The C. sativa microbiome includes many microorganisms, the majority of which are not pathogenic to plants or people; however, there are several toxigenic, allergenic, and/or pathogenic microbial species that have been identified on cannabis plants and flower products [49, 50]. Therefore, these organisms and their metabolites are likely present in cannabis cultivation and manufacturing environments. Studies investigating airborne microorganisms in hemp and cannabis facilities have identified many types of potentially harmful bacterial and fungal microorganisms, including but not limited to Actinobacteria, Pseudomonas aeruginosa, Staphylococcus, Escherichia coli, Enterobacter, Enterococcus, Botrytis, Aspergillus, Penicillium, Cladosporium, Alternaria alternata, Trichoderma viride, Wallemia, Mucor, Rhizopus, Candida albicans, and Stachybotrys chartarum [2, 43,44,45,46,47,48].

One report estimated levels of airborne mold spores in cannabis facilities at > 500,000 spores/m3. When air samples were processed to enumerate airborne CFU, many samples overloaded culture plates, and culturable airborne microorganisms could not be quantified (highest reported as > 11,300 CFU/m3). These authors cautioned that elevated mold spore levels warranted respirator use in these environments [43]. Genetic identification of fungal species within cannabis cultivation air samples also showed elevated and enriched mold species typically associated with water-damaged homes [44, 45]. Notably, airborne levels of culturable bacteria and fungi regularly exceeded 1,000,000 CFU/m3 within a hemp facility, and workers were provided with respirators for their protection [2]. Although there is evidence that waste collection workers exposed to environmental pathogens are at risk for infectious diseases [51], it is poorly understood how exposure to bioaerosols found in cannabis facilities may cause infection in cannabis workers. Indeed, there is no established OSHA OEL for airborne spores or bacterial/fungal CFU despite the World Health Organization (WHO) categorizing “high” levels of airborne culturable fungal species as ≥ 1,000 CFU/m3 [52]. Of note, this WHO categorization is not uniformly accepted in many regulatory circles, and lower levels have been suggested for airborne pathogens and thermophilic actinomycetes as they may be harmful at lower abundance [52,53,54].

Elevated airborne fungi and bacteria levels in these environments could also contribute to non-pathogenic and allergenic pulmonary diseases within cannabis workers due to respiratory exposures to associated airborne microbial breakdown products, metabolites, and/or allergens, as discussed below [3,4,5,6,7,8,9,10,11,12,13].

Endotoxin, also called lipopolysaccharide (LPS), is a macromolecule composed of a lipid and polysaccharide that is found within the Gram-negative bacterial outer membrane. In addition to its functional attributes in bacteria, it can be a potent endotoxin that activates the mammalian immune system and causes inflammation, fever, and septic shock; moreover, endotoxin is an established inhalation hazard linked to inflammatory lung diseases [3, 9,10,11, 25, 55, 56]. Airborne endotoxin levels are measured as endotoxin units normalized by air volume (EU/m3); however, there is no established OSHA OEL for airborne endotoxin. Recently, the Dutch Expert Committee on Occupational Safety (DECOS) proposed an OEL of ≤ 90 EU/m3 over an eight-hour weighed average work period, which is used for reference here [57].

There are limited reports measuring airborne endotoxin in hemp and cannabis operations, but they show that endotoxin levels range widely depending on the operational activity and the manufacturing environment. The highest levels of airborne endotoxin in two hemp processing studies were 1,600 EU/m3 and 59,801 EU/m3, suggesting hemp workers can be exposed to extremely high endotoxin levels [2, 58]. Reports in cannabis operations have generally observed airborne endotoxin below the DECOS exposure limit; however, in two cases endotoxin levels were elevated to 85 or 94 EU/m3 during grinding of dried cannabis flower [1, 45, 46, 59].

Importantly, exposure to airborne endotoxin levels below the DECOS limit has been associated with respiratory symptoms and a decline in lung function [56]. This is also acknowledged in the CDC report investigating a fatal cannabis occupational asthma attack, as the authors state, “Airborne respirable dust and endotoxin levels below occupational exposure limits do not exclude a sufficient level of airborne allergen to trigger asthma and other allergic symptoms” [1].

Although airborne endotoxin is a classic industrial hygiene measure, there are a multitude of other airborne microbial metabolites, breakdown products, and associated bioactive agents that may contribute to pulmonary disease following respiratory exposure, several of which are known to contribute to inflammation independent of endotoxin levels [8, 9]. Like bacterial endotoxin, ergosterol is a cell membrane component, and it is important for regulating fungal membrane fluidity. Inhalation exposure to this fungal immunologically active lipid is associated with asthma, and within mammalian cells it can induce pyroptosis, a type of necrotic and inflammatory programmed cell death [8, 25, 60, 61].

Peptidoglycan acts as the backbone of bacterial cell walls. Both Gram-negative and -positive bacteria contain this structural element, but it is present at much higher levels in the latter, and thus is utilized as a surrogate measure of Gram-positive bacteria within dust and air samples. These molecules are recognized by the mammalian innate immune system, inducing a pro-inflammatory response linked to lung inflammation, specifically in response to inhaling organic dusts [9, 62, 63].

The structural element (1➔3)-β-D-glucan is a component of fungal cell walls that is also present in some bacteria and plants. Mammalian phagocytosing cell receptors bind (1➔3)-β-D-glucan molecules, and these fungal molecules elicit pro-inflammatory and cytotoxic responses by disrupting cell cycle signaling, inducing cell death, and causing oxidative stress in vitro. Although broader epidemiological studies are lacking, the presence of (1➔3)-β-D-glucan within occupant air has been associated with airway inflammation and asthma symptoms [26, 27, 64].

Fungal organisms can produce hundreds of mycotoxins, which are low molecular weight molecules that act as virulence factors and defense chemicals. These mycotoxins can cause adverse health effects in humans by inhibiting essential cellular processes, resulting in immunotoxicity, organ-specific toxicity, and cancer [65, 66]. Indeed, parallel agricultural, animal husbandry, and indoor environments have demonstrated that airborne mycotoxins contribute to respiratory diseases as well as “sick building syndrome” [4, 66,67,68]. Many molds associated with cannabis production have the capacity to produce multiple mycotoxins, including Alternaria, Aspergillus, Penicillium, Stachybotrys, Wallemia, and Fusarium species [36, 43,44,45, 66, 69].

To the authors’ current knowledge, there are no published studies investigating the abundance of airborne ergosterol, peptidoglycan, (1➔3)-β-D-glucan, or mycotoxins within cannabis work environments or their connection to cannabis worker respiratory disease.

Allergens are most commonly protein macromolecules that activate the mammalian adaptive immune system, initiating a Type I Hypersensitivity (IgE-mediated) response. This IgE response is considered a “classic allergic” response that differs from other pro-inflammatory responses activated by other microbial metabolites described above. Airborne allergen exposure usually causes mild inflammation of the eyes, nose, inner ear, throat, and airways, but these aeroallergens can also have significant health impacts, triggering asthma attacks and even causing anaphylaxis [22, 28, 70].

Because of its complexity, direct aeroallergen monitoring is not commonly performed, and allergen abundance is often indirectly evaluated through surrogates, such as measuring dust levels, culturing airborne bacteria and fungi, microscopic spore counts, or by DNA analysis for target organisms known to produce allergens. Moreover, many bioaerosol collection methods can denature the proteins they are meant to assess; thus, appropriate collection methods must be used to preserve allergen protein structure to allow for accurate immunoassay quantification [22, 28, 70].

To date, data directly identifying and measuring aeroallergens in cannabis facilities are lacking. However, there have been several documented cannabis work-related asthma cases where exposure to airborne mold or plant materials have been contributing factors [1, 14, 15]. Cannabis and hemp work environments can present relatively high levels of airborne protein, organic dusts, molds, and plant matter, suggesting that aeroallergens may be found within these bioaerosols [2, 43,44,45,46,47]. In similar agriculture and CEA environments, allergens and allergenic responses are linked to a diversity of other worksite sources, including spider mite pests, predatory mites, paper pots, and growing substrates like coco coir and rockwool [6, 71,72,73].

Of the fungi associated with cannabis and cannabis facility air, several are known to produce allergenic proteins, including Aspergillus, Penicillium, Cladosporium, Alternaria, Rhizopus, and Fusarium species [36, 43,44,45, 69, 74]. The opportunistic pathogen Aspergillus fumigatus was recently designated by WHO as a Critical Priority Fungal Pathogen and is known to produce several mycotoxins; furthermore, A. fumigatus also produces at least 30 known allergens listed on the WHO/IUIS (International Union of Immunological Societies) Allergen Nomenclature Database (http://www.allergen.org) [74,75,76]. This mold species exemplifies the diverse bioaerosol constituents that a single organism can produce, which may cause a wide variety of respiratory exposure responses. In wood milling operations, A. fumigatus allergens can be found in wood and wood-derived dust, presenting potential respiratory hazards to workers [77]. Indeed, some allergens are secreted by germinating A. fumigatus mycelium and are found associated with spores, so they are likely present on A. fumigatus-laden cannabis flower, on growing substrates, and/or building materials in cannabis manufacturing environments, potentially posing an inhalation risk to workers if aerosolized [78].

Studies of some hemp operations found a high prevalence of respiratory symptoms among workers exposed to respirable organic dust, where levels ranged from 10–80 mg/m3 [2,