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Microbiological contaminants pose a potential threat to cannabis consumers. Bacteria and fungi may cause opportunistic infections in immunocompromized 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.
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Chapter 22
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 aatoxins, may pose a
problemunlikely, but possible. The Cannabis plant hosts a robust microbiome;
the identication 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 plantsthe 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.
22.1 Introduction
Cannabis (the plant) and cannabis (the plant product) may be contaminated by
microbes, heavy metals, or pesticide residues. The rst two contaminants, microbes
and heavy metals, present a Janus-face or ip-side of the coinin relation to
Cannabis. Some bacteria and fungi are part of the plants microbiome. They pro-
vide benets to Cannabis. See the book chapter by Parijat Kusari and Oliver Kayser
for more about the Cannabis microbiome. On the ip-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
K.J. McKernan
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,
DOI 10.1007/978-3-319-54564-6_22
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 efciency. 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 ip-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 Organicby the USA Department of Agriculture.
This chapter focuses on microbes, heavy metals, and pesticide residues in can-
nabis inorescences 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). Adulterantsdeliberately added contaminantsare 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-freecrop. 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 owering tops are caused by three ubiquitous fungiBotrytis 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 curedby 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. avus. 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 avus, 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). Aatoxins are the most
common mycotoxins.
Aspergillus species (A. avus, A. parasiticus) produce aatoxins in warm and
humid conditionsoptimally 33 °C (91.4 °F), and 0.99 water activity. Aatoxins
are acutely poisonous as well as carcinogenic. Llewellyn and ORear (1977)
identied aatoxins in cannabis, but under articial conditions. They added 15 ml
water to 5 g pulverized owering tops, autoclaved the material, and inoculated it
with A. avus or A. parasiticus. After 14 days at 25 °C (77 °F), the fungi sporu-
lated and produced moderateamounts of aatoxins. Importantly, no studies have
reported aatoxins in cannabis under normal storage conditions (McPartland and
Pruitt 1997).
Kurup et al. (1983) isolated three thermophilic actinomycetes from questionably
sourced material, Thermoactinomyces candidus, T. vulgaris, and Micropolyspora
faeni. These endospore-forming microbes cause farmers 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 cannabisspecies 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 oors (B. Thomas, pers. commun. 1999)an unacceptable method today.
A disease outbreak caused by another member of the Enterobacteriaceae
Salmonella muenchenwas associated with cannabis (Taylor et al. 1982). The
investigators concluded that the plant material, sourced from Mexico, was con-
taminated or adulterated by untreated manureanother 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
todays 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, todays 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 Ofce 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 nal 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 quantied 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 TYMCwhich is close
to sterility. Certain specic pathogens must be completely absentStaphylococcus
aureus,Pseudomonas aeruginosa, and bile-tolerant Gram-negative bacteria such as
Escherichia coli. Furthermore, the absence of fungal mycotoxins must be conrmed
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 specic tests for
Coliform bacteria (<3 MPN/g), and E. coli (absent). Their upper limit for aatoxins
B1, B2, G1, G2, and ochatoxin A is <20 µm/kg cannabis.
In the USA, medical cannabis was rst 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 certication of
organicmedical cannabis (NJMMP 2011).
The American Herbal Pharmacopoeia (AHP) issued specic protocols for
microbial testing (Upton et al. 2013). The AHPs 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 lm-based assays for bacterial, yeast,
and mold.
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 aatoxins. 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 aatoxin assays. The AHP proposed specic
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 specic testing protocols,
reviewed by Holmes et al. (2015). Colorados 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. Washingtons 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 specic pathogens: Escherichia coli, Salmonella
spp., and four species of Aspergillus:A. avus, A. fumigatus, A. niger, and A.
In 2015 Colorado changed its testing regimen: (1) total yeast and mold count
(limit <10
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. avus, A. fumigatus, and A. niger, although this was never
Aspergillus is a large genus with 250 species, and separating three specic
species from the others is not easy. Traditionally, identication required culturing
on Aspergillus-selective plating media, and morphological measurements by a
specialist (Samson et al. 2004). Due to the challenges associated with
species-specic 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 34 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 dened growth time (termed
enrichment) to accommodate for the subsampling.
Because of these difculties, 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 specic
Aspergillus species.
The drawback to qPCR is the methods 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 conrmed 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
lm-based detection systems: 3 M Petrilm, Simplate-Biocontrol Systems,
and BioLumix. They tested 17 dispensary-obtained cannabis samples. Six sam-
ples tested positive with the qPCR assay, ve samples tested positive with the
Biocontrol Systemsassay (>10,000 CFU/g), four samples test positive with the
3 M Petrilmassay (>10,000 CFU/g), and only one sample tested positive with
the BioLumixassay, which is a simple pass-fail test.
McKernan and colleagues then subjected ITS amplicons to DNA sequencing, to
identify specic fungi. All three Aspergillus species on the bad list turned up:
462 J.M. McPartland and K.J. McKernan
A. avus (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. lifera, E. repens, E. bicolor. Two of these produce toxins, A.
versicolor and A. terreus.
ITS amplicons identied 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 conrmed its presence with PaxPss1 and
PaxPss2 DNA primers. Paxilline has been shown to decrease the antiseizure ben-
ets 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 identied 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 1012% MC. Fungi and bacteria cannot grow below 15% MC. Herb dried below
10% MC becomes brittle and disintegrates easily. Hazekamp (2006) recommended
510% 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 books chapter by David Potter discusses GW
Pharmaceuticals methods of growing healthy Cannabisby 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)
used radioactive
Co gamma rays, a dose of 15,00020,000 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 badquality 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 controversialit 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. Quantication with ultra performance liquid chro-
matography (UPLC) and gas chromatography-ame 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 monoterpenoidsa-guaiene (10%), cis-ocimene (723%), b-myrcene (8
18%), terpinolene (1638%), and seven sesquiterpenoidsguaiol (6%), nerolidol
(7%), trans-b-farnesene (710%), b-caryophyllene (610%), c-selinene (1317%),
eudesma-3,7(11)-diene (14%), and c-emelene (819%). 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
CFU/g in
CC samples. Because screening tests do not identify species, one CC sample was
sent out for further testing, which identied 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. avus, 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 lters
(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 FaceEndophytes
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 rst
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) identied 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 identied by
their morphological and cultural characteristics. Gautam and colleagues identied
three Aspergillus species (A. niger, A. avus, A. nidulans), two Penicillium species
(P. citrinum, P. chrysogenum), and Rhizopus stolonifer. They also identied ve
other species known to be foliar pathogens of Cannabis: Curvularia lunata,
Alternaria alternata, Cladosporium sp., Colletotricum sp., Phoma sp. One plants
protective phylloplane fungus is another plants 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 identication: DNA
extraction and PCR amplication 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 identication. 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, identied 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-specic differences in endorhizae (root-inhabiting bacteria). Their study
was limited to drug-type hybrids; this work should be extended to ber-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 Hawaii,
where the volcanic soil contains naturally high levels of mercury. Siegel notes that
mercury is absorbed 10 times more efciently 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 signicant 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
cleanfertilizer 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 1050 ppm (N. Palmer, pers. commun. 2016).
Rockwool, a.k.a. mineral wool ber, used as hydroponic growth medium, may also
be contaminated.
In a study on hemp seeds, Mihoc et al. (2012) report a problem with cadmium
contamination; they measured levels of 1.34.0 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 FaceBioremediation
Cannabis is so efcient 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, sunower, 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
ber-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 itdespite the fact that
chloride is an essential nutrient, and sodium is benecial in trace amounts. Salty
breezes near the sea are sufcient 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 counterpesticides
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% naturalmiticide,
which contained undisclosed abamectinresulting 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, spiromesin, and several synthetic
pyrethroids), four fungicides (imazalil, myclobutanil, trioxystrobin, paclobu-
traxol), and three plant growth regulators (daminozide, paclobutraxol, chlormequat
Testing of medical cannabis products in central California identied 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
inorescences) from legal stores in Washington State, and passed the samples via a
witnessed chain to a state certied 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-
door grows.
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
formulated guidelines.
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 EPAmostly 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 difcult 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
cannabiszero in Colorado (N. Palmer, pers. commun., 2016).
There have been several high-prole 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 Maines largest medical cannabis dispensary revealed that
nine types of insecticides and fungicides were being applied to Cannabis; the
dispensary was ned $18,000 (Shepard 2013). Colorado regulators quarantined
thousands of plants grown by a dispensary chain that used myclobutanil, a turfgrass
fungicide; consumers led 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 rst 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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... Finally, a last group of Pseudomonas strains potentially relevant in cannabis cultivation concerns P. aeruginosa. Since this opportunistic human pathogen can cause important infections in immunocompromised and hospitalized patients, screening for contaminations in marijuana products is usually implemented during production quality control and may vary according to local regulations (McPartland and McKernan, 2017). Testing for P. aeruginosa contaminants is especially important for fresh raw plant products, since this Gram-negative non-sporulating bacterium is highly sensitive to heat and desiccation, and therefore unlikely to survive the processes of marijuana drying, curing, decarboxylation, extraction and/or smoking (Holmes et al., 2015). ...
... Various strategies can be explored to increase the yield and quality of cannabis crops. Several recent literature reviews can be consulted about the current opportunities and challenges associated with cannabis genetic diversity, cultivar breeding and agronomic traits improvement (Salentijn et al., 2015;Clarke and Merlin, 2016;Schluttenhofer and Yuan, 2017;Hesami et al., 2020), cannabinoid elicitation (Gorelick and Bernstein, 2017;Backer et al., 2019), disease management (Punja, 2021), production factors optimization Eichhorn Bilodeau et al., 2019), and biosafety practices to reduce contaminants (McPartland and McKernan, 2017;Montoya et al., 2020;Vujanovic et al., 2020). Within all these promising developments, microbiome engineering and beneficial microbial inoculants appear as a recurring prospective trend, potentially promoting plant growth and fitness (Kusari et al., 2017;Backer et al., 2019;Lyu et al., 2019;Söderström, 2020), enhancing cannabinoid production Taghinasab and Jabaji, 2020;, controlling diseases (Kusari et al., 2017;Lyu et al., 2019;Söderström, 2020;Punja, 2021), and improving product biosafety (Vujanovic et al., 2020). ...
... In both hemp and marijuana crops, managing biotic stresses is especially challenging because of the limited range of registered pesticides available (Punja, 2021), the lack of formal agricultural recommendations and mitigation strategies based on reliable research (Sandler and Gibson, 2019), the high susceptibility of modern cultivars to fungal pathogens (Clarke and Merlin, 2016), and the regional variabilities in pest and disease pressures (Thiessen et al., 2020). Additionally, for drug-type crops, finished products destined to human consumption must comply with strict regulations on pesticide residues and microbial contaminants (McPartland and McKernan, 2017). Therefore, in addition to good management practices and crop resistance breeding efforts reviewed recently (Punja, 2021), inoculation with beneficial Pseudomonas spp. ...
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Among the oldest domesticated crops, cannabis plants ( Cannabis sativa L., marijuana and hemp) have been used to produce food, fiber, and drugs for thousands of years. With the ongoing legalization of cannabis in several jurisdictions worldwide, a new high-value market is emerging for the supply of marijuana and hemp products. This creates unprecedented challenges to achieve better yields and environmental sustainability, while lowering production costs. In this review, we discuss the opportunities and challenges pertaining to the use of beneficial Pseudomonas spp. bacteria as crop inoculants to improve productivity. The prevalence and diversity of naturally occurring Pseudomonas strains within the cannabis microbiome is overviewed, followed by their potential mechanisms involved in plant growth promotion and tolerance to abiotic and biotic stresses. Emphasis is placed on specific aspects relevant for hemp and marijuana crops in various production systems. Finally, factors likely to influence inoculant efficacy are provided, along with strategies to identify promising strains, overcome commercialization bottlenecks, and design adapted formulations. This work aims at supporting the development of the cannabis industry in a sustainable way, by exploiting the many beneficial attributes of Pseudomonas spp.
... According to Krivokapic, the toxicity of heavy metals against soil organisms could be ranked following the descending order: Hg > Cd > Cu > Zn > Pb [23]. Heavy metals also contribute to controlling pesticides and pathogenic microorganisms [24]. To be specific, Figure 4 indicates that the control and Treatment 1 field show considerably higher Cu content in topsoil than that in Treatment 2. However, for soils collected at higher depth, Treatment 2 accumulated excessively high Cu, possibly because the metal content in the fertilizer was not washed away during irrigation and rainfall or due to accumulation from other unknown sources. ...
... According to Krivokapic, the toxicity of heavy metals against soil organisms could be ranked following the descending order: Hg > Cd > Cu > Zn > Pb [23]. Heavy metals also contribute to controlling pesticides and pathogenic microorganisms [24]. To be specific, In general, the highest heavy metal content was found in the topsoil of the control sample, followed by topsoil in Treatment 2 field and then in the Treatment 1. ...
... According to Krivokapic, the toxicity of heavy metals against soil organisms could be ranked following the descending order: Hg > Cd > Cu > Zn > Pb [23]. Heavy metals also contribute to controlling pesticides and pathogenic microorganisms [24]. To be specific, high concentrations of Cu have been to be responsible for the reduction of the number of bacteria and nematodes [25], and high accumulation of Zn would reduce the number of arthropodae, especially mites, fungi and bugs such as brown planthopper [26]. ...
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Widespread use of chemical fertilizers in agricultural activities poses a high risk of multi-micro metal contamination in soils and potentially causes health issues through consumption of contaminated foods. Bio-organic fertilizers from sewage sludge have been regarded as a suitable substitute for chemical fertilizer for rice farming. In this study, we investigated accumulation of heavy metals (Cu and Zn) in soil, water and rice plant in three pilot-scale rice paddy fields treated with different fertilization schemes. The control field was treated with conventional chemical fertilizers while the soil of two treatment fields was mixed with biological sewage sludge obtained from a local wastewater treatment system in Vietnam at different ratios (1% and 3%). Initial results showed that heavy metals accumulated in the soil, water, and rice plant at varying levels and most of the Cu and Zn contents found in soils, water and rice products exceeded permissible Vietnamese standards (QCVN 03: 2008) and US EPA 503. Notably, the rice field whose soil was treated with sludge at 3% ratio showed a significantly lower accumulation of heavy metals in soil, water and in rice plant. However, treatment of sludge at this level seemed to cause higher heavy metal retention in soil after one harvest. Semi-quantitative risk analysis revealed that the risk of metal contamination in soil and water of the control field ranged from medium (RQ index between 0.1 and 1) to high risk (RQ index higher than 1) and that fertilization methods would also affect the level of risk to the environment.
... risks are not mitigated by adequate testing programs in most states, though contamination is found by anecdotal studies(Lenton, et al., 2018). Pesticides have been shown to have carcinogenicity and teratogenicity in humans,(Dryburgh, et al. 2018).Additionally, heavy metals bioaccumulate in plants such hemp(McPartland, et al. 2017), and could potentially be coextracted and concentrated in products infused with THC. The chances of these adverse health incidences increase as the market and production of such products grows. ...
The increasing demand of adult-use cannabis is outpacing the regulatory stride of the states that legalize recreational marijuana. This work is the product of two studies that focus on those states’ agencies that closely work with their constituents. They include those who are invested or reliant on cannabis businesses and those who consume cannabis for medical or recreational purposes. State agencies and resources are funded by those same constituents’ taxes, for which they have the responsibility to protect the public’s best interests as the industry grows. Chapter 2 describes an inquiry on what information and support is shared with adult-use cannabis industry stakeholders. Land Grant Universities, Cooperative Extensions, and states’ Departments of Agriculture in American states with legalized recreational marijuana by January 2020 were investigated, including recruiting participants for an interview. The policies and norms of interaction with adult-use cannabis business-owners were compiled, and it was found that those agencies that participated in regulation were most likely to provide cannabis stakeholders with information. Regulating authorities and their approaches varied among states. Many agencies’ policies were unclear or unfinished. Reliance on federal funding is a barrier to support and research, which are both crucial to maintaining a safe and sustainable industry for the entire community. The University of Maine (a Land Grant University) prohibits all cannabis from its campus, even that which is legally purchased by a medical marijuana patient 18 years of age or older. This is due to the university’s reliance on federal funding for scholarship and other services. Chapter 3 focuses on the development of a cannabis harm reduction course for the University of Maine to utilize when disciplining students who violate its cannabis ban. A pilot course was delivered to student volunteers who also opted into an anonymous survey. The survey was designed to promote self-reflection on cannabis use habits and beliefs as well as to capture their valuable information as University of Maine students. Sharing reliable researched-based information on cannabis aligned with the principles of harm reduction with college students reduces the negative effects of consuming marijuana and acknowledges adult-use cannabis as a legitimate recreational drug as it is in an increasing number of states. The disparity between federal prohibition of marijuana and state adult-use cannabis causes tension and hinders adequate research and regulation. State-run research institutions struggle to advise and support their citizens without risking their standing with the federal government and its funding. To sufficiently fulfill their responsibilities, state agencies must have freedom to research and clarity on how to regulate adult-use cannabis. While states continue to legalize cannabis and the required infrastructures are built, a multifaceted approach is recommended. Ensuring the safety of products and efficient industry standards attends to the wellbeing of each member of the community.
... In this study, we analyze the connection between cannabis cultivation and mycotoxins outcomes. Aflatoxins become especially problematic if the drying and storage of cannabis flowers are inappropriate (27). ...
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Aim: The aim of this study was to investigate whether the cannabis extract obtained from cannabis flowers that contain the maximum allowed level of mycotoxins affects human safety and health. For that purpose, a novel liquid chromatography with tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of aflatoxins and ochratoxin A (OchA) in cannabis extracts to demonstrate that this analytical method is suitable for the intended experimental design. Methods: Experimental design was done by adding maximum allowed concentration of aflatoxins (B1, B2, G1, G2) and OchA according to the European Pharmacopeia related to cannabis flowers. The concentration of aflatoxins and OchA was determined using the same LC/MS/MS analytical method in the starting material (dry flower) before preparing the spiked sample and after obtaining decarboxylated extract with ethanol 96%. Results: The results obtained indicate that aflatoxins and OchA, primarily added to the cannabis dried flowers, were also determined into the obtained final extract in amounts much higher (m/m) than in the starting plant material. Conclusion: With this experiment, we have shown that mycotoxins, especially aflatoxins, which are extremely toxic secondary metabolites, can reach critical values in cannabis extracts obtained from dry cannabis flowers with the maximum allowed quantity of mycotoxins. This can pose a great risk to consumers and their health especially to those with compromised immune systems.
... One of these components of concern is a class of compounds called mycotoxins. Mycotoxins are thermally resistant compounds that exhibit carcinogenic, nephrotoxic, and neurotoxic properties that can remain in the resulting extract as they are extremely stable (Daley et al. 2013;McPartland and McKernan 2017). For example, aflatoxins-AFBs (AFB1 and AFB2s)-and fumonisins-FUMs (FB1 and FB2)-are all very highly soluble in the same solvents that are used to concentrate cannabinoids from cannabis plant material (Bennett and Klich 2003). ...
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The microbial testing of cannabis and cannabis products presents a unique set of challenges. Unlike food testing, cannabis testing has various routes of administration to take into account. Cannabis flowers express high levels of antimicrobial cannabinoids and terpenoids and thus represent a different matrix than traditional foods. It is currently estimated that 50% of cannabis is consumed via vaporizing or smoking oils and flowers while the other half is consumed as Marijuana Infused Products or MIPs which encompass a wide variety of matrices. In a testing landscape that consistently focuses heavily on chemical analysis, the microbiological testing of cannabis is often overlooked. However, it is truly one of the most important analyses in the context of product safety as the accidental ingestion or inhalation of these contaminants can cause severe illnesses, infections, or worse, death. The present chapter explores the microbial contaminants of interest in cannabis, current testing methodologies, and the challenges that testing laboratories face in this continuously evolving domain. Different perspectives for ensuring product safety are presented in the context of current regulations and their varying approaches. Tactics for the remediation of contaminated product and preventative strategies used by cultivators are also discussed in the context of the existing incongruent patchwork of regulatory framework. Microbial testing acceptance criteria, methods, and recommendations from various standards organizations are presented and efforts towards the standardization and development of reference methods are highlighted.
... Cannabis has gradually garnered attention as a "bioremediation crop" because of its strong ability to absorbing and storing heavy metals (McPartland and McKernan 2017). It can remove heavy metal substances from substrate soils and keep these in its tissues by means of its bio-accumulative capacity (Dryburgh et al. 2018). ...
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Interest in growing cannabis for medical and recreational purposes is increasing worldwide. This study reviews the environmental impacts of cannabis cultivation. Results show that both indoor and outdoor cannabis growing is water-intensive. The high water demand leads to water pollution and diversion, which could negatively affect the ecosystem. Studies found out that cannabis plants emit a significant amount of biogenic volatile organic compounds, which could cause indoor air quality issues. Indoor cannabis cultivation is energy-consuming, mainly due to heating, ventilation, air conditioning, and lighting. Energy consumption leads to greenhouse gas emissions. Cannabis cultivation could directly contribute to soil erosion. Meanwhile, cannabis plants have the ability to absorb and store heavy metals. It is envisioned that technologies such as precision irrigation could reduce water use, and application of tools such as life cycle analysis would advance understanding of the environmental impacts of cannabis cultivation.
... In addition, the combustion of Cannabis and its deep inhalation that is typical of marijuana smokers may expose the consumers to very high levels of metals (Moir et al., 2008). Other toxicological concerns are that some metals as Cd and Pb have a long biological half-life and bio-accumulate for years in the human body when chronically exposed, and the lungs (McPartland and McKernan, 2017) can absorb Hg 10 times more efficiently than the gut. ...
Recently, the cultivation of light Cannabis, with a total THC content less than 0.6%, has been encouraged due to its industrial and therapeutic potential. This has increased the consumption of hemp for both smoking purposes and food preparation. Even so, Cannabis inflorescences are not subject to EU regulations and standards provided for food and tobacco products. A study was carried out on thirty-one inflorescences samples, collected in different Italian regions, in order to determine cannabinoids, pesticides and metals and to evaluate the exposure of consumers to contaminants and ensure a safe consumption. Contents of THC were always below 0.5%, while CBD ranged between 0.3 and 8.64%. The determination of 154 pesticides showed that 87% of the samples contained fungicides and insecticides in the range 0.01–185 μg/g. The most found are spynosad and cyprodinil. The concentration of metals ranged from 1 to more than 100 μg/g, and As, Cd, Co, Cr, Hg, Cu, Mo, Ni and V exceeded the regulatory US limits for inhaled Cannabis products, while Pb exceeded them for both oral and inhaled products. These contaminants are intrinsically toxic and may affect public health. Actions are needed to establish regulatory measures and reduce the adverse effects caused by contaminants in Cannabis.
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The efficiency of hemp (Cannabis sativa L.) in remediating sites contaminated with heavy metals has received great attention in recent years. The main advantage of this technology relies on its inherent sustainability with a potential re-utilization of the significant amount of produced biomass which acts as a valuable flow resource. In this study, a combined system consisting of Cannabis sativa L. (hemp) and the blue-green alga Arthrospira platensis (spirulina) was tested to clean up soils contaminated with cadmium, chromium, copper, nickel, lead, and zinc. The application of non-targeted NMR methods combined with ICP-AES quantification provided an efficient strategy for detecting residual heavy metals within plant tissues and soil. Importantly, non-targeted metabolomic analysis helped to reveal the relationships between metabolites distribution in hemp tissues and the sequestered metals. It was demonstrated that hemp accumulates copper, chromium, nickel, and zinc preferentially in the leaves, while lead is distributed mainly in the stems of the plant. Moreover, it was found that, at higher concentrations, spirulina acts as a growth promoter, contributing to an increase in the final generated biomass. Results reported in this work indicate that the hemp/spirulina system represents a suitable tool for remediation of metal contaminated soils by modulating biomass production and metals uptake.
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Leonotis leonurusL. R. Br. (Lamiaceae) is a medicinal plant native to the South Africancontinent also employed as a recreational drug and a substitute to Cannabis sativaL. (Cannabaceae). Given the interest of the last mentioned species as a source of treatments for epilepsy among many other pathologies and its possible substitution for L. leonurus, the aim of this article is obtain anatomical and micrographical characters for its identification in chopped or powdered material and to survey the user ́s perceptions about this plant based in posts extracted from a recreational drug user Internet forum. L. leonurusleaves have pluricellular tector trichomes and two classes of pluricellular trichomes with unicellular and pluricellular heads, styloid crystals in its mesophyll among many other characters, while the flowers have wooly trichomes and characteristic pollen granules. Regarding the Internet forum survey, it was reported that L. leonurusleaves and flowers were the employed parts and that the mode of use was smoked. The reported effect was sedative. The anatomical data reported in this article may help to identify L. leonurusin pharmaceutical or forensic contexts.
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BACKGROUND Cannabis sativa L. (C. sativa; hemp) is a medicinal plant producing various cannabinoids. Its consumption is legalized for medical use due to their alleged positive health effects. To satisfy the demand C. sativa plants are grown in contained growth chambers. During indoor propagation pesticides are usually used for an efficient production. However, pesticide registration and safe application in C. sativa has not been investigated in detail. RESULTS With this study the metabolic degradation of pesticides in recently established C. sativa callus cultures is examined. Tebuconazole, metalaxyl-M fenhexamid, flurtamone and spirodiclofen were applied at 10 μM for 21 days. Results were compared with metabolism data obtained from Brassica napus L., Glycine max (L.) Merr., Zea mays L. and Tritium aestivum L. callus cultures as well as in metabolism guideline studies. The successfully established C. sativa callus cultures were able to degrade pesticides by oxidation, demethylation, cleavage of ester bonds in phase I as well as glycosylation and conjugation with malonic acid in phase II and III. Initial metabolites were detected after 7 days and were traced during the 21 day. CONCLUSION The resulting pathways demonstrate the same main degradation strategies as crop plants. Since metabolites could be the main residue, the exposure of consumers to these residues will be of high importance. We present here an in vitro assay for a first estimation of pesticide metabolism in C. sativa. This article is protected by copyright. All rights reserved.
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The Center for Disease Control estimates 128,000 people in the U.S. are hospitalized annually due to food borne illnesses. This has created a demand for food safety testing targeting the detection of pathogenic mold and bacteria on agricultural products. This risk extends to medical Cannabis and is of particular concern with inhaled, vaporized and even concentrated Cannabis products. As a result, third party microbial testing has become a regulatory requirement in the medical and recreational Cannabis markets, yet knowledge of the Cannabis microbiome is limited. Here we describe the first next generation sequencing survey of the microbial communities found in dispensary based Cannabis flowers and demonstrate the limitations in the culture based regulations that are being superimposed from the food industry.
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In several countries with a National medicinal cannabis program, pharmaceutical regulations specify that herbal cannabis products must adhere to strict safety standards regarding microbial contamination. Treatment by gamma irradiation currently seems the only method available to meet these requirements. We evaluated the effects of irradiation treatment of four different cannabis varieties covering different chemical compositions. Samples were compared before and after standard gamma-irradiation treatment by performing quantitative HPLC analysis of major cannabinoids, as well as qualitative GC analysis of full cannabinoid and terpene profiles. In addition, water content and microscopic appearance of the cannabis flowers was evaluated. This study found that treatment did not cause changes in the content of THC and CBD, generally considered as the most important therapeutically active components of medicinal cannabis. Likewise, the water content and the microscopic structure of the dried cannabis flowers were not altered by standard irradiation protocol in the cannabis varieties studied. The effect of gamma-irradiation was limited to a reduction of some terpenes present in the cannabis, but keeping the terpene profile qualitatively the same. Based on the results presented in this report, gamma irradiation of herbal cannabis remains the recommended method of decontamination, at least until other more generally accepted methods have been developed and validated.
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In the carnivorous plant genus Genlisea a unique lobster pot trapping mechanism supplements nutrition in nutrient-poor habitats. A wide spectrum of microbes frequently occurs in Genlisea’s leaf-derived traps without clear relevance for Genlisea carnivory. We sequenced the metatranscriptomes of subterrestrial traps versus the aerial chlorophyll-containing leaves of G. nigrocaulis and of G. hispidula. Ribosomal RNA assignment revealed soil-borne microbial diversity in Genlisea traps, with 92 genera of 19 phyla present in more than one sample. Microbes from 16 of these phyla including proteobacteria, green algae, amoebozoa, fungi, ciliates and metazoans, contributed additionally short-lived mRNA to the metatranscriptome. Furthermore, transcripts of 438 members of hydrolases (e.g. proteases, phosphatases, lipases), mainly resembling those of metazoans, ciliates and green algae, were found. Compared to aerial leaves, Genlisea traps displayed a transcriptional up-regulation of endogenous NADH oxidases generating reactive oxygen species as well as of acid phosphatases for prey digestion. A leaf-versus-trap transcriptome comparison reflects that carnivory provides inorganic P- and different forms of N-compounds (ammonium, nitrate, amino acid, oligopeptides) and implies the need to protect trap cells against oxidative stress. The analysis elucidates a complex food web inside the Genlisea traps, and suggests ecological relationships between this plant genus and its entrapped microbiome.
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Aims: This study is aimed at assessing the effectiveness of Cannabis sativa in the absorption of cesium and strontium elements from the soil. Materials and Methods: This study was conducted in 2011, in Tehran, Iran. We employed the phytoremediation technology to refine the contamination of soil with radioactive material such as cesium and strontium. Cannabis sativa was selected because of its capability for potential radioactive absorption. It was planted in various soils with different concentrations of cesium and strontium (20 ppm, 40 ppm, 60 ppm, and 80 ppm), and after sufficient growth for about six months, it was separated into root, stem, and leaves for measuring the absorption of these elements in the main parts of the plant. The samples were measured by using the Inductively Coupled Plasma (ICP) method. Results: Strontium absorption and the main parts of the plant showed a significant relationship. The percentage of strontium absorption was 45% in the root, 40% in the stem, and the minimum absorption was found in the leaves (15%), but the corresponding figure was not significant for the cesium element. A strontium concentration of 60 ppm was possibly the maximum absorption concentration by Cannabis. Conclusion: Our findings suggest that strontium can be absorbed by Cannabis sativa, with the highest absorption by the roots, stems, and leaves. However, cesium does not reach the plant because of its single capacity and inactive complex formation.
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Eight species of Phoma and Ascochyta are described and contrasted. Diplodina cannabicola, Diplodina parietaria f. cannabina, Erysiphe communis var. urticirum, Phyllosticta cannabis, and Ascochyta cannabis become synonyms of Phoma cannabis, comb. nov. (≡ Depazea cannabis). Other Phoma species on Cannabis include P. exigua (= P. herbarum f. cannabis, Plenodomus cannabis), P. herbarum, P. glomerata, and P. piskorzii. Ascochyta arcuata, sp. nov. is described as the anamorph of Didymella arcuata. Ascochyta prasadii and Ascochyta cannabina are also described. Didymella arcuata is contrasted with D. cannabis. The pathogen complex is compared to one involving a related host, stinging nettles, Urtica dioica. Host range and geographic distribution of the eight fungi are discussed.
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Medicinal cannabis is an invaluable adjunct therapy for pain relief, nausea, anorexia, and mood modification in cancer patients and is available as cookies or cakes, as sublingual drops, as a vaporized mist, or for smoking. However, as with every herb, various microorganisms are carried on its leaves and flowers which when inhaled could expose the user, in particular immunocompromised patients, to the risk of opportunistic lung infections, primarily from inhaled molds. The objective of this study was to identify the safest way of using medicinal cannabis in immunosuppressed patients by finding the optimal method of sterilization with minimal loss of activity of cannabis. We describe the results of culturing the cannabis herb, three methods of sterilization, and the measured loss of a main cannabinoid compound activity. Systematic sterilization of medicinal cannabis can eliminate the risk of fatal opportunistic infections associated with cannabis among patients at risk.