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The world of fungi, mold, and mycotoxins is explored and discussed.
Mold and fungi are found everywhere in the world. Due to their size and their spore’s mobility, fungi can spread throughout crops and food stores. When the conditions of humidity and temperature are favorable, the fungi can proliferate into colonies that can either destroy crops by consuming them or poison them with toxic secondary metabolites called mycotoxins. Just like some of the other potential agricultural contaminants (that is, pesticides and heavy metals), fungi, molds, and mycotoxins also must be of concern to the cannabis industry as a factor in product safety and quality. In this column, the world of fungi, mold, and mycotoxins is explored and discussed as they relate to the cannabis industry and the future of analytical testing.
The complexity and range of products produced by the cannabis industry makes it unique. The product range encompasses medicines, recreational agents, and food products. The industry has to worry not only about health effects of an inhalable product but also the effects of a medical preparation and food safety. The exposure risks of contamination from chemicals and microbials must be taken into consideration as the mode of ingestion of inhalation is used. The traditional industries of agriculture, food production, tobacco, and medicinal supplements all battle with contamination issues.
The traditional tobacco industry and now the vaping industry have been the object of scrutiny and testing, which has often showed significant levels of potentially dangerous contamination by chemicals and microbials (1). In the same vein, a large percentage of medical cannabis was found to be contaminated by some type of microbial organism in a study from UC Davis (2). In some cases, these microbial organisms were the cause of a patient’s death.
The food industry also struggles in the battle with contaminants. In the United States, it is estimated by the Centers for Disease Control and Prevention (CDC) that 48 million people get sick from foodborne illnesses and up to 3000 die from foodborne diseases (3). More than 250 agents are known to cause foodborne illness and are introduced through contamination, improper handling practices, and sanitation. These agents can be chemical, physical, or biological.
Biological contaminants are by far one of the greatest concerns for illness. Microbes are everywhere and can be beneficial or cause illness and death. The five types of microbes are bacteria, viruses, parasites, protozoa, and fungi. Fungi are a very diverse kingdom of organisms (single and multicellular) which once were considered plants. In the past, the study of fungi was a branch of botany. Now it is known that fungi are more closely related genetically to animals than plants. Fungi are nonphotosynthetic and must obtain nutrients from organic matter. Fungi that derive nutrients from decaying or dead matter are known as saprophytes; while a small percent of fungi derive nutrients from living organics and are called parasites. There are between 70,000–100,000 known species of fungi and possibly an estimated 3.8 million species in total (4).
The classification of Kingdom Fungi is constantly being debated with the influx of DNA data. Currently the kingdom contains seven phyla (Table I) (5), which span the different forms of fungal organisms from single-celled yeasts to multicellular mushrooms.
Many species of fungi produce biologically active compounds that are used in food production and medicine-where would the world be if we did not have yeast for bread or penicillin. But, there are also many toxic compounds produced by fungi.
The classical thinking of toxicity from fungus is of the mushroom hunter out in the forest foraging for wild mushrooms. The typical mushrooms most of us think about are from the phylum Basidiomycota or club mushrooms. Toxic members of these phylums are infamous such as the Death Cap and Skull Cap. They produce secondary metabolites like amatatoxins, phallotoxins, and ergotamines that can easily be avoided by just not picking and eating those mushrooms. The more insidious toxic fungi are from the phylum Ascomycota, which include the molds, yeasts, mildew, and so on. These fast spreading and fast growing fungi are the plague of agriculture. These fungi produce the most common mycotoxins associated with food contamination.
Mycotoxins are organic compounds and secondary metabolites produced by fungus that are capable of causing illness and death. Secondary metabolites are not needed for the normal life cycle of the organism, in many cases the reason for their production is unknown (6). One species of fungi may produce different mycotoxins and some mycotoxins are produced by multiple types of fungi (see Table II). Most of the major mycotoxins of concern in human beings come from a few dozen species from the phylum Ascomycota or the sac fungi.
Cannabis as a crop is particularly prone to the growth of mold and fungi because of cultivation conditions, some of the most common forms of mold are powdery mildew and bud rot. Late harvests can contain significant amounts of water which will increase rot. Cannabis can also be contaminated at many points of harvest and processing, exposing the product to dangerous mycotoxins. Pests can flourish in storage facilities and delays between harvesting and drying can increase mold damage. The amount of drying of the cannabis can affect the potential for mold growth. Plant materials with more than 14% moisture can encourage mold growth. Some mycotoxins, especially aflatoxins and ochratoxins, need oxygen to grow so the reduction of the oxygen in the storage areas can retard growth.
There are dozens of potential contaminants in cannabis including all of the toxins discussed in previous columns (7,8). However, mycotoxin contaminants can be some of the most dangerous contaminants. Toxic and lethal dosages can be quite small for acute poisonings. Aflatoxins: B1, B2, G1 and G2, and ochratoxin A are often of the most concern. Ochratoxin A is has a tolerable daily intake designated by the World Health Organization (WHO) of 5 ng/kg of body weight per day. Ochratoxin A is very toxic with a “Lethal Dose, 50%” (LD50) of 20–25 mg/kg of body weight. WHO recognizes products containing more than 1 mg/kg of aflatoxins as potentially dangerous or life threatening. The U.S. Food and Drug Administration (FDA) has limits for mycotoxins in human and animal feed up to 20 µg/kg for direct human exposure. In the United States, each state with cannabis regulations in place either follows a plan similar to the FDA limit of 20 ppb, but they apply it using various methods. Many of the states recognize and monitor the top five dangerous mycotoxins discussed previously. In states like Maryland and Illinois, each mycotoxin must measure less than 20 ppb. In New Mexico, all five mycotoxins together must be less than 20 ppb. Then, there are states such as Colorado which take an approach in between where the aflatoxins (B1, B2, G1, and G2) must total less than 20 ppb and ochratoxin A must be less than 20 ppb (9).
The analysis of mycotoxins in products has a lot of challenges. First, molds and fungus are ubiquitous to the environment and agricultural products. The amount of exposure changes with weather, growing conditions, agricultural practices, harvest, and storage conditions. The toxins can be distributed heterogeneously in a batch or harvest. The location and concentration of the toxins can vary greatly. The analytical targets, as we have discussed previously, are very low in the parts per billion ranges. The approach to analysis can be gaged either to screening or targeted analysis.
The simplest types of test are qualitative tests which provide a yes or no answer as to if a toxin is present. Samples are placed in test tubes, well plates, or dipped with testing strips for a cost effective answer if that particular sample tests positive for mycotoxins. For these types of tests, simple colorimetric chemical reactions are employed using a test tube or container with reactant or lateral flow material with reactant that color change in the presence of target analytes.
A second type of test is quantitative. These types of tests can range from quantitative test strips or methods to more complex and sensitive instrumental analysis. In all of these tests, the samples must be extracted and the resulting liquid becomes the testing matrix. Simple quantitative tests like testing machines and strips use readers that are able to calculate chemical changes and equate those changes to a quantitative measurement sometimes as low as parts per billion levels.
In many cases, testing laboratories prefer higher throughput of analysis and use more advanced techniques such as fluorometry, chromatography, and mass spectroscopy. Fluorometry is the study of the visible spectrum of fluorescence. The fluorimeter measures the intensity and wavelength distribution of an emission spectrum after excitation by light. Molecules that undergo fluorescence can be measured accurately and to low levels in the parts per trillion range using a fluorimeter. Many mycotoxins (B1, B2, G1, G2) at one time were detected by black light under which they would glow showing their fluorescence. In some mycotoxins they can be treated with a binding agent to become fluorescent and measurable by fluorometry. The first step of testing for mycotoxins with a fluorimeter is to isolate the mycotoxins from the rest of the extracted material using some method of separation like an immunoaffinity or other chromatography columns. Immunoinfinity columns use monoclonal antibodies to isolate target analytes by containing them in the column until they are washed with the proper solvent and concentrated into an extract. Other chromatography columns use substrates that select for size, chemistry, and polarity to retain analytes until the time they are washed into an extract.
Mycotoxin testing is often conducted in conjunction with other types of cannabis testing such as for terpenes, pesticides, and potency. In these multiple target methods, more complex analysis methods are used such as liquid chromatography–mass spectroscopy (LC–MS). These systems can test for multiple targets, but need a high level of expertise to run and are often costly to purchase and maintain unlike simpler test methods.
Cannabis, like every other agricultural product, is plagued by a variety of pests and contaminants. The agricultural practices and processes in the cannabis industry can lead to increased exposure to mycotoxins. There are significant health concerns regarding mycotoxin contamination of products that are not only processed into edible foods, but also used as inhalation products and medicines for children and patients with compromised immune systems. It therefore becomes critical to understand the nature of fungi and mycotoxins and how to reduce them in cannabis products.
Patricia Atkins is a Senior Applications Scientist with SPEX CertiPrep and a member of both the AOAC and ASTM committees for cannabis.
P. Atkins, Cannabis Science and Technology2(6), 20-23 (2019).