ATMOSPHERIC ENVIRONMENT: X 15 (2022) 100177

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Atmospheric Environment: X
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Characterization of sub-pollen particles in size-resolved atmospheric

aerosol using chemical tracers
Chamari B.A. Mampage a, Dagen D. Hughes a, Lillian M. Jones a, Nervana Metwali b,
Peter S. Thorne b, Elizabeth A. Stone a, c, *
a
Department of Chemistry, University of Iowa, Iowa City, IA, 52242, USA
b
Department of Occupational and Environmental Health, University of Iowa, Iowa City, IA, 52242, USA
c
Department of Chemical and Biochemical Engineering, University of Iowa, Iowa City, IA, 52242, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Pollen grains may contain allergens that exacerbate allergic respiratory diseases like asthma and rhinitis. In the
Bioaerosol presence of water, pollen grains (10–100 μm) can rupture to produce sub-pollen particles (SPP) with diameters
Primary biological aerosol particle (PBAP) <2.5 μm, which in comparison to intact pollen grains, have longer atmospheric lifetimes and greater penetration
Pollen fragments
to the lower lung. The current study examines SPP, fungal spores, and bacteria in size-resolved atmospheric
Fungal spores
particulate matter (PM) using chemical and biological tracers. During springtime tree pollen season in Iowa City,
Bacteria
Endotoxin Iowa, fine particle (PM2.5) concentrations of fructose (a pollen chemical tracer) increased on rainy sampling
Allergens periods, especially during severe thunderstorms, and peaked when a tornado struck nearby. Submicron fluo­
rescent particles, measured by single-particle fluorescence spectroscopy, were also enhanced during rain events,
particularly thunderstorms in agreement with the chemical tracer measurements. PM2.5 sucrose (a pollen
chemical tracer) concentrations were higher in early spring when nighttime temperatures were closer to freezing,
while fructose concentrations were higher in late spring with warmer temperatures, consistent with chemical
tracers being sensitive to seasonal temperature influences. The first co-located measurements of fructose and Bet
v 1 (birch pollen allergen), indicated that SPP ranged in diameter from <0.25 to 2.5 μm during rainy sampling
periods and that allergens and carbohydrates exhibited distinct size distributions. Meanwhile, mannitol (a fungal
spore tracer) peaked on warm, dry days following rain and was primarily in supermicron particles (>1.0 μm),
which is consistent with intact fungal spore diameters (1–30 μm). Bacterial endotoxins in PM also increased
during extreme weather events, primarily in supermicron particles. While the concentrations of fructose,
mannitol, and endotoxin all increased in PM2.5 μm during thunderstorms, the greatest relative increase in
concentration was observed for fructose. Together, these observations suggest that SPP containing starch
granules and allergens (Bet v 1) were released during rainy sampling periods. This study advances the use of
chemical tracers to track SPP and other bioaerosols in the atmosphere, by providing new insight to their size
distribution and response to extreme weather conditions.

1. Introduction
The impacts of bioaerosols on human health and the environment
depend highly on their size. Generally, intact pollen grains range
10–100 μm, SPP range 0.25–2.5 μm (Hughes et al., 2020), fungal spores
span 1–30 μm, bacteria are 0.25–8 μm and viruses are typically <0.3 μm
in diameter, but often exist as larger aerosols (Després et al., 2012; Jones
and Harrison 2004). Bioaerosols can be pathogens (Després et al., 2012),
inflammatory agents (Thorne 2021), and/or carriers of allergens that
can exacerbate asthma (WHO 2017). For example, endotoxin, found in
the cell walls of gram-negative bacteria, is implicated in asthma, bron­
chitis, toxic pneumonitis, chronic obstructive pulmonary disease, and
pulmonary function decline (Thorne et al., 2010). A particle’s size im­
pacts its atmospheric lifetime, transport, and the extent to which it
penetrates the human respiratory tract. Typically, particles >10 μm in
aerodynamic diameter are trapped in the upper respiratory tract, par­
ticles ≤10 μm can reach the thoracic region, and particles ≤2.5 μm can
penetrate the gas exchange region of the lungs (Brown et al., 2013;

* Corresponding author. Department of Chemistry, University of Iowa, Iowa City, IA, 52242, USA.
E-mail address: betsy-stone@uiowa.edu (E.A. Stone).

https://doi.org/10.1016/j.aeaoa.2022.100177
Received 29 December 2021; Received in revised form 9 May 2022; Accepted 4 June 2022
Available online 6 June 2022
2590-1621/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
C.B.A. Mampage et al.
Hinds 1999). Bioaerosols can also impact the Earth’s climate and pre­
cipitation as ice nuclei (IN) and cloud condensation nuclei (CCN)
(Murray et al., 2012; Sun and Ariya 2006). Due to the health and
environmental impacts of bioaerosols, it is important to understand the
factors affecting their atmospheric size distribution and abundance.
Anemophilous pollen grains are released from flowering plants in
large quantities to transport male genetic material to the female flower
for reproduction. The abundance and type of pollen in the Midwestern
United States varies seasonally. Tree pollen (e.g. birch, oak, alder, and
hazel) are released during springtime, whereas weed and grass pollen
are elevated in summer (Després et al., 2012; Targonski et al., 1995).
Meteorologically, warm and dry weather conditions favor the active
release of pollen grains, while rainfall typically lowers the overall pollen
count by scavenging (Taylor et al., 2004).
In response to rain and/or high humidity, many pollen types have
ruptured to release SPP including Chinese elm (Miguel et al., 2006),
grass (Taylor et al., 2002), giant ragweed (Stone et al., 2021), birch,
alder, and hazel (Grote et al., 2003). Processes of pollen rupture include
osmotic rupturing, abortive germination, and mechanical rupturing
(Miguel et al., 2006; Grote et al., 2003; Taylor et al., 2004; Visez et al.,
2015). Pollen rupturing in the atmosphere can increase bioaerosol
number concentrations by three orders of magnitude, as a single giant
ragweed pollen grain has been shown to rupture into 1400 SPP (Stone
et al., 2021). While intact pollen grains are retained in the head-airway
region, allergen-loaded SPP in the size range of 0.6–2.5 μm can pene­
trate the lower region of the lungs causing severe immunological re­
sponses in allergic individuals (Suphioglu et al., 1992; Wilson et al.,
1973; Schäppi et al., 1997a). “Thunderstorm asthma” or epidemic
asthma outbreaks coinciding with thunderstorms when pollen concen­
trations are high are hypothesized to involve SPP (D’Amato, 2017).
Individuals affected by these outbreaks were commonly outdoors when
the thunderstorm struck and were sensitive to the abundant pollen type
as demonstrated by the high levels of pollen-specific Immunoglobulin
(IgE) (D’Amato, 2019; Price et al., 2021). The proposed mechanism for
these asthma outbreaks involves thunderstorm updrafts lofting pollen
grains into the base of the clouds where high relative humidity triggers
osmotic rupture to release SPP that are transported to ground level by
downdrafts (Taylor and Jonsson 2004). This hypothesis is supported by
our recent publication demonstrating that a significant increase in the
SPP with diameters 0.25–2.5 μm during thunderstorms and rain events
in the springtime tree pollen season, with peak concentrations occurring
during convective thunderstorms with strong downdrafts, high rates of
rainfall, and lightning (Hughes et al., 2020).
Fungal spores are produced sexually or asexually by fungi enabling
them to colonize new areas in the environment. Ambient fungal spore
concentrations vary with season, time of the day, and weather (Elbert
et al., 2007; Bowers et al., 2013). The most abundant airborne fungal
spore genera are Cladosporium, Alternaria, Penicillium, Aspergillus, Epi­
coccum, yeasts, rusts, and basidiomycetes (Rockett and Kramer 1974;
Rotem and Aust 1991; Ataygul et al., 2007; Janine et al., 2009). Species
like Cladosporium depend on dry discharge by agitation during dry and
warm weather conditions (Herrero and Zaldivar 1997). In contrast,
some species of fungi (e.g., Ascomycota) disperse spores by wet discharge
(via liquid jets or droplets) under humid conditions at night, early
mornings, or after rain. Fungal spores have also been found to rupture
into particles ranging in diameter from 0.01 to 1 μm in size after
exposure to ~98% relative humidity for ~10 h followed by drying, but
not in response to short hydration and drying cycles (China et al., 2016).
Some of the affected individuals during epidemic thunderstorm asthma
events were sensitized to fungal spores like Alternaria alternata sug­
gesting a possible involvement of fungal spores in these events (Puli­
mood et al., 2007).
Like pollen and fungal spores, the variability of airborne bacterial
concentrations depends on seasonality and meteorological conditions.
Airborne bacteria are those commonly found in soil, such as Proteobac­
teria and Actinobacteria, but can also include Cyanobacteria,
2
Atmospheric Environment: X 15 (2022) 100177
Verrucomicrobia, Acidobacteria, and Bacillus (Després et al., 2012; Shaffer
and Lighthart 1997). Airborne bacteria are frequently attached to other
particles such as soil, vegetative detritus, or bacterial agglomerates
(Lighthart and Shaffer 1997). Typically, atmospheric bacterial counts
are higher in spring and summer compared to winter due to warmer
temperatures favoring the growth of vegetation which provide surfaces
for bacterial growth (Carty et al., 2003). However, bacterial endotoxin
concentrations can also peak when crops are harvested, due to aero­
solization of bacteria from plant surfaces and soil (Pavilonis et al., 2013;
Rathnayake et al., 2016). Artemisia sp. pollen grains (16.5–36.5 μm)
have been recorded as another carrier of bacterial endotoxin in coarse
PM during summer (Oteros et al., 2019). Bacteria can also be transferred
from soil to aerosol (<10 μm) via splashing caused by impacting rain­
drops, contributing to elevated airborne bacterial concentrations during
and after rain events (Joung et al., 2017, Robertson and Martin, 1994).
The time-varying concentrations of bioaerosols in the atmosphere
can be assessed via chemical and biological markers. Endotoxins, lipo­
polysaccharides present in Gram-negative bacteria can be used to track
bacteria in atmospheric PM samples (Thorne et al., 1996). Mannitol is a
chemical tracer for fungal spores (Bauer et al., 2008). It is used to store
energy in fungal spores, accounting for approximately 4–7% of spore
mass and its concentration correlates with airborne fungal spore counts
(Bauer et al., 2008; Witteveen and Visser 1995). Glucose, sucrose, and
fructose are energy storage materials found in pollen grains that con­
tributes to 5–14% of pollen mass and they have been used as chemical
tracers to quantify airborne pollen concentrations (Fu et al., 2012;
Rathnayake et al., 2017; Barnard 1975; Jia et al., 2010; Medeiros et al.,
2006). The concentrations of these carbohydrates in pollen grains vary
with developmental stage and external environmental conditions
(Vesprini et al., 2002). For example, the distribution of these molecules
across mono-, di-, and polysaccharide impacts the pollen water content
and turgor pressure (García et al., 2017; Pacini et al., 2006). Sucrose
further resists formation of ice crystals in freezing temperatures, pre­
serving pollen viability (Firon et al., 2012). Pollen antigens also can be
used to quantify specific pollen species in the atmosphere. For example,
Bet v 1 is quantified to find the airborne birch allergen concentration
(Schäppi et al., 1997b; Buters et al., 2012). Further insight to the
size-distribution of bioaerosols is gained by analysis of airborne particle
samples separated by size.
Our prior publication (Hughes et al., 2020) discusses the release of
SPP during spring rain events with a primary focus on single-particle
fluorescent concentrations and meteorology. The current manuscript
expands upon our prior study by Hughes et al. (2020), with a focus on
the use of chemical tracers to track SPP in the atmosphere and new
assessments of convective thunderstorms on the airborne concentrations
and size distributions of fungal spores and bacteria. The size resolved
measurements of SPP discussed in this manuscript are mainly focused on
five rainy sampling periods (May 8, 16, 17, 18, and 24) that were
selected based on the largest relative increase in chemical tracers and
fluorescent biological particles. The objectives of this study are to 1)
establish the distribution of pollen grains, SPP, fungal spores and bac­
teria across five particle size ranges (aerodynamic diameters <0.25,
0.25–0.50, 0.50–1.0, 1.0–2.5, >2.5 μm) during rain and exteme weather
events, 2) determine the seasonal and meteorological conditions coin­
ciding with elevated bioaerosol concentrations, and 3) provide recom­
mendations for the use of chemical tracers to track bioaerosols in future
atmospheric research. Together, these objectives advance the scientific
tools and knowledge surrounding the size distribution of atmospheric
bioaerosols and their response to changing meteorological conditions. A
separate manuscript is in preparation (Hughes et al. in preparation) to
describe the single-particle fluorescent data and meteorology during
other rain and non-rain events in spring 2019.
C.B.A. Mampage et al.
2. Methodology
2.1. Collection of ambient particulate matter samples and field data
2.1.1. Site description
Field measurements were conducted from April 06 – May 30, 2019,
at the University of Iowa Air Monitoring Site, Iowa City, Iowa, USA
(+41.6647, − 91.5845). This peri-urban site is located on the edge of the
urban areas of Iowa City and Coralville and is surrounded by woods,
meadows, prairie undergoing reconstruction, a miscanthus field, and a
parking lot.
2.1.2. Online measurements
Daily meteorological data with 1 min time resolution were collected
using a meteorological station (Vantage Vue, Davis Instruments)
installed on the rooftop of a trailer 3.5 m above ground level. Real-time
fluorescent particle concentrations with optical diameters 0.5–20 μm
were analyzed using Wideband Integrated Bioaerosol Sensor (WIBS-
NEO, Droplet Measurement Technologies) installed inside a wooden
shed at the field site. The particles were sampled using a total suspended
particulate sampling head (Mesa Laboratories) at 5 L min− 1 installed on
the shed roof 4 m above ground level. The sampled air was drawn
through stainless steel tubing. The WIBS NEO isokinetically sampled at
its designated flow rate of 0.3 L min− 1, while 4.7 L min− 1 make up air
was drawn by an auxiliary pump. Single particle data collected were
analyzed using WIBS toolkit in Igor Pro. Additional details on the WIBS
NEO set up and data analysis are available in the supporting information
to Hughes et al. (2020). PM mass measurements for PM2.5 and PM10
were obtained using beta attenuation monitors (BAMs) located at the
field site, following the methods described in Vaughn (2009).
2.1.3. Active sampling
23-h filter samples were collected from 08:00 to 07:00 the following
day. PM2.5 samples were collected onto pre-baked 90 mm quartz fiber
filters (Pall Life Sciences) using a medium volume sampler (URG Corp.)
equipped with a sharp-cut cyclone to select PM < 2.5 μm at a flow rate of
90 L min− 1. The sampler was mounted on to a wooden platform with the
inlet 2.5 m above ground level with a flow rate of 90 L min− 1 Size-
resolved PM samples were collected over 23 h by three 5-stage Sioutas
Cascade impactors (SKC Inc.) that were installed on the wooden plat­
form 2 m above ground level and collected particles with aerodynamic
diameters >2.5, 1.0–2.5, 0.50–1.0, 0.25–0.50, and <0.25 μm (50%
cutoff diameters). Samples were collected at a flow rate 9 L min− 1 onto
25 mm Teflon filters (SKC Inc.) for chemical and biological analyses. A
third impactor operating at a flow rate 8.5 L min− 1 collected particles
onto 25 mm polycarbonate filters (Isopore, Fisher Scientific, pore size
0.1 μm) for scanning electron microscopy (SEM) analysis. Pre-baked
quartz fiber filters (37 mm, Pall Life Sciences) were used as after-
filters in all three impactors. Teflon and quartz filters were stored in a
freezer at − 20◦ C while polycarbonate filters were stored in a desiccator
(relative humidity <20%) at ambient temperature.
2.1.4. Intact pollen collection and analysis
Intact pollen grain concentrations were measured using a volumetric
spore trap (Burkard Manufacturing Company) co-located with other
measurement platforms. The samples were collected on to a microscopic
slide (75 × 25 × 1.0 mm, Micro Slides, VWR) coated with a thin layer of
grease (Lubriseal, Thomas Scientific). The collected samples were
mounted and imaged using Olympus BX-61 light microscope with an
automated slide scanner (Leica Aperio Ariol). All pollen taxa are
included in the intact pollen grain concentrations. Longitudinal count­
ing method was used to obtain 1 h pollen counts. Birch pollen identifi­
cation was conducted for May 8, 16, 17, 18, and 24 by re-analyzing
higher resolution images of 1 h intervals starting at 15:00, 22:00 and
5:00 to differentiate birch/mulberry pollen grains from previous publi­
cation (Hughes et al., 2020). Further description of slide mounting,
3
Atmospheric Environment: X 15 (2022) 100177
instrumental analysis, pollen identification and determination of pollen
counts are detailed in the supporting information of Hughes et al.
(2020).
2.2. Analysis of carbohydrates
Glucose, sucrose, fructose, and mannitol were quantified using high-
performance anion-exchange chromatography with pulsed ampero­
metric detection (HPAEC-PAD, Dionex ICS 5000, Thermo Fisher, Sun­
nyvale, CA, USA) following the methods described by Rathnayake et al.
(2017).
2.2.1. Sample preparation
All glassware was cleaned with tap water (5 times), deionized water
(5 times) and ultrapure (UP) water with resistivity 18.2 MΩ cm− 1
(Barnstead EasyPure II, 7401; 5 times) and baked for 5.5 h at 500◦ C.
Plastic vials were cleaned with UP water (5 times). Half of the PM2.5
filter samples were subsampled using filter punches and placed inside
the plastic vials. Teflon filters were pre-wetted with 50 μL of acetone
(Sigma Aldrich). Substrates were extracted into 4.00 mL of UP water by
rotary shaking for 10 min at 125 rpm, sonication for 30 min at 60 Hz
(Branson 5510, Danbury, CT, US) and rotary shaking again for 10 min.
The extract was then filtered through 0.45 μm polypropylene syringe
filters (Whatman, GE Healthcare Life Sciences) before analysis.
2.2.2. Instrumental analysis
Samples were injected into the HPAEC-PAD equipped with a Car­
boPac PA20 analytical column (Dionex), a guard column (Dionex), and
an electrochemical detector consisting of a disposable gold working
electrode and a pH-Ag/AgCl reference electrode. Waveform A was used
for the pulsed amperometric detection (Jensen and Johnson 1997;
Rocklin et al., 1998). Instrumental control, data acquisition, and anal­
ysis were executed using Chromeleon 7 software. Mannitol (Sigma
Aldrich; > 98%), glucose (Sigma Aldrich; > 98%), sucrose (Fisher Sci­
entific; > 99%), and fructose (Sigma Aldrich; > 99%) were separated
isocratically using 10 mM sodium hydroxide with a flowrate of 0.5
mL/min. Initially, a 200 mM NaOH solution was prepared by diluting
(with UP water) a 50% w/w NaOH (Fisher Scientific) solution and the
needed 10 mM concentration was achieved using UP water with the
mobile phase proportioning in the instrument.
Eight-point calibration curves of fructose, glucose, sucrose, and
mannitol were linear from 0.0100 to 5.00 ppm with squared correlation
coefficient (r2) values > 0.995. Samples were analyzed in batches,
consisting of 7–8 samples, a field blank, a lab blank, and a spike recovery
sample. Spike recovery was assessed by spiking a blank filter with 50.0
μL of 25.0 ppm carbohydrate solution. The recovery percentages were in
the range of 86–114%. Uncertainty of the samples was propagated from
the standard deviation of the field blanks to account for precision error
and 14% of the measurement as a conservative estimate of inaccuracy
indicated by spike recovery analysis.
2.3. Analysis of endotoxins
For endotoxin analysis, substrates were transferred to pyrogen-free
15 mL tubes and stored at − 20 ◦ C until analysis. All glassware was
made endotoxin-free by heating overnight at 200 ◦ C prior to use. Filters
were extracted following a method previously described by (Sauvé et al.,
2020). Filters were extracted at room temperature using 2.00 mL of
Limulus Amebocyte Lysate (LAL) reagent water with shaking for 30 min.
This was followed by 30 min of sonication at 26 ◦ C, 10 additional min
shaking. and 5 min centrifuging (600×g, 4 ◦ C). A 0.45 mL aliquot of the
extract was transferred to pyrogen-free cryovials for endotoxin analysis
and the remaining extract was stored for allergen assay. The filter
extract was evaluated for endotoxin using the kinetic chromogenic LAL
assay as previously described by Thorne (2000). Briefly, eluents were
diluted into 4-fold serial dilutions from full strength; 1:4, 1:16.
C.B.A. Mampage et al. Atmospheric Environment: X 15 (2022) 100177

Absorbance was measured at 405 nm, every 30 s for 90 min using a

microplate reader (Molecular Devices SpectraMax Plus 384, Sunnyvale,
CA, with Softmax PRO 5.4 analysis software) and evaluated against a
12-point standard curve using E. coli 055:B5 for standard endotoxin
control. The minimum acceptable r2 value for the standard curve was
0.995.

2.4. Analysis of allergens

For the analysis of Bet v 1, a 0.450 mL aliquot of the PM extract and

0.05 mL sterile 10 × Phosphate Buffer Saline (PBS) at pH 7.2 with 0.5%
Tween-20 was added to yield 1x PBS with 0.05% Tween-20 and vor­
texed for 5 min. The extract was stored at − 80 ◦ C until the assay was
performed. A fluorescent multiplex array kit (MARIA, MRA-C3; Indoor
Biotechnologies, Inc.) was used following the manufacturer’s procedure.
Sample extracts were thawed, vortexed, and centrifuged for 2 min at
4 ◦ C at 16000×g. Each sample was assayed at full strength and at 1:5
dilution. A 12-point standard curve was prepared using 2-fold dilutions.
The plate was read by using an xMAP instrument (BioPlex200, BioRad,
Inc.). Each filter extract was assayed for the following allergens: Amb a 1
(ragweed pollen), Bet v 1 EP (birch pollen), and Phl p 5 (timothy grass).
Only Bet v 1 was detected.

2.5. Analysis of particles using SEM

Particles were analyzed by SEM in the Central Microscopy Research

Facility at The University of Iowa by a scanning electron microscope
with a field emission gun as the electron source (Hitachi S-4800).
Approximately 9 × 9 mm2 of PC filter from each impactor stage was
mounted on SEM stubs using carbon tape (Ted Pella Inc.). For
morphological analysis, the samples were coated with gold (Emitech
sputter coater). The microscope was operated at an accelerating voltage
of 5 kV for imaging.

3. Results and discussion

3.1. Pollen and sub-pollen particles (SPP)

This study was conducted from April 6-May 30, 2019, during the tree
pollen season in Iowa City, Iowa, USA, and coincided with the transition
from spring to summer. According to the National Allergy Bureau scale
for tree pollen (AAAAI 2020), the daily average intact pollen grain
concentration was high on 64% of the days (ranging 90–1499 grains
m− 3) and a very high pollen count (>1500 grains m− 3) occurred on April
16 at 1514 pollen grains m− 3 (Fig. 1b). Of the 55 sampling periods
having 23 h each, rain was recorded in 28 periods (Fig. 1a). The intact
pollen grain concentrations decreased with rain, likely due to scav­
enging (Lo and Levetin 2007; Pérez et al., 2009). SPP were observed
Fig. 1. Time series of a) cumulative rain and daily mean temperature, b) (total)
during many of these rain events, especially thunderstorms, by increases
intact pollen grain concentrations, PM2.5 concentrations of c) fructose, and d)
in submicron fluorescent particles detected by single-particle fluores­
sucrose. Missing data are marked by an asterisk. PM2.5 fructose concentrations
cence spectroscopy and by increases in fructose in size resolved particles from Apr 17-May 30 were previously discussed in Hughes et al. (2020).
<2.5 μm as described by Hughes et al. (2020). The influx of fluorescent
particles with the rain indicates that the rain delivered the submicron
concentrations were low in the extracted PM2.5 samples, near to the
particles (Figs. S1, S2, and S3) either in the downdraft or suspension of
lowest calibration point of 0.1 ng mL− 1. The lack of variability in PM2.5
biological material from the surface by splashing of rain. Elevated
Bet v 1 (Fig. S4) is likely due to the low birch pollen concentration
concentrations of fluorescent particles for limited time after rain sug­
leading to low Bet v 1 in the atmosphere. While our prior study sug­
gests a short window of exposure (Figs. S1, S2, and S3). In comparison
gested birch and/or mulberry pollen were among the major pollen types
offline measurement of chemical tracers with 23-h samples lacks the
(Hughes et al., 2020), reanalysis of microscope images indicated that
ability of providing the time-resolved information required to assess
only 0.14% of total pollen were birch with an estimated daily average of
exposure risks. Fluorescent particle data along with the chemical tracer
<2 grains m− 3. In addition to the low birch pollen counts, another
and allergen data provide evidence for release of SPP during thunder­
reason for low PM2.5 Bet v 1 concentrations observed in extracts of PM
storms supporting the Taylor and Jonsson’s hypothesis of thunderstorm
samples is the relatively low volumes of air analyzed (approximately 12
asthma (Taylor and Jonsson 2004).
m3). To quantify allergens like Bet v 1 in future studies, larger air vol­
The presence of SPP in daily PM2.5 samples was examined through
umes are needed.
measurements of birch allergen (Bet v 1; Fig. S4) and chemical tracers
Seasonally, fructose and glucose concentrations increased from early
previously associated with SPP (Fig. 1). The Bet v 1 allergen

4
C.B.A. Mampage et al.
spring to late spring (Figs. 1 and 2). This trend likely results from
increasing temperatures that promote maturation of pollen grains and
the interconversion of starch into soluble carbohydrates (Firon et al.,
2012). Sucrose concentrations were highest in early spring and
decreased in the late spring. This trend was opposite to the seasonal
variation observed for fructose and glucose and is likely driven by the
production of sucrose to prevent freezing. During early spring sampling
periods, daily average temperatures were low compared to late spring
(Fig. 1a) with night-time temperatures reaching below 0 ◦ C (Fig. S5).
Sucrose in pollen grains inhibits ice crystallization, preserving the pollen
viability in freezing temperatures and can be metabolically synthesized
from glucose and fructose (Firon et al., 2012). Thus, the prevalence of
sucrose early in the spring is likely due to freezing temperatures.
Daily PM2.5 fructose concentrations ranged 0.05–7.29 ng m− 3 and
Fig. 2. Time series of a) cumulative rain and daily mean temperature, PM2.5
concentrations of b) mannitol and c) endotoxin. Endotoxin was analyzed in a
subset of samples collected from May 3–30.
5
Atmospheric Environment: X 15 (2022) 100177
glucose concentrations ranged from 0.27 to 40.9 ng m− 3 with the
maximum concentrations of both tracers occurring on May 24 when a
strong convective storm struck and generated a tornado nearby (Hughes
et al., 2020). Meanwhile, sucrose concentrations ranged 0.07–12.9 ng
m− 3. The maximum sucrose concentration occurred on April 16, which
was a day with burning of prairie grasses nearby which could affect the
carbohydrate concentration in PM and rainwater (Medeiros and Simo­
neit 2008; Mullaugh et al., 2014). Among the carbohydrates measured,
fructose showed the greatest relative increase during rainy sampling
periods, especially May 18 and 24 when severe storms occurred (Hughes
et al., 2020). Elevation of submicron fluorescent particles with rain and
concurrent decreases in intact pollen concentrations were also observed
on April 17, 22, 27–29, and May 28–29. On five of these seven days,
fructose in PM2.5 was elevated relative to background days (Fig. 1c). The
lack of increase in PM2.5 fructose concentration on April 27 is likely due
to moderate rainfall rate not significantly impacting the ambient PM2.5
fructose concentrations when integrated over 23-h. Despite the heavy
rainfall rate, the ambient PM2.5 concentration did not increase on May
28. Similar observations were made during spring 2013 showing that
fructose is more sensitive to rain compared to glucose and sucrose at this
location (Rathnayake et al., 2017). On May 22, fluorescent particles
spiked at 09:27, persisted for 10 min, and particles >2 μm remained
elevated for 14 h (Fig. S7). No rain fell on this day and grass cutting
nearby the sampling site were expected to be the source of these fluo­
rescent particles. Relatively high concentrations of glucose, sucrose, and
fructose in PM2.5 (Fig. 1), were likely due to pollen, plant debris and dust
resuspension (Simoneit et al., 2004).
The mass of SPP in PM2.5 was roughly estimated from the fructose
enhancement on rainy days relative to background periods and by
assuming that the fructose above background levels was associated with
SPP. Mass concentrations of SPP were estimated by dividing the fructose
concentrations associated with SPP by the fructose-to-pollen mass ratios
observed for oak pollen in the literature (3.3% by average mass for red
and pin oak) (Rathnayake et al., 2017), due to the relatively high
abundance of oak trees in Iowa City area (Treeplotter Inventory, 2019).
Estimated SPP mass concentrations (averaged over 23 h) ranged
1.4–180 ng m− 3 and accounted for 0.1–2.2% of PM2.5 mass (Table S2).
The number of pollen grains ruptured into PM2.5 was approximated by
dividing the estimated SPP mass concentration by the measured mass of
an English oak pollen grain (10.8 ng grain− 1; Brown and Irving (1973)).
The estimated number of ruptured oak pollen grains during rain events
ranged from <1 to 17 grains m− 3, corresponding to rupturing of
<1–11% of pollen grains (Table S2). These estimates are limited by
available data on pollen grains and knowledge of what pollen types
undergo rupture. As an indication of the uncertainties in these estimates,
using data for the less abundant birch pollen (with a fructose-to-pollen
mass ratio of 1.8% by weight (Mueller et al., 2016) and pollen grain
mass of 7.9 ng grain− 1 (Schäppi et al., 1997b), would roughly double the
SPP mass concentrations and triple estimates of pollen grains ruptured.
Thus, these calculations provide only rough approximations of ambient
concentrations of SPP and extent of pollen grain rupture.
Even though glucose has previously been suggested as a pollen tracer
because of its relatively high mass fraction in pollen grains (Medeiros
et al., 2006; Speranza et al., 1997; Rathnayake et al., 2017), the data
collected during this study (Fig. 2b) suggest contributions from other
sources to its atmospheric concentration. The glucose concentration
strongly correlated with mannitol (R = 0.904, P < 0.001; Table S1). The
contribution from many other sources (e.g. growing leaves in spring,
fungal spores, and soil particles) (Simoneit et al., 2004; Medeiros et al.,
2006; Tereshina et al., 2000; Jia and Fraser 2011; Pashynska et al.,
2002) to glucose could lead to a diurnal variation of peak daytime
glucose and peak nighttime mannitol concentrations (Graham et al.,
2003) in 23 h samples. Together, these observations suggest that among
the measured pollen tracers, fructose is the most sensitive to rain during
late spring in Iowa City; however, further measurements are needed
prior to generalizing these findings to other locations.
C.B.A. Mampage et al. Atmospheric Environment: X 15 (2022) 100177

To more accurately determine the size distribution of bioaerosol
classes, PM samples collected by a Sioutas impactor were analyzed for
Bet v 1 and carbohydrates in five size ranges (with 50% cutoff di­
ameters): >2.5 (stage A), 1.0–2.5 (stage B), 0.5–1.0 (stage C), 0.25–0.50
(stage D), and <0.25 μm (E, after-filter) (Fig. 3). These measurements
were applied to five sampling periods with precipitation, elevated PM2.5
pollen tracer concentrations, and increases in ambient fluorescent bio­
logical particle concentrations (May 8, 16, 17, 18, and 24) (Hughes
et al., 2020). Additionally, three background periods (May 7, 15, and
23) were analyzed, which had relatively low fluorescent particle and
chemical tracer concentrations, no rain or light rain events (Table 1),
and close proximity in time to observations of SPP (Hughes et al., 2020).
In the impactor samples, pollen tracers observed on stage A were
interpreted as intact pollen grains and large SPP >2.5 μm, while pollen
tracers on stages B-E were interpreted as SPP <2.5 μm (Fig. 3). These
designations are supported by SEM experiments that showed a few
intact pollen grains on stage A (Fig. 4a and b), but no pollen grains on
stages B-D. Stage E was not analyzed by SEM due to measurement in­
compatibility with QFF. Our observation of only a few intact pollen
grains on stage A is consistent with this 5-stage impactor having low
collection efficiency for particles >10 μm (Misra et al., 2002).
Bet v 1 was elevated in PM < 2.5 μm on the five selected rainy
sampling periods, compared to background periods in which Bet v 1 was
not detected (Fig. 3). These observations provide additional evidence of
SPP release during springtime thunderstorms and further suggests that
birch pollen grains contributed to SPP, although to a small extent. Bet v
1 was most frequently detected in the PM size range <0.25 μm with
highest concentration in 0.5–1.0 μm size particles on May 24. The
appearance of Bet v 1 on rainy sampling periods agrees with prior ob­
servations in which light rainfall resulted in an increase in Bet v 1 in
particles <7.2 μm (Schäppi et al., 1997b). Bet v 1 was also observed in
submicron and <2.4 μm particles previously (Pehkonen and Rantio-­
Lehtimäki 1994) agreeing with our observations of Bet v 1 in particles
with diameters <2.5 μm (Fig. 3). Additionally, Grote et al. (2003) in­
dicates that Bet v 1 in birch pollen grains is mainly found in the cyto­
plasm and is released from the pollen grain during rupture in the
presence of rainwater, agreeing with the observation of the allergen in
the submicron particles during rainy sampling periods. Taylor et al.
(2004) observed that rupturing of birch pollen grains release allergen
loaded SPP distributed in size ranges 0.4–2 μm and 2–4 μm, agreeing
with our observations of Bet v 1 in these size ranges. Bet v 1 concen­
trations were low or below detection limit on top impactor stage, likely

Fig. 3. Mass concentrations of fructose (a chemical tracer for pollen), Bet v 1 (birch allergen), mannitol (a fungal spore tracer), and endotoxin from Gram-negative
bacteria, measured in ambient PM collected by a 5-stage impactor on 5 days with local rain events. Measurements are reported relative to background periods
corresponding to dry days. The mannitol concentration on May 18, and fructose concentrations on these 5 days were previously reported by Hughes et al. (2020).

6
C.B.A. Mampage et al. Atmospheric Environment: X 15 (2022) 100177

concentration (EU m− 3)
PM2.5 endotoxin

0.120

0.183

0.152

0.213

0.313
0.012

0.013

0.084
concentration (ng m− 3)
PM2.5 mannitol

5.55

11.8

7.70

25.0

24.3
0.71

6.18

4.8
concentration (ng m− 3)
PM2.5 fructose

Fig. 4. Scanning electron microscopy images of a) and b) Mulberry pollen

0.67

2.17

1.15

5.70

7.29
0.31

1.10

1.21

grains c) ascospore d) basidiospore (Particles a, b, c, and d were observed on the

stage A of cascade impactor on May 18). Intact pollen grains appear deflated as
the images were taken under vacuum or due to being stored in the desiccator
Daily average pollen
counts (grains m− 3)

until analysis.
Summary meteorology, daily average pollen counts, PM2.5 tracer concentrations, and background sampling periods in May 2019.

due to low collection efficiency for particles >10 μm (Misra et al., 2002)
coupled with the low atmospheric birch pollen concentration. In
185

449

159

117

627
611

145

350

contrast, Buters et al. (2012) collected size resolved samples with a

higher flow rate (800 L min− 1). They reported ~90% of the Bet v 1 in
PM > 10 μm fraction when atmospheric birch pollen counts were higher
than our study and no recorded thunderstorms. These observations
rainfall (mm)
Cumulative

suggest that the thunderstorms are the likely reason for the increase in
Bet v 1 concentration in submicron particles in Iowa City.
16.8

18.2

20.6

21.6
10.4
6.4
3.8

The fructose concentration was elevated in <0.25–2.5 μm particles

0

during the five selected rainy sampling periods compared to background

days (Fig. 3). The highest fructose concentration was recorded on May
Sample period begins at 08:00 on the date listed and ends at 07:00 the following day.

18 in 0.25–0.5 μm size particles (Fig. 3). These observations agree with

Temperature

previous observations of fructose concentration increasing in PM < 2.5

range (oC)

17.5–33.1

12.5–25.3

14.4–27.7
10.7–25.0

13.4–23.7
6.8–20.5

9.7–20.8
7.2–16.6

μm during periods with rain (Rathnayake et al., 2017). As fructose is

Rain events are defined based on rainfall rate (Bluestein and Howard, 1993)

associated with starch granules (0.5–2 μm) in the pollen grains (Sper­
anza et al., 1997, Matikainen and Rantio-Lehtimäki 1998), the appear­
Continued rain event from prior sampling period and light
Multiple rain events with light to heavy rain, thunderstorm

ance of fructose in PM < 2.5 μm during rainy sampling periods suggests

Multiple rain events with moderate to heavy rainfall, and

Morning and evening rain events with heavy rainfall and

the release of SPP due to pollen rupturing. The concentration of fructose

around mid- night and a severe thunderstorm at night

Multiple rain events with moderate to heavy rain and

and Bet v 1 often peaked in different PM size ranges. Bet v 1 was most
frequently detected in the PM size range <0.25 μm, while fructose
severe evening thunderstorm with a tornado

concentrations often peaked in particles 0.25–1.0 μm. This difference in

particle size likely occurs for several reasons. Bet v 1 is unique to birch
pollen, while fructose is common to many pollen types. Thus, the size
Early morning light rain on May 24
Early morning light rain on May 8

distribution of fructose is expected to be impacted by other pollen types

during the sampling period (Hughes et al., 2020), which is supported by
Description of meteorologyb

an afternoon thunderstorm

the relatively low concentrations of Bet v 1 compared to fructose

several thunderstorms

(Fig. 3). These data suggest that fructose is not an indicator of birch
rain in the afternoon

allergens, even when associated with SPP. Another reason for difference
No rain events

in size distribution could be the distribution of fructose and Bet v 1 in the

pollen grain. As previously mentioned, fructose is mainly found in the
starch granules, while Bet v 1 is present in the cytoplasm of pollen grains
(Grote et al., 2003). Although Bet v 1 is coated on starch granules it may
also be transferred into other types of particles and aerosolized during
rain events (Schäppi et al., 1997a) resulting in a differing size
(background)

(background)

(background)

distribution.
Sampling

Size-resolved sucrose concentrations increased in particles <2.5 μm

May 16

May 17

May 18

May 24
May 15

May 23
perioda
Table 1

May 8
May 7

on May 8, 16, 17, and 18 (Fig. S6). The increase in tracer concentration
b
a

7
C.B.A. Mampage et al.
in similar size particles indicates both fructose and sucrose are origi­
nated from the same source, likely SPP that are released due to pollen
rupturing events during the rainy sampling periods. Variations in the
relative concentration of fructose and sucrose are likely to result from
numerous factors, including seasonal temperature, pollen type, and
developmental stage (Carrizo García et al., 2017; Firon et al., 2012,
Carrizo García, 2010). The increase in concentration of both pollen
tracers in submicron particles provides further evidence of the release of
respirable SPP during rain events.
3.2. Fungal spores
During the spring 2019 sampling period, mannitol was used as a
tracer of fungal spores following Bauer et al. (2008). PM2.5 mannitol
concentrations gradually increased from April to May following the in­
crease in daily average temperature (Fig. 2c), agreeing with the warmer
temperatures promoting fungal growth (Jones and Harrison 2004). The
mannitol concentrations peaked during sampling periods after rain due
to favorable conditions for fungal growth (Jones and Harrison 2004; Van
Osdol et al., 2004). PM2.5 mannitol concentrations ranged 0.10–32.5 ng
m− 3 with the maximum concentration on May 30, a dry and warm
sampling period after several consecutive rainy sampling periods
(Fig. 2c).
According to the size-resolved mannitol concentrations during the
selected rainy periods, fungal spore mass was observed in the upper 3
size bins with more than 90% of mannitol mass in particles >1 μm
(Fig. 3). These observations are consistent with SEM observations of
fungal spores on the upper impactor stages (Fig. 4c and d) and intact
fungal spore diameters that range 1–30 μm (Després et al., 2012). Light
microscopy analysis also indicated the presence of ascospores and ba­
sidiospores in the atmosphere. However, spores could not be accurately
quantified because of limitations to magnification used. Elevated
mannitol concentrations during rainy sampling periods have been pre­
viously documented (Lyon et al., 1984; Pinkerton et al., 1998; Van Osdol
et al., 2004) and can be explained by active spore release during rain
(Lagomarsino Oneto, 2020). Mannitol observed in particles <1 μm is
likely due to the fungal spores reaching lower stages of the impactor as
only 50% of the particles on each stage are within the designed cutoff
diameter (Misra et al., 2002; Hinds 1999).
Co-located time-resolved fluorescent measurements showed an
influx of supermicron BC and ABC type particles, primarily at night and
early mornings during rainy sampling periods (Figs. S1, S2, S3, and S10).
Similar florescent pattern was observed on May 22 and May 30, a warm
and dry sampling periods following a rainy period with the high PM2.5
mannitol concentrations (Figs. S7 and S8). The fluorescent signature and
the size of the particles (optical diameter of 2–9 μm) agrees with pre­
vious fluorescent measurements of fungal spores (Hernandez et al.,
2016; Savage et al., 2017).
3.3. Bacteria
Endotoxin concentrations in PM2.5 samples ranged 0.008–0.332 EU
m− 3 and the concentrations increased during rainy sampling periods
(Fig. 2d). Size-resolved endotoxin concentrations show that, on back­
ground sampling periods endotoxin was primarily present in particles
>2.5 μm (Fig. 3) whereas on the rainy sampling periods there was a
bimodal distribution of endotoxin mass concentrations. The size mode
with particles <0.25 μm could be due to bacterial fragments (Wang
et al., 2007). Endotoxin in particles >0.5 μm most likely from intact
bacteria (Górny et al., 1999; Després et al., 2012).
The highest endotoxin concentrations were recorded on May 14
(0.332 EU m− 3) and May 24 (0.313 EU m− 3), which were both rainy
days (Fig. 2d). Prior to the rain event on May 14, there was an increase of
the wind speed which led to resuspension of particles evident by the
increased PM10-2.5 concentrations and an influx of fluorescent particles
(Fig. S9). Resuspension of particles due to high wind speeds during the
8
Atmospheric Environment: X 15 (2022) 100177
severe thunderstorm and tornado is a possible reason for the high
endotoxin concentration on May 24. On May 16, a sampling period with
severe thunderstorms, endotoxin concentrations elevated in particles
<2.5 μm, (Fig. 3). Coarse particle mass and fluorescent particle number
were elevated prior to the start of rain and dropped at the start of the
rain (Fig. S10). Meanwhile, the fine fluorescent particle concentration
increased (Fig. S10). Airborne bacteria are frequently attached to other
particles like soil, vegetative detritus, or agglomerates of bacterial cells
(Lighthart and Shaffer 1997) and it is expected that the resuspension of
such particles led to the elevated endotoxin concentrations in coarse
particles prior to rain. Additionally, rainfall can also transfer of bacteria
from soil and other surfaces by splashing and impaction of raindrops
releasing aerosols <10 μm (Joung et al., 2017), which provides a source
of fine particle endotoxin on rainy days. It is expected that the relative
increase in PM2.5 endotoxin concentration on May 22 (non-rainy sam­
pling period) (Fig. 2d) resulted from local grass cutting aerosolizing the
bacteria attached to plant matter and/or dust.
4. Conclusions
Through the combination of chemical and biological markers and
high time-resolution fluorescent particle measurements, new insight can
be gained on tracking pollen and SPP in the atmosphere. Elevated
fructose concentrations in fine particles during rainy sampling periods
provide evidence of the release of SPP by rupturing of pollen grains,
especially during extreme weather conditions like thunderstorms and
tornados. Due to its high sensitivity to rain and not showing any inter­
ference from other sources, we suggest fructose as a tracer for SPP in the
atmosphere. Sucrose concentrations in fine particles depend on tem­
perature. Low atmospheric temperature could lead to high sucrose
concentrations in pollen grains making sucrose a more sensitive tracer
when temperature is closer to freezing. As this temperature dependance
of sucrose was observed in Iowa City, IA, USA during spring, more
studies are needed to assess how generalizable these measurements are
in other locations. Although glucose has been used as a pollen tracer in
previous studies, our results from Iowa City suggest that it is affected by
other sources, suggesting glucose is not a reliable SPP tracer. As this
study focuses on spring tree pollen season and more analyses are
required to extend these observations to other locations and seasons.
Rain and thunderstorms affect SPP, fungal spores, and endotoxins
concentrations differently. While Yue et al. (2016) reported the contri­
bution of fungal spores and bacteria to the biological particles concen­
trations during rain, for the first time our study records elevation of
fructose and Bet v 1 concentrations in ambient PM providing evidence to
support the contribution of SPP to biological particles <2.5 μm during
rain events. SPP are highest in particles <2.5 μm during rain, while
fungal spore concentrations typically increased after rain events and
overnight, in particles >1 μm. Bacterial endotoxin concentrations
increased primarily in particles >1 μm prior to rain, likely due to dust
elevated by wind, and in particles <1 μm during rainy periods. Based on
the timing of the rain events, co-exposure to fungal spores, bacterial
endotoxin, and SPP are likely to occur. Other co-pollutants in thunder­
storms, like ultrafine particles (Wang et al., 2016), nitrogen oxides
produced by lighting (Choi et al., 2005), and/or ozone (Betts et al.,
2002) may further exacerbate the effects of allergenic bioaerosols. With
thunderstorms predicted to increase in intensity and severity (Maupin
et al., 2021), there is a need to advance public health preventive mea­
sures for susceptible individuals.
CRediT authorship contribution statement
Chamari B.A. Mampage: Investigation, Visualization, Formal
analysis, Writing – original draft. Dagen D. Hughes: Investigation,
Visualization, Writing – review & editing. Lillian M. Jones: Investiga­
tion, Writing – review & editing. Nervana Metwali: Investigation,
Writing – review & editing. Peter S. Thorne: Writing – review & editing,
C.B.A. Mampage et al.
Resources, Supervision. Elizabeth A. Stone: Conceptualization, Formal
analysis, Writing – review & editing, Supervision, Funding acquisition,
Project administration.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This research was funded by the National Science Foundation (AGS
1906091). We thank Patrick O’Shaughnessy, Ralph Altmaier, Tom Pe­
ters, the Environmental Health Sciences Research Center (NIH P30
ES005605) for loan of field sampling equipment; Vingie Ng and Matt
Sovers for assistance with field sampling; and Randy Nessler from Uni­
versity of Iowa Central Microscopy Research Facility for the assistance
with SEM imaging.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.aeaoa.2022.100177.
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