Major and trace elements Content in Cannabis Sativa – L cultivated in North of Morocco and Heavy metals health risk assessment

. The primary objective of this study was to assess the variation in the elemental composition of Cannabis Sativa seeds cultivated in Northern Morocco and to evaluate potential health risks associated with heavy metals (Cr, Cd, Pb, Ni, and As). We employed Inductively Coupled Plasma – Mass Spectrometry (ICP-MS) to identify and quantify both macro and trace elements in three seed samples. Our analysis revealed the presence of 20 elements in Cannabis seeds, with Mg, K, Ca, P, and Na present in substantial concentrations, while Cr, Zn, Fe, and other toxic elements were detected in trace amounts. Estimations of daily intake (EDI) indicated that Cr had the highest intake, followed by Ni, Pb, As, and Cd. However, Target Hazard Quotients (THQs) for individual metals and the Hazard Index (HI) all remained below 1, suggesting a relatively low risk associated with the consumption of Cannabis Sativa seeds from Northern Morocco. Furthermore, Carcinogenic Risk (CR) values were found to be at a tolerable level. In conclusion, our results suggest that the consumption of Cannabis Sativa seeds from this region poses minimal health risks."


Introduction
Cannabis Sativa-L, commonly Known in Morocco as "El-Kif" [1,2] , is the oldest known medicinal plant.The first historical reference to Cannabis dates back to 5000 Before Common Era (BCE) and place its origins in Central Asia, while its medicinal uses were recorded in stone and papyrus documents of ancient Egypt circa 1700-1600 BCE [1][2][3][4][5].
For centuries, cannabis cultivation has been a traditional practice in certain regions of Morocco, particularly in the Northern Rif Mountains [1, 2; 5, 6].Morocco has been one of the world's leading cannabis producers with approximately 47,000 tonnes of raw cannabis and 3080 tons of cannabis resins (hashish) in 2003 [6].
After years of debate, Morocco has finally legalized the use of cannabis according to Law No. 13-21 since May 2021.In March 2022, the Moroccan government had fixed the areas authorized for the cultivation, production and exploitation of cannabis, i.e. the provinces of Al-Hoceima, Chefchaouen and Taounate [7].Recently established after legalization, the National Agency for the Regulation of Activities related to Cannabis (ANRAC) is responsible for implementing the State's strategy in the field of cultivation, production, manufacturing, processing, commercialization, export, and import of cannabis and its products for medical, pharmaceutical, and industrial purposes [7].
Initially regarded as a byproduct following the extraction of "hashish" or other substances, the seeds of Cannabis Sativa were relegated to the status of animal feed.Nevertheless, with the subsequent realization of their exceptional nutritional value and health benefits, these seeds have now emerged as a significant and valued commodity [3; 8, 9].Seeds of Cannabis and seeds of Cannabis food products have become available to the general public in Canada and other countries [8].
Several studies have been conducted to assess the concentrations of macro and microelements, as well as heavy metals, in the leaves, stems, and seeds of cannabis plants cultivated around of the world.Zerihun et al were investigated the levels of heavy metals in Cannabis sativa L. leaves grown in Ethiopia.Samples from four regions were analyzed for calcium, zinc, nickel, copper, cadmium, lead, and chromium.Results showed regional variations, with the highest metal content in Metema and the lowest in Butajira region [9].Marcela Mihoc et al were analysed five varieties of Cannabis seeds approved in Romania in E3S Web of Conferences 469, 00027 (2023) ICEGC'2023 https://doi.org/10.1051/e3sconf/202346900027order to determine the nutritional value of Cannabis seed expressed by the oil content and by the concentration of metals.The study concludes that Romanian Cannabis seeds varieties offer a wealth of nutrients, including vital elements such as Ca, Mg, and K, along with easily digestible unsaturated oil.However, the presence of elevated Cd concentrations raises apprehensions regarding their suitability for use in food products.Moreover, Cannabis sativa demonstrates the capacity to extract metals from the soil, notably Fe, Mn, Zn, and Cd, hinting at its potential for phytoremediation in soil contaminated with cadmium, zinc, and iron [10].Zafeiraki et al conducted a study examining 29 different macro and minor elements, which encompassed both beneficial and harmful elements, such as heavy metals and metalloids.This investigation involved the analysis of 90 samples of Cannabis sativa L that were gathered in Greece [11].The findings from this research disclosed a wide range of concentrations for macro elements in the leaves and flowers of cannabis, with values spanning from 28 to 138.378 parts per million (ppm), as well as trace elements, which ranged from 0.002 to 1352.904 ppm.Although the concentrations of elements varied across the samples, there was a consistent pattern of element accumulation, with toxic elements contributing to less than 1% of the overall trace element concentration.Notably, the levels of the most hazardous elements identified in the samples remained within the safety thresholds set by the World Health Organization (WHO).Furthermore, the calculated Threshold Hazard Quotient (THQ) and Carcinogenic Risk (CR) values indicated that there was no risk, either in terms of carcinogenic or no-carcinogenic effects, for the population exposed to the tested cannabis samples.This underscores the safety of these samples.A separate study conducted by L.O.Eboh and Boye E. Thomas examined the distribution of heavy metals (specifically As, Cd, Cr, Fe, Ni, and Hg) in cannabis leaf and seed cultivated in Nigeria [12].
The results obtained in all studies confirm that Cannabis seeds offer a diverse range of macro-and micro-elements beneficial to human health, but also contain Heavy metals.Somme research findings have indicated that Cannabis serves as an effective phytoremediation agent for soil, given its capacity to extract heavy metals [13][14].Therefore, all Cannabis products used in food or pharmaceutical must undergo testing for heavy metal content to ensure product safety and prevent any unwanted contamination that could have adverse health effects on consumers.
In this study, ICP-MS was used to identify and determinate the macro and trace elements that are present in the Moroccan Cannabis Saliva seeds.
The primary objective of this study was to assess the elemental composition variability of Cannabis seeds cultivated in the Senhaja Srair region in Northern Morocco and to evaluate the potential health hazards associated with heavy metal contamination by estimating parameters such as Estimated daily intake (EDI) of these metals, the Target Hazard Quotient (THQ), the Hazard Index (HI), and the Carcinogenic Risk (CR).The area under investigation, Senhaja Srair, is situated in the El Hoceima province, which is a subdivision of the Tanger-Tetouan-El Hoceima, one of the twelve regions of Morocco established in the territorial division of 2015 [16] (Figure 1).Situated in the Central High Rif, the Senhaja Srair region offers various ecological and biological advantages due to its richly diverse flora..It is the part of Al Hoceima province historically known as a producer of cannabis [6][7].

Sample preparation
A dry mass of 0.3 grams of plant samples was accurately weighed and placed into digestion vessels.Each sample was then supplemented with 05 mL of concentrated nitric acid (HNO3) and 01 mL of 30% hydrogen peroxide (H2O2).The samples underwent predigestion, lasting for more than 2 hours or overnight, under a fume hood at room temperature before microwave treatment.To ensure precise temperature and pressure control during the microwave digestion process, the vessels were equipped with temperature and pressure sensors.
The microwave digestion of the plant samples, conducted in two steps each lasting 45 minutes, followed a carefully optimized program as outlined in After cooling, the entire digest was transferred into 50 mL plastic bottles made of Polypropylene and diluted to 25 mL with double deionized water.The blanks were prepared in a manner similar to that of the samples, and all sample solutions were clear prior to analysis by ICP-MS.

Standards:
Calibration and control element standard solutions were prepared by diluting a Multi-Element Standards stock solution at a concentration of 10 mg.L -1 (containing Na, Mg, P, K, Ca, Al, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Rb, Sr, Cd, Ba, Pb) and a mono-element standards stock solution at a concentration of 1000 mg.L -1 (containing Ga, Ir, Rh), both of which were supplied by INORGANIC VENTURES™.

Apparatus and method
The determination of major and trace elements was carried out using a Thermo ICAP-Q ICP-MS instrument, coupled with a Cetac ASX 520 Autosampler and controlled by Qtegra™ ISDS Software.Additionally, a Thermoflex 2500 Chiller was utilized.
The plasma power was set at 1550 W to maintain stable plasma during the analysis of the samples following acidic digestion.The flow rate of the plasma gas (99.9999% purity Argon N60) was increased to 14 L/min to accommodate the higher power usage.The ICP-MS ICAP-Q was outfitted with a concentric nebulizer, quadrupole mass analyzer, and collision chamber.Argon served as the plasma gas, while helium (99.999% purity) was employed in the collision chamber to mitigate molecular interferences.The ICP-MS system underwent calibration through the use of external standards with internal standards for control.The reagent blank solution contained 0.2% concentrated HNO3.Mixed standard solutions for calibration encompassed 20 elements, namely Na, Mg, P, K, Ca (ranging from 0.5 mg.L -1 to 5 mg.L -1 ), and Al, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Rb, Sr, Cd, Ba, Pb (ranging from 0.0010 mg.L-1 to 1 mg.L -1 ).For Gallium, Iridium, and Rhodium (Ga at 0.05 mg.L-1, Rh at 0.01 mg.L -1 , and Ir at 0.01 mg.L -1 ), these were employed as internal standards.

Quality Control
To evaluate the accuracy and precision of our analytical method, we prepared and analyzed the standard material SRM-1547 (Peach Leaves) from the National Institute of Standards and Technology (NIST) under the same conditions as the Cannabis Sativa Seeds samples.
A range of statistical approaches exists for appraising the precision and performance of analytical methods.In our investigation, we employed the En parameter, computed according to equations 1 [17]: Where Ulab, represents the uncertainty associated with the laboratory, while Ucert relates to the certified uncertainty values.

Health Risk Assessments of Heavy Metal
Cannabis Sativa is recognized for its capacity to accumulate heavy metals such as Cadmium, Lead, Arsenic, Mercury, Chromium, Nickel, Manganese and Cobalt in its roots, shoots, buds, and seeds.As a result, it has been used for soil decontamination through processes like phytoremediation and phytoextraction [13][14].However, this property can hinder the use of Cannabis in the food and medical industries.
In this study the health risk was assessed for 5 elements detected in seeds of Cannabis Sativa (Cr, Ni, As Cd, and Pb) cultivated in Senhaja Srair Region.Estimated daily Intake (EDI), non carcinogenic risk assessment (THQ, HI), and carcinogenic risk assessment (CR) were employed to explore the potential health impacts from heavy metal contamination in seeds of Cannabis Sativa.The description of all factors using for health risk assessment of toxic element was taken in the table 3.
Various pathways exist for human exposure to heavy metals and trace elements, but the predominant route is through dietary consumption (ingestion), accounting for approximately 90% of the overall health risk.Consequently, the current study focuses E3S Web of Conferences 469, 00027 (2023) ICEGC'2023 https://doi.org/10.1051/e3sconf/202346900027exclusively on risk assessment calculations pertaining to this particular pathway, and an average daily consumption of 3g of Cannabis Sativa seeds was assumed [11].

Non-Carcinogenic risk
The Target Hazard Quotient (THQ) serves as a quantitative assessment of the potential noncarcinogenic effects associated with individual toxic metals.The THQ calculation was derived from the following Equation 3: Where EDI is the daily average exposure dose (mg/kg/day) and RfD (mg/kg/day) represents the oral reference dose of metal.The following reference doses (RfD) for each elements was taken in the table 5.
The Hazard Index (HI) was employed to estimate the cumulative impact of toxic elements.HI is the total of the hazard quotients all heavy metals or toxic elements in plant, and it was estimated according to the equation 4: When the values of THQ and HI are below 1, the exposure dose is lower than the adverse reaction threshold.However, if the THQ and HI exceed 1, there is a possibility that toxic metals may have adverse effects on human health [18][19].

Carcinogenic risk (CR)
The CR model used in this study is based on the equation equations 5 and 6 where CRk represents the cancer risk for a specific heavy metal and CSf stands for the cancer slop factor defined as the upper-bound estimation of Cancer Risk (CR) linked to a substance over an individual's lifetime.The overal CR for Each Cannabis seed sample was obtained by summing up CRk values for all metals [11].

quality control results
The outcomes from our investigation for the NIST reference material, which are employed for quality control, and the En parameter are presented in Tables 4.. Performance levels were categorized as either acceptable (En ≤ 1) or unacceptable (En > 1).
As observed in Table 4, the majority of elements detected in the NIST reference material SRM-1547 (Peach Leaves) sample exhibited an En value below 1 (Mg, Br, Al, Ca, V, Mn, Ba, Fe, Cd, Cr, and Cu), except for sodium, potassium, and zinc.In general, these findings indicate a high degree of concordance between the measured concentrations and the certified values.

Major and Trace Elements in Cannabis sativa Seeds
The present study determined the concentrations of twenty major and trace elements in the Cannabis sativa seeds using ICP-MS (Table 5).
As seen in the table 5, the high mineral content of the seeds of cannabis is reflected in the concentrations of Ca, K, Mg, Na, Fe, Mn, and Al, while lower concentrations of Cr, Co, Cd, Pb and other elements were observed.

Essential Macro and micronutrient
The major macronutrients vital for human health and nutrition, including K, P, Mg, Ca, and Na, were found to have concentrations in the following order of decline: K>P>Mg>Ca>Na.The essential micronutrients were found to have concentrations in the following order: Fe>Mn>Zn>Cr.Essential elements such as potassium, calcium, sodium, chloride, and magnesium play crucial roles in maintaining human health.Potassium is vital for regulating blood pressure, salt sensitivity, and kidney stone risk, with hypokalemia being a significant concern in cases of deficiency.Calcium is essential for bone and teeth health, as well as various bodily functions.Sodium and chloride are necessary for maintaining fluid balance and plasma osmolality.Magnesium is involved in numerous enzymatic processes and contributes to bone health, potassium, and calcium regulation [20][21].
The seeds of cannabis sativa collected from Senhaja Srair analysed in this study showed significant concentrations of potassium, calcium, phosphorus, magnesium, and sodium.The concentrations of these elements in three samples range from (57,835±2,795) mg/kg for sodium to (19135 ± 325) mg/kg for potassium.
Zinc is an essential micronutrient that is involved in various enzymatic processes related to the metabolism of carbohydrates, proteins, and fats.Additionally, Zinc plays a crucial role in the immune system, influencing both cellular and humoral immunity, and the metabolism of other micronutrients [20][21].The concentration of zinc in the seeds of Cannabis were (66,926±1,082, 43,329±0,190, 48,589±0,842) mg/kg, falling within the range of concentrations typically found in highzinc foods like lean red meat, whole-grain cereals, pulses, and legumes, which provide the highest concentrations of zinc (25-50mg/kg) [21].
Iron is a vital element that performs various essential functions in the body.It exists in ferrous, ferric, and ferryl states, and acts as a carrier of oxygen from the lungs to the tissues through red blood cell hemoglobin, a medium for electron transportation within cells, and an integral component of important enzyme systems in different tissues [20][21].Three Seeds of Cannabis Sativa analyzed in this study contains a high concentration of iron (863,842±39,121, 572,836±15,871, 505,241±11,274) mg/kg.
Chromium is an important element that is found in foods in trivalent chromium or chromium II forms.The concentration of Cr found in the seeds of Cannabis sativa were (2,514±1,610; 1,531±0,028; 1,610±0,046) mg/kg.In the analyzed seeds of Cannabis Sativa, both highly toxic and less toxic elements, such as Al (aluminum), As (arsenic), V (vanadium), Hf (hafnium), Cd (cadmium), and Pb (lead), were identified.Among these, aluminum exhibited the highest concentration, measured at (358,302±9,758) mg/kg, while arsenic had the lowest concentration at (0,031±0,002) mg/kg.Lead (Pb) is a hazardous metal and is a major source of daily intake for humans and animals through food.The absorption of Pb is dependent on age and the degree of dissolution in the stomach.In adults, approximately 10% of Pb is absorbed from the gastrointestinal tract, while infants can absorb up to 40-50% [22].Chronic Pb poisoning in adults can lead to anemia, certain types of cancer, and male reproductive damage.In young children, exposure to Pb can lead to hormonal imbalances of vitamin D metabolites [22].

Toxic elements
The Pb concentration found in the seeds of cannabis sativa (0,069±0,003; 0,277±0,002; 0,026±0,002) mg /kg is lower than the Pb limits of 3, 5 and 10 mg/kg set by the EU, the European Pharmacopoeia and the WHO, respectively [23].Cadmium usually enters the body via oral, respiratory and skin routes.Several studies have demonstrated that exposure to Cadmium (Cd) or its compounds can result in osteoporosis and pediatric cancer.Cd toxicity can also lead to kidney damage, specifically in the proximal convoluted tubules, which is associated with mitochondrial dysfunction [23].
Arsenic is a toxic element that occurs naturally in the earth's crust, and exposure to high levels of arsenic can lead to a variety of health problems.Short-term exposure to high levels of arsenic can cause symptoms such as vomiting, abdominal pain, and diarrhea.Long-term exposure to lower levels of arsenic can lead to a variety of health problems, including skin lesions, diabetes, cardiovascular disease, and several types of cancer, including skin, lung, bladder, and kidney cancer [22].Results presented in table 4 show approximately the same concentration for three samples analyzed Cannabis N01, Cannabis N02 and Cannabis N03 respectively (0,031±0,002 -0,039±0,001 -0,037±0,003) mg/kg.

Daily Intake Estimations for Heavy Metals (EDIs)
Zafeiraki et al. adopted a precautionary approach by making an assumption founded on the highest reported cannabis consumption rate, which amounts to 3 grams per person per day [11].
In the current study, we have calculated the daily intake (in milligrams per day) of five heavy metals (arsenic, cadmium, chromium, nickel, and lead) under the same scenario (3 grams per day of Cannabis sativa seeds).The results presented in table 6 indicate that Cr had the highest estimated daily intake, followed by Ni, Pb, As, and Cd.

Non-carcinogenic Risk
Based on the results showed in Table 6, all THQ (Target Hazard Quotient) values were consistently below 1, indicating that HI (Hazard Index) values, an indicator of quantified risk, also remained below 1.This implies a negligible risk of non-carcinogenic effects for the population exposed to the current cannabis samples and their derived products.

Carcinogenic Risk
Carcinogenic risk quantifies the combined likelihood of developing cancer over an individual's lifetime due to exposure to a possible carcinogen.Consequently, two assessment parameters were taken into account: the EDI (Estimated Daily Intake) of each heavy metal and the Cancer Slope Factor (SF) for oral cancer.In this context, the cancer risk (CR) was evaluated through the utilization of equations 5 and 6.The Cancer Risk (CR) was considered as negligible when it was below 10-6 and as likely harmful when it exceeded 10-4.CR values falling within the range of 10-6 to 10-4 were indicative of an acceptable or tolerable risk level [11].
In this study, all three Cannabis sativa seed samples exhibit CR values within the acceptable tolerable range (Table 6), indicating that the risk associated with heavy metal through cannabis consumption is not alarming.However, ongoing monitoring and father research are recommended to ensure long term environmental and human health safety.

Conclusion
In this study, we assessed the elemental composition and potential health risks associated with five heavy metals (Cr, Cd, Pb, Ni, and As) in seeds of Cannabis Sativa cultivated in the North of Morocco.Utilizing Inductively Coupled Plasma-Mass Spectrometry (ICP-MS), We detected 20 elements in the seeds, with notable concentrations of Mg, K, Ca, P, and Na, while Cr, Zn, Fe, and other toxic elements were present in trace amounts.The estimation of daily intake (EDI) revealed that Cr had the highest intake, followed by Ni, Pb, As, and Cd.However, the calculated Target Hazard Quotients (THQs) for individual metals and the Hazard Index (HI) were all found to be less than 1, indicating that the consumption of Cannabis Sativa seeds cultivated in the North of Morocco poses relatively low health risks.Furthermore, the Carcinogenic Risk (CR) values were significantly in the range of 10-6 to 10-4 demonstrating an acceptable risk of cancer associated with the consumption of these seeds.
Overall, our findings suggest that the seeds of Cannabis Sativa grown in the studied region are relatively safe for consumption in terms of elemental composition and heavy metal contamination.However, continued monitoring of the elemental composition and potential contaminants in Cannabis Sativa seeds is essential to ensure ongoing safety and quality for consumers.

Fig 1 .
Fig 1. Map representation of the study area

Table 1 :
Optimized program for samples digestion.

Table 02 :
Optimal Operational Conditions for ICP-MS Analysis of the Investigated Samples.

Table 3 :
Essential Parameters Utilized in the Health Risk Evaluation of Toxic Elements.

Table 4 :
Results for NIST Reference Materials and En Parameter

Table 5 :
Elemental concentrations for Seeds of Cannabis sativa analysed.

Table 6 :
Heavy Metal EDIs, THQ, HI, CRk, and CR Values in Cannabis Sativa seeds.