Hello,

I hope that you're doing well and enjoying the summer. I also hope that you'll find the following articles on the health effects of inhaling fungal mycotoxins and on non-sporulating fungi both interesting and helpful.

If this is the first time you are reading our monthly technical newsletter for IAQ professionals, please take time to subscribe. It takes less than a minute to submit your information. Thank you for reading The Environmental Reporter.

With best wishes,
Dave Gallup

---

"Toxic mold" caused a flurry of media attention during the last 10 years and the resulting fear of "toxic mold" was a significant driver in the rapid growth of fungal IAQ investigations. In past issues of the Environmental Reporter we have discussed some of the adverse health effects that can be caused by fungi including allergic responses, hypersensitivity pneumonitis, and microbial volatile organic compounds. In this issue, we will discuss "toxic mold" and the risk of inhalation exposure to fungal toxins in residential and office environments. We will also focus on Stachybotrys, with a bias towards providing counterarguments to some of the claims of extreme adverse health effects that many of you have probably read or heard about.

Background
Many mycotoxins are secondary metabolites of fungi, meaning that they are not required for growth of the organism producing them. They are produced under suboptimal conditions for the fungi, such as when nutrients are limited. Production can vary significantly from one isolate to another and is dependent upon a poorly understood combination of many factors probably including temperature (Fusarium tricintum produces significant amounts of toxin when the temperatures is less than 15°C and produces very little when it is warmer), nutrient sources, competition with other organisms, relative humidity, growth rate, and maturity of the fungi^1,2. As a result, the mere presence of a toxigenic fungus, meaning a fungus that is capable of toxin production, does not assure that toxin is being produced at that location. Similarly, if a fungus is producing toxins in the field, that same organism may not produce toxins in the lab.

Mycotoxins have relatively high molecular weights and are not significantly volatile. Consequently, they are usually not airborne unless they are attached to a particle and there has been an aerosolization event and, as a result, significant exposure through inhalation is unusual.

Corn kernels infected with Fusarium moniliforme (upper row) showing "starburst" symptom (a white streaking of the kernel). Image used with permission. Source: "Mycotoxins in Corn."

Recognition of risk due to ingestion
Severe adverse health effects due to ingestion of moldy food are well documented in both humans and animals. Aflatoxin, one of the most well known fungal toxins in the IAQ community, has been classified as a type 1 carcinogen and is probably the most potent liver carcinogen for humans. In the 1960's, over 100,000 turkeys were killed in England due to aflatoxin contaminated peanuts. Ergotism is a mycotoxin disease caused by ingestion of moldy rye. The mycotoxin was responsible for outbreaks of "St. Anthony's Fire" in the middle ages². Ingestion of ochratoxin from moldy food remains a significant risk for cancer in some underdeveloped countries. Risks due to ingestion of moldy food are well recognized by the scientific community and should be avoided.

The fungus Aspergillus flavus sporulating on corn. Aspergillus flavus produces the mycotoxin known as aflatoxin. Image used with permission. Source: "Mycotoxins in Corn."

Risks due to inhalation of mycotoxins
There is some evidence for adverse health effects to humans in occupational environments where the exposure to mycotoxins is intense. However the available evidence and research regarding adverse health effects due to inhalation of mycotoxins supports the hypothesis that the risk is low in normal residential and office environments. This is primarily because the dosage is so low. The amount of mycotoxin contained in fungal spores is tiny. A Stachybotrys spore is roughly 9.5 x 7.5 µm in size. This is a volume of 2.8 x 10^-10cm³ per spore. Dust with 85% spores has been found to contain 9.5 nanogram (ng) of Satratoxin H (SH)/mg of dust, or 11ng/mg of spores. Note that one ng is just 0.000000001 grams. This yields 3.1x10^-15 grams of toxin per spore³. The implications of this are sometimes overlooked. In February of this year, Environmental Health Perspective published an article showing that the no effect dose in mice for intranasal instillation of Satratoxin G (SG) was 5x10^-6 grams/kilogram body weight and suggested that this was a low dose⁴. However, if we want to get a sense of what that means in the human model, and make the significant assumptions that the concentration of SG is the same as SH, that people have the same no effect dose as mice (this may be significantly off), and use a body weight of 70kg (154 pounds), then the no effect dose level for that person is 3.5x10^-4 grams. Using 3.1x10^-15 grams/spore, an intranasal instillation of 110 billion spores would be the no effect dose level.

Microscopic photo of Stachybotrys chartarum.

Another way to look at this is to make a mathematical model using conservative assumptions. A crude risk assessment can be made using the following set of data and conservative assumptions³:

• One nanogram of mycotoxin is enough to cause an adverse health effect in people. This is extraordinarily conservative. The European Commission Regulation on aflatoxins from 1999 required that total aflatoxin levels must be less than 4 micrograms/kg in products intended for human consumption⁵. That is more than 100 times higher than what we're permitting in this mathematical model.
• One Stachybotrys spore contains 3x10^-15 grams of mycotoxin, as calculated above.
• All the airborne spores have mycotoxin, are inhaled into the respiratory tract, all of the toxin is absorbed by the body, and accumulates over time with none of it being metabolized or broken down.
• A person breathes 30 m³ of air per day and is in this environment all day. This is probably a "reasonably" conservative estimate using data from the California Air Resources Board⁶ and reasonable assumptions about average activity levels throughout the day.
• A background level of 100 spores/m³ of Stachybotrys spores. Due to the stickiness of Stachybotrys spores, and their relatively fast settling rated, this is a conservative estimate since it would likely require an ongoing active disturbance of a source of Stachybotrys to maintain this level continuously.

Given the model above, it would take over 1,000 days for a person to reach the ten nanogram threshold. The above model provides support for an argument that the risk due to inhalation of fungal mycotoxins in normal office and residential environments is low. This is in contradiction to some of the anecdotal evidence that has been provided by the media. Both sets of data should be moderated by the fact that there are many other things to consider, such as the presence of other spores in the air besides Stachybotrys, the possible presence of other toxins in the air, non-exposure related effects such stress and psychological damage, etc. Taking these other factors into account is what makes fungal IAQ investigations challenging and interesting. Such investigations require skill, training, expertise, and compassion for our fellow human beings. Compassionate approaches require both that specific risks be identified, and that the absence of a specific risk is made clear.

Note that this is a complex topic and that due to space constraints this discussion is necessarily superficial and is not and should not be construed as medical or any other form of advice.

References:
1. Adverse Health Effects Associated with Molds in the Indoor Environment, American College of Occupational and Environmental Medicine, October 27, 2002.

2. D.M. Khun and M.A. Ghannooum, Indoor Mold, Toxigenic Fungi, and Stachybotyrs chartarum: Infectious Disease Perspective, Clinical Microbiology Reviews, January 2003, p144-172.

3. Adapted from Harriet A. Burge, Health Effects of Biological Contaminants, Indoor Air and Human Health, CRC Press, 1996, Chapter 10, p171-176.

4. Z. Islam, et. al., Satratoxin G from the Black Mold Stachybotrys chartarum Evokes Olfactory Sensory Neuron Loss and Inflammation in the Murine Nose and Brain, Environmental Health Perspectives, February 27, 2006.

5. http://www.micotoxinas.com.br/boletim34.pdf (PDF, 164kb)

6. Air Resources Board: How Much Air Do We Breathe?

---

Fungi that have not produced spores under the given environmental conditions (e.g., lack of appropriate light, moisture, temperature, or specific nutrient conditions) are grouped under the name "non-sporulating fungi." Many of these organisms never sporulate in culture (mycelia sterilia), but some represent non-sporulating colonies of common fungi (for example Cladosporium, Alternaria, or even Aspergillus). As all the fungi are capable of producing a non-sporulating state, the distribution of non-sporulating fungi is cosmopolitan in nature. Identification of these non-sporulating fungal species is not possible using standard microscopic techniques which require information on spore bearing structures.

Because the term "non-sporulating" is more of a reference to a "state of being" that essentially all fungi are capable of generating, non-sporulating fungi have been recovered from a wide range of regions and substrates. For example, seven species were isolated from soil samples collected from the dark zone of six caves in South India, a survey of the normal fungal flora of dogs carried out over a calendar year indicated that non-sporulating hyphomycetes (mycelial fungi) were usually a dominant component, and colorless and dark non-sporulating fungi were dominant among isolates from healthy and diseased corals in Andaman and Nicobar islands in India (PubMed).

Under laboratory conditions, many non-sporulating colonies of common hyphomycetes can be induced to sporulate by use of near ultraviolet light (black light; wavelength 300-380nm) and a temperature range of 21-28°C. Effects of black light can be lost if the temperature exceeds 30°C. Although black light may affect factors such as pigmentation or spore morphology, these effects are not sufficient to interfere with identification process that has been enabled by the generation of spores. Additionally, sometimes exposing the culture to diurnal patterns of light and dark might stimulate sporulation.

In general, sugar rich media, like potato dextrose agar (PDA), usually promote excessive amounts of mycelium and generate higher numbers of non-sporulating colonies, with a resulting reduction in the ability to identify the fungi recovered and a corresponding reduction in the usefulness of the data. Many fungi sporulate most successfully on nutritionally weak media (Global plant clinic). Frequently non-sporulating colonies are produced by Basidiomycetes (mushrooms), which usually do not produce fruiting structures or spores on laboratory media. They may produce clamp connections and /or arthrocondia (spores resulting from the fragmentation of hypha) within their mycelia (mass of hyphae constituting the body of fungus). Clamp connections are produced only by basidiomycetes. However, arthroconidia can be produced by many different kinds of fungi.

Fungi that do not sporulate in culture do produce spores in nature, and can produce allergens, irritants and can cause hypersensitivity pneumonitis, dermatitis and systemic infection in immunocompromised patients. A case of aspergilloses caused by a non-sporulating (in culture) strain of Aspergillus fumigatus was reported in a pregnant woman (M. E. Callister et al., 2004) and a case of an invasive infection with non-sporulating Chrysosporium species was reported in a patient who was treated with chemotherapy for relapsed acute lymphoblastic leukemia (Gan G.G. et al., 2002).

Some fungi are rarely found even in nature with spore bearing structures. On tape lift samples these non-sporulating mycelia may appear colorless or pigmented (brown), septate (with cross-walls) or non-septate. They may have clamp connections (basidiomycetes). Further identification is not possible unless sporulation can be induced. Sometimes, incubating a bulk sample under high humidity for weeks are months results in the production of spores. This is rarely done for environmental investigations, but is an excellent and interesting research technique.

References:
1. PubMed

2. Global Plant Clinic

3. A fatal case of disseminated aspergilloses caused by a non-sporulating strain of Aspergillus fumigatus. M.E. Callister etal. Journal of Clinical Pathology 2004; 57:991-992.

4. Non-sporulating Chrysosporium: an opportunistic fungal infection in a neutropenic patient. G.G. Gan, et. al. Medical Journal of Malaysia, March 2002, 57(1):118-122.

5. EMLab.com