Levels of essential and toxic metals in fenugreek seeds ( Trigonella Foenum-Graecum L . ) cultivated in different parts of Ethiopia

This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited Levels of essential and toxic metals in fenugreek seeds (Trigonella Foenum-Graecum L.) cultivated in different parts of Ethiopia Níveis de metais essenciais e tóxicos em sementes de fenacho (Trigonella Foenum-Graecum L.) cultivados em diferentes partes de Etiópia

Fenugreek leaves and seeds are consumed in different countries around the world for different purposes, such as medicinal uses (anti-diabetic, lowering the blood sugar and cholesterol levels, anti-cancer, anti-microbial, against breast cancer (IBRAHIUM; HEGAZY, 2009), avoiding blood poisoning from wounds (HOODA; JOOD, 2005), making food (stew with rice in Iran, flavouring cheese in Switzerland, syrup and bitter rum in Germany, mixed seed powder with flour for making flat bread in Egypt, curries, dyes, young seedlings for eating as a vegetable), roasted grain as a coffee-substitute (in Africa), controlling insects in grain storages and perfume industries.The seeds of this ancient herb have been used as both a spice and a herbal remedy in the Middle East, India, and Egypt and later on in Europe, China and other parts of the world (SUSHMA; DEVASENA, 2010).Fenugreek seeds have been reported for their pharmaceutical properties in treating such human diseases as diabetes and hypercholesterolemia (ZIA et al., 2003).
In Ethiopia fenugreek is cultivated between altitudes of 1600 and 2300 m above sea level all over the country (AHARI et al., 2009) and is used in preparing "Hilbet", a delicious, traditional, soft white food in the Tigray regional state.It is also used to make tea, "Enjera", flat bread mostly made of "tef" flour, as a spice in milk and in traditional medicine.
Recently some studies have been carried out on the mineral composition of selected spices (thyme, fennel, ginger, korarima, and red pepper) cultivated in Ethiopia (DERBIE;CHANDRAVANSHI, 2011;ENDALAMAW;CHANDRAVANSHI, 2015;WAGESHO;CHANDRAVANSHI, 2015;MEKASSA;CHANDRAVANSHI, 2015;TEFERA;CHANDRAVANSHI, 2016).Several global studies were also been done on Trigonella foenum-graecum (fenugreek) especially in the areas of medicine and to determine the metal content.However, none of these studies were done in Ethiopia.Therefore, it seemed worthwhile to study the levels of essential and non-essential metals in T. foenum-graecum (fenugreek) cultivated in Ethiopia.
The assessment of the major metals (Ca, K, Na and Mg), trace metals (Fe, Cr, Ni, Zn, Mn, Cu, Co) and toxic metals (Pb, Cd) in T. foenum-graecum (fenugreek) samples collected from the Amhara and Tigray regional states of Ethiopia was carried out in order to produce experimental data which could be used to identify the nutritional use as well as the toxicological status of the spice.It was also considered helpful to determine the sources of the metals and to assess the pollution level.
The main objective of this study was (i) to determine the levels of the major (Ca, K, Na, Mg), trace (Fe, Cr, Ni, Zn, Mn, Cu, Co), and toxic (Pb, Cd) metals in Trigonella foenum-graecum L. (fenugreek) cultivated in Ethiopia using flame atomic absorption spectrophotometry (FAAS), (ii) to compare the levels of the metals in fenugreek seeds from different regions of Ethiopia with that of the data in the literature, and (iii) to compare the levels of the metals in fenugreek seeds with that of the data on other spices, available in the literature.

Apparatuses
A Gallenkamp Kjeldahl apparatus (UK) was used to digest the fenugreek samples.A flame atomic absorption spectrophotometer (Buck Scientific Model 210 VGP AAS, East Norwalk, USA) using a deuterium background corrector with an air-acetylene flame was used to determine the concentrations of the metals Ca, K, Na, Mg, Fe, Cr, Ni, Zn, Mn, Cu, Co, Pb, and Cd in the fenugreek samples.

Reagents
All the chemicals and reagents used were of analytical grade.Sulfuric acid 98%, H 2 SO 4 (Fine Chem, Mumbai, India), perchloric acid 70%, HClO 4 (Fine Chem, Mumbai, India), hydrogen peroxide, 30%, w/w, extra pure (Spain), nitric acid, HNO 3 (pure 69-72%) (Spectrol, BDH, England) were used for the digestion of the fenugreek seed samples.Standard solutions (1000 mg/L in 2% HNO 3 ) of Ca, K, Na, Mg, Fe, Cr, Ni, Zn, Mn, Cu, Co, Pb, and Cd (Buck Scientific Puro-Graphic tm ) were used to prepare the calibration curves.LaNO 3 .6H 2 O (98%, Aldrich, USA) solution was used to prevent chemical interference from phosphates and other anions in the measurements of Ca and Mg.Chemically pure deionized water (with electrical conductivity < 2 μs cm -1 ) was used for cleaning the digestion and volumetric flasks and to dilute the samples.
Fenugreek seeds were collected from the different parts of the Amhara and Tigray regional states of Ethiopia.They were bought from the corresponding local markets from peasants living in that area.Half a kilogram was bought at most sample sites, packed into clean, dry polyethylene plastic bags, and finally transported to the Chemistry Department of the Science Faculty of Addis Ababa University for further study.

Sample preparation
The fenugreek seeds samples were washed with deionized water and then allowed to sundry in the open air.Each sample was then oven dried at 80 °C for 24 h.The dried samples were ground in a stainless steel blender (Moulindex, France) and the flour allowed to pass through a 1.4 mm mesh and kept in clean labelled plastic bags.

Optimization of the digestion procedure
There are several different methods to decompose organic samples using inorganic acids in an open vessel, and one of these is wet ashing.Wet ashing is the oxidative decomposition of organic samples by liquid oxidizing reagents such as HNO 3 , H 2 SO 4 , HClO 4 or mixture of these acids.This decomposition process converts the organic sample to carbon dioxide and water.Non-metallic elements such as halogens, sulfur and nitrogen, are wholly or partially lost by volatilization in an open vessel (SKOOG et al., 1996;SOYLAK et al., 2004).
To optimize the digestion of the fenugreek seed samples, different oxidizing agents and/or acid combinations were tested, using 0.5 g samples.The first and second combinations were not good and only the last combination gave good results.Therefore, to optimize the digestion procedure for fenugreek seeds, different ratios of HNO 3 /HClO 4 were used and the best result was obtained with a 5:1 ratio.The optimum condition for digestion was found using a total volume of 1.5 mL of the acid mixtures, temperature of 150 °C, 30 min pre-digestion time and 45 min total digestion time.The optimization of the digestion procedures for this study was done based on the parameters of time, volume, temperatures and reagent compositions, and the optimized condition was selected based on the criteria of minimum reagent costs, minimum time and a clear solution.Each of the 14 fenugreek seed samples was digested in triplicate.

Digestion of the fenugreek seed samples
A 0.5 g aliquot of powdered fenugreek seed was transferred to a 100 mL digestion flask followed by a total volume of 1.5 mL of the HNO 3 /HClO 4 acid mixture, i.e. 5:1 ratio.The mixture was allowed to remain in the open air for 30 min pre-digestion time and then digested at 150 °C for 45 min using a Gallenkamp Kjeldahl apparatus.The sample in the digestion flask was allowed to cool for about 40 min and then filtered through 70 mm Whatman filter paper into a 50 mL volumetric flask. 1 mL of a lanthanum nitrate solution and deionized water were added to the filtrate and then diluted to volume with distilled deionized water.The solution in the volumetric flask was thus ready for further analysis using flame atomic absorption spectrophotometry.
To determine the concentration of metals in the acid mixtures, blank samples were digested in the same manner as the fenugreek seed samples.A total of 10 blanks were prepared and digested.

Instrument calibration
The atomic absorption spectrometer was calibrated using four series of working standards.The working standard solutions of each metal were prepared freshly by diluting the intermediate standard solutions.A 10 mg L -1 intermediate standard solution of each metal was prepared by diluting a 1000 mg L -1 stock standard solution.The solution thus prepared was used to calibrate the instrument before determining the metal concentrations in the samples.

Method detection limit
A very important quantity in chemical analysis is the limit of detection.The LOD is the smallest concentration or amount that can be detected with reasonable certainty by a given analytical procedure (ENKE, 2000).In this study, the method detection limit of each element was calculated as three times the standard deviation of the blank (n = 10).The method detection limits were found to be low enough (<0.1 mg kg -1 ) to detect the trace quantities of selected metals in the fenugreek seed samples.

Precision and accuracy
The errors in analytical results can be expressed in terms of accuracy and precision (SKOOG et al., 1996).In this study the precision of the results was evaluated from the standard deviation of triplicate samples with triplicate measurements of each sample (n = 9), while the accuracy and the validity of the analytical method were determined by spiking the samples with known concentrations of standard solution.

Method validation
The efficiency of the optimized procedure was checked by spiking a known concentration of analyte into the sample.In this study 20% of the metal concentration determined was first spiked into the sample, and then digested in the same way as the un-spiked sample.The spiked concentrations of these metals were 0.307, 13.6, 0.906, 30.7, 23.1, 0.35, 0.13, 0.06, 0.056, 0.05, 0.03, 1.8 and 0.6 mg L -1 for Ca, K, Na, Mg, Fe, Cr, Ni, Zn, Mn, Cu, Co, Pb and Cd, respectively.50 μL each of Na, Fe, Ni, Mn, Cu, Co and Pb were spiked into one 100 mL digestion flask containing 0.5 g of fenugreek seed sample, and 50 μL each of Ca, K, Mg, Cr, Zn and Cd were spiked into another flask and digested using the optimized digestion procedure.Both the spiked and non-spiked samples were digested and analysed under similar conditions.Finally the percent recovery (% R) of the analyte was calculated.The percent recoveries were found to be in the range from 94-109%, which is within the acceptable range.

Distribution pattern of the metals in fenugreek samples from the different sampling sites
The concentrations of thirteen metals (Ca, K, Na, Mg, Fe, Cr, Ni, Zn, Mn, Cu, Co, Pb and Cd) were determined by digesting fenugreek seed flour and analysing by flame atomic absorption spectrometry, and the results are given in Table 1.The most abundant major element in fenugreek seed was Ca, the main trace element Fe and the major toxic element Pb.The concentrations of the major elements, i.e.Ca, K, Na and Mg in the fenugreek seed samples were found in the ranges from 1553-36771 mg kg -1 , 6789-11517 mg kg -1 , 201-1559 mg kg -1 and 31-102 mg kg -1 ; respectively.The concentrations of the trace elements, i.e.Fe, Cr, Ni, Zn, Mn, Cu and Co were found in the ranges from 6041-18585 mg kg -1 , 3-552 mg kg -1 , 31-108 mg kg -1 , 15-33 mg kg -1 , 16-28 mg kg -1 , N.D-35 mg kg -1 and 4-15 mg kg -1 ; respectively.Finally the concentrations of the toxic metals Pb and Cd were found in the ranges from 615-1814 mg kg -1 and 285-464 mg kg -1 ; respectively.Of the trace elements Fe, had the highest concentration due to the relatively higher Fe contents in Ethiopian soils.To the contrary, of the major plant elements, Mg was the metal with the lowest concentration.This may be due to the plant having a greater affinity for metals other than Mg.However, unlike other spices studied in Ethiopia, the concentrations of the toxic metals Pb and Cd were found to be much higher in the fenugreek seeds.The sources of these concentrations could be from the soil where the plants were cultivated or from the fertilizers used by the farmers.
The metal contents in the fenugreek seeds were found in the following decreasing order: Ca > Fe ≈ K > Pb > Na > Cd > Cr > Ni > Mg > Zn > Mn > Cu > Co. Mineral uptake in plants is a function of the mineral concentrations in the soils, soil pH, cation exchange capacity, organic matter content, types and varieties of plants and age of the plant.The reason why the Ca concentration was very high in the fenugreek seeds at all the sample sites may be due to the presence of the thick mesozoic limestone and gypsum sequences in the Blue Nile river area in central Ethiopia, and the proterozoic marbles that occur in northern (Tigray) and western (Gojam) Ethiopia.The marble and limestone could be the main sources of Ca in the soil from these sample areas.Moreover, Ca is amongst the major elements required by plants.In the above series, the concentration of K is higher than that of Na, since plants generally have higher K than Na contents (DEMAN, 1999).Trace elements are ubiquitous in our environment and are found in all the foods we eat.In general, the abundance of trace elements in foods is related to their abundance in the environment and the most abundant trace element in the soil is Fe.In this study Fe was the trace element found in the highest concentration.Similarly, as many authors have mentioned, the Ethiopian soil is rich in Fe minerals, e.g.hematite (Fe 2 O 3 ), and this may be the reason why the Ethiopian fenugreek seeds are rich in iron contents.However trace elements can get into foods by different pathways.The most important source is from the soil, but they can also be absorbed in aqueous solution through the roots.Another minor source is foliar penetration (industrial air pollution and vehicle emissions) and other possible sources are fertilizers, agricultural chemicals and sewage sludge (DEMAN, 1999).

Comparison of the metal levels between the sample sites
Of the thirteen elements analysed in the fenugreek seeds in this study, Ca showed the highest concentration.The mean concentration of Ca in this study was 26364 mg kg -1 , and the highest Ca concentration (36771 ± 1938 mg kg -1 of dry mass of fenugreek seed) was recorded in the sample from the Edagaarbi site.The three sample sites with the highest Ca concentrations were Edagaarbi, Denboskie and Koladiba, with concentrations of 36771 ± 1938 mg kg -1 , 36085 ± 1434 mg kg -1 and 32612 ± 770 mg kg -1 , respectively and that with the lowest Ca concentration was Hatsebo, with 15353 ± 1758 mg kg -1 . The RDA and RDI for Ca for people in the 25-50 year age range are 800 mg per day and 1000 mg per day, respectively (WARDLAW, 1997) and 10451 ± 220 mg kg -1 , respectively.The site with the lowest K concentration in the fenugreek seeds was Hatsebo (6789 ± 155 mg kg -1 ).The estimated minimum K requirement for a healthy person above 18 is 2000 mg per day (WARDLAW, 1997).In Canada, the median dietary intake range is from 3.2 to 3.4 g per day for men and 2.4 to 2.6 g per day for women (JENNIFER et al., 2006).The three sample sites with the highest Na concentrations per dry mass were Mesretgamra, Korem and Edagaarbi, with 1559 ± 41 mg kg -1 , 1532±12 mg kg -1 and 1172 ± 6 mg kg -1 , respectively, and the site with the lowest Na concentration was Koladiba (201 ± 6 mg kg -1 ).The estimated minimum Na requirement for a healthy person above 18 is 500 mg per day (WARDLAW, 1997).The AI (Adequate Intake) for Na for older and elderly adults has been set at 1.3 g per day for men and women from 50 to 70 years of age, and at 1.2 g per day for those over 70 (JENNIFER et al., 2006).
The three sample sites with the highest Mg concentrations were Korem, Ambagiorgis and Bichena with concentrations of 102±6 mg kg -1 , 99 ± 2 mg kg -1 and 96±5 mg kg -1 , respectively, and the site with the lowest Mg concentration in its fenugreek seeds was Koladiba (31 ± 4 mg kg -1 ).The amount of Mg that can be obtained from fenugreek seeds is very small and hence people are recommended to eat other foods to attain the required amount of Mg.
The three sample sites with the highest Fe concentrations in their fenugreek seed samples were Mayadrasha, Denboskie and Adet with concentrations of 18584 ± 1339 mg kg -1 , 17807 ± 1109 mg kg -1 and 17318 ± 1049 mg kg -1 , respectively.Conversely, the sample site with the lowest Fe concentration was Keteteekli, with 6041 ± 745 mg kg -1 . The recommended dietary allowance for Fe for those in the 23-50 year old group is 10 mg per day for men and 18 mg per day for women (WARDLAW, 1997).The Fe concentration determined in fenugreek is very high when compared to the above mentioned values, and therefore people are recommended not to consume this seed regularly.Koladiba, Dabat and Debrebirhan were amongst the sample sites with the highest Cr concentrations, with values of 552 ± 9 mg kg -1 , 419 ± 42 mg kg -1 and 413 ± 37 mg kg -1 , respectively.The lowest Cr concentration (3 ± 0.5 mg kg -1 ) per dry mass in fenugreek seed was recorded at the Korem site.The AI for Cr is 35 μg per day and 25 μg per day, for young men and young women, respectively (EFSA, 2014).In addition to the above, the ESADDIs for Cr for adults is 50-200 μg per day (WARDLAW, 1997).Fenugreek seed is rich in Cr, and should therefore not be taken daily in large quantities, so as to minimize the risk of Cr contamination.The three sample sites with the highest Ni concentrations were Ambagiorgis, Dabat and Jehaniagiorgis with concentrations of 117 ± 15 mg kg -1 , 115 ± 5 mg kg -1 and 108 ± 12 mg kg -1 , respectively, and that with the lowest Ni concentration was Keteteekli (31 ± 5 mg kg -1 ).The adequate intake of Ni for people above the age of 19 is 2.3 mg per day for males and 1.8 mg per day for females (JENNIFER et al., 2006).
The sample sites with the highest Cu concentrations were Mayadrasha (35 ± 1 mg kg -1 ) and Edagaarbi (35 ± 1 mg kg -1 ) followed by Debrebirhan with 28 ± 4 mg kg -1 of fenugreek seed per dry mass.However Cu was not detected at the Jehaniagiorgis sample site.The ESADDI for Cu for adults is 1.5-3 mg per day (WARDLAW, 1997).The three sample sites with the greatest Co concentrations were Hatsebo, Jehaniagiorgis and Edagaarbi with values of 15 ± 0.4 mg kg -1 , 15 ± 0.6 mg kg -1 and 14 ± 0.7 mg kg -1 , respectively, and that with the lowest Co concentration was Keteteekli, with 4 ± 0.3 mg kg -1 per dry mass.
The Meseretgamra, Debrebirhan and Edagaarbi sites recorded the highst Pb concentrations when compared with the other sample sites, with 2624 ± 101 mg kg -1 , 1814 ± 67 mg kg -1 and 1776 ± 145 mg kg -1 Pb per dry mass, respectively, and the Bichena sample site showed the lowest Pb concentration (422 ± 42 mg kg -1 ).The three highest Cd concentrations (464 ± 14 mg kg -1 , 463 ± 10 mg kg -1 and 396 ± 10 mg kg -1 ) were recorded at the Dabat, Bichena and Keteteekli sample sites, respectively, whereas the sites at Adet and Meseretgamra recorded the lowest Cd concentrations of 285 ± 22 mg kg -1 and 285 ± 32 mg kg -1 per dry mass, respectively.Of all the metals determined in fenugreek seeds in this study, Co showed the lowest concentration with a mean value of Co 11 mg kg -1 per dry mass.

Comparison of the metal concentrations found in fenugreek seeds in this study with the data found in the literature
Table 2 shows the comparison between the metal contents found in the Ethiopian fenugreek samples with the data found in the literature.The Ca concentration found in fenugreek seed was highest in Ethiopia (26364 mg kg -1 ) followed by Turkey (2341±146 mg kg -1 ), UAE (84 mg kg -1 ) and the lowest value in Saudi Arabia (1.28 mg kg -1 ).The K concentration found in Ethiopia was intermediate (9120 mg kg -1 ) between the K concentrations found in Saudi Arabia (272 mg kg -1 ) and in UAE (18203 mg kg -1 ).The Na concentration found in the fenugreek seeds in UAE (37280 mg kg -1 ) was considerably higher than the concentrations found in Ethiopia (744 mg kg -1 ) and in Saudi Arabia (20.3 mg kg -1 ).The Mg concentration found in Ethiopia was the lowest (69 mg kg -1 ) as compared to the concentrations found in UAE (168 mg kg -1 ) and in Turkey (1373 ± 45 mg kg -1 ).
Thus the concentrations of the toxic metals Cd and Pb were higher in the Ethiopian fenugreek seed than in the fenugreek seeds from the other countries, except from India.Hence, the government or authorized bodies should make people aware of the risk of ingesting these metals from the regular intake of fenugreek seed in foods.

Comparison of the metal concentrations found in fenugreek seeds with those found in other spices
The metal concentrations determined in the fenugreek seeds were compared with the values found in other spices in Ethiopia (Table 3).These spices were red ).The decreasing order of the Ca concentrations in these spices was as follows: fenugreek seed > fennel > cardamom > thyme > ginger > korarima > red pepper.The K concentration in fenugreek seed was (6789-11517 mg kg -1 ) and in red peppers (2378-2486 mg kg -1 ) while its concentration was not reported in the other spices.The Na concentration in fenugreek seed was (201-1559 mg kg -1 ) and in red peppers (75-93 mg kg -1 ) and again its concentration was not reported in the other spices.The highest Mg concentration was observed in ginger (2700-4094 mg kg -1 ) and the lowest in fenugreek seed (31-102 mg kg -1 ) while its concentration was not reported in red peppers.The decreasing order for the concentration of Mg in these spices was as follows: ginger > fennel > cardamom > korarima > thyme > fenugreek seed.
The highest Fe concentration was recorded in fenugreek seed (6041-18584 mg kg -1 ) and the lowest in korarima (37-46.5 mg kg -1 ).The decreasing order for the concentration of Fe in these spices was as follows: fenugreek seed > thyme > fennel > red pepper > ginger > cardamom > korarima.The Cr concentration was highest (3-552 mg kg -1 ) in fenugreek and lowest in korarima (3.8-5.8 mg kg -1 ) and its concentration was not reported in thyme.The decreasing order for the concentration of Cr in these spices was as follows: fenugreek seed > fennel > red pepper > ginger > cardamom > korarima.The highest Ni concentration was recorded in fenugreek seed (31-108 mg kg -1 ) and the lowest in red peppers (2.7-6.7 mg kg -1 ).The decreasing order for the concentration of Ni in these spices was as follows: fenugreek seed > fennel > thyme > cardamom > korarima > ginger > red pepper.The highest Zn concentration was recorded in ginger (38.5-55.2mg kg -1 ) and the lowest in korarima (12-18 mg kg -1 ) while the Zn concentration in fenugreek seed was between these two concentrations.The lowest Mn concentration in these spices was recorded in fenugreek seed (16-28 mg kg -1 ) and the highest in ginger (184-401 mg kg -1 ).The decreasing order for the Mn concentration in these spices was as follows: ginger > cardamom > korarima > thyme > fennel > red pepper > fenugreek seed.The highest Cu concentration was recorded in fennel (23.9-103 mg kg -1 ) and the lowest in red peppers (2.1-3.7 mg kg -1 ).The decreasing order for the concentration of Cu in these spices was as follows: fennel > fenugreek > thyme > cardamom > korarima > ginger > red pepper.The Co concentration was the highest in fennel (26.2-70.8mg kg -1 ) and the lowest in red peppers (1.0-2.2 mg kg -1 ). and the Co concentration in fenugreek was between these two concentrations.The decreasing order for the concentration of Co in these spices was as follows: fennel > fenugreek > ginger > thyme > cardamom > korarima > red pepper.
However, the Pb concentration in fenugreek was very high (615-1814 mg kg -1 ) whereas it was not detected in the other spices.Cd was studied in all the spices and the highest concentration was recorded in fenugreek (285-464 mg kg -1 ) whilst the lowest was found in red peppers (0.20-0.23 mg kg -1 ).The decreasing order for the concentration of Cd in these spices was as follows: fenugreek > fennel > thyme > korarima > cardamom > ginger > red pepper.

Analysis of variance
ANOVA is an extremely powerful statistical technique which can be used to separate and estimate the different causes of variation (MILLER; MILLER, 2000).In this study a one way ANOVA was used with the help of the SPSS software program to compare the means of more than two groups of samples.ANOVA uses the F-test statistic to compare whether the differences between the sample means are significant or not (MILLER;MILLER, 2000).
In the present study Trigonella foenum-graecum L. (fenugreek seed) samples were collected from 14 different regions of Ethiopia and the metal levels of 13 metals determined using FAAS.However, the sample preparation and digestion process could result in random errors during the instrumental analysis.Therefore a one way ANOVA was used with the help of the SPSS software program to analyse the variance that could result from the differences in the metal contents of the soils from the different sample sites, the acidity of the soil, the atmospheric condition of the environment, water, fertilizers, etc.
Table 4 shows that all the sample means had significant differences at F 13,28 at a 95% confidence level.Nevertheless, no significant difference was observed between the sample means in the case of Zn.The significant differences in the means of the samples could be due to differences in the metal contents of the soils at these sample sites, the pH of the soils and the type of fertilizers used at these sites.However, the difference between the sample means for Zn was not significant at F 13,28 for a 95% confidence level, most probably due to random errors in the analytical procedures and instrumental errors.

Pearson's correlation for the metals in the fenugreek seed samples
The product moment-correlation coefficient (r) has values that range from -1 ≤ r ≤ +1.The r value of -1 describes a perfect negative correlation, i.e. all the experimental points lie on a straight line with a negative slope.Similarly, the r value of +1 describes a perfect positive correlation, all the points lying exactly on a straight line with a positive slope.However, a zero r value does not mean that the two components (e.g.y and x in the formula) are entirely unrelated; it only means that they are not linearly related (MILLER; MILLER, 2000).Table 5 shows the values obtained for the correlation coefficients between the metal concentrations found in the fenugreek seed samples.The fenugreek seed metal vs. fenugreek seed metal system showed good positive correlations for Na-Pb, Mn-Co and Fe-K with corresponding r values of 0.659, 0.521 and 0.526, respectively.However, an inverse  relationship occurred between Na and Cr (-0.698).All the other relationships showed weak positive or negative linear relationships, which implies that the presence of one metal did not affect the presence of the other metal within the plant.

Conclusions
The levels of the major (Ca, K, Na, Mg), trace (Fe, Cr, Ni, Zn, Mn, Cu, Co) and toxic (Pb, Cd) metals in Trigonella foenum-graecum L. (fenugreek seed) grown in different parts of Ethiopia, were determined by flame atomic absorption spectrometry.
The levels of these metals were found in the following ranges and order in the fenugreek seeds; Ca (15353-36771 mg kg -1 ) > Fe (6041-18584 mg kg -1 ) ≈ K (6789-11517 mg kg -1 ) > Pb (615-2624 mg kg -1 ) > Na (201-1559 mg kg -1 ) > Cd (285-464 mg kg -1 ) > Cr (3-552 mg kg -1 ) > Ni (31-108 mg kg -1 ) > Mg (31-102 mg kg -1 ) > Zn (15-33 mg kg -1 ) > Mn (16-28 mg kg -1 ) > Cu (ND-35 mg kg -1 ) > Co (4-15 mg kg -1 ).The result of this study shows that the fenugreek seeds cultivated in Ethiopia are rich in all the above mentioned essential and toxic metals.The one-way ANOVA results at a 95% confidence level showed that there were significant differences amongst the mean concentrations of all the metals with the exception of Zn.This result can be attributed to differences in the pH of the soils, mineral composition of the soils and type of fertilizers used.Furthermore, Pearson's correlation coefficient (r) was determined and used to correlate the metal concentrations amongst the fenugreek seed samples via the metal level vs. metal level system, and except for a few cases, most showed weak positive or negative linear relationships, indicating that the presence of one metal in the plant did not influence the presence of the other.
Fenugreek seed has many benefits for both producers and consumers in Ethiopia.However, some of the very toxic elements were found in high concentrations in this study, e.g.Pb and Cd, and the prolonged accumulation of heavy metals ingested via foodstuffs may lead to chronic effects on the kidney and liver of humans and cause the disruption of numerous biochemical processes, leading to cardiovascular, nervous, kidney and bone diseases.Therefore, government organs and other concerned bodies should strengthen the studies of metal levels in most of the widely used spices and herbal medicines.
Levels of essential and toxic metals in fenugreek seeds (Trigonella Foenum-Graecum L.) cultivated in different parts of Ethiopia Hagos, M.; Chandravanshi, B. S.
Levels of essential and toxic metals in fenugreek seeds (Trigonella Foenum-Graecum L.) cultivated in different parts of Ethiopia Hagos, M.; Chandravanshi, B. S.
Levels of essential and toxic metals in fenugreek seeds (Trigonella Foenum-Graecum L.) cultivated in different parts of Ethiopia Hagos, M.; Chandravanshi, B. S.
Levels of essential and toxic metals in fenugreek seeds (Trigonella Foenum-Graecum L.) cultivated in different parts of Ethiopia Hagos, M.; Chandravanshi, B. S.

of essential and toxic metals in fenugreek seeds (Trigonella Foenum-Graecum L.) cultivated in different parts of Ethiopia
. The three sample sites with the highest K concentrations per dry mass were Debrebirhan, Edagaarbi and Mayadrasha, with concentrations of 11517 ± 294 mg kg -1 , 10807 ± 375 mg kg -1 Levels Hagos, M.; Chandravanshi, B. S.

Table 1 .
Concentrations (mean ± SD, n = 3, mg kg -1 of dry mass) of the major, trace and toxic metals in fenugreek seeds from the different sample sites in Ethiopia.

Levels of essential and toxic metals in fenugreek seeds (Trigonella Foenum-Graecum L.) cultivated in different parts of Ethiopia Hagos
, M.;Chandravanshi, B. S.

Table 2 .
Comparison of the metal concentrations found in the fenugreek seeds used in this study with results found in the literature.

Table 3 .
Comparison of the metal concentrations found in fenugreek seed with the values found in other spices as reported, in the literature.

of essential and toxic metals in fenugreek seeds (Trigonella Foenum-Graecum L.) cultivated in different parts of Ethiopia Hagos
, M.;Chandravanshi, B. S.

Table 4 .
Analysis of variance between and within samples of fenugreek seed samples at a 95% confidence level.

Table 5 .
Pearson's correlation of the fenugreek seed metals vs. fenugreek seed metals system.