Detecting Mycotoxins in the Cannabis Supply Chain

Moldy cannabis is much more than just an eye sore – it may be contaminated with dangerous mycotoxins.

Mycotoxins are secondary metabolites generated from molds that can negatively affect human health. Many mycotoxins are associated with kidney failure, carcinogenesis, respiratory illness, and immune system impairment. Acute overexposure to mycotoxins can even result in death.

The major mycotoxin classes responsible for contamination in cannabis biomass are aflatoxins and ochratoxins. Of the aflatoxin class, aflatoxin B1 is considered the most toxic. However, variants B2, G1, and G2 are also associated with serious health issues from chronic exposure. Aflatoxins are primarily produced by Aspergillus flavus and Aspergillus parasiticus, whereas ochratoxins are produced by Pencillum verucosum and Aspergillus ochraceus.

In a study of microbial contaminants sourced from cannabis samples in Massachusetts and Amsterdam in 2015, researchers found multiple strains of mycotoxic fungi, including Aspergillus, Cryptococcus, and Penicillium species. Aflatoxins have also been detected in both cannabis preparations and smoke.

Given the resistance of aflatoxins to thermal degradation, investigators have suggested that they may survive heat treatment and smoking processes. However, contemporary research into the presence of mycotoxins and mycotoxic fungi in cannabis remains limited and requires further studies.

Moldy grows

Because most biomass is processed soon after it has been harvested, there are few opportunities for molds to grow on cannabis.

“Once you’ve gotten rid of the water, the ability for mold to propagate is low,” Dr Justin Crumrine, chief scientific officer at cbdMD, a North Carolina-based CBD company, told Analytical Cannabis. “If there’s no water, Aspergillus and other molds are not going to colonize and propagate.”

However, if the plant material has been left out in the rain or kept in a high humidity environment during harvest, storage, or transportation, mold spores can easily take root and begin to proliferate.

“In general, the presence of mold is dependent on the geography from which you receive the biomass,” Dr Crumrine continued.

“Wet climates where a lot of hemp and cannabis is grown, such as the Willamette Valley of Oregon, have humid and wet fall seasons right before harvest. Excess rain in these regions contributes to poor air flow and high moisture, which is the perfect environment for mold and other microorganisms to start growing.”

Biomass that contains more than 14 percent moisture content is more susceptible to mold growth. Any plant matter that has been heavily contaminated with moist dirt or other mold-rich material can also become a breeding ground for mold.

Changing regulatory landscapes

The presence of mycotoxins in cannabis poses a challenge to the young cannabis industry. Any mycotoxin contamination could pose a serious threat to the health of cannabis users downstream, especially if inhaled. Inhaled mycotoxin contaminants are subject to minimal metabolism or degradation before they reach the circulatory system.

Case reports of fungal spore contaminants often derive from isolated reports and personal communications between growers. Currently, the health effects of inhaling heated mycotoxins from contaminated cannabis are yet unknown. Much research into mycotoxin toxicity comes from the food and agriculture industry, where mycotoxins are ingested or inhaled from dust particulates formed during the processing of cereals.

The United States Food and Drug Administration (FDA) imposes a limit on the level of mycotoxins in human consumables at 20 parts per billion. However, regulation of mycotoxins at the state level remains an ongoing challenge. As states across the country decriminalize the sale and production of cannabis products, they must create regulatory policies to the control for the levels of mycotoxins at different stages of production and distribution.

“A lot of CBD products are sold online, from one state to many other states through direct to customer sales,” said Dr Crumrine.

While products should comply to the mycotoxin regulations of the state where they are currently sold in and received, the logistical challenges involved in continuously testing products as they move across state lines makes enforcement difficult. But inconsistencies with standard practices and methodologies exist from state to state.

“There are potentially a ton of false positives and false negatives out there thanks to improperly selected methodologies,” said Dr Robert Brodnick, CEO at Titan Analytical, a California-based cannabis lab service provider. “Validation needs to be performed correctly.”

Many analytical laboratories are springing up to meet the growing needs of the fledgling cannabis industry. This evolving regulatory landscape requires ongoing validation and process development of detection methods as a once-illicit industry comes to the forefront. While many of these strategies have been adopted from the food, pharmaceutical, and agriculture industries, the cannabis fungal microbiome is unique from other consumable products and has not yet been well-studied.

The challenges of mycotoxins in cannabis processing

“Mycotoxin contamination is not likely to occur, but if it does, it’s going to shape the cannabis industry if it causes any damage to human health,” Rebecca Hobden, founder and CEO of ECC Test Lab, a Virginia-based cannabis lab, told Analytical Cannabis. “If something does get through and somebody gets hurt or sick, it’s going to be a huge detriment to the industry.”

Regulations do currently exist to screen for mycotoxins derived from fungal contamination in both recreational and medicinal marijuana. However, routine methods employed to assure product quality and safety are underdeveloped compared to those used by the food and pharmaceutical industries.

Indeed, the effects of the steps used to process cannabis products into concentrates, tinctures, distillates, and oils on mycotoxins are still unclear.

“If the mycotoxins are in the biomass, they’re likely to be in the finished product depending on your process,” Hobden said.

Direct solvent extraction could potentially concentrate soluble mycotoxins, leading to a more hazardous end-product. On the other hand, vacuum distillation of cannabis materials is unlikely to carry over mycotoxins as their boiling points are higher than CBD and THC.

“A lot of the industry is now using isolate, which is purified, recrystallized cannabinoids that come from distillate fractions with greater than 90 percent purity,” Dr Crumrine added.

The purification steps used in these highly refined cannabinoids are assumed to separate the desired components from fungal contaminants. However, further investigation is required to confirm the presence or absence of mycotoxins in cannabis end-products produced from different processing methods.

Immunochemical assays for mycotoxin detection

Enzyme-linked immunosorbent assays (ELISA) are the one of the simpler methods to detect mycotoxins in the food industry. Here, any mycotoxin present in a sample is immobilized to an antigen on a solid surface and then complexed with an antibody covalently linked to an enzyme. The substrate of the enzyme is added, which produces a visible signal that can be quantified to the level of mycotoxin in a sample.

While ELISA is relatively straightforward, these immunological assays have limited resolution. Proper sample preparation and dilution steps are necessary to ensure a sufficient concentration of mycotoxins is available for ligand binding.

“You’re looking for a needle in a haystack,” said Dr Brodnick. “The haystack needs to be a lot bigger if you want to find the needle. You’re probably missing the needle from the sample size.”

High-resolution analytical techniques for mycotoxin detection

According to Dr Brodnick, liquid chromatography coupled to mass spectrometry (LC-MS) is the gold standard for mycotoxin confirmation. LC-MS relies on the combined physical separation of molecules through a packed column under high pressure with the analytical capabilities of mass spectrometry. The technique requires the use of an internal standard to reduce potential interference and increase analytical accuracy.

Here, sample extraction and preparation remain a challenge. Matrices range from fresh marijuana plant material to medicinal oils and salves. Cannabis materials embedded in edible matrices, including baked goods, confectionaries and beverages, are also appearing on the commercial market. Inadequate dissolution of the mycotoxins in these heterogenous samples and interference from other matrix components can contribute to poor results.

“You have to account for the differences in matrices. You have to make sure you’ve got the dilution right. You have to make sure you have the right standards,” Dr Brodnick added.

The process of dilution and preparation require multiple steps that can limit the number of samples to be tested due to cost, time, and available instrumentation. Materials, validation, and expertise are also barriers for cannabis companies to completely rely on LC-MS. However, LC-MS is still one of the most powerful techniques available to ensure cannabis products are free from mycotoxins.

The future of mycotoxin detection

The current research literature regarding cannabis mycotoxins remains limited. Further studies are necessary to evaluate the types of mycotoxins produced from cannabis under varying environmental conditions and if they remain in processed materials at detectable levels.

Analytical methods used to detect mycotoxins in cannabis matrices must also be rigorously validated and adopted as standard practice across the United States. As the regulatory environments across states become more settled and unified, a codified repository for evaluating mycotoxins in cannabis products will become an important resource moving forward.

“We’re years away from having all the proper methods published and having that wealth of knowledge on hand,” said Dr Brodnick. “Right now, that’s going to be the hardest thing.”

This article was amended on February 4, 2021, to clarify a quote from Dr Justin Crumrine.