Document Type : Original Article


1 Departments of Animal and Poultry Health and Nutrition, Faculty of Veterinary Medicine, Tehran University, Tehran, Iran.

2 Institute of Agricultural Education and Extension, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran


Background: This research aims to evaluate heavy metals contamination of Alfalfa forage and soil in industrial and non-industrial
regions of Tehran province. The soil and plant samples were collected from Alfalfa fields situated in industrial and non-industrial
areas during two harvests of Alfalfa harvest.
Methods: The Electrothermal atomizers spectroscopy technique was employed to measure the heavy metals (lead, arsenic, and
cadmium) levels in the samples. A flame atomic absorption spectrometer equipped with acetylene-air flame was employed.
Results: The highest heavy metals concentrations were detected in the soil and Alfalfa samples from the industrial regions. Lead had
the highest level in the soil followed by arsenic and cadmium; the same trend held for the Alfalfa as well. Moreover, the heavy metal
content in Alfalfa declined from the first to second harvest, while it increased in the soil. The heavy metals content of Alfalfa was
below the maximum permitted concentration for feedstuffs and livestock. The highest soil-plant transfer factor of heavy metals was
detected in the first harvesting. The highest arsenic transfer factor was recorded in the industrial areas.
Conclusion: The findings of the current study can be helpful in the determination of healthy forage in various areas



The heavy metals (HMs) accumulation in the soil and agricultural crops have become one of the major challenges throughout the world. HMs refer to the environmental contaminants capable of influencing the agricultural plants which can indirectly or directly enter the human food chain through livestock. HMs (lead, arsenic, and cadmium) contaminations of the plant used as the livestock feed will contaminate the human diet (1). As one of the prominent classes of pollutants, HMs can enter the environment through various industrial and agricultural activities (2). The significance of HMs has resulted in the establishment of strict regulations regarding dietary exposure in husbandry practices, dietary ingredients, soil ingestion or spurious soil contamination in foliage (3,5). In this regard, the animal feed contaminants impacts on animal and human health have been comprehensively reviewed (5). Such information is essential in terms of the natural amount / background of trace elements in the soil, biological indicators of the chemical status of the environment, soil remediation and excessive accumulation of trace metals in the soil. (6). The soil is the major medium for root activity and irrigation with the industrial effluent could be potential for significant accumulation of toxic metal in plant (7,8). Several studies have addressed the content of potentially toxic elements in the plants cultivated near or far from the cities (9,10). Soil contamination of the agricultural fields in the vicinity of large cities has become a serious environmental challenge (11). Some elements such as cadmium, lead, and arsenic are more important in terms of toxicity. Lead poisoning in livestock is very common which is originated from several industrial activities (e.g. lead paint, lead accumulator batteries and mines) (12). Cadmium contamination is primarily due to the direct uptake from the cadmium-polluted soils (13). The European food safety authority (EFSA) panel evaluated the risk of arsenic contamination of the food chain on human health (5). As one of the highly toxic metalloids, arsenic could be released to nature through a wide variety of industrial activities (14). Upon HMs exposure, plant adapts several defense mechanisms among which the elevated proline synthesis, osmotic regulation to prevent the metal transfer to the aerial organs, preservation of the enzymes involved in maintaining the protein synthesis, and enhanced the activities of the antioxidant enzymes (e.g. catalase, peroxidase) can be mentioned (15,16). As one of the prominent forage crops, Alfalfa (Medicago sativa) can reach high biomass yields with superior nutritional value applicable for the livestock feed. Alfalfa is a perennial herb of the legume family that can be harvested about 5 in cold climates and 15 per year in tropical of the most important forage plant in the world (17).Industrial activity In addition to the accumulation of several heavy elements (zinc, lead, chromium, nickel, copper, , etc.) in various plant components, it may alter your nutrient soil which can be beneficial for some and dangerous for Others (4,10). The use of HM-contaminated plants as the feed of domestic animals can decrease their milk and meat production or decline their resistance against diseases (18).Exposure of livestock and humans to bioavailable heavy metals has shown the accumulation of HMs hazards in the soil and their transfer to plants.(19,20). A limited number of works however investigated the HMs accumulation in Alfalfa in different harvest times in industrial and non-industrial regions. In this context, the current study is devoted to assessing the influence of HMs (lead, arsenic, and cadmium) in the industrial and non-industrial areas and their content in Alfalfa cut in different periods (first to second).

2. Material and methods

This study measured the HMs levels (lead, cadmium, and arsenic) in the agricultural soil and Alfalfa grown in two regions of Tehran province. 36 Alfalfa and soil samples were collected in 2 harvests. First, the selected agricultural farms of industrial and non-industrial regions were divided into 3 farms; then, each farm was separated into three parts (locations first, middle and end) to randomly sample Alfalfa and soil. The sampling of each area was independent during the first and second Alfalfa harvestings. Moreover, water samples were obtained from two areas, HMs levels were lower than the limit of detection of the sensors. Soil samples were collected from an approximate depth of 15 cm. The collected samples were put in polyethylene bags, washed with detergent, and preserved in a container including 5% nitric acid, and water to be sent to the laboratory.

2.1 Industrial Area

The industrial area is located near Ray city at South of Tehran. This area is surrounded by small polluting industries (for example, copper smelters, battery factories and oil and gas storage tanks) and large Tehran oil refineries. Ray city landfill also polluted the agricultural farms in the mentioned area. Moreover, the Tehran cement factory was also near this industrial.

2.2 Non-Industrial Area

An area near Varamin (south of Tehran province) was considered as the non-industrial area as it was situated far from the industrial factories.

2.3 Samples Preparation

Alfalfa samples were dried at 105 °C in an oven for 24 h. The dried samples were then milled by a 2-mm grid and kept in a desiccator to determine their HMs contents (21). Furthermore, the soil samples were dried at 70 °C for 24 h. They were also sieved by a 2-mm sieve to eliminate the stones and other solids as described in Sposito (1989) methods (22).

2.4 Measurements of heavy metals

HM content of the soil and plant samples was assessed using an atomic absorption instrument. For this purpose, a flame atomic absorption spectrometer equipped with acetylene-air flame was employed. Detection limit was 0.1 ppm in the digested solution.

2.5 Calculation of transfer factor

The soil-plant transfer factor of HMs (TF) was also computed by the following schemes (23, 24):


C PLANT= Concentration of HMs in plant (mg/kg)

C SOIL = Concentration of HMs in soil (mg/kg)

3. Data Analysis

Data analysis was conducted by SPSS 18 software through ANOVA. Duncan's multiple range tests were also employed for the comparison of the mean values if the variances were uniform. A t-test was also applied for comparing the mean of two harvests in the industrial and non-industrial areas in which P

4. Result

  Heavy metals concentration in agricultural soil

 According toTable 1, the concentration of the investigated HMs in the soil of three farms in the two harvest stages showed no significant difference

The order to the mean accumulation levels of HMs in the soil of the two areas was as follows: Pb> As > Cd. The variations in the HMs of the soils sampled in 2 harvests are shown in (figure 1). Despite the incremental trend of the mean HMs contents of the soils from first to the second harvests,this trend was not significant.

Moreover, a significant difference was observed in the mean As, Pb, and Cd accumulations in the Alfalfa agricultural soil in the industrial and non-industrial regions in each harvesting time (Table 2).


 Heavy metals concentration in Alfalfa

The HM contents of the Alfalfa grown in farms located in the two areas in both harvests are presented in (Table 3). No significant difference was detected in the 3 farms with respect to lead, cadmium, and arsenic.

The mean HM accumulation in Alfalfa grown in the two areas varied in the following order: Pb > As > Cd. The variation of HM in alfalfa is shown in (figure 2). As can be seen, HMs concentration showed a declining trend from the first to the second harvest in each area. No significant difference was detected in the average Pb and Cd contents in the two harvests; the mean arsenic content, however, exhibited a reduction from first to the second harvesting in the non-industrial area (P≤0.05).

Moreover, a significant difference was observed in the mean As, Pb, and Cd accumulations in the Alfalfa cultivated in the industrial and non-industrial regions in each harvesting time (Table 4). The average Pb accumulation was higher in Alfalfa grown in the industrial area for both harvests. In addition, the two regions were significantly different in terms of Cd and As (P≤0.05).


 5. Transfer factor

Similar HMs transfer factors were calculated for the non-industrial and industrial regions.  Comparing the harvests, HMs of the first harvest were higher in both areas. The arsenic transfer factor was the highest in the industrial area compare with other heavy metals (Figure 3).

6. Discussion

6.1 Heavy metals in agricultural soil

The lead, cadmium, and arsenic contents of the farms were below the maximum permitted content in first harvesting for the non-industrial area (Table. 1). Esmaeili et al. in Iran (2014) assessed the lead and cadmium content of agricultural soils of industrial and non-industrial areas, 319.3 and 8.68 mg/kg and 7.2 and 0.11 mg/kg respectively (25). According to Kelepertzis et al in Greece (2014), Pb, Cd, and As contents of agricultural soils ranged in 3.17-48.49, 0.07-6.1, and 2.7-12.8 mg/kg, respectively in which the highest Cd concentration exceeded the maximum permissible concentration (5 mg/kg) (26). Furthermore, the mean heavy metal contents of non-industrial regions were lower than the permissible limit. Pb concentration of the three investigated farms of the industrial area exceeded the maximum permitted concentration (Table.5); as concentration was however below the maximum permitted concentration. Concerning Cd, its concentration exceeded the maximum permitted concentration (1-5 mg/kg) in the second harvest for the industrial area. Moreover, the mean Pb concentration of the industrial area fell above the maximum concentration. In addition, HMs contents of the agricultural soils exhibited during two harvesting an enhancement from the first to second harvest (Figure 1). Some of study were shown that The accumulation distribution HMs in the soil was not uniform and depended on several factors including fertilization and irrigation, precipitation and physical features of soil (27, 28). Chemical factors like pH, soil organic matter, interfering ions, and soil texture as well as the solubility of HMs, their bonding with organic matter, and the capacity and mobility combined with the availability of these elements for the roots and their translocation from soil to Alfalfa play a key role in the HMs accumulation in the alfalfa.

6.2 Heavy metals in Alfalfa

According to Table 3&5 concentration of heavy metals on alfalfa in industrial and non-industrial area HMs content of the studied fields was below the permitted concentration for animal feed and other standard limits. Comparing the total HMs concentrations, HMs content of in Alfalfa grown in industrial areas was higher than those cultivated non-industrial areas for both harvesting stages (Table 5). According to Al-Rashidi and Sulayman in Oman (2013), the element concentrations in Alfalfa plants are closer to Blatter and their distributions were consistent in two distances from the same industrial areas (3). GhaziFard and Sharifi (2003) studied Pb accumulation (1.25 mg/kg dry matter) in different plants near the Iran around IRANKUH Lead and Zincmine. They also compared the Alfalfa (in different harvesting stage) with the rest of the plants and Alfalfa plants grown in the Cd-free areas. These results were in line with the present study (29). Pb and Cd concentrations in leguminous plants vary in different countries and range in 1-18.8 and 0.22-0.2 mg/kg dry matter, respectively (7). In general, As residual concentration in the plants and crops cultivated on non-contaminated soils is less than 0.5 mg/kg dry matter (30). Arsenic content in non-contaminated straw and Alfalfa feedings were recorded

6.3 Evaluation of the transfer of heavy metals from soil to plant

The mean HMs transfer factors in the first and second harvests were similar in the non-industrial and industrial regions. Al-Rashidi and Suleiman (2013) declared similar findings in line with this study (3). Some studies expressed that the transfer factors HMs of elements depends on various factors including dissolving ability, element capacity, soil texture, and plant capture ability in the harvesting stage (7). In general the concentration of metals is not directly or automatically reflected from the soil in plants. It depends on the fraction of the metals that are bioavailable to plants by root uptake (Figure 3). In the industrial area, as ranged from 0.05 to 0.1 (the maximum standard limit) (36).

7. Conclusion

HMs on agricultural and environmental pollutants can directly or indirectly affect the human food chain through animal husbandry. Some studies have been published on the accumulation of HMs in soil and their transfer to plants. HMs-contaminated livestock feedstuffs can threaten public health by influencing the disease development in the animals. This research revealed the significant HMs accumulation in the soil of industrial areas. Thus, Alfalfa crops cultivated in such regions are hazardous to livestock, in particular, for ruminant and other spices of animals kept in the vicinity of the industrial areas. Apparently, enhancement of the HMs content of the soil in the industrial regions did not elevate their accumulation in Alfalfa. It depends on the fraction of the metals that are bioavailable to plants by root uptake. Regarding a decline in the pollution of heavy metals in alfalfa for the second harvest (both industrial and non-industrial regions), this research recommends employing the second harvest for livestock feeding.


The current study was partially supported by the agricultural research, education and extension organization (AREEO) (project code: 2-01-01-005- 960566. (


Conflict of interest

None to be declared

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