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