution of fungi to primary biogenic aerosols in the atmosphere: wet and dry discharged spores, carbohydrates, and inorganic ions[END_REF] Fungi can actively discharge their spores via liquid jets into the air, known as actively wet discharged spores (e.g., Ascomycota and Basidiomycota). [START_REF] Elbert | Contribution of fungi to primary biogenic aerosols in the atmosphere: wet and dry discharged spores, carbohydrates, and inorganic ions[END_REF][START_REF] Pringle | The captured launch of a ballistospore[END_REF] Concentrations of these wet discharged spores tend to increase during humid conditions such as during the wet season in the Amazon basin. [START_REF] Elbert | Contribution of fungi to primary biogenic aerosols in the atmosphere: wet and dry discharged spores, carbohydrates, and inorganic ions[END_REF] Chemical compositions of primary biological particles are highly variable and remain insufficiently characterized even at a phenomenological level due to difficulties distinguishing between biological and other carbonaceous particles. [START_REF] Despreś | Characterization of primary biogenic aerosol particles in urban, rural, and high-alpine air by DNA sequence and restriction fragment analysis of ribosomal RNA genes[END_REF][START_REF] Martin | Sources and properties of Amazonian aerosol particles[END_REF][START_REF] Poschl | Atmospheric aerosols: composition, transformation, climate and health effects[END_REF] Therefore, it is currently assumed that actual contributions of primary biological particles to the total atmospheric aerosol are underestimated. [START_REF] Jaenicke | Omnipresence of biological material in the atmosphere[END_REF][START_REF] Hummel | Regional-scale simulations of fungal spore aerosols using an emission parameterization adapted to local measurements of fluorescent biological aerosol particles[END_REF] Biological materials are primarily carbonaceous and produced from metabolic activity of fungi and bacteria. [START_REF] Di Filippo | Fungal contribution to size-segregated aerosol measured through biomarkers[END_REF][START_REF] Nirmalkar | Arabitol and Mannitol as Tracer for Fungal Contribution to Size-Differentiated Particulate Matter of Rural Atmospheric Aerosols[END_REF] Sugar alcohols such as mannitol, arabitol, and ergosterol are commonly used as tracers for source apportionment measurements of primary biological particles, such as fungal spores. [START_REF] Di Filippo | Fungal contribution to size-segregated aerosol measured through biomarkers[END_REF][START_REF] Nirmalkar | Arabitol and Mannitol as Tracer for Fungal Contribution to Size-Differentiated Particulate Matter of Rural Atmospheric Aerosols[END_REF] For example, the average concentrations of mannitol were almost 2-3 times higher in the Amazon basin compared to extratropical locations. [START_REF] Elbert | Contribution of fungi to primary biogenic aerosols in the atmosphere: wet and dry discharged spores, carbohydrates, and inorganic ions[END_REF] Previous studies reported that coarse pollen grains (5-150 μm) rupture under high humidity and release cytoplasmic material ranging in size from several nanometers to several micrometers. [START_REF] Grote | Abortive pollen germination: a mechanism of allergen release in birch, alder, and hazel revealed by immunogold electron microscopy[END_REF][START_REF] Taylor | Release of allergens as respirable aerosols: a link between grass pollen and asthma[END_REF][START_REF] Steiner | Pollen as atmospheric cloud condensation nuclei[END_REF][START_REF] Taylor | Birch pollen rupture and the release of aerosols of respirable allergens[END_REF] However, these submicrometer or nanoparticles are difficult to detect by traditional bioaerosol sampling and analytical techniques due to their small size. [START_REF] Goŕny | Fungal fragments as indoor air biocontaminants[END_REF] Pollen grains are often covered with amorphous layers and Raman spectroscopy measurements show that the chemical composition of these layers significantly differs between species. [START_REF] Pummer | Chemistry and morphology of dried-up pollen suspension residues[END_REF] Many of the fragmented submicrometer particles (subpollen) are starch granules that contribute to atmospheric organic carbon. [START_REF] Steiner | Pollen as atmospheric cloud condensation nuclei[END_REF][START_REF] Pummer | Chemistry and morphology of dried-up pollen suspension residues[END_REF][START_REF] Laurence | Airborne starch granules as a potential contamination source at archaeological sites[END_REF][START_REF] Suphioglu | Mechanism of grass-pollen-induced asthma[END_REF] These subpollen particles can act as cloud condensation nuclei 7 and ice nuclei. [START_REF] O′sullivan | The relevance of nanoscale biological fragments for ice nucleation in clouds[END_REF][START_REF] Pummer | Suspendable macromolecules are responsible for ice nucleation activity of birch and conifer pollen[END_REF] In contrast, the environmental impact and cloud formation potential of expelled particles from relatively smaller sizes (1-6 μm) fungal spores has not been recognized.

Because of the smaller size of fungal spores compared to pollens, they have longer atmospheric lifetimes and can be lofted to the mid and upper troposphere. Their abundance depends on environmental factors such as annual season, rain, thunderstorms, wind and temperature. [START_REF] Despreś | Primary biological aerosol particles in the atmosphere: a review[END_REF][START_REF] Elbert | Contribution of fungi to primary biogenic aerosols in the atmosphere: wet and dry discharged spores, carbohydrates, and inorganic ions[END_REF][START_REF] Miguel | Meteorological influences on respirable fragment release from Chinese elm pollen[END_REF][START_REF] Visez | Wind-induced mechanical rupture of birch pollen: Potential implications for allergen dispersal[END_REF] For example, high ambient concentrations of biological particles are associated with rainfall events. [START_REF] Huffman | High concentrations of biological aerosol particles and ice nuclei during and after rain[END_REF] Aircraft measurements over central China showed higher concentrations of fungal spore tracers in spring than in summer. [START_REF] Fu | Aircraft measurements of polar organic tracer compounds in tropospheric particles (PM10) over central China[END_REF] They have relatively high number concentrations (∼10 4 m -3 ) in the continental boundary layer. [START_REF] Despreś | Characterization of primary biogenic aerosol particles in urban, rural, and high-alpine air by DNA sequence and restriction fragment analysis of ribosomal RNA genes[END_REF] Fungal spores and other primary biological particles contribute up to 80% of coarse mode particles in the Amazon basin and significantly impact hydrological cycle. [START_REF] Elbert | Contribution of fungi to primary biogenic aerosols in the atmosphere: wet and dry discharged spores, carbohydrates, and inorganic ions[END_REF][START_REF] Huffman | Size distributions and temporal variations of biological aerosol particles in the Amazon rainforest characterized by microscopy and real-time UV-APS fluorescence techniques during AMAZE-08[END_REF] Here, we show evidence of rupturing of fungal spores collected in the Amazon. The rupturing and release of submicrometer subfungal fragments are observed after exposure to water vapor and subsequent drying. We discuss the previously unexplored climatic implications of these submicrometer fragments and links to new particle burst observations in central Amazonia.

■ EXPERIMENTAL SECTION

Samples of atmospheric particles were collected during the wet season (January and February, 2015) at the ZF2 Tower (02°35.3517′ S, 60 06.8333′ W), a pristine rainforest site in Central Amazonia located 40 km North of Manaus margins. Samples were collected from both below (∼2 m above ground) and above the canopy (39 m above ground). The residence time of air parcel within the forest canopy is on the order of minutes but it can vary depending on the turbulence level. During nighttime turbulence level is low and that limit the transport of air parcel from above to within the canopy. However, during transition of nighttime to daytime, turbulence progressively increased and reaches the lower half of the canopy ∼1.5 h after sunrise. [START_REF] Fuentes | Linking meteorology, turbulence, and air chemistry in the Amazon rainforest[END_REF] Particles were collected onto 400 mesh transmission electron microscopy (TEM) grids coated with Carbon Type-B films (Ted Pella, Inc.) using 10-stage Micro-Orifice Uniform Deposition Impactors (MOUDI; model 110-R, MSP, Inc.). This study focuses on the samples from stage 4 (size range: 3.2-5.6 μm) where the relative abundance of biological particles is higher than on the other stages. An environmental scanning electron microscope (ESEM, Quanta 3D model, FEI, Inc.) with a Peltier cooling stage was used for water vapor exposure experiments. Fungal spores on the substrate were investigated before water vapor exposure experiments and only intact particles were monitored during experiments. We note that potential caveats of substrate based approach cannot be avoided such as possible damage and morphology modifications of particles upon impaction. A scanning transmission electron detector was used for the ESEM imaging. [START_REF] Laskin | Analysis of individual environmental particles using modern methods of electron microscopy and X-ray microanalysis[END_REF] ESEM experiments were conducted at 278-280 K and 0.08-6.50 Torr of water vapor, corresponding to sample exposures of 1-99% relative humidity (RH). Samples were systematically exposed to high RH (∼98%) for time periods of 0.5-10 h. Between exposures, samples were dried (to RH ∼ 1%) by decreasing the water vapor pressure (to 0.1 Torr) of the ESEM chamber for an interval of 1-2 h. In this study a total of eight samples were investigated. Samples from above and below canopy were collected simultaneously for same duration at the same location. Table S1 (Supporting Information (SI)) shows the sampling time, duration and duty cycle. After prolonged exposure to water vapor, fungal spores ruptured and released submicrometer fragments. We dried the sample inside the ESEM chamber after rupturing of fungal spores. Later we characterized the fragments in dry condition to determine their elemental compositions and morphologies. Furthermore, we conducted dynamic hydration experiments using ESEM to study the hygroscopic behavior and growth of fungal fragment particles. Hydration experiments were conducted up to 96% RH (6.32 Torr of water vapor). Computer-controlled scanning electron microscopy with energy-dispersive X-ray (CCSEM/ EDX) [START_REF] Laskin | Heterogeneous chemistry of individual mineral dust particles with nitric acid: A combined CCSEM/EDX, ESEM, and ICP-MS study[END_REF] was used to investigate the morphology and elemental composition of biological particles. TEM imaging and electron energy loss spectroscopy (EELS) measurements were performed in scanning mode using an aberration-corrected transmission electron microscope (FEI, Inc. model Titan 80-300) operated at 300 kV. Scanning transmission X-ray microscopy (STXM) [START_REF] Moffet | Fundamentals and Applications in Aerosol Spectroscopy[END_REF] was used to map specific elements (e.g., carbon, sodium) within individual biological particles prior to and post exposure to high relative humidity and subsequent drying.

Furthermore, we used size distribution data from a scanning mobility particle sizer (custom-built SMPS system, Lund University) collected at the ZF2 site during the wet season (Jan-Jun) in the years of 2008-2009, comprising measurements from 133 days [START_REF] Artaxo | Atmospheric aerosols in Amazonia and land use change: from natural biogenic to biomass burning conditions[END_REF] to investigate observed bursting events of nanoparticles in the Amazon basin. Particle size distribution measurements were performed 10 m above the canopy top. All measurements were performed under dry conditions (RH 30-40%), assured by an automatic diffusion dryer. [START_REF] Tuch | Design and performance of an automatic regenerating adsorption aerosol dryer for continuous operation at monitoring sites[END_REF] ■ RESULTS AND DISCUSSION Size Distribution and Chemical Composition of Fungal Spores. Typically, fungal spores are a few micrometers in size, often spherical, rod-like or spheroidal (prolate) in shape. [START_REF] Huffman | Size distributions and temporal variations of biological aerosol particles in the Amazon rainforest characterized by microscopy and real-time UV-APS fluorescence techniques during AMAZE-08[END_REF][START_REF] Gabey | Measurements and comparison of primary biological aerosol above and below a tropical forest canopy using a dual channel fluorescence spectrometer[END_REF][START_REF] Hussein | Species traits and inertial deposition of fungal spores[END_REF] Figure 1 shows representative morphologies of fungal spores found in the Amazonian samples. Spheroidal fungal spores are 3-7 μm long and 2-4 μm wide. Sizes (area equivalent diameters) of the fungal spore particles range from 1.1 to 5.9 μm with an average diameter of 2.8 μm. Previous studies found presence of several species of fungal spore in the Amazon rainforest canopy with different sizes. [START_REF] Womack | Characterization of active and total fungal communities in the atmosphere over the Amazon rainforest[END_REF][START_REF] Froḧlich-Nowoisky | Biogeography in the air: fungal diversity over land and oceans[END_REF] These studies applied culture-independent approaches to measure the composition of total and active atmospheric fungal spores over the Amazon forest canopy. An RNA-based approach was also applied to measure metabolically active microbial communities in the atmosphere. Phyla Ascomycota and Basidiomycota were most abundant in total airborne fungal communities with relative abundance of Basidiomycota over 90%. Within the Basidiomycota, Agaricomycetes were most abundant class. However, Ascomycota found to be major fraction (mean relative abundance∼ 80%) within the active community in the atmosphere over the Amazon forest canopy. Sordariomycetes (∼27%) and Lecanoromycetes (∼18%) were most abundant classes within the Ascomycota community. Overall, these studies found presence of potentially active fungi in the atmosphere, including lichen fungi (class Lecanonomycetes) and the following genera: Agaricus; Amanita; Aspergillus; Boletus; Cladonia; Lepsita; Mortierella; Puccinia; and Rhizopus. [START_REF] Womack | Characterization of active and total fungal communities in the atmosphere over the Amazon rainforest[END_REF][START_REF] Froḧlich-Nowoisky | Biogeography in the air: fungal diversity over land and oceans[END_REF] Elemental composition analysis by CCSEM/EDX shows that fungal spores are primarily composed of C and O. Other frequent elements included N, Na, P, K, S; some fungal spores also contain Cl and Mg. Similar elemental compositions of biological particles have been reported previously. [START_REF] Ebert | The chemical composition and complex refractive index of rural and urban influenced aerosols determined by individual particle analysis[END_REF] SI Figure S1 shows an example of EDX elemental maps of a typical fungal spore particle along with its corresponding EDX spectrum. The C map indicates the fungal spore contains significant carbonaceous content. Within this primary unprocessed particle, the elements are relatively homogeneously distributed, consistent with measurements of other fungal spores found in the samples. However, the fractions of different elements vary considerably among fungal spore particles (SI Figure S2).

In this study, fungal spores were identified based on their characteristic shapes and chemical composition, which includes a high carbonaceous component and the presence of other elements such as phosphorus. [START_REF] Huffman | Size distributions and temporal variations of biological aerosol particles in the Amazon rainforest characterized by microscopy and real-time UV-APS fluorescence techniques during AMAZE-08[END_REF][START_REF] Artaxo | Atmospheric aerosols in Amazonia and land use change: from natural biogenic to biomass burning conditions[END_REF][START_REF] Artaxo | Physical and chemical properties of aerosols in the wet and dry seasons in Rondonia, Amazonia[END_REF] Fungal spore particles contributed up to 56% and 17% of the total number of particles in the size range of 3.2-5.6 μm (aerodynamic diameter) below and above the canopy, respectively.

Rupture of Fungal Spores. Rupturing of fungal spores and subsequent expulsion of fungal fragments was observed after exposure to high RH conditions (∼98%) for ∼10 h and subsequent drying. Representative examples of fragmented and expelled fungal spores after wet and drying cycles are shown in Figure 2. Substantial rupturing was not observed in the samples exposed to high RH for shorter time periods that were subsequently dried. These results suggest that rupture occurred during the prolonged wet environment, likely repeated several times, and not due to surface tension forces during a single hydration/dehydration cycle. [START_REF] Steiner | Pollen as atmospheric cloud condensation nuclei[END_REF] Rupturing of fungal spores at high humidity may impact the culturability of the fungal spores. Hydrated fungal spores may contain significant amount of osmotically active solutions. At high humidity osmotic pressure can be developed across fungal walls and once it is high enough then spores can rupture. [START_REF] Grote | Release of allergen-bearing cytoplasm from hydrated pollen: a mechanism common to variety of grass (Poaceae) species revealed by electron microscopy[END_REF] Figure 3 shows the size (area equivalent diameter) distributions of original fungal spores and expelled fungal fragments after rupture. Here, only individual particles that did not agglomerate during the drying process were used for the analysis. Fungal spores release fungal fragments in a broad range of sizes, from 10s of nanometers up to a micrometer. Fragmented particles smaller than 10 nm may also be present, but they cannot be unambiguously detected due to the imaging limits of ESEM. This study motivates the need for further investigation of rupturing of airborne biological spores that can provide quantitative information about size and number concentration of fragments, as well as more accurate information about the environmental conditions for the rupturing process. Previous studies found that cytoplasmic material from ruptured pollen grains can be in the range of 0.05-2 μm in size. [START_REF] Taylor | Birch pollen rupture and the release of aerosols of respirable allergens[END_REF][START_REF] Miguel | Meteorological influences on respirable fragment release from Chinese elm pollen[END_REF] The size and chemical composition of fragmented particles significantly influences their cloud activation potential. ESEM images reveal that during rupture the number of particles released ranges from 10 to ∼10 3 individual fragmented particles per fungal spore. Fragmented particles show distinguishable variations in their compositions and morphologies (Figure 2). In the Amazon, the presence of a variety of fungal spores with different compositions and chemical aging results in a wide range of fragmented particles. Figure 4a-c shows representative STXM images at the Na pre edge (1070 eV), peak (1078 eV) and optical density map of biological particle after exposure to high RH. Na pre edge image (Figure 4a) represents the optical density for non-Na elements over the investigated sample area and Na peak represents the maximum Na absorption. The optical density (-ln(I( d )/I 0 ) is calculated based on the measured intensity (I d ) using Beer-Lambert's law. [START_REF] Moffet | Scanning X-ray transmission microscopy: applications in atmospheric aerosol research[END_REF] Transmission intensity through a particle free region of the substrate is used to obtain I 0 . The STXM map shows the presence of Na containing fragments and inclusions of fungal spores. Furthermore, EDX spectra of fragmented particles show signatures of inorganic salts, for example, such as Na and Cl (Figure 4d,e) and relatively low C compared to primary spore. We hypothesize that inorganic salts within the primary spore will be in liquid phase at high humidity and these salts can disperse from the primary fragments. This process may result in relatively higher inorganic salt and less carbon in the fragmented spores compared to the primary spore. A previous study reported an elemental composition of C and O in submicrometer (200 nm in diameter) fragmented pollen particles and the presence of carbohydrates and proteins in organic pollen solutions. [START_REF] Steiner | Pollen as atmospheric cloud condensation nuclei[END_REF] Previously, an amorphous film around fragmented pollen was reported. [START_REF] Pummer | Chemistry and morphology of dried-up pollen suspension residues[END_REF] Chemical characteristics of the fungal spore walls and their thickness may influence their rupturing process. Water vapor can diffuse and penetrate the film thickness of the wall and facilitate the expansion of the layer, resulting in an increase of the adhesion forces, thus affecting the rupturing process. [START_REF] Goŕny | Fungal fragments as indoor air biocontaminants[END_REF] Hence, understanding their wetting behavior requires characterization of the fungal spore walls. For example, pollen walls (exines) are composed of several types of proteins, lipids, and sporopollenin. [START_REF] Laurence | Airborne starch granules as a potential contamination source at archaeological sites[END_REF] Previous studies found glucuronic acid, uronic acid, and heteropolymers of mannose, galactose, glucose, and glucuronic acid in several kinds of fungal spore walls. [START_REF] Bartnicki-Garcia | Cell wall chemistry, morphogenesis, and taxonomy of fungi[END_REF][START_REF] Brown | Polysaccharides from cell walls of Aureobasidium (Pullularia) pullulans. II. Heteropolysaccharide[END_REF] These components and their concentrations vary among fungal spore types. The measured width of the film around the spore wall from SEM images ranged from 0.03 to 0.58 μm and increased to 0.11-0.78 μm at elevated relative humidity (SI Figure S4). Saccharide compounds in the fungal spore wall, such as glucose, can lead to the growth of the film at high RH. Similar to other carbonaceous particles, fungal spores contain high carbon, thus TEM/EELS analysis can provide further information on chemical bonding. EELS analysis shows considerable differences in aromatic carbon (exhibited by the intensity of π* peak) between fungal spores and carbonaceous soot particles (SI Figure S3). Fungal spores show relatively weaker π* peak compared to soot particles. Furthermore, substantial differences in the intensity of the carbon π* peak between various fungal spores are also noticeable. For example, our experimental observations suggest that fungal spores with weaker π* peak (less aromatic) are more susceptible to rupture and release higher number of fragments compared to those with stronger π* peak (SI Figure S3). Overall, TEM/EELS analysis suggests that structural variability within various fungal spores and their walls may determine the extent and specific conditions for rupturing.

Spore morphology can also influence the rupturing process and the release of materials. For example, elongated (high aspect ratio) fungal spores (e.g., Aspergillus and Penicillium) are more susceptible to stress compared to spores with less elongated structures (Cladosporium). [START_REF] Goŕny | Fungal fragments as indoor air biocontaminants[END_REF] Similarly, our limited observations support the hypothesis that elongated particles are more likely to rupture than less elongated particles (Figure 2). Hence, elongated particles release a higher number of submicromter particles.

Climatic Impacts. Aircraft measurements suggest that fungal spores can be uplifted from the ground surface to the mid-troposphere [START_REF] Fu | Aircraft measurements of polar organic tracer compounds in tropospheric particles (PM10) over central China[END_REF] and potentially impact climate. In locations where the ambient relative humidity is high enough (such as the Amazon) fungal spores can rupture and release fungal fragments. Expelled submicrometer particles can potentially be transported to the free troposphere by tropical convection and further impact cloud properties. The number, mass, size, and chemical compositions of fine biological particles are insufficiently quantified and remain highly uncertain for the Amazon basin. [START_REF] Despreś | Characterization of primary biogenic aerosol particles in urban, rural, and high-alpine air by DNA sequence and restriction fragment analysis of ribosomal RNA genes[END_REF][START_REF] Martin | Sources and properties of Amazonian aerosol particles[END_REF][START_REF] Poschl | Atmospheric aerosols: composition, transformation, climate and health effects[END_REF][START_REF] Fuzzi | Overview of the inorganic and organic composition of size-segregated aerosol in Rondonia, Brazil, from the biomass-burning period to the onset of the wet season[END_REF] Our results suggest that expelled fungal fragments may contribute to the fine aerosol fraction. We note that temperature and ambient pollutants (e.g., O 3 ) during the laboratory experiments were not similar to those in the Amazon basin. Other environmental conditions (e.g., temperature, wind speed and concentrations of ambient pollutants) may also influence rupturing spores under wet conditions in the Amazon basin. However, the release of the small fungal fragments may not be easily detected by in situ techniques such as fluorescence UV-APS due to instrument detection limits. [START_REF] Huffman | Size distributions and temporal variations of biological aerosol particles in the Amazon rainforest characterized by microscopy and real-time UV-APS fluorescence techniques during AMAZE-08[END_REF] The hygroscopicity of submicrometer fungal fragments was investigated using ESEM. From ESEM images collected in the hydration experiments, area equivalent diameter growth factors (diameter wet /diameter dry ) of fungal fragments were estimated in the initial (dry) size range of 150 to 600 nm (SI Figure S5). Since fungal fragments contain inorganic salt elements (e.g., Na and Cl) and fungal spore walls contain sugars, growth factors of fungal fragments are compared with those of NaCl and D-glucose. Figure 5 shows the growth factors of fungal fragments compared with hygroscopic characteristics of pure NaCl and D-glucose submicrometer particles. [START_REF] Mochida | Hygroscopic properties of levoglucosan and related organic compounds characteristic to biomass burning aerosol particles[END_REF] Results suggest that the hygroscopicity of fungal fragments lies between NaCl and D-glucose particles within an RH range of 70-96%, indicating that fungal fragments are more hygroscopic than D-glucose particles. The growth factors of fungal fragments approach those of NaCl particles at high RH (∼96%). The growth factors of fungal fragments are 1.18, 1.35, 1.64, and 2.31 at RH of 60, 76, 85, and 96%, respectively (Figure 5). As discussed earlier, with increasing RH, the fungal spore film wall width also increases (SI Figure S4). However, previous studies found aerodynamic diameter growth factors of 1.06-1.27 at 98% for 1.6-3.3 μm fungal spore particles. [START_REF] Despreś | Primary biological aerosol particles in the atmosphere: a review[END_REF][START_REF] Madelin | Fungal and actinomycete spore aerosols measured at different humidities with an aerodynamic particle sizer[END_REF][START_REF] Reponen | Effect of relative humidity on the aerodynamic diameter and respiratory deposition of fungal spores[END_REF] Overall, our results indicate that fungal fragments are hygroscopic in nature and can participate in cloud formation. Similarly, a recent study showed that fragmented pollen grains are active as cloud condensation nuclei, requiring supersaturations of 0.81 and 0.12 for 50 and 200 nm particles, respectively. [START_REF] Steiner | Pollen as atmospheric cloud condensation nuclei[END_REF] The increase in light scattering cross section due to hygroscopic growth of the fragmented particles at elevated RH can be estimated using Mie theory. [START_REF] Bohren | Absorption and Scattering of Light by Small Particles[END_REF][START_REF] Maẗzler | MATLAB functions for Mie scattering and absorption, version 2[END_REF] For these calculations a wavelength of 550 nm was used. The refractive indices of the hydrated particles are derived using the volume mixing rule, [START_REF] Abo Riziq | Optical properties of absorbing and non-absorbing aerosols retrieved by cavity ring down (CRD) spectroscopy[END_REF] with a real part of the refractive index of 1.40 for biological particles [START_REF] Ebert | The chemical composition and complex refractive index of rural and urban influenced aerosols determined by individual particle analysis[END_REF] and 1.33 for water. [START_REF] Seinfeld | Atmospheric Chemistry and Physics: From Air Pollution to Climate Change[END_REF] At 60% RH, the scattering cross section of fungal fragments increased by a factor of 1.2, compared to dry conditions. The scattering cross section of fragments increases with increasing RH, meaning that a larger growth factor leads to a larger scattering cross section. The increases in scattering cross section of fungal fragments are 1.2, 1.9, 4.3, 8.6, and 10.3 at relative humidities of 60, 76, 85, and 96%, respectively (SI Figure S6). These properties could allow fragmented particles to have a direct radiative effect on climate.

Mechanisms of the new particle formation in the Amazon basin remains insufficiently understood and occurs much less frequently than reported in a boreal forest. [START_REF] Kulmala | Direct observations of atmospheric aerosol nucleation[END_REF] Furthermore, the Amazon basin has one of the lowest aerosol concentrations in any continental regions, [START_REF] Martin | Sources and properties of Amazonian aerosol particles[END_REF][START_REF] Artaxo | Atmospheric aerosols in Amazonia and land use change: from natural biogenic to biomass burning conditions[END_REF] and as such the release of nanoand submicrometer particles from fungal spores can strongly influence aerosol concentration and thus its role on clouds. Nucleation events and subsequent growth of nucleated nanoparticles (3-10 nm) to larger sizes are regularly observed in other continental locations. [START_REF] Martin | Sources and properties of Amazonian aerosol particles[END_REF][START_REF] Artaxo | Atmospheric aerosols in Amazonia and land use change: from natural biogenic to biomass burning conditions[END_REF] Bursting events of nanoparticles (SI Figure S7) in the size range of 10-50 nm occur frequently during the wet season in Amazonia. [START_REF] Artaxo | Atmospheric aerosols in Amazonia and land use change: from natural biogenic to biomass burning conditions[END_REF] However, near surface measurements revealed no significant evidence of regional scale new particle formation from gas-particle nucleation events in the Amazon basin. [START_REF] Martin | Sources and properties of Amazonian aerosol particles[END_REF] A large amount of water vapor emitted from the forest makes the Amazon basin very humid, often >70% RH and in addition deep vertical convection is notorious in this tropical area. [START_REF] Artaxo | Atmospheric aerosols in Amazonia and land use change: from natural biogenic to biomass burning conditions[END_REF] Figure S7 (c)) and no photochemistry occurs. Figure 6 illustrates that the occurrence of bursting events increases with increasing RH, which is in accordance with our observation of rupturing of spores at high RH. Expulsion of the fine particles from fungal spores under wet conditions in the Amazon basin, and/or outflow from deep convective clouds (in-cloud processing of fungal spores) could be common and may provide insight into new particle formation (see discussion in the SI).

The rupturing process and release of fungal fragments may impact the complex land-atmosphere exchange process (emissions and deposition). The lifetime of the fungal spore particles can increase after their rupturing and release of submicrometer fragments. This process potentially affects the deposition of biological particles. Deposition of biological particles may influence metabolic activity and trigger reproduction of biological spores. These activities can either facilitate or hinder further emission of biological particles, ultimately may affect biogeochemical cycle and related feedback loops. [START_REF] Poschl | Multiphase Chemistry at the Atmosphere-Biosphere Interface Influencing Climate and Public Health in the Anthropocene[END_REF] Finally, it may alter the feedback loops of the exchange processes between the atmosphere and soil, and long-range transport of particles. Furthermore, release of compounds from the biological particles by a rupturing process is typically ignored, 2 but can substantially influence budget calculations of biological particles. Future studies should focus on (1) detailed characterization and improved understanding of the chemical composition of different types of fungal spores and the variability of their fragments; (2) systematic evaluation of specific environmental conditions promoting rupturing events (e.g., RH, temperature, atmospheric pressure, humidity cycling rates).

■ ASSOCIATED CONTENT

* S Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.6b02896.

Additional information as noted in the text (PDF)

■

Figure 1 .

 1 Figure 1. Representative SEM images of fungal spores collected in the Amazon basin. Scale bar is 2 μm.

Figure 2 .

 2 Figure 2. Representative SEM images of ruptured fungal spores and expulsion of submicrometer fragments. Panel (c) shows an example of germination pores of fungal spores as indicated by arrows. Scale bar is 2 μm.

Figure 3 .

 3 Figure 3. Size distribution of fungal spore particles (blue line) and fragmented subfungal spore particles (green line). A total of 711 individual fungal spores and 2041 fragment particles were identified by microscopy and used for the analysis.

Figure 4 .

 4 Figure 4. Representative STXM images at the (a) Na K pre-edge (1070 eV), (b) peak (1078 eV) and (c) optical density map of Na-containing biological particle (optical density difference between Na K peak and pre-edge). SEM image (d) of a ruptured fungal spore particle and its fragments after water exposure; (e) the EDX spectra acquired over different regions of the particle (marked by boxes in the SEM image) show the presence of elemental components representative of salts (i.e Na and Cl) in the fragmented particles. The Cu peak in the EDX spectrum is from the substrate.

  Figure 6 shows the occurrence of bursting events in Amazon basin measured at the ZF2 site during the wet season of 2008-2009. The median diurnal variation of N 50 concentrations (SI Figure S7 (b)) indicates that particles with diameters less than 50 nm are more frequently observed at nighttime, when ambient relative humidity reaches its highest values close to saturation (SI

Figure 5 .

 5 Figure 5. Area equivalent diameter growth factors (diameter wet / diameter dry ) of fragmented subfungal spore particles (green line). Gray line indicates the growth factor of the laboratory generated NaCl particles. Black line indicates the growth factor of D-glucose from Mochida and Kawamura 55 using tandem differential mobility analyzer.

Notes

DOI: 10.1021/acs.est.6b02896 Environ. Sci. Technol. 2016, 50, 12179-12186

The authors declare no competing financial interest. ■ ACKNOWLEDGMENTS The Pacific Northwest National Laboratory (PNNL) group acknowledges support from the Chemical Imaging Initiative of the Laboratory Directed Research and Development program at PNNL. The Lawrence Berkeley National Laboratory (LBNL) group acknowledges support from the U.S. Department of Energy's Atmospheric System Research program, an Office of Science, Office of Biological and Environmental Research (OBER). The CCSEM/EDX and ESEM analyses were performed at Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by OBER at PNNL. PNNL is operated by the U.S. Department of Energy by Battelle Memorial Institute under contract DE-AC06-76RL0. STXM/NEXAFS analysis at beamline 11.0.2 of the Advanced Light Source at Lawrence Berkeley National Laboratory is supported by the Director, Office of Science, Office of Basic Energy Sciences of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. Beamline 11.0.2 also acknowledges support from the Office of Basic Energy Sciences Division of Chemical Sciences, Geosciences, and Biosciences by the Condensed Phase and Interfacial Molecular Sciences Program of the U.S. Department of Energy.

Brazilian group acknowledges support from the FAPESP projects 08/58100-2, 2013/25058-1 and 2013/05014-0. We thank Dr. Erik Swietlicki's group at Lund University for the lending the SMPS system and Simone R. da Silva for assistance with sampling. We also thank INPA (Instituto Nacional de Pesquisas da Amazonia) for the logistical support.


Joel Brito

Florian Wurm

João Ana Maria Yáñez-Serrano

Vicente De Assunção

José Marcus Godoy

Paulo Artaxo

Ana Maria Yańẽz-Serrano

Vicente Joaõ

De Assuncaõ

Josémarcus Godoy

Vehicular Emission Ratios of VOCs in a Megacity Impacted by Extensive Ethanol Use: Results of Ambient Measurements in São Paulo, Brazil

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

■ INTRODUCTION

Emissions in megacities in both the developed and developing parts of the world are strongly impacted by the transportation sector. The Saõ Paulo Metropolitan Area (SPMA), located in the southeast of Brazil, has a population of nearly 20 million people and 8 million vehicles, many of which are fueled by a considerable amount of ethanol. [START_REF] Brito | Physicalchemical characterisation of the particulate matter inside two road tunnels in the Saõ Paulo Metropolitan Area[END_REF] Such a fleet is considered a unique case of large-scale biofuel usage worldwide. Current inventories attribute over 80% of hydrocarbons emissions within the SPMA to vehicular sources. [START_REF]Annual Report of Air Quality in the Sao Paulo State[END_REF] Despite the large impact on human health and atmospheric chemistry dynamics, many uncertainties are found in terms of gas and particulate matter emissions from vehicles and their corresponding atmospheric reactivity, for example, secondary organic aerosol formation. [START_REF] De Abrantes | Emission of polycyclic aromatic hydrocarbons from gasohol and ethanol vehicles[END_REF][START_REF] Ferreira Da Silva | Characterization of metal and trace element contents of particulate matter (PM10) emitted by vehicles running on Brazilian fuels-hydrated ethanol and gasoline with 22% of anhydrous ethanol[END_REF][START_REF] Kirstine | The global atmospheric budget of ethanol revisited[END_REF][START_REF] Salvo | Reduction in local ozone levels in urban Saõ Paulo due to a shift from ethanol to gasoline use[END_REF] Ethanol was first introduced as a commercial alternative to fossil fuels in Brazil in the early 1980s and its use has strongly increased since the 2000s. For a decade now a generation of cars termed flexible fuel vehicles (FFV) has allowed the use of any content of anhydrous ethanol from 27% (mixed with gasoline, E27) to pure hydrous ethanol (E100). Both fuels (E27 and E100) are available at the petrol station and the consumer can choose whether to use E27 or E100, or combine them to reach an intermediate ethanol fraction (E50 or E90, for example). [START_REF] Salvo | Reduction in local ozone levels in urban Saõ Paulo due to a shift from ethanol to gasoline use[END_REF] Most of motorcycles are not flexible fuel vehicles, and thus can only be fueled by E27. Heavy-duty vehicles are fueled by diesel with 7% biodiesel (B7). During the period of our study (austral summer 2013), the state of Saõ Paulo consumed on total 10% more ethanol than gasoline, namely 6.98 × 10 5 m 3 of ethanol versus 6.35 × 10 5 m 3 of gasoline per month. [START_REF]Oil, Natural Gas and Biofuels Statistical Yearbook[END_REF] Aiming to better assess the role of vehicular emissions in the SPMA, a comprehensive project entitled FONTES ("sources" in Portuguese) was carried out. The project encompasses vehicular exhaust emissions characterization using standard dynamometer test cycles, tunnel 1 and ambient measurements, [START_REF] Almeida | Measured and modelled cloud condensation nuclei (CCN) concentration in Saõ Paulo, Brazil: The importance of aerosol size-resolved chemical composition on CCN concentration prediction[END_REF] as well as numerical modeling. Ambient measurements were largely based on filter collection (over 1200 filters collected at four sites). Furthermore, intensive campaigns were performed using a suite of real-time instrumentation, including instrumentation for aerosol and gas-phase characterization. For several of these instruments, this was the first deployment in such unique environment.

Among the innovative aspects of the FONTES ambient measurements, we include real-time measurements of a suite of volatile organic compounds (VOCs), including oxygenated VOCs (OVOCs). VOCs serve as fuel for the photochemical production of ozone, [START_REF] Atkinson | Atmospheric chemistry of VOCs and NOx[END_REF] influence the oxidizing capacity of the atmosphere [START_REF] Singh | High concentrations and photochemical fate of oxygenated hydrocarbons in the global troposphere[END_REF] and have been increasingly identified to contribute to the aerosol burden of the atmosphere. [START_REF] Allan | Airborne observations of IEPOX-derived isoprene SOA in the Amazon during SAMBBA[END_REF][START_REF] Jimenez | Evolution of organic aerosols in the atmosphere[END_REF][START_REF] Xu | Effects of anthropogenic emissions on aerosol formation from isoprene and monoterpenes in the southeastern United States[END_REF] In urban areas, VOC emissions are dominated by anthropogenic sources and, as such, their quantification is a first critical step to develop effective abatement strategies, as well as determine their environmental impacts.

Previous laboratory studies have shown a significant change in the emission profile with varying levels of ethanol content in gasoline. [START_REF] Durbin | Effects of fuel ethanol content and volatility on regulated and unregulated exhaust emissions for the latest technology gasoline vehicles[END_REF][START_REF] Hochhauser | Summary of Research on the Use of Intermediate Ethanol Blends in On-Road Vehicles[END_REF][START_REF]Assessing the Effect of Five Gasoline Properties on Exhaust Emissions from Light Duty Vehicles Certified to Tier 2 Standards: Final Report on Program Design and Data Collection[END_REF] Acetaldehyde, for example, is an important OVOC with well-known impact on human health [START_REF] Woutersen | Inhalation toxicity of acetaldehyde in rats. III. Carcinogenicity study[END_REF] and ozone formation. [START_REF] Atkinson | Atmospheric chemistry of VOCs and NOx[END_REF] Acetaldehyde was observed to increase with increasing ethanol usage. [START_REF] Yanowitz | Impact of adaptation on flex-fuel vehicle emissions when fueled with E40[END_REF] Benzene, also a VOC with important effects on human health [START_REF] Dean | Recent findings on the genetic toxicology of benzene, toluene, xylenes and phenols[END_REF] and a relevant aerosol precursor, [START_REF] Li | Optical properties of secondary organic aerosols generated by photooxidation of aromatic hydrocarbons[END_REF] showed the opposite trend, that is, emission reduction with increasing ethanol content in gasoline. 21 The objective of this study is to provide the quantification of a suite of (O)VOCs in a megacity largely impacted by biofuel use, and furthermore, to assess under real-world conditions their anthropogenic emission ratios. The observed VOC emission ratios will then be compared with observations in megacities worldwide, seeking to identify trends in the VOC emission profile linked to the extensive biofuel use.

■ MATERIALS AND METHODS

The measurements described here were carried out from 08 February to 23 April 2013 at a sampling site in downtown Saõ Paulo, surrounded by 12-35 km of dense urban occupation. The site is located at the Public Health Faculty (23°33′13.87″S 46°40′23.46″W), at the crossing of Dr. Arnaldo and Teodoro Sampaio avenues, one of the most heavily impacted areas by traffic emissions within the SPMA. 22 A quadrupole Proton-Transfer-Reaction Mass Spectrometer (PTR-MS, Ionicon Analytic, Austria) was used to provide realtime VOC mixing ratios. The system was operated under standard conditions (2.2 mbar drift tube pressure, 600 V drift voltage, E/N 140 Td). Every 60 min air was diverted through a catalytic converter (Supelco, Inc. with platinum pellets heated to 400 °C) to assess the background signal for each species. A system calibration was performed during the campaign using permeation tubes stabilized at 40 °C for isoprene, benzene, toluene, and p-xylene. [START_REF] Brito | An unheated permeation device for calibrating atmospheric VOC measurements[END_REF][START_REF] Jost | Calibration with permeation devices: is there a pressure dependence of the permeation rates?[END_REF] Furthermore, methanol, acetaldehyde, acetone, isoprene, MVK+MACR, MEK, benzene, toluene and p-xylene were postcampaign calibrated using a set of gravimetrically prepared multicomponent mixtures (Apel Riemer Inc., USA) for a large range of humidity values. Concentration estimates of compounds calibrated by both methods agreed well within instrumental accuracy. [START_REF] De Gouw | Measurements of volatile organic compounds in the earth's atmosphere using proton-transfer-reaction mass spectrometry[END_REF] The PTR-MS was deployed on the third floor of the Public Health Faculty building, roughly 15 m above street level. Air was sampled through an unheated 5 m long 1/8 in. O.D. PTFE tube to the sampling system.

The instrument detection limit (DL) was calculated according to Taipale et al. ( 2008 Although the PTR-MS technique allows for real-time measurement of VOCs down to very low mixing ratios, its species separation is solely based on the compound molecular mass, which results in limited speciation capability. [START_REF] De Gouw | Measurements of volatile organic compounds in the earth's atmosphere using proton-transfer-reaction mass spectrometry[END_REF] Therefore, interference from other species cannot be completely ruled out, in particular for acetone and isoprene, which have previously been reported to suffer interferences in urban areas. [START_REF] De Gouw | Measurements of volatile organic compounds in the earth's atmosphere using proton-transfer-reaction mass spectrometry[END_REF][START_REF] Valach | Concentrations of selected volatile organic compounds at kerbside and background sites in central London[END_REF] Acetone has been previously identified to suffer an interference from propanal, however under a large set of environments the contribution of the former was limited to 10%. [START_REF] De Gouw | Measurements of volatile organic compounds in the earth's atmosphere using proton-transfer-reaction mass spectrometry[END_REF] Isoprene has been observed to suffer from interferences from furans and cycloalkanes. Whereas the former is only significantly emitted from biomass burning, [START_REF] De Gouw | Measurements of volatile organic compounds in the earth's atmosphere using proton-transfer-reaction mass spectrometry[END_REF] the latter can be significant under ambient environments. [START_REF] Valach | Concentrations of selected volatile organic compounds at kerbside and background sites in central London[END_REF] The data for ozone (O 3 ), nitric oxide (NO), nitrogen dioxide (NO 2 ), NO x (NO+NO 2 ), and carbon monoxide (CO) reported here was provided by the Saõ Paulo State Environmental Agency (CETESB), from the Cerqueira Ceśar station, some 50 m from the sampling site.

■ RESULTS AND DISCUSSION

Time Series and Meteorology. The campaign took place during a southern hemisphere summer/fall, with an average temperature of 21.9 ± 3.6 °C and average humidity of 76 ± 15% (Figure 1). Close to 80% of the days were considered sunny (24 h integrated insolation >10 000 kJ m -2 ). Mean mixing ratios of the monitored species are depicted in Table 1. The observed values of CO, NO x and ozone are comparable with observed values in other megacities such as Mexico City, [START_REF] Shirley | Atmospheric oxidation in the Mexico City Metropolitan Area (MCMA) during[END_REF] Paris, [START_REF] Zhang | Formation of organic aerosol in the Paris region during the MEGAPOLI summer campaign: evaluation of the volatility-basis-set approach within the CHIMERE model[END_REF] Houston 30 and New York, [START_REF] Ren | {HOx} concentrations and {OH} reactivity observations in New York City during PMTACS-NY[END_REF] under comparable meteorological conditions. It is interesting to notice that observed ozone values are significantly lower at the sampling site, in a downtown area, than usually found in the SPMA (40-70 ppbv). [START_REF] Salvo | Reduction in local ozone levels in urban Saõ Paulo due to a shift from ethanol to gasoline use[END_REF] This difference is mainly attributed to the abundance of NO x from vehicular sources, and as such a reduction of ozone is expected in a VOC-limited regime. [START_REF] Salvo | Reduction in local ozone levels in urban Saõ Paulo due to a shift from ethanol to gasoline use[END_REF] The diurnal pattern of CO and NO (as well as NO x ) present a distinct peak in the morning rush hour consistent with a dominant vehicular source (Figure 2). Ozone levels are observed to increase around 09:00 and reach a plateau around noon until 16:00. Diurnal patterns of NO x and CO show a similar behavior, suggesting a homogeneous source profile throughout the day. Furthermore, the average NO x /CO ratio (0.11 ± 0.03) is comparable with values seen for light-duty vehicles recently derived from tunnel measurements (0.049 ± 0.05), but is much lower than rates seen for heavy-duty vehicles (2.4 ± 1.6). [START_REF] Peŕez-Martínez | Emission factors of air pollutants from vehicles measured inside road tunnels in Saõ Paulo: case study comparison[END_REF] The wind pattern during the campaign is shown in Figure 3, depicting a predominance of easterly winds during the morning and night hours, and well-defined NW and SE components in the afternoon. Whereas the sampling site is largely impacted by vehicular sources from all directions, biogenic VOCs (BVOCs) are likely to originate from Ibirapuera Park. Comprising an area of 1.5 km 2 and over 15 000 tress, the park is located 4 km southeast of the sampling site. Such wind direction is dominant in the afternoon and shall be considered when interpreting the partitioning of primary anthropogenic or biogenic/secondary Emission Ratios. Real-time measurement of ambient VOCs provides a powerful data set for quantification of urban emission factors, although photochemical processing and dilution/mixing must be accounted for. The latter is considered via the normalization of a given VOC by a long-lived anthropogenic marker, usually CO or acetylene. Photochemical processing can be corrected by an estimation of the plume age and estimated reaction rates. [START_REF] Warneke | Determination of urban volatile organic compound emission ratios and comparison with an emissions database[END_REF] However, the sampling station is found within the SPMA, and thus plume age is considered highly uncertain. Another approach recently developed [START_REF] Borbon | Emission ratios of anthropogenic volatile organic compounds in northern mid-latitude megacities: Observations versus emission inventories in Los Angeles and Paris[END_REF] relies on applying a linear regression fit to calculate the slope of the scatterplot of nighttime data (20:00-06:00 local time) between a given VOC and CO. During the nighttime, atmospheric processing of most VOCs can be considered negligible and, furthermore, the emission ratios of VOCs associated with plant emissions during photosynthesis, such as isoprene, are carried out in the absence of light. [START_REF] Borbon | Emission ratios of anthropogenic volatile organic compounds in northern mid-latitude megacities: Observations versus emission inventories in Los Angeles and Paris[END_REF] Interestingly, during the nighttime most of the VOCs have correlated well with CO (R 2 > 0.8), including those usually associated with biogenic emissions and/or secondary formation, such as acetone, acetaldehyde and MVK+MACR (Figure S1). Such a high correlation indicates that such VOCs share a common source with CO (e.g., combustion) in regard to evaporation, for example. The correlation of MEK with CO is an exception (R 2 ≈ 0.5), possibly due to varying degrees of biogenic contributions during night hours of such long-lived species. [START_REF] Chew | OH radical formation yields from the gas-phase reactions of O3 with alkenes and monoterpenes[END_REF] Curiously, toluene also yielded a moderately good correlation (R 2 = 0.65), which can indicate whether some impact of evaporation, which is independent of CO, or the result of heterogeneity on emission ratios emissions throughout the campaign for this particular compound.

The observed emission ratios are found in close agreement with other megacities (Table 2), despite the specificity of the Brazilian fuel. Acetaldehyde, for example, which could be expected to have a significantly high emission ratio due to the ethanol content in gasoline, [START_REF] Yanowitz | Impact of adaptation on flex-fuel vehicle emissions when fueled with E40[END_REF] had a calculated emission ratio of 3.93 pptv/ppbv CO, whereas in Los Angeles the emission ratio is 5.42 pptv/ppbv CO [START_REF] Borbon | Emission ratios of anthropogenic volatile organic compounds in northern mid-latitude megacities: Observations versus emission inventories in Los Angeles and Paris[END_REF] and in Mexico City 1.0 pptv/ppbv CO. [START_REF] Bon | Measurements of volatile organic compounds at a suburban ground site (T1) in Mexico City during the MILAGRO 2006 campaign: measurement comparison, emission ratios, and source attribution[END_REF] Benzene, on the contrary, has been reported to decrease with increasing ethanol usage, 21 had a higher emission ratio (1.03 pptv/ppbv) than the average of 28 U.S. cities (0.70 pptv/ ppbv CO [START_REF] Baker | Measurements of nonmethane hydrocarbons in 28 United States cities[END_REF] ), but a lower emission ratio than Los Angeles (1.30 pptv/ppbv CO 34 ), Paris (1.07 pptv/pppbv CO [START_REF] Borbon | Emission ratios of anthropogenic volatile organic compounds in northern mid-latitude megacities: Observations versus emission inventories in Los Angeles and Paris[END_REF] ) and Mexico City (1.21 pptv/ppbv CO [START_REF] Bon | Measurements of volatile organic compounds at a suburban ground site (T1) in Mexico City during the MILAGRO 2006 campaign: measurement comparison, emission ratios, and source attribution[END_REF] ). Interestingly, at high fuel distillation temperatures, benzene emission has also been reported to increase with increasing ethanol content. [START_REF] Durbin | Effects of fuel ethanol content and volatility on regulated and unregulated exhaust emissions for the latest technology gasoline vehicles[END_REF] A relatively high emission ratio of anthropogenic isoprene has been observed, yielding a ratio of 1.17 pptv/ppbv CO. The emission ratios of anthropogenic isoprene for Los Angeles were calculated to be 0.30 pptv/ppbv CO [START_REF] Borbon | Emission ratios of anthropogenic volatile organic compounds in northern mid-latitude megacities: Observations versus emission inventories in Los Angeles and Paris[END_REF] and for Mexico City were 0.08 pptv/ppbv CO, [START_REF] Apel | Chemical evolution of volatile organic compounds in the outflow of the Mexico City Metropolitan area[END_REF] respectively. Such a high emission ratio derived here could be the result of interference of other species, such as cycloalkanes, as previously discussed, and certainly motivates further studies.

The lack of a well-defined trend in the VOC emission profile in Saõ Paulo in regard to other megacities indicates that driving conditions and fleet composition play a more important role than the ethanol content in gasoline. Large scale studies in Saõ Paulo, including events of abrupt changes in the ethanol consumption, [START_REF] Salvo | Reduction in local ozone levels in urban Saõ Paulo due to a shift from ethanol to gasoline use[END_REF] will certainly aid in elucidating the effects of biofuel under real-world conditions. Biogenic/Secondary Components. By using the nighttime derived emission ratios to the entire CO data set (including daytime), one can estimate in a simple way the primary anthropogenic component of the studied VOCs eq 1:

= × PA ER CO (1)
Where PA is the primary anthropogenic component of a given VOC and ER is the emission ratio. By extending such an equation to daytime, two ground assumptions are made: (i) the fleet composition is comparable between daytime and nighttime (i.e., same ER) and (ii) the anthropogenic component of the VOCs is dominated by local emissions, with little reaction time in the atmosphere. Argument (i) is supported through CO/NO x analysis, indicating comparable values from daytime and nighttime. Argument (ii) arises from knowledge of local traffic, and will be supported by results presented below.

The difference between the PA and actual ambient mixing ratios, i.e., the "excess", is then considered the contribution of other sources to the analyzed VOCs. Such sources are thought in this case to be mainly dominated by biogenic sources or secondarily formed in the atmosphere:

= - biogenic/secondary VMR PA (2)
Where VMR is the volume mixing ratio of a given VOC. The diurnal pattern of VMR, PA, and the biogenic/secondary component is depicted in Figure 4.

The ambient mixing ratios of benzene, toluene, and C8 aromatics are almost entirely apportioned to the PA component. The fact that daytime concentrations of these compounds can be explained by the nighttime derived ER multiplied by daytime CO mixing ratio allows us to conclude that for this particular site: (i) There is a homogeneous source profile throughout the day, as already indicated by a relatively constant CO/NO x ratio; (ii) Fuel evaporation of the studied species is negligible; (iii) Reaction time in the atmosphere can also be considered negligible.

Other species besides aromatics have shown a more significant contribution of the biogenic/secondary component. Furthermore, isoprene, acetaldehyde and, to a less extent, acetone, have depicted peaks of biogenic/secondary component in the afternoon of comparable magnitude to the PA component morning peak. It is important to notice that local winds in the afternoon suggest transport from Ibirapuera Park, a large source of BVOCs within SPMA, as previously discussed. Interestingly, the biogenic/secondary component of isoprene and MVK+MACR depict a well-defined mid-day peak characteristic of plant emissions, [START_REF] Serrano | Diel and seasonal changes of biogenic volatile organic compounds within and above an Amazonian rainforest[END_REF] however with comparable magnitude between them (0.8 ppbv for the former and 0.6 ppbv for the latter) suggesting a relatively processed air mass. [START_REF] Stroud | Isoprene and its oxidation products, methacrolein and methylvinyl ketone, at an urban forested site during the 1999 Southern Oxidants Study[END_REF] Many of the BVOCs emissions are associated with photosynthesis, which in turn are directly or indirectly linked to ambient temperature. [START_REF] Serrano | Diel and seasonal changes of biogenic volatile organic compounds within and above an Amazonian rainforest[END_REF] Therefore, the biogenic/secondary component can be further analyzed considering its variation with daytime ambient temperature. As shown in Figure 5, only isoprene (R 2 = 0.40) and MVK+MACR (R 2 = 0.31) depict some correlation with temperature, indicating that these are the only species studied here which are directly linked with biogenic emissions. The temperature dependence for the biogenic component of isoprene and MVK+MACR is 101 ± 6 pptv K -1 and 80 ± 5 pptv K -1 , respectively. While the results shown here provide robust evidence for the nature of species

Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

This work was supported by the Research Foundation of the State of Saõ Paulo (FAPESP, process 2013/25058-1), by the National Council for Scientific and Technological Development (CNPq) and PETROBRAS (project 6000.006831.11.9). We acknowledge Nelson Alves and Gisele Birman Tonietto for the PETROBRAS coordination and Dr. Carsten Warneke for the scientific discussion. We thank the support from Prof. Peŕola Vasconcellos in calibrating the system. We thank Ana Lućia Loureiro, Alcides Ribeiro, Fernando Morais and Fabio Jorge for their valuable support before and during the campaigns. We thank the support from the School of Public Health staff that made the campaign possible. We gratefully acknowledge the Saõ Paulo State Environmental Agency (CETESB) for providing NO x , CO and ozone data.

Figure 1 .

 1 Figure 1. Time series of meteorological parameters and ambient mixing ratios of several species. Weekends are marked in light blue. Ozone, CO, NO, and NO 2 data were provided by the Saõ Paulo State Environmental Agency, station Cerqueira Ceśar.

  ), i.e., calculated as two sigma of the baseline corrected background signal. The species reported here are methanol (m/z 33 -DL 314 pptv), acetaldehyde (m/z 45 -DL 179 pptv), acetone (m/z 59 -DL 40 pptv), isoprene (m/z 69 -DL 38 pptv), MVK+MACR (m/z 71 -DL 19 pptv), MEK (m/z 73 -DL 20 pptv), benzene (m/z 79 -DL 14 pptv), toluene (m/z 93 -DL 27 pptv) and C8aromatics (m/z 107 -DL 28 pptv).

Figure 2 .

 2 Figure 2. Diurnal profile of CO, NO, NO 2 , NO x , and ozone from the Cerqueira Ceśar site during the time of the campaign.

Figure 3 .

 3 Figure 3. Wind rose during the sampling period. Morning hours range from 06:00-12:00, afternoon hours range from 12:00-18:00 and night hours from 18:00-06:00. Data from the National Institute of Meteorology.

Figure 4 .

 4 Figure 4. Diurnal profile of the mixing ratios of the monitored VOCs, the component apportioned to primary anthropogenic (PA) and the biogenic/ secondary component.

Table 1 .

 1 Mean Mixing Ratios Observed during the Campaign, Numbers Are Given in ppbv

	parameter	mean	std
	ozone	14.0	12.8
	ozone 12:00-18:00	25.8	14.0
	CO	702	389
	NO	35.4	36.9
	NO 2	43.1	21.3
	NO x	78.5	50.1
	methanol	4.37	1.57
	acetaldehyde	3.42	1.98
	acetone	2.82	1.31
	isoprene	1.06	0.68
	MVK+MACR	1.26	0.77
	MEK	1.09	1.01
	benzene	0.67	0.44
	toluene	2.11	2.06
	C8 aromatics	1.52	1.47

Table 2 .

 2 Urban Emission Ratios of VOCs Given in pptv/ ppbv CO

	VOC	Saõ Paulo	Los Angeles a Paris a	Mexico City b	London c	US Cities d
	methanol	5.39	21.72		2.1	-
	acetaldehyde	3.93	5.42		1.0	4.14
	acetone	3.59	11.78		0.51	3.18
	isoprene	1.17	0.30		0.08 e	1.13
	MVK+MACR	1.63	0.36
	MEK	1.42			0.29
	benzene	1.03	1.30	1.07	1.21	1.59	0.70
	toluene	3.10	3.18	12.30	4.2	3.09	2.70
	C8 aromatics	2.15	2.45	4.75	4.3	3.69	1.40

a Borbon et al., (2013). b Bon et al., (2011). c Valach et al., (2014). d Baker et al., (2008). e Apel et al., (2010).

DOI: 10.1021/acs.est.5b03281 Environ. Sci. Technol. 2015, 49, 11381-11387