A Survey of The Effect of Some Heavy Metals in Plant on The Composition of the Essential Oils Close to Veshnaveh-Qom Mining Area

The purpose of this study was to evaluate the effect of copper and silver in the plants on the mining area and the ingredients of essential oils. Plants collected from the same family, but they were collected randomly from various regions. The concentration of heavy metal in plant samples collected from Veshnave, Qom, Iran have been determined by Flame Atomic Absorption Spectrometry (FAAS). The chemical composition of essential oil isolated by hydrodistillation from Tanacetum polycephalum and was determined using Gas Chromatography/Mass Spectrometry (GCMS). Results obtained from unidirectional variance analysis suggest that there is a meaningful statistical difference between various regions.


INTRODUCTION
The plants for growing need relatively low amount of mental ions.Natural soil consists of great amount of metal elements.Despite the selective absorption in root cellular membrane, it is possible a few metal ions are found in plant tissues ¹.Heavy metals are important environmental pollutants and they will be toxic as the result of human activities in natural and agricultural areas.The reason of toxicity of heavy metals in higher concentration is that they disturb some vital factors of body.Some metals are found naturally in the body and are essential for health.Naturally, low-concentration heavy metals are found and they are considered low-quantity metals.They are toxic in high concentration or lead to inefficiency and defect in other low-quantity metals ³.In high concentration of metals, substitution with essential metals is occurred and as essential metals play important role in formation of pigments and enzymes, the pigments are disrupted and existing elements in soil will be unsuitable for plant growth and biological variety is eliminated 13,14 .Heavy metals have no role in the body as a metal and they may be extremely toxic 4 .
Copper has roles in proteins structure, photosynthesis electrons transport, mitochondrial respiration, oxidative tension, cell wall metabolism.Copper (Cu) is a cofactor in enzymes as copper/zinc superoxide, cytochrome-c oxidize, amino oxidize, lactase, plastocyanin, and polyphenol oxidize.
Copper is effective in movement of iron in cells¹.The plants need copper as a micronutrient matter for growth.Silver soluble salts namely silver nitrate is toxic in concentration above two grams.Silver compounds could be absorbed easily by body tissues and blue or black pigments are created in the skin.One of the suitable methods to measure metals in various materials is using FAAS technique 15 .The present study applied this method to measure heavy metals in plant tissue.Determining low quantities of metals in plants was accomplished by digestion with acid or mixture of acids by microwave.The elements were measured by suitable apparatus techniques 6 .Microwave digestion technique is used for multiple samples and short digestion time 7,8,9 .

Reagent and Standard
All reagents were of analytical grade.Double deionized water was used for all dilution.Nitric acid (HNO 3 ) with purity 65%, H 2 O 2 with purity 30% were of pure quality (Merck, Germany).Cu (1.0-5.0 mg/L) and Ag (0.5-5.0 mg/L) were prepared by dilutions from stock solutions of 100 mg/L.

Collection of plants
Plants used in the research from close, 5 and 10 Km around the Veshnave Mine, Qom, Iran in May 2012.Plants were of family Corpositae, a genus Tanacetum polycephalum.After the plants were collected, dried and powdered, it was homogeneous 10 .

Essential oil extraction
The dried parts from Tanacetum polycephalum were subjected to hydrodistillation for 3 hours.The essential oil obtained was separated from water with 0.5 ml hexan normal.To analyze the essence, GC-MS is used.

Gas Chromatography -Mass Spectrometry (GC-MS)
GC-MS analysis was performed by Hewlett-Packard equipped with a HP-5MS column (30m × 0.25 mm, film thickness 0.32 mm).Helium used as the carrier gas (1 ml/min).The oven temperature increased from 60°C to 220°C at a rate of 6°C/min.

Identification of components
Retention indices (RI) were calculated by using retention times of n-alkanes (C6-C24) that were injected after the oil at the same temperature and conditions.The compounds were identified by comparing their RI with those reported in the literature, and their mass spectrum was compared with those reported in Wiley Library 17 .

Sample digestion and analysis
To digest the plant samples, microwave is used.7 mL of HNO 3 (65% v/v) and 1 ml hydrogen peroxide (30% v/v) were added to 0.5 g dry weight (DW) of tissue and digestion was performed.The extract was filtered and diluted to 25 ml.The metals (Cu and Ag) in the surface water and the plant extracts were analyzed by FAAS (Varian-spectraAA-240) 7,11 .

RESULTS AND DISCUSSION
The solutions after digestion are used for analysis of copper and silver.The samples (n=9) were collected in particular, from three different areas.Mean values and standard deviations of Cu and Ag in different areas were tabulated in Table 2. RSD of method for 9 successive measurement of copper was achieved 0.36 % and for silver as 1.13%.LOD for copper is 0.01% and for silver 0.025% and R for copper is ranging 90.15% to 103.9% and ranging 93% to 106% for silver.According to the chart observed this plant susceptible to absorb Cu more than Ag.

Essence analysis by GC-MS
The chemical composition of each oil was tabulated in table 2.
Mean values and standard deviations of Cu and Ag in different areas were tabulated in Table 5.The highest concentration of Cu and Ag was observed near the mine and the lowest value was observed at distances of 10 km from the mine.Our results indicate the significant differences between the mean values of Cu and Ag in the different regions (F(Cu) =3025.3;F(Ag) = 171.3;p<0.001); alternatively, by increasing distance from the mine, the concentration of Cu and Ag decreases.The results of Duncan test showed that the average of Cu and Ag concentration near the mining areas was significantly higher than 5 km and 10 km from the mine (p<0.05);moreover, mean Cu and Ag concentration was significantly higher in the 5 km area of mine than 10 Km area.
The values of MH in different areas shown in Table 3.As shown in the charts, by increasing the copper and silver concentration, monoterpene hydrocarbon concentration is increased, too.Mean values and standard deviations of MH in different areas shown in Table 4.The highest concentration of MH was observed at distances of 10 km from the mine and the lowest value was observed near the mine.Our results indicate the significant differences between the mean values of MH in the different regions (F =954.2;p<0.001); on the other hand, by increasing distance from the mine, the concentration of MH would be increased.

Fig. 1 :Fig. 2 :Fig. 3 :Fig. 4 :Fig. 5 :
Fig. 1: The investigation of changes of metals concentration with distance from mine.*Region 1 is in the mine, region 2 is 5km from the mine and region 3 10km from the mine

Table 2 : The identified compositions in essence of different regions
Each value in this table shows compositions in essence in dry plants.* KI: Kovats Index, **region 1, is in the mine, region 2, is 5km from the mine and region 3, 10km from the mine.

Table 3 : Identified terpens in various regions essence
Each value in this table shows terpens in essential oil, * MH: Monoterpene Hydrocarbon; OM: Oxygenated Monoterpene; SH: Sesquiterpene Hydrocarbon; OS: Oxygenated sesquiterpene.**Region 1 is in the mine, region 2 is 5km from the mine and region 3 10km from the mine.

Table 4 : Comparison of mean concentrations (Zn, MH, OM and SH) in different regions using unidirectional variance analysis and Duncan's test results
*Values "Mean ± SD" is shown; **MH: Monoterpene Hydrocarbon; OM: Oxygenated Monoterpene; SH: Sesquiterpene Hydrocarbon; OS: Oxygenated sesquiterpene; ***region 1 is in the mine, region 2 is 5km from the mine and region 3 10km from the mine.