March 2005

Volume 3 | Issue 3

I hope that you are enjoying spring and that you will find the attached articles about endotoxins and Rhizopus helpful. Separately, I am pleased to introduce the newest member of our team, Dr. John Shane, who will lead EMLab's Midwest region. Dr. Shane is a well known instructor in the field of spore trap analysis for environmental particles and mold service in our industry. For the past 10 years Dr. Shane was Professor and Director of Research of the McCrone Research Institute in Chicago, IL. He will be a regular contributor in future Environmental Reporters, as well as an instructor at upcoming Mold University seminars.

With best wishes,
Dave Gallup
Chairman

Endotoxins
By: Dr. Harriet Burge and Dave Gallup

Endotoxin
Endotoxins are bacterial products that are ubiquitous in the environment and, although they do take part in some disease processes, exposure at some level is probably essential for the normal development of the human (and animal) immune systems.

The nature of endotoxin
Bacteria produce two general types of toxins: exotoxins and endotoxins. Exotoxins are soluble proteins that are excreted from the bacterial cell. Anthrax, tetanus, botulism, and toxic shock syndrome are a few of the diseases caused by bacterial exotoxins. Endotoxins, on the other hand, are lipopolysaccharides (LPS) that are part of the bacterial cell structure. The toxin is minimally soluble and effects occur directly from contact with the bacteria. Endotoxins are produced by Gram negative bacteria such Escherischia coli, Salmonella, Shigella, Pseudomonas, Neisseria, Haemophilus, and many others. All Gram negative bacteria have lipopolysaccharides as part of the cell wall. However, the lipopolysaccharides are not always toxic and those that are vary in potency.

Health Effects
Endotoxins are potent inflammatory agents, stimulating the release of many chemicals that activate the immune system. In high concentrations, such as would occur during a Gram negative bacterial infection, fever, inflammation, coagulation of platelets, hemorrhage, and shock may result. At the much lower concentrations encountered during inhalation exposures, inflammation is the primary outcome.

In some work environments in which organic material is handled or large amounts of water are used (e.g., swine confinement, fiberglass manufacturing) work-related respiratory illnesses may occur. These illnesses are characterized by fever, chills, muscle aches, shortness of breath, and cough. These symptoms may be worse on Monday and may gradually abate during the week, only to return following a weekend of no exposure. In residential and office environments, effects are less clear. Some studies relate dust or air levels of endotoxin to respiratory inflammation.

In residential environments, endotoxin exposure appears to be related to a decrease in the risk of developing asthma. This effect is called the hygiene hypothesis, which considers that the reduction in exposure to endotoxin caused by increasing standards for home hygiene has led in part to the rising incidence of asthma.

Sampling and measurement technologies

Analytical methods

The most commonly used analytical method for endotoxin detection and measurement is the Limulus amoebocyte assay. This assay measures endotoxin in units of toxic activity as measured by the ability to clot the blood of Limulus (the horseshoe crab). Results from this assay are highly dependent on the lot of Limulus lysate being used, and the assay is affected by many factors, including materials that may be present in a sample. In general, this means that data from different laboratories cannot be directly compared and, even within a laboratory, data from different days or different batches of chemicals are difficult to compare. The ACGIH Bioaerosols Committee has proposed a relative standard for airborne endotoxin based on the Limulus assay that requires outdoor measurements for comparison. The comparisons are made on data produced by the same assay using the same lot of reagents.

Total lipopolysaccharides can be measured using gas chromatography. This method is stable and interlaboratory comparisons can be made. However, the method measures both toxic and non-toxic lipopolysaccharides. Since the non-toxic forms may be immunostimulants, this approach may actually be the most relevant in the long run. LPS is measured in concentration of the actual compound (e.g., nanograms/gram of dust). Endotoxin is measured in units of potency-endotoxin units (EU). In some literature, endotoxin concentrations are listed as nanograms (ng) or micrograms (ug)/ unit of measure. In these cases, either GCMS has been used to measure total LPS, or the authors are referring to units of the reference LPS standard used in the assay. However, because the type of lipopolysaccharide in the unknown sample may differ from the standard, EU is the preferred unit for Limulus assay data.

Sampling methods
Samples for endotoxin analysis can be collected from air or dust. For air samples, endotoxin-free membrane filters can be used. For dust, a variety of vacuum-based methods have been used with variable results. It is important to use the method by which any reference data that is to be used for comparison was collected.

Distribution in the environment

Outdoor air
Endotoxin is always present in outdoor air. Measured levels have ranged from 0.01-more than 5 EU/m³ of outdoor, with geometric means generally in the 0.02-0.5EU/m³. Endotoxin concentrations in outdoor air near farming activities can be much higher, reaching levels in excess of 1,000 EU/m³. In residential and other supposedly "clean" environments, measured levels of airborne endotoxin are rare. Concentration ranges for the few available studies are in the range 0.5-2,500 EU/m³, with the higher part of the range possibly indicating unusually high levels. Most residential measures are dust concentrations, which range from 8,000-250,000 EU/g of dust. Repeated measurements of endotoxin in residential dust tend to be correlated. The presence of a dog in the house tends to be strongly correlated with endotoxin concentrations in dust.

Most of the data on endotoxin exposure comes from occupational environments where organic material or water is used in the manufacturing process. In these types of environments, extremely high endotoxin concentrations have been measured. A summary of measured levels are listed in Table 1.

Table 1

Role in environmental investigations
In occupational environments where organic material is handled, or water is a part of the manufacturing process, airborne endotoxin measures are important and air samples should be collected in both the occupational environment and in outdoor air. There is a large amount of literature on such occupational exposures, and reference to the medical literature is an important component of interpretation. The National Library of Medicine is a free and very useful resource: www.pubmed.org.

There is little indication that endotoxin levels in residential and office environments are likely to be related to ongoing problems. Measuring endotoxin in house dust has been done often enough that there is reference data that can be used for interpretation of whether levels are low, moderate, or high. How the data is interpreted with respect to health is less clear.

Fungus of the month: Rhizopus
By: Dr. Payam Fallah

In past issues of the Environmental Reporter we have discussed fungal species that belong to a large class of fungi known as the Ascomycetes. The types of spores they produce are known as ascospores (spores produced sexually) and conidia (spores produced asexually). In this issue, members of a totally different and smaller class of fungi called the Zygomycetes are discussed. The Zygomycetes are distinguished from other classes of fungi by the production of sexual spores known as zygospores and by the production of asexual spores called sporangiospores The sporangiospores are usually produced in an enclosed capsule called a sporangium (sporangia- pl.). Each sporangium may contain up to 100,000 sporangiospores or as few as 50. Sporangiospores are the most abundant spores produced and are primarily disseminated by wind. Animals, insects, and rain can also contribute to dissemination. The sexual zygospores are large and considered to be resistant to environmentally adverse conditions therefore, they are not widely disseminated. Perhaps the most striking characteristic of this class of fungi is the presence of a coenocytic mycelium (a mycelium that lacks regular septation, i.e. no cross walls). This is an especially important characteristic of identification during direct microscopic examinations where mycelium from many different fungi may be present and there are few spores.

In indoor environments, one of the most common zygomycete genera detected is Rhizopus. The genus Rhizopus includes approximately 10 species, the most common of which is R. stolonifer. The identification of this species in culture or on direct exam is straightforward. The combination of rhizoids (root-like hyphae) and striations on sporangiospores are the most easily identified characteristics. The sporangiospores of this species occasionally appear on spore traps and, because of their relatively distinct shape and surface texture, an experienced analyst can identify the spores. Other members of the genus Rhizopus do not have this spore characteristic. Although experienced analysts can recognize Rhizopus stolonifer spores, counts of these spores tend to underestimate cultural recoveries.

In culture, Rhizopus species are usually very fast growing. Culture plates may be entirely covered with this fungus within 24 to 48 hours. The fast growth often causes problems with cultures because it may inhibit other fungi from growing in the Petri dish. Additionally, during analysis of the sample, colony counts and identification of other fungi may be difficult because the massive amount of mycelia from Rhizopus can substantially obscure and hide other colonies of fungi. The radial growth rate of Rhizopus (but not spore germination) is inhibited by high water activity media such as DG-18, which can be used in environments where Rhizopus is expected to be abundant.

Rhizopus species are common on wet fruits such as berries (the grey fuzz that spoils strawberries so quickly is often Rhizopus). The fungus is also capable of growth on a variety of other substrates including soil, grains, vegetables, and wallboard paper when sufficient water is present.

It is worth noting that some Rhizopus species are used to increase the digestibility of vegetable materials like soybeans, wheat, and rice. These materials are inoculated with Rhizopus, which partially digests the proteins and carbohydates. The resulting metabolites can produce a meat-like flavor. Miso, made in Japan from rice, and tempeh and sufu, made in Indonesia and China from soybeans, are foods produced by digestion with Rhizopus.

The data and other information contained in this newsletter are provided for informational purposes only and should not be relied upon for any other purpose. Environmental Microbiology Laboratory, Inc. hereby disclaims any liability for any and all direct, indirect, punitive, incidental, special or consequential damages arising out of the use or interpretation of the data or other information contained in, or any actions taken or omitted in reliance upon, this newsletter.

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