Indoor Molds and Mycotoxins Estelle Levetin, PhD Faculty of
Biological Science University of Tulsa
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Fungi Abundant in the natural environment and able to grow on
many environmental and synthetic substrates Capable of producing
secondary metabolites, mycotoxins and VOCs Small percent are plant
or animal pathogens In terms of human exposure, fungi can be
responsible for allergic, infectious, or toxic diseases
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Major Groups of Fungi Myxomycetes - slime molds Oomycetes -
water molds Zygomycetes - bread molds, sugar fungi Ascomycetes -
sac fungi, morels, yeast Basidiomycetes - mushrooms, puffballs
Asexual fungi (Deuteromycetes)- molds, microfungi, anamorphic
fungi, mitosporic fungi asexual stages of ascomycetes
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Fungal Spores Fungi reproduce by spores Majority of spore types
adapted for airborne dispersal Spores unicellular to multicellular
from 1 to 100 m always microscopic
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Outdoor Fungal Spores Amazing diversity of spores in atmosphere
Concentrations up to 200,000 spores/m 3 Daytime dominated by Dry
Air Spora Cladosporium, Alternaria, Drechslera, Curvularia,
Pithomyces, and smut spores Late night and early morning dominated
by basidiospores and ascospores Rainy periods dominated by
ascospores
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Fungal Spores in Outdoor Air
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Mold Spores in the Indoor Environment Spores occur in all
indoor environments Considered contaminants indoors Fungi can
colonize countless substrates indoors HVAC system can also become
contaminated and even help disperse spores
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Environmental factors that influence indoor fungal
contamination Outdoor concentration and type Type and rate of
ventilation Activity levels Modern building materials Indoor
moisture levels
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Typical Yearly Spore Levels
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Environmental factors that influence indoor fungal
contamination Outdoor concentration and type Type and rate of
ventilation Activity levels Indoor moisture levels Modern building
materials
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Environmental factors that influence indoor fungal
contamination Outdoor concentration and type Type and rate of
ventilation Activity levels Indoor moisture levels Modern building
materials
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Environmental factors that influence indoor fungal
contamination Outdoor concentration and type Type and rate of
ventilation Activity levels Indoor moisture levels Modern building
materials
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Indoor Moisture Levels Availability of moisture most critical
factor in determining if fungi will grow Leaks and moisture seeping
through walls, ceilings, basements obvious source of problems
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Courtesy of Terry Brennan
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Moisture Problems Become worse in past 20 - 30 yrs increased
use of washing machines, dishwashers vaporizers and humidifiers
actively spray droplets into the air (often contaminated) tighter
buildings for energy conservation trap moisture Anytime moisture
available fungi will grow
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Humidity Indoor relative humidity Below 30% R.H. no mold growth
and Above 70% optimal for mold Usually above 50% mold growth can
occur Humid air condenses on cool surfaces Cold windows in winter -
molding and sills become wet and suitable for fungal growth Cold
floors in winter Cooling coils in AC units in summer Humid air
allows hygroscopic materials to absorb water
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High humidity in home where subslab ducts failed
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Environmental factors that influence indoor fungal
contamination Outdoor concentration and type Type and rate of
ventilation Activity levels Indoor moisture levels Modern building
materials
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Modern Building Materials Ceiling tiles, sheetrock, and other
building materials seem to be especially prone to fungal
contamination High cellulose content Many fungi are cellulose
degraders in the natural environment - perfect substrate to
culturing fungi
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Objective of Study What happens when new ceiling tiles are
soaked with water? How long does it take before contamination is
visible? What fungi are present?
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Methods New ceiling tiles aseptically cut into 6 x 6 cm squares
and placed in sterile petri dishes Ceiling Tile Squares (CTS)
saturated with 33 ml water: sterile distilled water tap water rain
water CTS in triplicate, experiment repeated, and extra sterile
distilled water saturated CTS
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Methods Dishes sealed with parafilm and allowed to incubate at
room temperature for a minimum of 10 weeks CTS were regularly
evaluated by direct microscopy of surface growth At the end of the
experiment randomly selected CTS were ground up and cultured
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Direct Microscopy CTS within sealed petri dishes were evaluated
with a dissecting microscope Fungi were identified After 10 weeks
estimates made of percent surface area covered by fungal
growth
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Results of Direct Microscopy All CTS showed fungal
contamination CTS saturated with tap-water had the greatest
contamination had a mean surface coverage 65% (range 50-90%) CTS
saturated with sterile distilled water had a mean coverage of
60%(range 5-100%) CTS saturated with rainwater showed the lowest
contamination with a mean coverage of 10% (range 1-25%). 10 genera
of fungi identified plus yeast
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Fungal Growth on CTS after 10 Weeks Incubation Sterile
Distilled Water Tap Water Rainwater
Additional Observations Visible colonies of Alternaria and
Epicoccum appeared within 4 days after water was added Other fungi
appeared much later After 10 weeks incubation, dominant fungal
types of CTS varied greatly Alternaria dominant on some
Stachybotrys dominant on some Chaetomium dominant on some
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Culture Analysis One CTS was randomly selected from each water
type from each experiment 2 sterile distilled water saturated CTS 2
tap saturated CTS 2 rainwater saturated CTS Three additional CTS
from sterile distilled water group also selected at random
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Culture Analysis Methods Each CTS was placed in a sterile
blender cup with 50 ml of sterile distilled water CTS blended on
high for two 15 sec intervals Resulting slurry was filter through 4
layers of sterile cheese cloth Resulting suspension was dilution
plated on MEA + strep and Cellulose Agar Incubated at room temp for
7 to 10 days
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Results of Culture Analysis Overall Penicillium most abundant
genus on culture plates - found on all water types Several fungi
that were dominant on tile surfaces were low or absent in culture
Quantification difficult when plates overgrown with Penicillium
Results of all media and all dilutions combined
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Summary of Fungi Identified on CTS
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Indoor Fungi Indoor spores generally reflects outdoors unless
there is a source of contamination Many different types of fungi
occur - 160 spp Most common genus is Cladosporium - just like it is
outdoors some species difference indoors BUT Penicillium and
Aspergillus often exist at higher concentrations indoors
Stachybotrys has received most media attention over the past 4
years
Cladosporium Common fungal genus occurring both indoors and
outdoors Most abundant outdoor spore type with a worldwide
distribution Normally exists as a saprobe or weak plant pathogen
Spores are known to be allergenic
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Cladosporium spp.
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Cladosporium on diffuser
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Penicillium One of the most common soil fungi in natural
environment Over 250 species Well known allergen Some species
produce mycotoxins Some species produce antibiotics Produce
VOCs
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Aspergillus Also common soil fungi Cause rot of stored grain
Over 150 species Well known allergens Several species form
mycotoxins Some species can grow at high temperatures Several
species cause infections in lung, sinuses, and hypersensitivity
pneumonitis
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Penicillium and Aspergillus Small spores passively aerosolized
when spore clusters disturbed Spores extremely buoyant, remain
airborne for extended time Penicillium and Aspergillus spores look
alike distinguished in culture
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Pen/Asp Concentrations Previous and on-going studies in my lab
have focused on trying to determine base- line levels of
Penicillium/Aspergillus levels Collected multiple air samples from
12 indoor locations during Sept, Nov, Feb along with outdoor
controls Andersen samples for culturable fungi Spore trap samples
for total spores
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Pen/Asp Concentrations Penicillium and Aspergillus identified
in all locations Culture analysis identified 23 species of
Penicillium (mean 39.1 CFU/m 3 ) 12 species of Aspergillus (mean
14.1 CFU/m 3 ) Spore trap samples found 332 spores/m 3 as the mean
level of Penicillium/Aspergillus type spores indoors
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Stachybotrys chartarum Soil fungus in nature Commonly found
indoors on wet materials containing cellulose, such as wallboard,
jute, wicker, straw baskets, and paper materials Spores in slimy
mass Thought to be allergenic although little is known May produce
potent mycotoxins
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Stachybotrys
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Indoor Air Sample (Spore Trap)
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Fusarium Common saprobe and important plant pathogen Normally
found in the soil Indoors it is often found in the bathroom or
other areas with high moisture Allergenic Some species produce
mycotoxins
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Building Related Diseases Allergic diseases Allergic rhinitis
(Hay fever) Asthma Allergic sinusitis Hypersensitivity pneumonitis
Infectious diseases Human pathogens Opportunistic pathogens Toxic
disease
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Secondary Metabolites Fungi produce remarkable diversity of
secondary metabolites Often confined to one species or one strain
Antibiotics, volatile organic compounds (VOCs), toxins, glucans
Fungal toxins harmful to humans or other animals
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Volatile Organic Compounds (VOCs) Produced by many fungi Earthy
odor of some mushrooms Musty, moldy smell of basements and attics
Health effects of VOCs not well studied Some suggest VOCs
responsible for headaches, dizziness, and eye and mucous membrane
irritation Possibly many Sick Building symptoms caused by VOCs
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Fungal glucans Fungal cell wall carbohydrates Studies suggest
they have inflammatory and/or immunomodulatory properties
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Fungal Toxins Harmful to humans or other animals May provide
the fungus some advantage in natural environment Currently we are
doing some competition assays with Stachybotrys and other fungi
Toxin types: - Mushroom toxins formed in the fleshy fruiting bodies
of higher fungi - Mycotoxins formed by common molds growing under a
variety of conditions
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Mycotoxins Produced by many fungi in contaminated foods and
other substrates Can develop in grains or nuts in the field
Generally, mycotoxins develop in storage and remain within the food
after processing and cooking Many common indoor fungi are toxigenic
Some studies revealed significant levels of mycotoxins in airborne
spores
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Indoor Fungi Capable of Forming Mycotoxins Some Aspergillus
species Aspergillus versicolor most widely isolated in buildings
with moisture damage Some Penicillium species Some isolates of
Stachybotrys chartarum Some Fusarium species Many other fungi that
occur occasionally
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Health Effects of Mycotoxins Acute and chronic effects on both
humans and livestock Many are potent carcinogens Majority of
research focused on health effects following consumption of
contaminated food Effects range from immediate toxic responses and
immunosupression to potential long-term carcinogenic effects
Possible health effects due to airborne exposure (exposure to
airborne spores with mycotoxins)
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Health effects from airborne exposure to toxins? Clinical
studies not completed yet Association of Stachybotrys with health
effects in contaminated buildings but no experimental studies with
human exposure Animal studies suggest effects of respiratory
exposure very important Possible effects: immune suppression, rash,
headache, fatigue, sore throat, pulmonary hemorrhage (in infants),
memory loss??? We need more research studies and data!
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Toxic Black Mold in the Media Refers to Stachybotrys chartarum
Media frenzy started with Cleveland baby deaths and the initial CDC
report in 1997 CDC retraction in 2000 is seldom mentioned Media
frenzy stirred up again following the 1999 lawsuit by Melinda
Ballard in Dripping Springs, Texas Media frenzy has not
stopped!
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USA Weekend Cover Stories
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Dorr Dearbon, MD - Cleveland Dorr Dearborn was one of the
physicians involved in the Cleveland baby case and has continued
studies of Stachybotrys "There is a negative health impact of
living in a moldy environmentBut the details as to what the health
effects are and how much mold it takes that is what we don't
know."
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Malina Bill Introduced in Congress Oct 2002 The Malina bill
will: Require the EPA to define what dangerous toxic mold levels
are Create an emergency federal insurance program, like FEMA, to
pay the astronomical costs of black mold clean up Create a national
database of homes infected with black mold