Levels of selected metals in leaves of Cannabis sativa L. cultivated in Ethiopia
Forensic Investigation Directorate, Ethiopian Federal Police Commission, Addis Ababa, Ethiopia
Department of Chemistry, College of Natural Sciences, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia
Abstract
Background
Cannabis sativa L. is one of the illicit drug bearing plants. Cannabis products are the most widely trafficked drugs worldwide. The highest levels of cannabis production in the world take place in the African continent. A small volume of cannabis is produced in rural areas of Ethiopia, of which a small portion is exported to neighboring countries and the majority is consumed at home. The literature survey revealed that there is no report on the metal contents in cannabis cultivated in Ethiopia. The main objective of this study is to determine the level of selected metals in leaves of Cannabis sativa L. cultivated in Ethiopia.
Methods
Cannabis sativa L. samples were collected from Metema (Amhara Region), Mekelle (Tigray Region), Sheshemene (Oromia Region) and Butajira (South Nations Nationality and Peoples (SNNP) Region) of Ethiopia. After proper sample pretreatment, the volumes of reagents used, digestion temperature and digestion time were optimized and using the optimized conditions the levels of metals were determined by flame atomic absorption spectrometry.
Results
The accuracy of the optimized procedure was evaluated by analyzing the digest of the spiked samples with standard solution and the percentage recoveries varied from 88 to 103%. The levels of metals determined (µg/g dry weight) were in the ranges Ca (657–1,511), Zn (321–380), Ni (124–172), Cu (122–176), Cd (3–10), Pb (8–10), and Cr (4–8). Zn was with the highest concentration among trace metals.
Conclusion
A statistical analysis of variance (ANOVA) at 95% confidence level indicated that there is a significant difference in the levels of all the metals between the four sample means except Pb. The results indicate that the content of Pb and Cd exceeds the permissible amount for medicinal plants which form the raw materials for the finished products set by World Health Organization (WHO).
Background
Cannabis (Cannabis sativa L.) is an annual herbaceous plant. It is a dioecious plant (Suurkuusk 2010). In many countries, cannabis is cultivated as a narcotic substance or a source of narcotic substances like hashish and hashish oil. Now a day cannabis is cultivated on the large areas with the mild and tropical climate for the cannabis oil and fibre (United Nations on Drugs and Crimes 2009). The highest levels of cannabis production in the world take place in the African continent. The crude drug can be obtained from leaves, flowers, seeds and stem of cannabis. The female plant yield more drug than the male. It can be smoked in cigarettes or pipes and can be snuffed or added to food (Eboh and Thomas 2005).
Cannabis products are the most widely trafficked drugs worldwide. Practically all countries in the world are affected by cannabis trafficking (United Nations on Drugs and Crimes 2009). Ethiopia does not play a major role in the production, trafficking or consumption of illicit narcotics or precursor chemicals associated with the drug trade. A small amount of cannabis is produced in rural areas of Ethiopia, of which a small portion is exported, primarily to the neighboring countries; the majority is consumed at home, but absolute quantities in both cases are moderate (INCSR 2006).
The main areas of cannabis cultivation in Ethiopia are Alemaya district of eastern Hararghe, Shebedino district of Sidamo, Sheshemene district and other areas in Oromia region, and Debre Berhan district in Amhara region. Now, Sheshemene is famous for the quality of its cannabis produced and its involvement in the commercial cannabis trade in Ethiopia and surrounding countries (Sensi 2014).
The chemical composition of cannabis varies with the type, age and part (flower, root, leaf, fiber, etc.) of cannabis plant as well as with the type of preparation. While ∆9-tetrahydrocannabinol is responsible for psychoactive properties of cannabis some of the other components modulate its activity.
Cannabis sativa L. is known for millennia for its therapeutic properties and as a recreational psychoactive drug. The adverse health effects of cannabis are a source of contention in debates about policies towards the drug. Cannabis can adversely affect some users, especially adolescents who initiate use early and young adults who become regular users (Hall 2009).
Herbal medicines are likely to be contaminated with heavy metals. In trace amounts some heavy metals are essential for the human body however they may be toxic if present in a higher concentration. They have the ability to bioaccumulate and disrupt functions of vital organs and glands in the human body such as brain, kidney and liver (Dzomba et al. 2012).
The high concentration of heavy metals in soils is reflected by higher concentrations of metals in plants, and consequently in animal and human bodies (Buszewski et al. 2000). The uptake mechanism is selective, plants preferentially acquiring some metals over others (Angelova et al. 2004).
Recently some studies have been carried out on levels of essential and non-essential metals in a psychoactive khat leaves (Atlabachew et al. 2010) and some medicinal plants cultivated in Ethiopia (Derbie and Chandravanshi 2011; Gebre and Chandravanshi 2012; Aregahegn et al. 2013; Mekebo and Chandravanshi 2014; Endalamaw and Chandravanshi 2015; Dubale et al. 2015; Wagesho and Chandravanshi 2015).
Levels of selected heavy metals have been determined in cannabis in some countries. In Pakistan heavy metals (Cr, Pb, Cu, Cd, Ni and Zn) were determined in Cannabis Sativa L. and the soil of the area from where the plant was collected using AAS. The plant parts including root, stem and leaves were found to have the quantity of heavy metals corresponding to their contents in the soil (Khan et al. 2008).
Another research conducted on the distribution of heavy metals content in cannabis leaf and seed were carried out using AAS in Nigeria. The results showed that As, Cd., Cr, Fe, Ni, Pb and Hg levels in cannabis leaf exceeded those of the cannabis seed. Therefore cannabis leaves seem to be more dangerous to health than the seeds (Eboh and Thomas 2005).
The levels of some heavy metals (Pb, Ni, Cd and Cr) in eight different medicinal plants samples including Cannabis sativa L. along with soils were collected from two different locations from salt range of Punjab, Pakistan in order to evaluate those vital metals involved in human health implications. The findings suggest that the use of these plant species for the management of diseases will not cause heavy metal toxicity (Khan et al. 2013).
The levels of selected heavy metals (Fe, Mn, Cu, Zn and Pb) and macronutrients (Na, Ca, Mg, K and P) in eight useful herbal plants including Cannabis sativa L. in Pakistan were determined by AAS. The levels of heavy metals determined in the analyzed samples were found below the maximum allowable limit. The analyzed samples were good source of important macro elements (Ghani et al. 2012).
The literature survey revealed that there is no report on the metal contents in cannabis cultivated in Ethiopia. The main objective of this study is to determine the level of selected metals in leaves of Cannabis sativa L. cultivated in Ethiopia. The specific objectives are: (1) to develop an optimum working procedure for digestion of leaves of Cannabis sativa L. samples to determine selected metals by flame atomic absorption spectrophotometry (FAAS), (2) to determine the concentrations of Ca, Cr, Ni, Cu, Zn, Cd and Pb in leaves of Cannabis sativa L., (3) to compare the levels of the identified metals in leaves of Cannabis sativa L. from different regions of Ethiopia, and (4) to compare the levels of the identified metals in leaves of Cannabis sativa L. in Ethiopia and with that of the data in the literature.
Methods
Equipments
Polyethylene bags were used during sample collection, sample drying and preserving the ground and homogenized samples. Electronic blending device, Moulinex (Ecully Cedex, France) was used for grinding and homogenizing the sample. Electronic series balance, WL 3000, Adam Equipment Co. Ltd. (Milton Keynes, UK), with precision of ±0.0001 g was used for weighing samples. 100 mL round bottom flasks fitted with reflux condensers were used in Kjeldahl, Fisher Scientific UK Ltd (Leicestershire, UK) apparatus hot plate to digest the dried and powdered cannabis samples. Borosilicate volumetric flasks (25, 50 and 100 mL) were used during dilution and storage of samples and preparation of metals standard solutions. 1, 2, 5 and 10 mL pipettes, Pyrex (St. Louis, MO, USA) were used for measuring the solutions. Refrigerator, Hitachi (Tokyo, Japan) was used for the preservation of digested samples before AAS analysis was made. Micropipettes (10–100 µL and 100–1,000 µL), Dragonmed (Shangai, China) were used for measuring different volumes of metal standard solutions for spiking. Flame atomic absorption spectrophotometer, Analytik Jena ZEEnit 700P (Jena, Germany) equipped with deuterium arc back ground connectors and hollow cathode lamps with air-acetylene flame was used for the determination of the analyte metals (Ca, Cr, Ni, Cu, Zn, Cd and Pb) in the samples.
Reagents and chemicals
The chemicals and reagents used in the study were HNO3 69.5% (Scharlau Chemie S.A., European Union, Spain), HClO4 70% (Reaserch-Lab Fine Chem Industries, Mumbai, India), H2SO4 98% (Research-Lab Fine Chem Industries, Mumbai, India), La(NO3)3.6H2O 98% pure (BDH Chemicals Ltd, Poole, England), 1,000 mg/L, in 2% HNO3, standard of the Ca, Cr, Ni, Cu, Zn, Cd and Pb (BDH Chemicals Ltd Spectrosol®, Poole, England) and H2O2. Deionized water was used for dilution of sample and intermediate metal standard solutions prior to analysis and rinsing glassware.
Sample collection
All the cannabis samples analyzed in this investigation were confiscated during 2013. Three of the samples were whole fresh plants collected from the growing plots by the police before they had been harvested except samples from Mekelle which was collected from the users. Leaves of cannabis samples caught by police were collected from the four sites. The four sites are Butajira (SNNP Region), Metema (Amhara Region), Sheshemene (Oromia Region) and Mekelle (Tigray Region). About 200 g of fresh leaves of cannabis was taken from each sites except Mekelle (Tigray Region) and the collected samples were packed into polyethylene plastic bags, labeled and transported to the laboratory for further treatment. The geographical locations of these sampling sites are described in Table 1.No. Sample sites Approximate geographical locations Latitude Longitude Altitude in meters (above sea level) Distance in kilometers and directions from Addis Ababa 1 Butajira (SNNP Region) 8°10′N 38°50′E 1,900 132 km South West 2 Metema (Amhara Region) 12°58′N 36°12′E 684 898 km North West 3 Sheshemene (Oromia Region) 7°12′N 38°36′E 2,009 240 km South East 4 Mekelle (Tigray Region) 13°29′N 39°28′E 2,037 787 km North
Sample preparation for elemental analysis
The cannabis samples were washed with a running tap water so as to remove adsorbed soil particulates and then rinsed with deionized water. The samples were exposed to sun light until the weight becomes constant for several days to reduce the moisture content so as to express the result in terms of dry mass basis. The dried sample was ground using electronic blender and sieved to prepare fine powder of cannabis for digestion. Then the fine powder of cannabis samples were kept in properly washed, dried and cleaned polyethylene plastic bags until appropriate amounts of the samples were taken for digestion.
Optimization of digestion procedure
The basic requirements for sample preparation for analysis are to get an optimum condition for digestion. The optimum condition is the one which required minimum reagent volume consumption, minimum reflux time, clarity of digests, and ease of simplicity (Soylak et al. 2004; Tuzen and Soylak 2007; Uluozlu et al. 2007; Demirel et al. 2008).
In wet acid digestion the optimized digestion procedure is attained based on changing different digestion parameters like volume ratio of reagents added, digestion temperature and digestion time. In this procedure the organic components are decomposed in the different gaseous forms and other metallic elements are left in the solution except those volatile metals like Hg. The digestion is assumed to be complete if the solution is clear and colorless (Endalamaw and Chandravanshi 2015; Wagesho and Chandravanshi 2015). Based on this fact different digestion procedures were optimized using the HNO3:HCl:H2O2:HClO4 acid mixtures by varying parameters such as volume of the acid mixture, digestion time and digestion temperature. The results of optimization procedures for the analysis of Cannabissativa L. samples for its metals content are shown in Tables 2, 3, 4.No. Total volume (mL) Volume ratio (mL) HNO3: HCl:H2O2:HClO4
Observation 1 4 2:0:1:1 Pale yellowish solution with small residue 2 4 2:1:0:1 Yellowish solution with residue 3 4 2:0.5:0.5:1 Pale yellow solution with residue 4 5 2:2:0:1 Pale yellow solution with residue 5 5 2:1:1:1 Yellow solution with residue 6 5 3:1:1:0 Deep yellow solution with small residue 7 6 3:1:1:1 Yellow solution with small residue 8 6 3:2:0.5:0.5 Colorless solution but not clear 9 7 4:1:1:1 Pale yellow solution with small suspension 10 7 4:2:0.5:0.5 Yellow solution with residue 11 8 5:1:1:1 Slightly clear and colorless solution with residue 12 8 5:2:0.5:0.5 Pale yellow solution with small suspension 13 9 6:2:0:1 Slightly clear and colorless solution with residue 14 9 7:0:1:1 Slightly clear and colorless solution with small residue 15
10
9:0:0:1
Clear and colorless solution
16 10 9:0.5:0:0.5 Clear and colorless solution 17 10 9:0:0.5:0.5 Clear and colorless solution No. Temperature (oC) Observation 1 180 Slightly colorless solution with residue 2 210 Slightly colorless solution with residue 3 240 Slightly colorless solution with residue 4
300
Clear and colorless solution
No. Time (h) Observation 1 2:00 Colorless solution with some residue 2 2:30 Colorless solution with some residue 3 3:30 Clear and colorless with some residue 4
4:00
Clear and colorless
Digestion of leaves of Cannabis sativa L. samples
Applying the optimized conditions (Tables 2, 3, 4), 0.5 g of dried and homogenized cannabis samples were transferred into a 250 mL round bottom flask. Then 10 mL of a mixture of HNO3 (69.5%) and HClO4 (70%) with a volume ratio of 9:1 (v/v) was added and the mixture was digested on a Kjeldahl digestion apparatus fitting the flask to a reflux condenser by setting the temperature at 300°C for 4:00 h. The digest was allowed to cool to room temperature for 30 min without dismantling the condenser from the flask and for 10 min after removing the condenser. To the cooled solution 10 mL of deionized water was added to dissolve the precipitate formed on cooling and to minimize dissolution of filter paper by the digest residue while filtering with Whatmann (110 mm diameter) filter paper into 25 mL volumetric flask. The round bottom flask was rinsed subsequently with 5 mL deionized water until the total volume reached around 15 mL. To this final solution, about 0.67 g of La(NO3)3.6H2O was added to prevent the precipitation of Ca+2 with the SO4−2 and PO4−3 and the solution was filled to the mark (25 mL) with deionized water. The digestion was carried out in triplicate for each bulk sample. Digestion of a reagent blank was also performed in parallel with the cannabis samples keeping all the digestion parameters the same. The digested samples were kept in the refrigerator, until the level of all the metals in the sample solutions were determined by FAAS.
Instrument calibration
Metal standard solutions for calibration were prepared for each of the metals from an intermediate standard solution containing 10 mg/L which was prepared from the atomic absorption spectroscopy standard stock solutions containing 1,000 mg/L. These intermediate standards were diluted with deionized water to obtain four working standards for each metal of interest. The absorbances of the working standard solutions were measured and the calibration curves for each of the analyte metal (Ca, Cr, Ni, Cu, Zn, Cd and Pb) were constructed. The levels of each metal in all the samples were determined with FAAS using external calibration curve after the instrument parameters were optimized for maximum signal intensity. Three replicate determinations were carried out on each samples and standards. The same analytical procedure was employed for the determination of elements in digested blank solutions.
Concentration of working standards, correlation coefficient of the calibration curves and equation for calibration curves for each metal are listed in Table 5. The correlation coefficients in Table 5 for each metals show that the change in absorbance with concentration is in good positive correlation and linear fit.Metal Wavelength (nm) Method detection limit (μg/g dry weight) Concentration of working standards (mg/L) Correlation coefficient (r) Equation for calibration curves Ca 422.7 25 0.25, 0.5, 0.75, 1.0 0.9997 Y = 0.49265 X − 2.64 × 10−3
Cr 357.9 1 1.0, 2.0, 3.0,4.0 0.9997 Y = 0.09698 X + 5.00 × 10−4
Ni 232.0 8 1.0, 2.0, 3.0,4.0 0.9998 Y = 0.04318 X − 6.22 × 10−3
Cu 324.8 19 0.25, 0.5, 1.0, 2.0 0.9999 Y = 0.20258 X − 2.80 × 10−4
Zn 213.9 31 0.25, 0.5, 0.75, 1 0.9999 Y = 0.66746 X − 1.13 × 10−3
Cd 228.8 0.4 0.25, 0.5, 0.75, 1 0.9997 Y = 0.49265 X − 2.64 × 10−3
Pb 217.0 6.0 1.0, 2.0, 3.0,4.0 0.9995 Y = 0.09786 X + 4.52 × 10−3
Method detection limits (MDL)
The method detection limit is the minimum concentration that can be detected by the analytical method with a given certainty. It is also the smallest concentration or amount of an analyte that can be reliably shown to be present or measured under defined condition, and the limit of detection is the lowest concentration level that can be determined statistically different from a blank (99% confidence). The MDL/LOD is typically determined to be in the region where the signal-to-noise ratio is greater than 3 but not necessarily quantified as an exact value (Chen 2007). It can be calculated by multiplying the pooled standard deviation of the reagent blank (Sblank) by three (MDL = 3 × Sblank, n = 12). Method detection limits of the metals of interest are shown in Table 5 which indicates that the presence of trace amounts of metals of interest in the sample can be detected by the method.
Validation of optimized procedure
The validity of the optimized procedure was assessed by spiking experiments. For this purpose standard solution of 1,000 mg/L was used and intermediate standards of 10 mg/L were prepared. For recovery measurement Amhara sample was selected randomly. The spiking was done in three groups in triplicate.
In the first group 1.4 µL of 1,000 mg/L of Ca and 240 µL of 10 mg/L of Zn were spiked. In the second group 12.2 µL of 1,000 mg/L of Cu, 40 µL of 10 mg/L of Cr and 7.4 µL of 1,000 mg/L of Ni were spiked. In the third group 210 µL of 10 mg/L of Pb and 80 µL of 10 mg/L of Cd were spiked.
The spiked, un-spiked and reagent blank samples were digested and analyzed in similar condition using the optimized procedure as used for sample analysis. The results of recovery analysis are shown in Table 6 and the percentage recoveries lies within the range 88–103%. The percentage recovery for cannabis samples are within the acceptable range for all the metals.Metal Amount spiked (mg in 0.5 g) Amount recovered (mg in 0.5 g) Percent recovery (% R) Ca 0.0014 0.0013 92.9 Cr 0.0004 0.0004 100 Ni 0.0074 0.0072 97.3 Cu 0.0122 0.0125 102.5 Zn 0.0024 0.0022 91.7 Cd 0.0008 0.0007 87.5 Pb 0.0021 0.0019 90.5
Results and discussions
Statistical analysis
In analytical work two or more mean results are compared to check whether there is a significant difference between them or not. The variation between the means of different samples can result from random and controlled sources of error. This kind of variation and the variation due to the difference in the original sample type can be separated and estimated by a powerful statistical technique known as analysis of variance (ANOVA) (Miller and Miller 2005). In this study, a one way ANOVA and Pearson correlation coefficient was used to decide the variation between samples analyzed was significant or not for the mean concentrations of each metal in triplicate analysis.
Analysis of variance
The variation in samples means come from different sources such as experimental procedure or heterogeneity among the samples (i.e. difference in mineral contents of soil, pH of soil, water, atmosphere; variation in application of agrochemicals like fertilizers, pesticides, herbicides, etc. or other variations in cultivation procedures). In this study the variation in samples means of the analyte was tested whether they have significant difference or not by using ANOVA of SPSS 20 (IBM) software (Table 9).df F Significance Ca Between samples 3 55 0.000 Within samples 8 Total 11 Zn Between samples 3 6 0.022 Within samples 8 Total 11 Ni Between samples 3 5 0.034 Within samples 8 Total 11 Cu Between samples 3 6 0.022 Within samples 8 Total 11 Cd Between samples 3 19 0.001 Within samples 8 Total 11 Pb Between samples 3 4 0.068 Within samples 8 Total 11 Cr Between samples 3 19 0.001 Within samples 8 Total 11
From Table 9 one can see that there is significant difference at F3,8 at 95% confidence level in mean concentration for all metals except Pb. This is due to variations in the other factors than in the experimental procedure. The source for this significant difference between sample means may be the difference in mineral contents of soil or pH of soil which predict the extent of mineral absorption by cannabis.
Conclusion
In this study the levels of metal in Cannabis sativa L. collected from four regions of Ethiopia were analyzed for their contents of Ca, Cr, Ni, Cu, Zn, Cd and Pb using flame atomic absorption spectrometer. The optimized wet digestion method for the analysis of cannabis was found efficient for all the metals and it was evaluated through the recovery experiment and a good percentage recovery of 88–103% was obtained for all the metals identified.
The level of metals investigated in cannabis is in the following order: Ca (657–1,511 μg/g) > Zn (315–380 μg/g) > Ni (124–172 μg/g) > Cu (122–176 μg/g) > Pb (7.9–10.2 μg/g) > Cr (3.6–7.6 μg/g) > Cd (3.2–4.7 μg/g).
Statistical analysis by using one way ANOVA indicates that there is significant difference in mean concentration of metals in Cannabis sativa L. in the four sampling sites except Pb. This may be attributed to differences in soil composition, use of different fertilizers, pesticides, etc. and for Pb, the difference may only be attributed to random errors in the experimental procedures. Pearson correlation matrices indicate that most of the metals have negative correlation with each other except Ca having positive correlation with Cr and Pb; Zn with Ni, Cu, Cd; Ni with Cu; Cu with Cd; and Pb with Cr. This clearly indicates that most metals have different sources of accumulation in Cannabis sativa L.
Based on the results of this study, it can be concluded that Cannabis sativa L. contains higher levels of essential metals Ca, Zn and Cu. But highly toxic metals like Pb and Cd were also detected in Cannabis sativa L. The content of both toxic metals are beyond permitted limits set by WHO. This will further lead to the harmful effect of Cannabis sativa L. on human health. It is necessary to monitor the levels of these toxic metals in medicinal herbs.
In general, the levels of metals in the Ethiopian cannabis are comparable to those reported in the literature from other countries.