1. Introduction
Contamination of agricultural soils with heavy metals or petroleum hydrocarbon is considered the foremost global problem of industrial and non-industrial societies [1]. The most common type of environmental contamination is petroleum hydrocarbon. Petroleum hydrocarbons quickly alter the natural environment characteristics and lower the ecosystem’s functionality. Multiple plants or organisms are killed or damaged by the imported hydrocarbons, changing the ecosystem’s and the microbial community’s functional capacity [2]. 
Moreover, heavy metals entering the environment due to industrial activities and technological developments are significantly increasing and considered a severe threat to the environment and human health [3]. Heavy metals enter the soil through soil preparation processes or human activities. Organic and inorganic fertilizers also contain different amounts of heavy elements such as lead (Pb), nickel (Ni), cadmium (Cd), copper (Cu), and arsenic (As), which may be absorbed by the animal body through fodder plants and enter the human body by consuming the milk and meat of these animals [4, 5].
The growth and development of a healthy society mainly rely on providing safe and healthy food for nutrition [6]. Unhealthy food leads to people’s sickness, inactivity, and other people’s involvement in their care, causing irreparable damage to society [7]. The proper health of a community is safeguarded when healthy and sufficient food is available, and the first condition for achieving this is the safety of food raw materials [8].
Unlike unchangeable organic pollutants, heavy metals are non-degradable and stable in the soil. With the development of industries, mining, metal casting, and chemical fertilizers containing heavy elements, soil contamination with heavy metals has become one of the significant environmental problems in human societies. Accumulation of heavy elements such as Cd, Ni, and chromium (Cr) in the soil, especially in agricultural fields [9], gradually reaches a level that threatens human food safety. When heavy metal ions are present in large amounts in the environment, they are absorbed by the roots of plants and transferred to aerial organs, which causes disturbance in plant metabolism and growth [10]. In this regard, finding solutions such as heavy metals immobilization or inoculating plants with endophytic fungi such as Piriformospora indica (P. indica) [11] or arbuscular mycorrhizal fungi (AMF) [12] may help alter the absorption of heavy metals by plants.
 Among the heavy elements, Cd and Pb are very important because of their long half-life in human and animal bodies. About 95% of the Pb entered into the human body is deposited in the bones in the form of Pb phosphates with a half-life of 20 to 30 years, and the rest is in the tissues in an exchangeable form. The accumulation of these elements in the body affects the hematopoietic, nervous, digestive, and renal systems [3, 13]. 
Heavy metal absorption from contaminated land by plants and especially agricultural products is one of the principal ways for these elements to enter the food chain. Furthermore, in many cases, a combination of petroleum compounds with heavy metals can double its adverse effects [14]. In the meantime, assessing the risk of heavy metal entry can be a suitable solution to investigate the carcinogenic and non-carcinogenic risks caused by the entrance of heavy metals [15].
Risk assessment is a process that estimates the probability and magnitude of damage caused by a risk and its potential threat to health. Meanwhile, risk management results are considered from various economic, political, legal, and ethical aspects, and environmental management decisions are made based on risk assessment [16]. There have been many studies on the risk assessment of heavy metals in different parts of the world, including Iran [17-19]. However, these studies have been conducted with various plants at different times and places and should be evaluated separately. 
This research aimed to investigate the effect of inorganic and organic Zn sources and P. indica fungus on reducing the risk of Cd and Pb for lettuce consumption cultivated in soil polluted with crude oil. Because the edible part of the lettuce plant accumulates heavy elements [20, 21], a risk assessment for heavy metals is required to avoid the possible risks caused by the consumption of this plant. 

2. Materials and Methods
This research is a pot experiment to investigate the effect of the inorganic and organic Zn fertilizers on the Cd and Pb risk factors in lettuce plants cultivated in soil contaminated with crude oil, Pb (800 mg/kg), and Cd (15 mg/kg) naturally. 
Treatments consisted of applying organic and inorganic Zn fertilizers in the form of Zn sulfate, Zn oxide, and Zn-EDTA sources at the rates of 0, 20, and 40 kg Zn/ha in a Cd and Pb contaminated (naturally) soil that was simultaneously polluted with crude oil (4% and 8% w/w) under cultivation of lettuce inoculation with P. indica. 
The soil used in this experiment was non-saline soil (EC=0.8 dS/m) with low organic carbon (OC <0.1%) that was collected from the soil surface layer (0–15 cm) around Esfahan City, central Iran. The studied soil (naturally polluted with Pb and Cd) was air-dried and ground to pass a 2 mm sieve. The soil was contaminated with crude oil at 0%, 4%, and 8% w/w and incubated for one month to reach equilibrium. Furthermore, the soil was treated with organic and inorganic Zn fertilizer at the mentioned rates and incubated for another month. During this period, to reach the equilibrium, the treated soil was constantly wetted and dried until it reached the field capacity (FC) point. Then, five lettuce seedlings were planted in each pot (54 treatments in three replications). After the establishment of the plants, the number of plants was reduced to one. During the plant growth period, irrigation was based on the plant’s water needs, and weeding operations were performed uniformly every 3 to 4 days. The average temperature of the greenhouse was between 18°C and 20°C.
Lettuce plants were harvested after 4 months, washed, and air-dried. Afterward, the samples were placed in an oven at 60⁰C for 24 hours to reach 0 humidity. The samples were powdered by an electric mill and stored in plastic containers. Then, the samples were burned at 480°C. After that, the concentration of plant Cd and Pb concentration was measured [22] after extracting the samples with HCl 2N using atomic absorption spectroscopy (AAS) (Perkin Elmer model 3030). A tri-acid solution (HNO3:H2SO4:HClO4 5:1:1) was used [23] to extract the total Pb and Cd from the soil and organic additions. The amount of Pb and Cd was then measured using AAS  [23].
To calculate the risk of contracting non-cancerous diseases, the Equations provided by the US Environmental Protection Agency (USEPA) were used in such a way that by using the Equation 1, the amount of pollutant absorption through food (lettuce) was calculated [24]:
1. ADD=(CF×IR×FI×EF×ED)/(BW×AT)
Where:
ADD=Pollutant absorption rate through food consumption (mg/kg/d)
CF=Contaminant concentration in food (mg/kg)
IR=Ingestion rate (kg/meal)
FI=Fraction ingested from a contaminated source (Unit less), the amount of pollutants absorbed by the body through food (This coefficient varies between 0.25 and 0.4. In this study, this coefficient was considered to be 0.4)
EF=Exposure frequency (meals/year)
ED=Exposure duration (years); this factor calculates the risk of non-cancerous diseases for 70 years.
BW=Body weight (kg)
AT=Averaging time (period over which exposure is averaged/days).
In this study, we assumed that an adult person’s daily lettuce (IR) intake is about 76 g [25].
Then, non-carcinogenic effects were determined by dividing the intake from Equation 1 by the reference dose (RfD) of each contaminant shown in Equation 2.
2. HQ=ADD/RFD
Where:
HQ=Hazard quotient. 
Regarding RFD, each element has a specific value (Pb=0.001 and Cd=0.004 [26]). This value was obtained by experimenting on animals and indicates the maximum element concentration that did not cause problems for the organisms (mg/kg/d). 
The total non-cancer hazard quotient (THQ) was also calculated as Equation 3:
3) THQ=∑HQi
Where: i=Pb or Cd element.

Statistical analysis
The experiment was set in factorial arrangements in a completely randomized design. SAS software, version 9.1 was used to perform statistical analyses following the ANOVA technique. The least significant difference (LSD) test was used to assess how the differences be +P.indica and –P.indica indicated the presence and  
tween the means differed. The significant difference was set at P<0.05.

3. Results

Soil Cd and Pb availability
The maximum Cd and Pb concentrations belonged to the soil with the highest level of crude oil under the cultivation of non-inoculated plants (Tables 1 and 2). Accordingly, increasing soil pollution with crude oil from 0% to 8% w/w significantly increased (P<0.05) the soil Cd and Pb concentration by 10.4% and 11.9%, respectively. Applying organic and inorganic Zn fertilizers considerably reduced the available concentration of Cd and Pb in soil (P<0.05).



However, using Zn from organic sources (Zn-EDTA) was more efficient than using it from inorganic sources. Soil application of pure Zn fertilizer from Zn-EDTA, Zn-oxide, and Zn-sulfate sources (40 kg/ha pure Zn) significantly and respectively decreased the Pb available concentration by 11.9%, 10.3%, and 9.8%, and for Cd concentration by 10.8%, 9.2%, and 8.8% (Tables 1 and 2).



Cd and Pb concentrations in plants and their daily intake 
The maximum Cd concentration was observed in the population who consumed the plants with the highest Cd concentration. According to this, the maximum plant Cd concentration belonged to the non-inoculated plants cultivated in the soil with the highest level of crude oil (8% w/w) (Table 3). Accordingly, increasing soil pollution with crude oil from 0% to 8% (w/w) significantly increased the Cd concentration of non-inoculated plants by 13.1% (P<0.05) (Table 3).



The P. indica inoculated plant increased by 11.5% (Table 3). Soil application of Zn fertilizer significantly reduced Cd concentration in plants (P<0.05). The same trend was observed also for Pb concentration (Table 4).



The daily intake amount has also shown a similar trend (Table 5).



Accordingly, consumption of plants cultivated in crude oil-polluted soil (8% w/w) relative to non-petroleum hydrocarbon polluted soil significantly increased the Pd intake (daily intake factor) by 13.8% (P<0.05) (Table 6). Using organic and inorganic Zn fertilizers significantly decreased the Pb and Cd intake factor (P<0.05). Soil application of pure Zn fertilizer from Zn-sulfate, Zn-oxide, and Zn-EDTA sources (40 kg/ha pure Zn) significantly decreased the Pb and Cd daily intake factor by 12.7%, 13.1%, and 14.3%, respectively (Tables 5 and 6). The plant Pb and Cd concentration was significantly related to the soil Pb and Cd concentration (P<0.05).



Pb and Cd hazard quotients (HQ)
The lowest Cd HQ factor due to the lettuce consumption belonged to the inoculated plant with P. indica, which was cultivated in the non-polluted soil with crude oil, while the highest was obtained from non-inoculated plants grown in contaminated soil with 8% w/w crude oil (Table 7). Plant inoculation with P. indica significantly reduced the HQ factor of Cd (P<0.05), as the results of our study have shown that inoculation of plants grown in the crude oil-contaminated soil (8% w/w) with P. indica significantly increased the HQ factor of Cd by 13.1%. Furthermore, using inorganic and organic fertilizers significantly decreases the Cd HQ factor (P<0.05). Accordingly, 40 kg/ha pure Zn from Zn-sulfate, Zn-oxide, and Zn-EDTA significantly decreased the HQ factor by 10.7%, 11.2%, and 15.3%, respectively. 



The Pb HQ factor showed similar results. According to this, increasing soil pollution with crude oil significantly increased the HQ factor for Pb (P<0.05) (Table 8). Based on the results of our study, increasing soil pollution with crude oil from 0% to 4% and 8% w/w significantly increased the HQ factor for Pb by 12.1% and 15.6% , respectively. However, the consumption of plants inoculated with P. indica has a lower HQ factor than non-inoculated plants (Table 8).



The highest amount of THQ due to the lettuce consumption belonged to the none-inoculated plant, which was cultivated in the polluted soil with the highest rate of crude oil (8% w/w), while the lowest was obtained from P. indica inoculated plants, which was grown none polluted soil with crude oil (Table 9). Plants inoculated with P. indica considerably reduced the THQ factor (P<0.05). The results of our research have indicated that P. indica inoculation of plants grown in crude oil-contaminated soil (8% w/w) significantly increased the THQ factor by 15.6%. Furthermore, using inorganic and organic fertilizers significantly decreases the THQ factor (P<0.05). Accordingly, using 40 kg/ha pure Zn from Zn-sulfate, Zn-oxide, and Zn-EDTA significantly decreased the THQ factor by 12.4%, 13.2%, and 17.1%, respectively (Table 9). 



4. Discussion 
The highest plant Pb and Cd concentrations belonged to the plants cultivated in the soils with the highest rate of crude oil pollution (8% w/w). It can be concluded that increasing soil pollution with crude oil significantly increases the soil heavy metal availability and thereby increases the heavy metal uptake by the plant, which is dangerous for food chain safety. Increasing the available concentration of Pb and Cd in soil with raisin the rate of petroleum hydrocarbon in the soil can be attributed to the effect of these compounds on the soil salinity and consequently increases the heavy metals solubility [27]. Sodium chloride in the soil forms chloride complexes with heavy metals, such as Pb and Cd. Thus, the availability of heavy metals in the solution phase of soil increases and raises the absorption rate of these metals by the plant [28].
Due to the extensive cultivation of lettuce in central Iran, the simultaneous contamination of heavy metals and petroleum compounds can increase the risk of entering heavy metals by consuming such vegetables, which should be considered in environmental studies. Numerous studies have shown that lettuce can absorb a large amount of heavy metals, such as Pb and Cd, without reducing the yield or showing signs of toxicity [29]. Some researchers have shown that the amount of heavy metal accumulation in plant tissues depends on the type of metal, soil conditions, and plant species. However, the amount of accumulation in aerial organs, especially leaves and stems, is more than in other organs and is much less in seeds than in leaves and stems [30, 31]. Heavy metal concentrations in leafy plants are higher than in other food plants [32, 33]. Lettuce (Lactuca sativa) is a plant that has many nutritional values. The extract of this plant contains various vitamins (mostly in greener leaves), water, and minerals. Considering that lettuce profoundly accumulates heavy metals and its consumable part for humans is its leaves [34, 35], it is necessary to study the cultivation conditions of this plant to reduce the possible risk to human health.
Based on the results of this study, consumption of 76 g none-inoculated lettuce cultivated in the crude oil-polluted soil (4% w/w) can increase the Cd and Pb daily intake significantly (Tables 5 and 6) by 14.1% and 12.6%, respectively. Therefore, it seems necessary to provide appropriate solutions to reduce the absorption of heavy metals by plants. One approach could be using endophytic fungi such as P. indica, which optionally communicate with symbiotic plants. In this regard, it has been reported that endophytic fungi like P. indica considerably impact the growth of various plants [36]. Colonization of P. indica with plant roots increases the intake of nutrient elements [37]. In contrast, P. indica can use different mechanisms to enhance the host plant’s resistance against biotic and abiotic stresses, such as heavy metal contamination [38, 39]. Overall, P. indica can increase the immobilization of heavy metals in roots and limit their translocation to host plants’ shoots by binding them to the hyphal cell wall [40]. Additionally, the symbiosis interactions between P. indica and plant roots activated the antioxidant system of plants, thereby enhancing plant tolerance to non-biological and biological stress [36, 41]. The important point is that in the country’s central regions, low nutrient availability in soil reduces plant growth and has also caused the accumulation of dangerous heavy metals such as Pb and Cd in plants. This condition can increase the risk of different carcinogenic and non-carcinogenic diseases [42, 43].
It has been reported that applying Zn fertilizers in wheat, barley, corn, and oat plants has reduced the concentration of dangerous heavy metals in these plants [44]. These results show that heavy metal accumulation is higher in Zn-deficient soils, and appropriate Zn prevents Cd accumulation in the soil. Also, Liu et al. studied the effect of Cd and Zn on the yield and nutritional quality of Brassica Junicea. They mentioned that human health is less endangered by consuming plants grown in Cd-contaminated soils when the Zn concentration in the soil is sufficient [45]. In this regard, using fertilizers containing micronutrient elements can reduce the absorption of heavy metals by plants by improving the plant’s nutritional conditions [46]. According to the results obtained in this research, the application of 40 kg/ha of pure Zn in contaminated soil with 8% w/w crude oil reduced the Pb and Cd concentration in the plant by 13.1% and 15.6%, respectively, which can be attributed to the competitive effect of Zn with heavy metals. Accordingly, the Cd and Pb HQ have been significantly decreased by 13.1% and 14.5%, respectively, an essential point in environmental studies. Considering the importance of the studied factors on the concentration of Pb and Cd in the plant and the effect on the amount of HQ, it is necessary to investigate the impact of these factors on the absorption of heavy metals in the soil and, consequently, the human food chain.
The antagonistic effect of Zn with heavy metals has previously been reported by different researchers [47, 48]. It is well known that heavy metal toxicity is the cause of oxidative cell damage resulting from plant-related reactive oxygen species (ROS). Therefore, the ROS elimination role of plants under heavy metal pollution is a significant mechanism against heavy metal toxicity [49, 50]. Zn plays different protective roles in reducing plant heavy metal damage [50]. For example, Zn improves the activities of antioxidant enzymes such as Zn-containing enzyme superoxide dismutase and competes with heavy metals to bind to –SH groups and membrane proteins of enzymes to protect plants against heavy metal toxicity [51]. Zhou and Zhang showed that increasing the application of Zn fertilizer reduced the Cd toxicity and membrane damage of barley, and the plant growth was enhanced [52]. However, applying organic chelates has been more effective in reducing the concentration of Pb and Cd in plants and, as a result, reducing their daily intake and risk. This outcome can be due to the considerable effect of chelate on Zn solubility [53]. In general, chelates have different functional groups on their surface that bond with metals, increasing their permeability and causing them to be more absorbed by plants [54]. Additionally, the results of our study showed that with a reduction of Pb and Cd concentration in plants, the daily intake of these heavy metals by the human body and the HQ value reduced significantly, something necessary for human health and safety. 

5. Conclusion
Based on the results of this study, using organic Zn sources such as Zn-EDTA has the most significant effect on decreasing the plant Cd and Pb availability that reduces the non-carcinogenic effects of heavy metals (HQ factor) in consumers of the lettuce. Accordingly, using 40 kg/ha Zn pure from Zn-EDTA sources significantly (P≤0.05) decreases the HQ factor for the lettuce cultivated in the soil polluted by 8% w/w crude oil. Soil pollution with crude oil and heavy metals had interaction effects. Our results show that increasing soil pollution with crude oil has risen significantly in the Pb and Cd HQ factors. On the other hand, plant inoculation with P. indica has significantly decreased the Pb and Cd HQ factors. However, this finding should be investigated in the field research. 

Study limitations
Considering that greenhouse results can be different from field results, conducting this research in field studies is advised to obtain the correlation coefficient between greenhouse results and field results. Because the type of pollutant and the physical and chemical characteristics of different soils can affect the availability of heavy metals, it is better to investigate this research with soils with other physical and chemical characteristics.

Ethical Considerations

Compliance with ethical guidelines
This study was approved by the Ethics Committee of Islamic Azad University, Arak Branch (Code: IR.IAU.Arak.REC1401.037).

Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflict of interest
The authors declared no conflict of interest.

Acknowledgements
The author gratefully acknowledges the Arak Branch,  Islamic Azad University for analyzing samples.



References