Indoor Molds and Mycotoxins Estelle Levetin, PhD Faculty of Biological Science University of Tulsa

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

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

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

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

Fungal Spores in Outdoor Air

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

Environmental factors that influence indoor fungal contamination Outdoor concentration and type Type and rate of ventilation Activity levels Modern building materials Indoor moisture levels

Typical Yearly Spore Levels

Environmental factors that influence indoor fungal contamination Outdoor concentration and type Type and rate of ventilation Activity levels Indoor moisture levels Modern building materials

Environmental factors that influence indoor fungal contamination Outdoor concentration and type Type and rate of ventilation Activity levels Indoor moisture levels Modern building materials

Environmental factors that influence indoor fungal contamination Outdoor concentration and type Type and rate of ventilation Activity levels Indoor moisture levels Modern building materials

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

Courtesy of Terry Brennan

Moisture Problems Become worse in past 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

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

High humidity in home where subslab ducts failed

Environmental factors that influence indoor fungal contamination Outdoor concentration and type Type and rate of ventilation Activity levels Indoor moisture levels Modern building materials

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

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?

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

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

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

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

Fungal Growth on CTS after 10 Weeks Incubation Sterile Distilled Water Tap Water Rainwater

Fungi Identified by Microscopy Alternaria Aspergillus Chaetomium Cladosporium Drechslera Epicoccum Mucor Penicillium Stachybotrys Ulocladium Yeast

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

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

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

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

Summary of Fungi Identified on CTS

Indoor Fungi Indoor spores generally reflects outdoors unless there is a source of contamination Many different types of fungi occur 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

Sampling Andersen (N-6) samplerVarious portable spore trap impactors

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

Cladosporium spp.

Cladosporium on diffuser

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

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

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

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

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

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

Stachybotrys

Indoor Air Sample (Spore Trap)

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

Building Related Diseases Allergic diseases Allergic rhinitis (Hay fever) Asthma Allergic sinusitis Hypersensitivity pneumonitis Infectious diseases Human pathogens Opportunistic pathogens Toxic disease

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

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

Fungal glucans Fungal cell wall carbohydrates Studies suggest they have inflammatory and/or immunomodulatory properties

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

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

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

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)

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!

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!

USA Weekend Cover Stories

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 environment…But the details as to what the health effects are and how much mold it takes – that is what we don't know."

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