A New Approach to Bioaerosol Monitoring in Ireland
Summary 1. Particulate Matter (PM) in the Atmosphere 2. Primary Biological Atmospheric Particles (PBAP) 3. BioCheA: EPA 2007 CCRP Project 4.4.6.b 4. Where To and What Next? Future Applications of
the WIBS Technology
Professor John Sodeau Department of Chemistry and Environmental Research Institute
University College Cork
ACKNOWLEDGEMENTS Dr David Healy
David O’Connor (BIO-POSTER)
Dr Stig Hellebust
Ian O’Connor (CHEM-POSTER)
Dr Paul O’Driscoll
EPA for funding via BioCheA (2007 CCRP Project 4.4.6.b)
and a Doctoral Scholarship to David O’Connor
David Dodd
Airborne particles range in size, chemical (and biological) composition and morphology. Some might be mainly
composed of sea-salt whereas others might contain toxic transition metals such as cadmium in bioavailable forms.
PM contains biological components
FUNGAL CELLS/SUB-POLLEN: CONTRIBUTE 12-22% TO ORGANIC COMPOUNDS,OC
RESPONSIBLE FOR 4-11% OF TOTAL MASS
Approximate chemical composition of URBAN fine Particulate Matter (PM2.5)
The UCC group have monitored PM10 and PM2.5 composition, EC/OC splits, sulfates, SO2, ozone, NOX as well as atmospheric Hg in Cork harbour as part of a variety of air quality receptor-modelling projects funded by EPA, EU and SFI since 2006.
Primary Biological Aerosol Particles PBAP
POLLEN
BACTERIA VIRUSES
FUNGAL SPORES
ALGAE PLANT
DEBRIS
~0.02 – 0.5 mm ~0.5-3.0 (-30) mm
~1.5-4.0 (-30) mm ~10-100 mm
CRYPTOSPORIDIUM
OOCYSTS
~4-6 mm
On-line monitoring of PBAP
for the EPA
2006-2010
EPA CCRP/STRIVE
REPORT (2012)
Analysis of the development & occurrence of
BIOlogical & CHEmical Aerosols (2008-2011)
Pollen and fungal spores can cause allergies such as hayfever
Why monitor PBAPs in the Atmosphere?
1. HEALTH
3. TERRORISM
Anthrax attacks in USA, 2001
2. CLIMATE
Can act as ice nuclei and initiate ice-crystal formation in clouds
4. WELFARE
Irish potato famine due to the fungus, Phytophthora infestans
Traditional PBAP Characterization: Many Days of Intensive Labour
Impaction
POLLEN
SporeWatch
“Eyeball” analysis
Optical Microscopy
However PBAP fluoresce intrinsically
PBAP are known to fluoresce because they contain intrinsic bio-fluorophores such as:
Trytophan, NAD(P)H and the Flavinoids We utilize this property in our real-time detection method
The fluorescence spectra can be measured
0.00E+00
2.00E-08
4.00E-08
6.00E-08
8.00E-08
1.00E-07
1.20E-07
1.40E-07
400 450 500 550 600 650 700
Fluorescence
Wavelength (nm)
Grasses
Chlorophyll-a peak
Excitation at 370 nm
Collection of the fluorescence on-line
•Single particles analysed by a compact device
•Excitation at both 280 nm and 370 nm
•Fluorescence captured at (300-400 &
420-650 nm)
•Diode laser gives scatter signal to allow
determination of particle size (0.5-30 mm) and
asymmetry factor (“shape”), the AF value
•Time-resolution of secs-msecs REAL-TIME
Side scatter
54°-126°
Forward
scatter
6°-25°
Xe2
(370nm)
Xe1
(280nm)
Diode
Laser
635nm
Beam
dump
Aerosol in
FL2
(~420-650nm)
FL1
(~300-
400nm)
Copyright: Dr W Stanley
Waveband-Integrated Bioaerosol Sensor
WIBS 4 Real-Time Data Display:
SHAPE
SHAPE SHAPE
SIZE
FL1
FL2
FL3
PARTICLE TYPE
Differing PBAP show differing patterns of the 5 signals depending on identity
An alarm can be incorporated when “high” levels of a chosen signal are
exceeded
Individual PBAP have WIBS “signatures”
Normalised fluorescence laboratory measurements of FL1 and FL3 plotted
against optical particle size (median values) for each sample type coloured
according to its corresponding AF value. (The AF colour-bar indicates:
navy/blue as a particle that is more spherical in shape and red-brown
represents a more rod-like particle).
Optical Microscopy cannot
distinguish between these
important compost
bioaerosols
WIBS-4 Field Testing: Real-time vs Traditional
Rural Setting: environment with
very low contributions from human
activities and anthropogenic
sources
Four week campaign
WIBS-4, SporeWatch, Weather
Station etc deployed
Killarney National Park
(KNP)
August 2010
WIBS-4: Filtering the Data (MILLIONS of particles observed in the campaign)
SET THRESHOLDS: •Instrumental lower limit of fluorescence defined for each FL1, FL2 and FL3 •Defined fluorescence thresholds •Power performance of the lamps
• Size range, e.g. ignore > 20 mm • By asymmetry, e.g. spherical or rod-like? • Fluorescence signals, normalised to their FL ratios • Campaign Site
IGOR Pro (Wavemetrics)
OTHER FILTERS:
Examples: Dust particles are large but non-fluorescent; oil (PAH) droplets are fluorescent but small and spherical
Focus Period: 24 to 27-August-2010 F
L3
-SIZ
E
FL
2-S
IZE
• Hourly median values vs time of day PLUS Relative Humidity Data • Following diurnal trend of fluorescent particles in channels FL2 & FL3
Some Conclusions • Sum of Ascospores, Badiospores
& Ganoderma fungal spore numbers track well the diurnal trends observed in FL2 and FL3
• Fungal spores appear at night-time when 80-100 %RH
• FL2 data indicate two PBAP groups (~1.2 mm and ~ 3 mm: median values); former not pronounced in FL3 and latter not pronounced in FL1)
• ~0.8-1.5 mm group:
Actinomycetes / Streptomycetes from soil?
Sp
ore
Wa
tch
Tra
p D
ata
2.5x102
2.0
1.5
1.0
0.5
0.0
Co
nce
ntr
atio
n (
m-3
)
20151050
Time of Day (hr)
1.5x104
1.0
0.5
0.0
Co
nce
ntra
tion
(m-3
)
Ganoderma Basidiospores Ascospores NFL3
2.5x102
2.0
1.5
1.0
0.5
0.0
Con
cent
ratio
n (m
-3)
20151050
Time of Day (hr)
BioChea Sampling Campaigns
KarlsruheMunich
Uni. Of Hertfordshire
Port of Cork
Killarney National Park
Where To and What Next?
Future applications of the WIBS technology in light of BioCheA
discoveries
Planning and permitting bodies such
as councils and the Environment
Agency (EA) now require risk
assessments and regularly monitor
bioaerosol emissions on sites that fall
under the "potential to cause local
health concern to the workers and
surrounding public" category of
facilities/sites. How easy is this
currently?
The British Standards Institute (BSI)
recognises the increasing levels of
concern and interest in bioaerosols
and has released a technical
specification (CEN/TS 16115-1:2011)
on the measurement of moulds in
ambient air to identify, quantify and
characterise bioaerosol pollution in
ambient air resulting from emissions
from different sources.
WASTE MANAGEMENT
Current preferred method
employs direct impaction
(Anderson Sampler) where
Petri dishes of appropriate
media are loaded into the
sampler. The dishes are then
incubated in a laboratory and
the bacteria/fungal spore
colonies (CFU) counted later
by optical microscopy
Not real time data:
“snapshot” sampling.
Impactor can become
overloaded quickly
Farmers’ lung and Aspergillosis are caused by the inhalation of thermophilic Actinomycetes (0.5-1.5 mm) or Aspergillus (2-3.5 mm) species in decomposing compost, hay, or sugar cane. Exposure to large quantities of contaminated hay is the most common source. This most commonly occurs during the winter months due to the cold, damp climate. Incidence is highly variable but it is estimated to affect 0.4%-7% of the farming population.
OCCUPATIONAL LUNG DISEASES
8
6
4
2
FL
1/F
L3
8642
Size (microns)
2.0
1.5
1.0
0.5
0.0
FL
2/F
L3
WIBS can distinguish between PBAP in HAY using SIZE
Previous studies show that the size range for Aspergillus AND Penicillium is 2-3.5 mm.
2.5 mm
3.5 mm
Two PBAP clusters of similar size (2- 3 mm) and similar FL2:FL3 ratio but differing FL1:FL3 ratio.
Cluster ~0.5-1.5 mm with different fluorescence character from both the other two clusters: Actinomycetes?
2.0 mm
WIBS can distinguish between PBAP in HAY using SHAPE
AF values closer to 0 indicate SPHERICAL shape. Closer to 100 indicates ROD-LIKE shape. Aspergillus and Penicillium are close to spherical/ovoid.
Two PBAP clusters of similar size (~2 mm) and spherical shape particles but differing FL1:FL2 and FL2:FL3 ratios
Cluster includes smaller sizes (~1 mm) with FL ratios similar to one of the larger clusters: Actinomycetes?
Our optical microscope picture of hay “dust”
8
6
4
2
FL
1/F
L2
80604020
AF (shape factor)
5
4
3
2
1
0
Siz
e (m
icro
ns
)
IRRITANTS AND KILLERS
And what about cryptosporidium oocysts (4-6 mm)
aerosolized release from contaminated water and
"biosolids"?
The annual "Pollen Count" for Ireland
shown on the Irish Health PollenAlert app is
compiled entirely from monitoring
measurements made in the UK. They are
sold to us as a computer model prediction
by the University of. Worcester.
Although Ireland was a pioneer in this field
many years ago, the labour intense nature
of the work coupled with the high level of
expertise required using the traditional
impaction/microscopy approach has meant
that we no longer provide a service for
ourselves.
Can the WIBS provide an on-line approach
to supply real-time measurements of the
pollen count?
An outbreak of Legionaires Disease
broke out in Scotland in early June
2012. It was ascribed to the
aerosolized release of Legionella
pneumonia, a bacterium that
possesses a distinctive rod-like
shape. (0.5-0.7 x 2 mm long)
Poolbeg power station, for example,
has such water coolers. If Dublin
were ever to experience a similar
outbreak, could WIBS help to provide
a rapid, on-line, on-site analysis?
Pollen event in the Yew Forest at KNP: February 2010
Pollen Event Date Start time Finish time
28/02/2010 11:44.49 16:26.55
Pollen event.....is mainly YEW pollen
WIBS 4
SporeWatch
0
100
200
300
400
500
600
700
Po
llen
Co
un
ts (
grai
ns
per
m3)
28/02/2010 SporeWatch Data Yew counts hazel counts alder counts
2 hour resolution
Seconds resolution
11.45 16.30
WIBS can measure sizes up to 30 mm: Yew pollen
Size AF TOF FL1_280 FL2_280 FL2_370
Mean 26.70 18.75 8.75 973.17 2044.73 1954.31
Median 26.85 17.57 8.75 892.00 2077.00 1967.00
Mode 30.55 19.86 8.94 2116.00 2077.00 1967.00 Standard Deviation 2.60 7.95 0.67 398.42 141.47 109.98 Sample Variance 6.78 63.18 0.45 158741.33 20012.89 12096.67
Kurtosis -0.32 0.52 2.01 0.35 50.59 169.67
Skewness -0.36 0.65 -0.34 0.73 -6.36 -12.27
Range 11.94 48.27 5.56 2094.00 1706.00 1713.00
Minimum 18.61 1.01 5.46 22.00 371.00 254.00
Maximum 30.55 49.27 11.02 2116.00 2077.00 1967.00
Count 582 582 582 582 582 582
0
50
100
18 19 20 21 22 23 24 25 26 27 28 29 30 31
Par
ticl
e C
ou
nt
Yew pollen size distribution
0
50
100
150
200
5 15 25 35 45 55
fre
qu
en
cy
AF values
AF
INDOOR AIR
In a given space, concentrations of fungal
spores (Aspergillus/Penicillium) in indoor
environments are highly variable and depend
upon climate, season and the sampling
methods employed, which make studies to
date difficult to compare with any validity.
Problems of indoor air quality are
recognized as important risk factors for
human health. In hospitals, day-care
centres, retirement homes and schools,
microbial air pollution affects population
groups that are particularly vulnerable.
Pseudomonas aeroginosa
Pseudomonas baby infection deaths in
Belfast's Royal Jubilee Hospital,
January 2012
WIBS studies from BioCheA on PSEUDOMONAS
Ice nucleation experiments
performed at the AIDA
chamber in Germany
• Bacterial species isolated from
cloud water
(Pseudomonas Do~ 0.7 mm in
“diameter”)
• Sampled from Puy de Dôme
station (Clermont-Ferrand,
France)
Real-time detection of bacteria pulse
BIOCHEA RESULTS SHOW: The WIBS technique offers a comprehensive, one-stage, real-time, remotely operated monitoring methodology for bioaerosols of environmental concern both outdoors (and indoors): 1. It discriminates between chemical and biological aerosols. 2. It discriminates between bioaerosols by size and “shape”
and their fluorescence characteristics. 3. It can distinguish between bioaerosols that cannot be
distinguished by optical microscopy. 4. It has a high throughput: up to 125 particles per sec 5. It employs cheap, robust xenon flash-lamp sources used in
cameras and a diode laser rather than expensive laser excitation sources used in earlier and alternative approaches.
THANK YOU
Question and Answer Slides
Particulate Matter (PM)
Fine fraction (PM2.5)
Coarse fraction (PM2.5-PM10)
Approximate chemical composition of Particulate Matter
Elemental and
Organic Carbon
Sulphate
Nitrate
Ammonium
Chloride
Insoluble minerals
Na, K, Mg, Ca
PM is a complex mixture of extremely small particles and liquid droplets. Chemically it comprises a number of components including acids (such as nitrates and sulfates), organic compounds (OC), elemental carbon (EC), transition metals, soil, fugitive dust and sea-spray.
PM size is directly linked to their potential for
causing health problems. The
smaller the more lethal.
Overview of WIBS operating principles
Size:
~0.5 µm to 12 μm
~3 µm to 31 μm
Index of shape:
Uses scatter intensity values received
by each quadrant of a quadrant PMT
detector to calculate an AF value
Fluorescence
FL1:
280 nm excitation; emission ~310 – 400 nm
FL2:
280 nm excitation ; emission ~420 – 650 nm
FL3:
370 nm excitation; emission ~420 – 650 nm
Single particle measurements
Combines particle UV fluorescence,
particle sizing & ‘shape’
assessment in one sensor
Wide Issue
Bioaerosol Sensor
(WIBS)
Overview of the Killarney National Park Campaign
Focus Period
FL1
FL2
FL3
λex = 280nm λem = ~310 – 400 nm
λex = 280nm λem = ~420 – 650 nm
λex = 370nm λem = ~420 – 650 nm
SPOREWATCH DATA
0.5-12 mm range of
sizes
February 2010 campaign in KNP: Particles > 20μm
NF
(cm
-3)
“Pollen” events
1 2
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