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The initial Colorado regulations are quite different than the Washington state ones. The list of mold species, in particular, appears to originate with a series of papers on Cannabis microbiology published by one of the co-authors of the present white paper (JW McPartland)7-10. They are all molds that have been isolated at one time or another from Cannabis plants. However, spores of these species are ubiquitous, and they were mentioned in these publications in the context of experimental studies that assessed or replicated poor storage conditions. Studies of contaminants of marijuana in the 1970s and 1980s primarily investigated cannabis smuggled from Latin America. Their relevance to current, domestically-produced cannabis is very limited. The product was sweat-cured, then compressed into bricks for smuggling, under conditions not controlled for temperature or humidity. These conditions gave rise to “storage molds” that are easily discernable and frankly unacceptable by today’s consumers. Some organisms reported in these studies, such as Mucor sp., thermophilic actinomycetes, and Dienerella arga, indicated a highly deteriorated condition. In another study that identified several of these mold species on Cannabis11, the authors analyzed street samples submitted by cannabis smokers, and isolated Aspergillus, Mucor, and Penicilliumspecies. The Cannabis in this study, as well, was likely smuggled into the country under inappropriate storage conditions. The study used nonselective culture media, which actually selects for the growth of fast-growing and ubiquitous fungi such as the Mucorand Penicilliumspecies that were found. No quantification of these molds was provided, and allergy testing of each of the Cannabis smokers with Mucorand Penicilliumantigens showed no greater sensitization
7amongst smokers than control subjects. At least one of these molds (Aspergillus) is a genus that does indeed contain species that are a health threat likely to be present on Cannabis. Aspergillusis ubiquitous in soil and on many plants, which means that the initial requirement in Colorado that there be “none detected” on Cannabis is not feasible. On the other hand, there are hundreds of Aspergillusspecies, and very few ofthese cause human disease, so a general test for Aspergillusis inappropriate. Another problem with requiring tests for these molds is that there is no adequate existing test that is specific to them. There is no selective mediaor commercial plate available that allows only these species to grow and be quantitated. Molecular methods have been developed for some of these species, but have not been generally commercialized. Non-commercial plate-culture methods do exist, but these require a trained mycologist to identify the mold species by eye in the presence of many different types of mold.Colorado has since modified its list of required microbiological tests, but it remains to be seen how they will approach many of these issues.
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Living Cannabis plants do not support high levels of bacterial growth, and pathogenic bacteria are unlikely to be associated with living Cannabis plants. There is also some evidence for anti-fungal activity of certain cannabinoids20, but fungal growth is not at all uncommon on Cannabis plants. Most of these mold and mildew species are plant pathogens, and not human ones; molds such as Botrytis cinereamay harm the Cannabis plant, but they are unlikely to harm humans.Nonetheless, mold is perhaps the single most important quality issue in Cannabis production. Outdoor plants are exposed to a wide variety of fungal species. Indoor plants are exposed to less of these, and can potentially be kept cleaner. Inpractice, however, many indoor plants are exposed to inappropriate watering, humidity, fertilizer, or ventilation conditions. All of these can contribute to very high levels of mold.Even under ideal conditions, it is possible that small numbers of cellsor spores capable of causing human disease may be present on plant material from contact with air, soil, or water. If any of these species are capable of replicating aggressively either on dried plant material or upon contact with humans, they could theoretically prove to be a threat.
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These are the ones that remain:* Bacteria resistant to low water-activity. Pathogenic bacteria that are extremely resistant to drying could potentially live on Cannabis, and be transferred to humans or to other items and then to humans. They would not survive the heat of smoking or decarboxylation, but they could nonetheless be carried into homes and come into contact with hosts through their presence on Cannabis. The only organism of concern in this categoryis Salmonella.* Fungal spores.These are extraordinarily resistant to heat, and could survive the heat of smoking or decarboxylation. These are not known to cause disease through the oral route, but the spores of certain species in the genusAspergilluscan enter the lungs, germinate, and cause invasive lung diseasein susceptible individuals.* Bacterial spores.In theory, these could pose a danger just as fungal spores might. Bacterial spores could survive on plant material or in infused edible products, and enter the lung or stomach. However, there are no such spores that pose a threat under the conditions Cannabis is subject to. This will be discussed below in the section dealing with Clostridium Botulinum.* Toxins. In theory, either bacterial or fungal toxins could be present on Cannabis because of the earlier presence of high levels of toxin-producing organisms. These could then be transferred to the lungs by smoking, or transferred into foods and delivered to the stomach. Alternatively, toxin-producing organisms could be present in food products and produce toxins there that remain a threat. We will deal with each of these possibilities below. In short, none of them are possible with Cannabis, because the conditions required for the high level of replication needed for toxin production are never available. In addition, the potential toxins of concern are all rapidly degraded rapidly by heat.
These are the categories that potential dangers could fall into. In the sections below we will cover each of the potential organisms or toxins that could mediate these threats. We have included only those that have plausible relevance, based on the public health histories of the food and agriculture industries. We know of no other microorganisms that should be of concern. Of course there are many other human pathogens we have not mentioned here, but they fall into the same categories as those that are safe or irrelevant.
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Fungal pathogens
Mold, mildew and yeast are all types of fungi. Mold in particular is very common on agricultural products. Certain types can grow on live plants; others, termed saprophytes, generally grow on dead plant material. Cannabis is host to many mold species of both types. The molds that are common on living Cannabis, such as Botrytis cinerea, are plant pathogens, not human ones130. Non-pathogenic molds can be a source of allergic hypersensitivity reactions131-133, but there is no evidence associating such reactions with smoking. As discussed above, a number of pathogenic mold species have been isolated from Cannabis kept under extremely poor conditions11,81. Spores of these species are ubiquitous, and Cannabis presents no special risk for fungal infections caused by them. However, certain molds of the genus Aspergillus do present a risk.
Aspergillus
Aspergillus is a mold that produces extremely hardy spores, and is capable of replication at much lower water activity levels than most organisms134-136. It is also ubiquitous; Aspergillus spores are though
to exist in soil and on plants essentially everywhere137-139. Gardeners and farmers in particular are believed to breathe in thousands of spores every day140-142. Under normal conditions, the human immune system removes these from the lungs143-146. In the immunocompromised, however, certain Aspergillusspecies can cause invasive lung disease147-156. Invasive pulmonary aspergillosis is extremely hard to diagnose and to treat, and the mortality rate is quite high157-162. In addition, there is a known clinical association between Cannabis smoking and pulmonary aspergillosis. Cannabis smoking is considered a clear risk factor for this disease, and there are many cases on record163-170. It appears likely that the spores can survive the heat of smoking and are mobilized by the smoking process and transferred to the lungs. In the absence of a healthy immune system, the spores can germinate and colonize the lungs.This is particularly significant in the case of the modern medical marijuanaindustry. Pulmonary aspergillosis is the one serious documented microbiological safety threat to Cannabis smokers. It usually takes hold only in the immunocompromised, but many medical marijuana patients have diseases –such as cancer or HIV infection –that result in damaged immune systems. In addition there is thought to be a dose effect171,172. Plant material that was improperly dried or handled and has higher mold levels could potentially present a higher risk.Aspergillusis ubiquitous, but the majority of Aspergillusspecies are not pathogenic. There are hundreds of species in this genus, and most of them are harmless. The species thought to be responsible for the vast majority ofcases of human aspergillosis are these: A. fumigatus, A. flavus, A. terreus, A. niger, and potentially A. nidulans147,162,173,174. A. fumigatusalone is likely responsible for about 75% of Aspergillusinfections in the U.S175. All four of these species should be tested for. Samples that test positive for any of them should be returned to the producer. Returned samples cannot easily be sterilized because of the hardiness of fungal spores, and they should not be sold. However, they can reasonably be used for concentrate production destined for edibles.Cannabis users should know the risks involved in smoking a substance that can contain viable Aspergillusspores. Those who are immunocompromised should be counseled to avoid smoking in general. Edible cannabis products are now widely available and will be safer for this population, as Aspergillussporesgenerally do notcause disease when ingested orally.It isimportant for legislators to understand that no data is available on the environmental burden of Aspergilluson Cannabis. When this data becomes available it may turn out that pathogenic Aspergillusspecies are quite rare on Cannabis. The opposite is more likely, however. It may be the case that A. fumigatus, in particular, is so common that all Cannabis samples (at least outdoor-grown varieties) contain some level of it. If this is the case, it will NOT make sense to require that all Cannabis be tested for Aspergillus. Healthy people have extremely high innate immunity to Aspergillus176, and there is no advantage in testing for ubiquitous organisms.However, choosing not to test for this pathogen could only be done in parallel with a concerted public health education campaign to alert immunocompromised patients to the danger of Cannabis-mediated Aspergillosis. There may also be some middle ground in which it would be reasonable to identify a threshold below which some A. fumigatusis acceptable if samples are clearly labeled with testing results. Nonetheless, given the information that is now available, we strongly recommend that all Cannabis be tested for A. fumigatus, A. flavus, A. terreus, and A. niger, and failed if positive for any of these.
Penicillium
Penicilliumis a genus of fungal mold species, and it has been isolated from Cannabis plants. It is ubiquitous on plants and in soil, and it is fast-growing and extremely likely to predominate on the agar culture plates used for fungal culture in the 1980s. Although one Penicilliumvariety is an opportunistic pathogen of immunocompromised HIV patients in Southeast Asia177,178, except in very rare cases179,180the genus is otherwise not a cause of human disease. MucorThis is a large genus of fungi containing over 3000 separate species181,182. As with Penicillium, they are ubiquitous, fast-growing, and very easy to recover on culture plates183,184. A very small number of these can cause human disease. This disease, known as mucormycosis, is extremely rare, and typically presents in non-immunocompromised patients only in cases where the spores are introduced to the body through “traumatic inoculation”185
Pulmonary forms of mucormycosis are know to occur, but usually only in patients with underlying hematological malignancy188-191, and these are not associated with increased inhalation exposure.Botrytis cinerea This is one of the most common fungal plant pathogens. It infects many different crops, but is particularly an issue with wine grapes192-195(where it is on occasion a positive influence) and Cannabis (where it is not). It does not infect humans, and although allergic hypersensitivity reactions to it have been described, they are only two reports of it in the existing scientific literature196,197.Microbial toxinsAflatoxinsAflatoxins are a variety of mycotoxin produced mainly by two species of Aspergillus (A. flavus, andA. parasiticus)198,199. Because Aspergillusis ubiquitous, aflatoxins are as well, and many industries have set baseline levels for acceptable amounts of aflatoxin contamination200-203. However, the conditions necessary for the production of significant levels of aflatoxin are not present on Cannabis. In order for aflatoxin production to occur, Aspergillusmust initiate a successful colonization of some substrate that supports hyphae production and robust replication204. Aspergillusis a saprophyte, meaning it commonly grows on dead and decomposing plant matter205-207. It can also grow on living plants, but it requires high levels of oils and other nutrients for robust growth and aflatoxin production208-210. The agricultural crops capable of fulfilling these conditions are high-oil-content seeds, and certain grains and nuts208. Aspergillusreplication on Cannabis would be possible only on extremely moldy post-harvest plant material, or on the seeds themselves. Because Cannabis flowers sold indispensaries today are produced entirely from un-fertilized female plants that do not produce seeds, this is not a concern. In addition, even with permissive nutrient sources, aflatoxin production is halted at low water activity levels211-213. This is the case as water activity approaches Aw 0.9. Cured Cannabis is much dryer than this, typically under Aw0.6.
Data do exist that could be interpreted to indicate a risk posed by aflatoxins on Cannabis. For instance, Aspergillusflavusis extremely widespread in soil, and some plants that cannot support Aspergillusgrowth are still capable of aflatoxin uptake from the environment214. There is no evidence that this is likely to lead to aflatoxin levels above established international exposure thresholds, and there is no evidence that it takes place in Cannabis. Another possible concern is that the heat applied to Cannabis during smoking or decarboxylation would not remove all aflatoxins. The aflatoxin molecule is somewhat heat-labile; it is degraded by exposure to heat levels above 160°C215,216. But decarboxylation and smoking temperatures are not always this high, and though they would lead to some degradation of aflatoxin, it would not be complete. Finally, Cannabis seeds have high oil content, and they would not be an unusual host for Aspergillus. There is a reasonable concern that Aflatoxins may be present in hemp seed products. It is also true that the hermaphroditic tendency of the Cannabis plant sometimes leads to the occasional seed in commercially sold Cannabis flowers, and these could potentially be colonized be Aspergillus.Taken together, these concerns do not warrant batch testing of all commercial Cannabis for aflatoxins.The presence of detectable levels is highly unlikely, and after many decades of popular use, aflatoxin poisoning has never been linked to Cannabis use. Aflatoxins have been mentioned as a concern with respect to Cannabis, because aflatoxins do contaminatemany other food products, and because Aspergillus itself is clearly a concern. But in this case it is invasive fungal disease that is a threat, rather than aflatoxin poisoning. Only one paper in the existing literature describes the isolation of aflatoxinfrom Cannabis217. In this study, the authors themselves added large amounts of Aspergillusflavus and Aspergillusparasiticus to Cannabis confiscated by police. They mixed the Cannabis with the Aspergillusin large amounts of water, and assessed aflatoxin production 14 days later. They then reported levels of aflatoxin production that were extremely low compared to other growth substrates. It is worth pointing out that cannabinoids have been found to have strong antifungal properties20,218, and that the Cannabis used in this 1977 paper had a THC content of 1.5%.
