Contaminants of Concern in Cannabis:
Microbes, Heavy Metals and Pesticides
John M. McPartland and Kevin J. McKernan
Abstract Microbiological contaminants pose a potential threat to cannabis con-
sumers. Bacteria and fungi may cause opportunistic infections in immunocom-
promized individuals. Even dead organisms may trigger allergies and asthma.
Toxins from microbial overloads, such as Shigla toxin and aﬂatoxins, may pose a
problem—unlikely, but possible. The Cannabis plant hosts a robust microbiome;
the identiﬁcation of these organisms is underway. Cannabis bioaccumulates heavy
metals in its tissues, so avidly that hemp crops have been used for bioremediation.
Heavy metals cause myriad human diseases, so their presence in crops destined for
human consumption must be minimized. Pesticide residues in cannabis pose a
unique situation among crop plants—the Environmental Protection Agency
(EPA) will not propose pesticides guidelines, because Cannabis is illegal on the
federal level. The use of illegal pesticides is a rising crisis, and a breakdown in
ethics. Testing for pesticide residues and maximal limits are proposed.
Cannabis (the plant) and cannabis (the plant product) may be contaminated by
microbes, heavy metals, or pesticide residues. The ﬁrst two contaminants, microbes
and heavy metals, present a Janus-face or “ﬂip-side of the coin”in relation to
Cannabis. Some bacteria and fungi are part of the plant’s microbiome. They pro-
vide beneﬁts to Cannabis. See the book chapter by Parijat Kusari and Oliver Kayser
for more about the Cannabis microbiome. On the “ﬂip-side of the coin,”other
bacteria and fungi cause disease, and these must be controlled.
J.M. McPartland (&)
GW Pharmaceuticals place, 1 Cavendish Place, London W1G 0QF, UK
Courtagen Life Sciences, 12 Gill Street Suite 3700, Woburn, MA 01801, USA
©Springer International Publishing AG 2017
S. Chandra et al. (eds.), Cannabis sativa L. - Botany and Biotechnology,
Heavy metals are harmful to humans, and these contaminants must be mini-
malized in cannabis destined for human consumption. Cannabis pulls heavy metals
from soil with great efﬁciency. Therein lies a second Janus face: the plant has great
potential as a tool for bioremediation. Bioremedial plants extract pollutants from
soil and accumulate the pollutants in their tissues, for harvesting and removal.
Pesticide residues have no “ﬂip-side of the coin,”they are just bad. Growth in
the cannabis industry, from outdoor hippie gardens to indoor commercial ware-
houses, has multiplied pesticide usage. Pesticide regulation in the USA is primarily
a responsibility of the Environmental Protection Agency (EPA). The EPA will not
register pesticides for use on Cannabis or set tolerance levels because the crop is
illegal on the federal level (Stone 2014). For that same reason, no cannabis can be
labeled as “Organic”by the USA Department of Agriculture.
This chapter focuses on microbes, heavy metals, and pesticide residues in can-
nabis inﬂorescences and seed oil. Other contaminants exist, such as butane residues
in cannabis extracts. For these, the reader is directed elsewhere (Upton et al. 2013;
Farrer 2015). Adulterants—deliberately added contaminants—are a separate issue,
particularly hashish diluents and psychoactive adulterants (Bell 1857; Dragendorff
and Marquis 1878; Indian Hemp Drugs Commission 1894; Perry 1977; Wilson
et al. 1989; McPartland and Pruitt 1997; McPartland 2002; Caligiani et al. 2006;
McPartland et al. 2008; Busse et al. 2008; Venhuis and de Kaste 2008; Scheel et al.
22.2 Microbial Contaminants
Cannabis is often characterized as a “disease-free”crop. In fact, a plethora of plant
pathogens attack the plant. At least 88 fungal species cause diseases in Cannabis
(McPartland 1992), as do eight pathovarieties of plant pathogenic bacteria
(McPartland et al. 2000). Some phytopathogens are unique to Cannabis
(McPartland 1984), and some organisms are ubiquitous. The most threatening
diseases of ﬂowering tops are caused by three ubiquitous fungi—Botrytis cinerea
(the cause of gray mold), Trichothecium roseum (white mildew or pink rot), and
Alternaria alternata (brown blight).
Phytopathogens cannot infect humans, except perhaps immunocompromized
individuals. Opportunistic infections by A. alternata have been reported in patients
receiving chemotherapy, recent organ transplant patients, and people with AIDS.
Airborne conidia (spores) of B. cinerea and A. alternata cause mold allergies and
asthma, particularly in greenhouse workers (Jurgensen and Madsen 2009).
From a consumer perspective, a separate population of bacteria and fungi is of
greater concern than phytopathogens: post-harvest storage microbes (McPartland
1994a). Storage organisms are saprophytes, rather than pathogens. They can only
invade dead plants after harvest. Fungi are the primary cause of storage contami-
nation. They thrive under low oxygen levels, limited moisture, and intense com-
petition for substrate.
458 J.M. McPartland and K.J. McKernan
The spectrum of post-harvest storage fungi has changed in the past 40 years.
Most black-market cannabis available in the 1980s came from Latin America. It
was “sweat cured”by drying herb in a pile, covered by cloth. Heat arising from
fermentation quickly cured the product, but allowed storage organisms to gain a
foothold. Then the cannabis was compressed into bricks for smuggling, and stored
under ambient humidity and warm temperatures. Under these conditions, fungi
from four genera commonly contaminated the product: Aspergillus, Penicillium,
Rhizopus, and Mucor (Fig. 22.1).
Kagen et al. (1983) isolated three worrisome Aspergillus species from marijuana:
A. niger, A. fumigatus, and A. ﬂavus. Schwartz (1985) scraped an aspergilloma
(“fungus ball”) caused by A. niger from the sinuses of a marijuana smoker suffering
severe headaches. Llamas et al. (1978) implicated A. fumigatus-contaminated
marijuana in a case of bronchopulmonary aspergillosis. Aspergillosis is an invasive
disease, unlike an aspergilloma. It usually stays localized (e.g., a pneumomycosis)
but sometimes becomes systemically disseminated. Chusid et al. (1975) reported A.
fumigatus causing near-fatal pneumonitis in a 17-year old. They noted that the
patient buried his marijuana in the ground for “aging.”Penicillium, Rhizopus, and
Mucor have also been cultured from moldy cannabis (Kagen et al. 1983; Kurup
et al. 1983; Bush Doctor 1993).
Mycotoxins produced by fungi are hepatotoxic, nephrotoxic, and carcinogenic.
Ochratoxins, citrinin, and patulin are produced by Aspergillus and Penicillium
species. Paxilline is produced by Penicillium paxilli. Trichothecenes gained noto-
riety for their reputed use in biological warfare (“yellow rain”). Trichothecenes are
Fig. 22.1 Common storage fungi in the 1980s. From left to right:Rhizopus stolonifer, Mucor
hiemalis, Penicillum chrysogenum, P. italicum, Aspergillus ﬂavus, A. fumigatus, and A. niger.Top
row sporophores cross-sectioned to reveal internal structures (400x). Bottom row natural habitat
(25x). From McPartland (1989), reprinted with permission
22 Contaminants of Concern in Cannabis …459
produced by Fusarium oxysporum, a biological control fungus deployed against
illegal Cannabis cultivation (McPartland and West 1999). Aﬂatoxins are the most
Aspergillus species (A. ﬂavus, A. parasiticus) produce aﬂatoxins in warm and
humid conditions—optimally 33 °C (91.4 °F), and 0.99 water activity. Aﬂatoxins
are acutely poisonous as well as carcinogenic. Llewellyn and O’Rear (1977)
identiﬁed aﬂatoxins in cannabis, but under artiﬁcial conditions. They added 15 ml
water to 5 g pulverized ﬂowering tops, autoclaved the material, and inoculated it
with A. ﬂavus or A. parasiticus. After 14 days at 25 °C (77 °F), the fungi sporu-
lated and produced “moderate”amounts of aﬂatoxins. Importantly, no studies have
reported aﬂatoxins in cannabis under normal storage conditions (McPartland and
Kurup et al. (1983) isolated three thermophilic actinomycetes from questionably
sourced material, Thermoactinomyces candidus, T. vulgaris, and Micropolyspora
faeni. These endospore-forming microbes cause “farmer’s lung,”which is a
hypersensitivity reaction rather than an infection.
Turning to bacteria, Ungerleider et al. (1982) cultured several members of the
Enterobacteriaceae from NIDA-sourced cannabis—species of Klebsiella,
Enterobacter, and Enterococcus (group D Streptococcus). It should be noted that
NIDA marijuana at that time was sweat cured by placing harvested material on
concrete ﬂoors (B. Thomas, pers. commun. 1999)—an unacceptable method today.
A disease outbreak caused by another member of the Enterobacteriaceae—
Salmonella muenchen—was associated with cannabis (Taylor et al. 1982). The
investigators concluded that the plant material, sourced from Mexico, was con-
taminated or adulterated by untreated manure—another unacceptable method today.
Some of these organisms, particularly Rhizopus, Mucor, and thermophilic acti-
nomycetes, reduce cannabis to a deteriorated state that is no longer acceptable by
today’s consumers. The product is dark brown, crumbly, smells musty or moldy,
and produces a brown or sooty smoke (McPartland et al. 2000). Although methods
of sweat curing are still promoted on websites, today’s product is carefully air dried,
often vacuum-sealed (sometimes under nitrogen), and stored in cold, dry condi-
tions. This process maintains potency and also prevents the growth of storage
Here in the 21st century, Aspergillus- and Penicillium-contaminated cannabis
still poses a problem (Rechlemer et al. 2015; Cescon et al. 2008; Szyper-Kravitz
et al. 2001; Verweij et al. 2000). Martyny et al. (2013) sampled grow operations in
Colorado for airborne fungal spores. Aspergillus and Penicillium spp. predominated
indoors, and Cladosporium spp. predominated outdoors. Cladosporium may be an
emerging problem; this fungus also infests hemp mills (McPartland 2003). About
1% of cannabis supplies received by Harborside Medical Cannabis Dispensary in
Oakland, California were returned to vendors because of unacceptable levels of
Aspergillus contamination (DeAngelo 2010).
460 J.M. McPartland and K.J. McKernan
22.3 Microbial Testing
The Ofﬁce of Medicinal Cannabis in the Netherlands initiated microbial testing
(Hazekamp 2006,2016). Bedrocan BV, the primary supplier of medical cannabis in
the Netherlands, tests harvested plants as well as ﬁnal packaged products. They use
two petri plate-based screening tests recommended by the European Pharmaopoeia—
one for total aerobic microbial count (TAMC), the other for total yeast and mold
count (TYMC). Degree of contamination is quantiﬁed by counting the number of
colony-forming units arising from one gram of plated cannabis (CFU/g). They placed
upper limits of <100 CFU/g for TAMC, and <10 CFU/g for TYMC—which is close
to sterility. Certain speciﬁc pathogens must be completely absent—Staphylococcus
aureus,Pseudomonas aeruginosa, and bile-tolerant Gram-negative bacteria such as
Escherichia coli. Furthermore, the absence of fungal mycotoxins must be conﬁrmed
by additional quality control testing (Hazekamp 2016).
Health Canada (2008) mandated similar tests, with different upper limits:
<100 CFU/g for TAMC, and <100 CFU/g for TYMC, as well as speciﬁc tests for
Coliform bacteria (<3 MPN/g), and E. coli (absent). Their upper limit for aﬂatoxins
B1, B2, G1, G2, and ochatoxin A is <20 µm/kg cannabis.
In the USA, medical cannabis was ﬁrst legalized by California in 1996.
Microbial testing was not mandated until 2011, when New Jersey instituted sample
testing for pests, mold, mildew, heavy metals and pesticides, and the certiﬁcation of
“organic”medical cannabis (NJMMP 2011).
The American Herbal Pharmacopoeia (AHP) issued speciﬁc protocols for
microbial testing (Upton et al. 2013). The AHP’s protocols were based on tests for
commodity food products issued by the EPA and the Food and Drug
Administration, as well as assays for cannabis used in Holland (Hazekamp 2006).
The tests consist of a series of petri plate- or ﬁlm-based assays for bacterial, yeast,
For orally consumed cannabis, the AHP recommended four tests: (1) total yeast
and mold count, (2) total coliforms, (3) Escherichia coli, (4) Salmonella spp. In
addition, they recommended immunochemical methods to screen for aﬂatoxins. For
products to be inhaled, more stringent tests were recommended: (1) total yeast and
mold count, (2) total aerobic count, (3) bile-tolerant gram-negative bacteria,
(4) E. coli and Salmonella spp., and aﬂatoxin assays. The AHP proposed speciﬁc
limits in CFU/g counts, but emphasized that these values did not represent pass-fail
criteria. Rather they were recommended levels when plants are cultivated and
harvested under normal circumstances.
The states of Colorado and Washington issued speciﬁc testing protocols,
reviewed by Holmes et al. (2015). Colorado’s list of fungi required for testing was
based on publications from the 1980s, including some species that may not be
relevant to current, domestically-produced cannabis. Washington’s protocols were
adopted from the AHP. Holmes et al. (2015) criticized the use of screening tests,
noting they are based on guidelines for food product facilities (and not necessarily
the testing of end products). Some of the tests are quite outdated (e.g., bile-tolerant
22 Contaminants of Concern in Cannabis …461
gram negative bacteria). Furthermore, anonymous CFU/g counts do not identify
relevant pathogens, or the threat of fecal contamination. Instead Holmes recom-
mended testing herbal cannabis for speciﬁc pathogens: Escherichia coli, Salmonella
spp., and four species of Aspergillus:A. ﬂavus, A. fumigatus, A. niger, and A.
In 2015 Colorado changed its testing regimen: (1) total yeast and mold count
CFU/g), (2) Salmonella (limit <1 CFU/g), (3) Shiga-toxin producing
E. coli (STEC, limit <1 CFU/g). Colorado recommended testing for three species of
Aspergillus:A. ﬂavus, A. fumigatus, and A. niger, although this was never
Aspergillus is a large genus with 250 species, and separating three speciﬁc
species from the others is not easy. Traditionally, identiﬁcation required culturing
on Aspergillus-selective plating media, and morphological measurements by a
specialist (Samson et al. 2004). Due to the challenges associated with
species-speciﬁc detection, Colorado changed their testing requirements again in
2016, to a 10,000 CFU/g total yeast and mold test, but left in place single CFU/g
testing for E.coli and Salmonella spp.
Microbial tests that require CFU/g detection are prone to sampling bias, since the
cannabis sample (usually 250 mg to 1 g) is usually wetted with 3–4 ml of Tryptic
Soy Broth (TSB), a general purpose culture medium. This large volume cannot be
placed into a given petri dish, PCR reaction, or culture based detection device. Thus
a subsample of the large volume is taken after a deﬁned growth time (termed
enrichment) to accommodate for the subsampling.
Because of these difﬁculties, and to accelerate testing turn-around time, some
laboratories now use quantitative polymerase chain reaction (qPCR) assays. This
method detects DNA sequences in cannabis samples. Primers for 18S rDNA ITS
(Internal Transcribed Spacer) are particularly useful for identifying speciﬁc
The drawback to qPCR is the method’s indifference to living or non-living
DNA. To accommodate this, an enrichment step is performed, where the cannabis
samples are incubated overnight in TSB broth prior to qPCR detection. Overnight
growth in TSB ensures only live organisms are measured, but raises questions over
preferential culture conditions for broader total yeast and mold tests. To address this
conundrum, some labs perform a qPCR on total yeast and molds, and positive
results are conﬁrmed with an additional test extracted 24 h later to ensure the signal
from the pre-incubation test was from live organisms.
McKernan et al. (2015) compared results between qPCR and three petri plate- or
ﬁlm-based detection systems: 3 M Petriﬁlm™, Simplate-Biocontrol Systems™,
and BioLumix™. They tested 17 dispensary-obtained cannabis samples. Six sam-
ples tested positive with the qPCR assay, ﬁve samples tested positive with the
Biocontrol Systems™assay (>10,000 CFU/g), four samples test positive with the
3 M Petriﬁlm™assay (>10,000 CFU/g), and only one sample tested positive with
the BioLumix™assay, which is a simple pass-fail test.
McKernan and colleagues then subjected ITS amplicons to DNA sequencing, to
identify speciﬁc fungi. All three Aspergillus species on the bad list turned up:
462 J.M. McPartland and K.J. McKernan
A. ﬂavus (one sample), A. fumigatus (one sample), and A. niger (three samples).
Twelve other Aspergillus/Emericella species were detected: A. candidus, A.
ostianus, A. sepultus, A. sydowii, A. tamari, A. terreus, A. versicolor, E. rugulosa,
E. nidulans, E. ﬁlifera, E. repens, E. bicolor. Two of these produce toxins, A.
versicolor and A. terreus.
ITS amplicons identiﬁed 17 Penicillium species. The most common fungus was
P. paxilli, surpassing all Aspergillus species. This species has not previously been
reported in association with Cannabis or cannabis. P. paxilli produces paxilline
toxin, so McKernan and colleagues conﬁrmed its presence with PaxPss1 and
PaxPss2 DNA primers. Paxilline has been shown to decrease the antiseizure ben-
eﬁts of cannabidiol in a mouse epilepsy model (Shirazi-Zand et al. 2013).
Although Holmes et al. (2015) questioned the need to test cannabis for E. coli,
Listeria spp., and Pseudomonas aeruginosa, McKernan (unpublished study 2016)
has identiﬁed several Pseudomonas species in cannabis with DNA testing. The
most dangerous pathogen, P. aeruginosa, was not seen. The array of organisms that
need to be screened is not yet formalized.
Screening herbal cannabis for moisture content (MC) is another approach. Bush
Doctor (1993) and McPartland et al. (2000) recommended drying herbal cannabis
to 10–12% MC. Fungi and bacteria cannot grow below 15% MC. Herb dried below
10% MC becomes brittle and disintegrates easily. Hazekamp (2006) recommended
5–10% MC. The AHP monograph recommended not more than 15% MC (Upton
et al. 2013). Holmes et al. (2015) used water activity (a
) as a metric; a
the partial vapor pressure of water in a substance. The a
of pure distilled water
equals 1.0. Bacteria usually require a minimum of 0.9 to grow, and fungi require a
minimum of 0.7. Holmes and colleagues recommended a maximum a
of 0.65 for
herbal cannabis, approximately 13% MC.
22.4 Microbial Harm Reduction
Prevention is the best strategy to avoid microbial contamination. Growers must
harvest disease-free Cannabis. This book’s chapter by David Potter discusses GW
Pharmaceutical’s methods of growing healthy Cannabis—by controlling humidity,
using biological controls and natural predators, and without resorting to pesticides.
The use of pesticides is addressed below.
To kill microbial contaminants in medical cannabis, Ungerleider et al. (1982)
Co gamma rays, a dose of 15,000–20,000 grays. Dutch and
Canadian medical cannabis is treated with 10,000 grays (Hazekamp 2006; Health
Canada 2008). Microbial counts in Dutch cannabis are tested before and after
irradiation, because “bad”quality cannabis should not be rescued by irradiation
(Hazekamp 2016). In comparison, packaged meat and poultry may be irradiated
with up to 70,000 grays. Gamma radiation remains controversial—it may destroy
terpenoids, and it does not destroy mycotoxins (Lucas 2008).
22 Contaminants of Concern in Cannabis …463
Hazekamp (2016) evaluated the effects of 10,000 grays in four cultivars of THC-
or CBD-dominant Cannabis. Quantiﬁcation with ultra performance liquid chro-
matography (UPLC) and gas chromatography-ﬂame ionization detector (GC-FID)
showed that levels of total THC and/or CBD were not altered by irradiation
treatment in any of the cultivars tested, compared to controls. Irradiation decreased
four monoterpenoids—a-guaiene (10%), cis-ocimene (7–23%), b-myrcene (8–
18%), terpinolene (16–38%), and seven sesquiterpenoids—guaiol (6%), nerolidol
(7%), trans-b-farnesene (7–10%), b-caryophyllene (6–10%), c-selinene (13–17%),
eudesma-3,7(11)-diene (14%), and c-emelene (8–19%). Hazekamp compared these
reductions to similar decreases arising from short term storage in a paper bag (Ross
and Elsohly 1996).
Hazekamp (2006) compared the inoculum load of irradiated medical-grade
herbal cannabis (MC) to that of untreated recreational coffeehouse cannabis (CC).
An Enterobacteriaceae assay revealed <10 CFU/g in MC samples (n = 2), and a
mean of 1.4 !10
CFU/g in CC samples (n = 11). An assay for molds and aerobic
bacteria revealed <100 CFU/g in MC samples, and a mean of 5.4 !10
CC samples. Because screening tests do not identify species, one CC sample was
sent out for further testing, which identiﬁed E. coli and Aspergillus, Penicillium,
and Cladosporum spp.
Ruchlemer et al. (2015) tested three other ways to sterilize cannabis: gas plasma,
autoclaving, and ethylene oxide. These methods decreased THC content 12.6, 22.6,
and 26.6%, respectively. Levitz and Diamond (1991) killed condia (spores) of A.
fumigatus, A. ﬂavus, and A. niger in marijuana by baking herb at 150 °F (300 °C)
for 15 min. Water pipes do not prevent the transmission of fungal spores from
contaminated cannabis (Moody et al. 1982), not even water pipes with ﬁlters
(Sullivan et al. 2013). Fungi and bacteria are capable of passing through vaporizers
(Ruchlemer et al. 2015). Some toxins produced by fungi and bacteria, such as Shiga
toxin, are resistant to heat treatment (pasteurization).
22.5 Janis Face—Endophytes
A microbiome is the ecological community of commensal, symbiotic, and generally
non-pathogenic microorganisms that inhabit plants, animals, and us. The plant
microbiome is a key determinant of plant health and productivity, and has gained
attention recently (Turner et al. 2013). Over a century ago, however, botanists ﬁrst
recognized mutualistic associations between plants and fungi, termed mycorrhizae.
Emil Arzberger, a USDA scientist, discovered fungi living in the roots of healthy
Cannabis plants back in 1925. He died shortly thereafter, without reporting his
results; they were rediscovered in USDA archives (McPartland et al. 2000). The
endorhizal (root-inhabiting) microorganisms that colonize Cannabis improve plant
nutrition and disease resistance (McPartland and Cubeta 1997; Citterio et al. 2005;
Winston et al. 2014).
464 J.M. McPartland and K.J. McKernan
Researchers have turned their attention to phylloplane organisms, which live in
nooks and crannies above the leaf epidermis (epiphytes) or in spaces below the
epidermis (endophytes). Phylloplane organisms protect their plant hosts by repel-
ling pathogenic organisms. The yeast-like fungus Aureobasidium pullulans is a
ubiquitous epiphyte, and it has been isolated from Cannabis (Ondrej 1991). It oozes
chitinases and other enzymes that attack other fungi, including the dreaded gray
mold fungus, Botrytis cinerea.
Gautam et al. (2013) identiﬁed a number of Cannabis endophytic fungi. They
eliminated epiphytes from their study by surface-sterilizing plant material with
sodium hypochlorite (bleach) for 40s. They rinsed material with sterile distilled
water, and plated it on agar with antibacterial antibiotics. Fungi were identiﬁed by
their morphological and cultural characteristics. Gautam and colleagues identiﬁed
three Aspergillus species (A. niger, A. ﬂavus, A. nidulans), two Penicillium species
(P. citrinum, P. chrysogenum), and Rhizopus stolonifer. They also identiﬁed ﬁve
other species known to be foliar pathogens of Cannabis: Curvularia lunata,
Alternaria alternata, Cladosporium sp., Colletotricum sp., Phoma sp. “One plant’s
protective phylloplane fungus is another plant’s latent pathogen”(McPartland et al.
Kusari et al. (2013) tested plants obtained from Bedrocan BV. Samples were
surface sterilized with ethanol and bleach, and cultured on agar with antibiotics.
Kusari and colleagues used molecular methods for species identiﬁcation: DNA
extraction and PCR ampliﬁcation using primers for ITS1, 5.8S, and ITS2 regions of
ribosomal DNA. Amplicons were sequenced, and the sequences were BLASTed for
matches in the EMBL nucleotide database. The predominant endophyte was
Penicillium copticola. Other species included P. meleagrinum, P. sumatrense,
Eupenicillium rubidurum, Chaetomium globosum, Paecilomyces lilacinus, and
Aspergillus versicolor. None of these fungi have previously been associated with
Cannabis except for C. globosum (McPartland et al. 2000). Kusari and colleagues
demonstrated that these endophytes antagonized in vitro growth of two common
Cannabis pathogens, Botrytis cinerea and Trichothecium roseum.
The aforementioned study by McKernan et al. (2015) highlighted the predom-
inance of Penicillium species in a majority of samples tested. They proposed that a
number of these were endophytes. They likely isolated epiphytes as well as
endophytes, because they dispensed with surface sterilization and plating, and went
straight to molecular identiﬁcation. Five organisms they isolated were phy-
topathogens previously reported causing Cannabis diseases: Diplodia
spp. (McPartland 1994b), Pestalotiopsis spp. (McPartland and Cubeta 1997),
Botryosphaeria dothidea (McPartland 1994c), Fusarium oxysporum (McPartland
and Hillig 2004a), and Pseudomonas syringae (McPartland and Hillig 2004b).
These studies reveal a surprisingly depauperate Cannabis foliar microbiome.
A recent study of Genlisea species, using similar methods, identiﬁed 92 genera of
organisms (Cao et al. 2015). See Delmotte et al. (2009) for rich microbiomes in
other plant species. Many of the 97 species of fungi that Gzebenyuk (1984) isolated
from hemp stems in Russia may be phylloplane organisms.
22 Contaminants of Concern in Cannabis …465
Phylloplane research should be extended to a comparison of indoor crops and
outdoor crops. Outdoor crops may show a seasonal community succession.
Comparing the microbiome in Cannabis from different climates and continents
would be informative. Winston et al. (2014) demonstrated Cannabis
cultivar-speciﬁc differences in endorhizae (root-inhabiting bacteria). Their study
was limited to drug-type hybrids; this work should be extended to ﬁber-type cul-
tivars and wild-type plants.
22.6 Heavy Metals and Radionucleotides
Contamination by heavy metals is a health concern because these elements accu-
mulate in the body. They are toxic, carcinogenic, and cause a variety of diseases.
Particularly dangerous elements include cadmium, mercury, lead, arsenic, and
nickel. Radionucleotides present in the environment may also contaminate plants,
and contribute to the risk of lung cancer.
Siegel et al. (1988) measured 440 ng mercury per gram of cannabis in Hawai’i,
where the volcanic soil contains naturally high levels of mercury. Siegel notes that
mercury is absorbed 10 times more efﬁciently by the lungs than by the gut. He
calculated that smoking 100 g of volcanic cannabis per week could lead to mercury
Volcanic soil also contains signiﬁcant levels of cadmium. Grant et al. (2004)
attribute this to elevated levels of cadmium in Jamaican-grown tobacco and can-
nabis. However, anthropogenic emissions, from fossil fuel combustion and
mining/smelting activities, are the primary source of cadmium.
Tainted fertilizer is another source of heavy metal contamination. Safari Singani
and Ahmadi (2012) showed that C. sativa readily takes up lead and cadmium from
soils amended with contaminated cow and poultry manures. Even reportedly
“clean”fertilizer seems to increase the uptake of cadmium by C. sativa (Ahmad
et al. 2015). Phosphate ions are the main carriers of heavy metal contamination, and
hydroponic fertilizers are particularly vulnerable to contamination (Karadjov 2014).
Phosphate fertilizers targeted at Cannabis growers (“bud blooms”) have particular
problems with arsenic, in some cases 10–50 ppm (N. Palmer, pers. commun. 2016).
Rockwool, a.k.a. mineral wool ﬁber, used as hydroponic growth medium, may also
In a study on hemp seeds, Mihoc et al. (2012) report a problem with cadmium
contamination; they measured levels of 1.3–4.0 mg/kg. Eboh and Thomas (2005)
showed that concentrations of arsenic, cadmium, chromium, iron, nickel, lead and
mercury were greater in leaf material than in seeds. Moir et al. (2008) measured
heavy metals in marijuana smoke, including mercury, cadmium, lead, chromium,
nichel, arsenic, and selenium. Deep inhalation, typical of marijuana smokers,
doubled the exposure to heavy metals.
Health Canada (2008) mandated upper limits for arsenic (0.14 µm/kg body
weight per day), cadmium (<0.09 µm/kg), lead (<0.29 µm/kg), and mercury
466 J.M. McPartland and K.J. McKernan
(<0.29 µm/kg). The AHP proposed maximal limits for orally consumed cannabis
products: mercury 2.0 µm/day, arsenic 10.0 µm/day, and cadmium 4.1 µm/day
(Upton et al. 2013).
22.7 Janis Face—Bioremediation
Cannabis is so efﬁcient at absorbing and storing heavy metals that it has gained
attention as a “bioremediation crop.”Bioremediation uses plants or microorganisms
to remove pollutants. Plants such as Thlaspi caerulescens (= T. alpestre) extract
toxins from soil and accumulate the toxins in their tissues. The plants are harvested
and the toxins removed. Cannabis is an excellent candidate for bioremediation (Shi
and Cai 2009), although the amount of metal taken up by Cannabis pales in
comparison to T. caerulescens (Giovanardi et al. 2002;Löser et al. 2002; Citterio
et al. 2003; Meers et al. 2005).
Jurkowska et al. (1990) measured high levels of lithium in hemp (1.04 mg/kg),
higher than the other crop plant tested, including barley, maize, mustard, oats,
radish, rape, sorrel, spinach, sunﬂower, and wheat. Cannabis has been sown on
toxic waste sites contaminated with cadmium and copper in Silesia. The metals are
recovered by leaching the harvested seed with hydrochloric acid (Kozlowski 1995).
Other studies have shown that hemp accumulates heavy metals in its roots
(Giovanardi et al. 2002; Citterio et al. 2003; Shi and Cai 2009), and in leaf material
(Giovanardi et al. 2002; Arru et al. 2004). Plants with mycorrhizal fungi growing in
their roots show greater translocation of heavy metals from roots to shoots (Citterio
et al. 2005). Perhaps mycorrhizal-inoculated plants are healthier, and therefore can
better tolerate heavy metal stress.
Ciurli et al. (2002) showed potential for bioremediation using ‘Fibranova’
ﬁber-type plants, which tolerated growth in zinc-contaminated soil. They also
showed that experiments of this type need to be done in soil, and not in a
hydroponic-based screening test. The plants tolerated zinc salts much better in soil
than in hydroponic culture.
Cannabis bioaccumulates sodium chloride, which kills it—despite the fact that
chloride is an essential nutrient, and sodium is beneﬁcial in trace amounts. Salty
breezes near the sea are sufﬁcient to stunt hemp crops. Italian accessions are being
tested for tolerance to salt water, 2.5% NaCl (G. Grassi, pers. commun. 2016).
Cannabis can extract toxic polycyclic aromatic hydrocarbons from soil, such as
benzo[a]pyrene and chrysene (Campbell et al. 2002). Cannabis also extracts
radioactive caesium-137 and strontium-90 from contaminated soil (Vandenhove
and Van Hees 2005; Hoseini et al. 2012). Hemp crops were planted near the
Chernobyl site for the purpose of removing radionucleotides (Anonymous 2000).
Löser et al. (2002) were not impressed with the ability of C. sativa to uptake
heavy metal-polluted river sediment. Although the plants took up zinc, cadmium,
22 Contaminants of Concern in Cannabis …467
and nickel, about 95% of the plants died within a week. Apparently different
cultivars vary in their ability and tolerance in taking up cadmium from contami-
nated soils (Shi et al. 2012).
22.8 Pesticide Residues
Pesticide residues pose a uniquely unpredictable risk to consumers, because can-
nabis is usually smoked and inhaled, unlike most agricultural products. Up to
69.5% of pesticide residues remain in smoked cannabis (Sullivan et al. 2013). The
use of illegal pesticides is a rising crisis, and a breakdown in ethics. Voelker and
Holmes (2015) estimated that pesticide residues are found on close to half of the
cannabis sold in Oregon dispensaries.
Sloppy and unscrupulous Cannabis growers utilize “over the counter”pesticides
available in garden supply stores. Some of these are only approved for landscape
plants, not food plants. Hydroponic shops repackage pesticides for ornamental
plants, such as bifenazate and abamectin, for sale to Cannabis cultivators (McLean
2010). A dubious corporation marketed Guardian, a “100% natural”miticide,
which contained undisclosed abamectin—resulting in the recall of cannabis in
several states (Associated Press 2016).
McPartland et al. (2000) published a list of pesticides used by growers, derived
from anecdotal reports and the literature. This veritable witches brew included
abamectin, acephate, benomyl, carbaryl, carboxin, chlorpyrifos, chlorothalonil,
chlorpyrifos, diazinon, dichlorvos, dicofol, dimethoate, fenbutatin oxide, iprodione,
malathion, maneb, parathion, vinclozolin, and a slew of synthetic pyrethroids. The
Centre for Disease Control in British Columbia studied former marijuana grow
operations in residential homes. Their list of pesticide residues found in former
grows included chlorpyrifos, diazinon, and 11 synthetic pyrethroids (NCCEH
Medical cannabis products in southern California have been contaminated with
diazinon, paclobutrazol, and synthetic pyrethroids (Sullivan et al. 2013). The AHP
published a list of pesticides that are most likely to be used on Cannabis, including
12 insecticides/miticides (abamectin, acequinocyl, bifenazate, etoxazole, fenoxy-
carb, imidacloprid, spinosad, spiromesifen, spiromesiﬁn, and several synthetic
pyrethroids), four fungicides (imazalil, myclobutanil, triﬂoxystrobin, paclobu-
traxol), and three plant growth regulators (daminozide, paclobutraxol, chlormequat
Testing of medical cannabis products in central California identiﬁed 12 pesti-
cides and growth regulators, in up to 49.3% cannabis samples (Wurzer 2016).
Myclobutanil led the list (40%), followed by bifenazate (20%), spiromesifen (15%),
imidacloprid (4.6%), and spinodad (1.3%), as well as abamectin, acequinocyl,
bifenazate, daminozide, fenoxycarb, pyrethrum, and spirotetramat.
A survey of 389 cannabis samples obtained from Oregon dispensatories found
residues of 24 pesticides and growth regulators: abamectin, azadirachtin, bifenazate,
468 J.M. McPartland and K.J. McKernan
bifenthrin, carbaryl, chlorfenapyr, chlordane, chlorpyrifos, coumaphos, cyperme-
thrin, diazinon, dichlorvos, ethoprophos, imidacloprid, malathion, metalaxyl,
mevinphos, myclobutanil, paclobutrazol, permethrin, piperonyl butoxide, propoxur,
and 4-4’-DDE (Voelker and Holmes 2015). Two percent of the samples contained
>100,000 ppm pesticides. Piperonyl butoxide was the most commonly seen con-
taminant, with up to 407,000 ppm in one sample. This is a synthetic compound
linked with human disease.
Russo (2016) purchased 26 cannabis samples (24 concentrates, 2 cannabis
inﬂorescences) from legal stores in Washington State, and passed the samples via a
witnessed chain to a state certiﬁed legal licensed laboratory. Pesticides residues
were detected in 22 samples (84.6%), including 24 distinct agents of every class:
insecticides (organophosphates, organochlorides, carbamates, neonicotinoids),
miticides, fungicides, an insecticidal synergist, and growth regulators. One sample
was contaminated with nine agents, include the fungicide boscalid (112,033 ppb)
and the extremely toxic insecticide carbaryl (25,483 ppb). Samples obtained from
indoor grows had a higher risk of contamination than samples obtained from out-
Fertilizers may also contaminate Cannabis. Spraying plants with liquid fertil-
izers may result in the formation of N-nitrosamines, which are potent carcinogens
(Farnsworth and Cordell 1976). Ramírez (1990) reported four policemen con-
tracting pulmonary histoplasmosis while pulling up marijuana plants. The plants
were likely fertilized with bird guano contaminated with the fungus Histoplasma
capsulatum. The use of human dung has been associated with outbreaks of hepatitis
viral infections (Cates and Warren 1975; Alexander 1987).
The EPA claims its failure to act in the interests of the American public is simply
because it “has yet to receive any applications for pesticide use on marijuana and,
therefore, we have not evaluated the safety of any pesticide on marijuana”(EPA
2016). In the absence of federal regulations, individual stakeholders and states have
In the spirit of harm reduction, the Maine legislature allowed the application of
25(b) pesticides on Cannabis (State of Maine 2013). These are minimal-risk pes-
ticides exempted by the EPA—mostly botanicals (e.g., rosemary oil, thyme oil,
garlic oil, corn gluten meal, eugenol), and other substances such as 2-phenylethyl
propionate and potassium sorbate (EPA 2015). The Colorado Department of
Agriculture and the Washington Department of Agriculture released larger lists of
allowable pesticides (CDA 2016, WSDA 2016). Most of these pesticides are per-
mitted in The National List of materials designated by the Organic Foods
Production Act of 1990. They include botanical poisons (e.g., neem oil, garlic oil,
azadirachtin, pyrethrins), minerals (e.g., potassium salts, copper, sulfur), and bio-
logical control organisms (e.g., Bacillus thuringiensis, Streptomyces griseoviridis).
Both states allowed piperonyl butoxide. All these materials are described at
book-length elsewhere (McPartland et al. 2000).
Assaying for pesticide residues is more difﬁcult than microbial testing. Each
pesticide must be tested individually, and the secretive use of pesticides leaves
regulators in the dark (Stone 2014). The Oregon Health Authority posted a list of 59
22 Contaminants of Concern in Cannabis …469
pesticides required for testing before cannabis can be release for sale (Farrer 2015).
Voelker and Holmes (2015) suggested testing for 123 pesticides, with tolerance
limits of 100 ppb. Feldman (2015) documented pesticide regulations in other states.
Detecting pesticides requires expensive analytical methods, such as GS-MS and
HPLC (Upton et al. 2013). Adequate pesticide testing costs around $400; labora-
tories charging only $100 are substandard (T. Flaster, pers. commun., 2016). To
wit, few independent laboratories have been accredited for pesticide testing in
cannabis—zero in Colorado (N. Palmer, pers. commun., 2016).
There have been several high-proﬁle cases of cannabis removed from sale due to
pesticides. In 2011 California issued a cease-and-desist order against the sale of
cannabis contaminated with daminoside and paclobutrazol (Upton et al. 2013). In
2012, a whistleblower at Maine’s largest medical cannabis dispensary revealed that
nine types of insecticides and fungicides were being applied to Cannabis; the
dispensary was ﬁned $18,000 (Shepard 2013). Colorado regulators quarantined
thousands of plants grown by a dispensary chain that used myclobutanil, a turfgrass
fungicide; consumers ﬁled a lawsuit against the corporation (Wyatt 2015). This was
only one of nine marijuana recalls in Denver that year (Baca and Migoya 2015).
Mikuriya et al. (2005) reported the ﬁrst case of hospitalization due to concealed
pesticide use. The case report involved a bud trimmer working with cannabis con-
taminated with avermectin (abamectin), which a grower used against spider mites.
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