Assessment of heavy metals and potential health risks associated with the consumption of vegetables grown on the roadside soils

Vegetables cultivated near roads absorb toxic metals from polluted soil, which enter into the human body through the food chain and cause serious health problems to humans. The present study investigated the contamination level of lead (Pb) and nickel (Ni) in soils and vegetables grown along the roadside of District Swat, Pakistan, and the health risks associated with the consumption of the tested vegetables have been investigated. The plant samples were collected from the cultivated eld within a 120-meter range from the roadside. Spinacia oleracea, Allium cepa, Rumex dentatus, and Trachyspermum ammi were selected based on their importance as vegetables. In results, Pb concentration was higher in plants located at the distance between 0-10 m away from the roadside than the WHO permissible limit. In such plants, Pb concentration was higher than Ni. Rumex dentatus contained the highest concentration of Pb among the tested vegetables while Ni concentration was highest in Trachyspermum ammi as compared to other plants. Concentration and accumulation of both the metals decreased in soil and plants with increasing distance from the road. Similarly, target hazard quotient values noted for Pb and Ni were greater than unity, which shows that there is a potential risk associated with the consumption of tested vegetables near the road. Moreover, the values of target cancer risk were greater than 0.0001, which shows that there is a risk of cancer with the consumption of tested vegetables. In conclusion, the consumption of the tested vegetables was very dangerous as it may lead to higher risks of cancer.


Introduction
Fossil fuel combustion in automobiles releases toxic heavy metals into the air, which are then deposited on the soils nearby roadsides (Suzuki et (Komjarova and Blust, 2009). Plants may uptake Pb through several pathways. High negative potential on the surface of plant roots is required for Pb absorption. Due to similarity with calcium (Ca), Pb enters into the plant mainly through channels speci ed for Ca transport (Wang et al. 2007).
No biological role of Pb has been reported until now and thus it is a non-essential element for living organisms. On contrary, it has been considered as the second most hazardous metal after arsenic (Maestri et al 2010; Mehmood et al. 2018Mehmood et al. , 2019. Besides plants, Pb also adversely affects human health. A decrease in the number of erythrocytes has been reported during Pb exposure because of the inhibition of enzymes (coproporphyrinogen and ferrochelatase, delta-aminolevulinic acid ALAD) needed for the synthesis of hemoglobin and red blood cell (Sipos et al. 2003; Warren et al. 2003). It has been found that Pb exposure increased the excretion of amino acids and glucose in urine due to its toxic effect on the proximal convoluted portion of the nephron (Loghman-Adham, 1997). Long-term Pb exposure causes kidney failure, increased blood uric acid concentration, high blood pressure, and joint infection (Alasia, 2010). The highest amounts of Pb (about 1/3rd of the entire body) were accumulated in the liver (Mudipalli, 2009). As Pb is analogous to Ca, therefore it can cross the blood-brain barrier easily where it can replace and activate calcium-mediated activities, which disturb brain physiology and development (Sanders et al. 2009). People working in the lead storage industry, show disorders of the male gonads (testes), reduced testosterone production, and low sperm count in semen (Queiroz and Waissmann 2006).
Threshold limit of Pb that can cause reproductive abnormalities is about 40 µg dL -1 (Quintanilla- Vega et al. 2000). Lead affects the menstrual cycle, reduces the duration of pregnancy, and causes abnormal birth (Han et al. 2000).
Ni is the 22nd most abundant element found in the earth's crust, occurring mostly in rocks as a free metal or bonded with iron metal. Ni is a hard, ductile, and silvery-white metal (McIlveen and Negusanti, 1994). Concentrations of Ni in soil and drinking waters are lower than 100 and 0.005 ppm, respectively (Naveed et al. 2020(Naveed et al. , 2021b. Anthropogenic sources of Ni pollution include vehicular emissions, fossil fuel burning, mining, smelting, municipal and industrial wastes (Alloway, 1995). Ni concentrations may reach up to 26000 ppm in contaminated soils (Alloway, 1995).
Ni is absorbed by plants mainly through the root system via both passive and active transport (Seregin, 2006). The ratio of Ni uptake between active and passive transport changes with plant species, Ni form, and Ni concentration in the soil (Dan et al. 2002;Naveed et al. 2020). Moreover, soluble compounds of Ni might also be absorbed through the Mg ion transport system, due to the similar charge/size ratio of the two metal ions (Oller et al. 1997). However, its concentration in most of the plant species is extremely low i.e. 0.05-10 ppm (Nieminen et al. 2007). Extremely high concentrations of Ni have made some farmland soils unsuitable for growing crops, vegetables, and fruits (Naveed et al. 2021).
In humans, Ni is an abundant metal commonly responsible for skin allergies and is one of the greatest causes of allergic contact dermatitis, as revealed by positive dermal patch tests (Cavani, 2005). Ni is a carcinogenic element in several animal species but the basic mechanisms behind are still unknown (Chang, 1996). Ni can act as a tumor inducer by inhibiting natural killer cell activity (Costa and Klein, 1999). Another possible way by which Ni induces cell death and/or damage is through lipid peroxidation (Misra et al. 1990;Chen et al. 1998;Janicka and Cempel, 2001).
Based on the above discussion, it has been found that both Pb and Ni cause toxic effects on plants as well as humans. The increasing needs of the human population for food have resulted in intensive farming, even near the roadsides. In addition, there is an increasing trend regarding the production of vegetables along the roadside near urban areas. Therefore, it is necessary to evaluate the contamination level of heavy metals especially Pb and Ni in soils as well as plants growing along the roadsides, as it would help in exploring the contamination level of these metals in those soils and plants and to calculate health risks associated with the consumption of these edible plants growing along the roadside.
Moreover, it would also help in identifying the safe distance for the production of different crop plants along the roadside. Up to our knowledge, no study has focused on this aspect of the present research work conducted. Based on these hypotheses, the present study was conducted to investigate the contamination level of Pb and Ni in soils and plants that grew along the roadside of Bara Bandai, Ningolai, and Ghalegay, District Swat, Khyber Pakhtunkhwa. Moreover, different parameters related to the health risks associated with the consumption of the tested vegetables have been investigated.

Study area
The present study was conducted to investigate the contamination level of Pb and Ni in soils and plants that grew along the roadside of Bara Bandai, Ningolai and Ghalegay, District Swat, Khyber Pakhtunkhwa. Plants and soil samples were collected from the cultivated elds near the roadside at Bara Bandai, Ningolai, and Ghalegay.

Collection of soil and plant samples
Plant samples were collected from the cultivated eld within a 120-meter range from the roadside. Collection of plants was made in four groups based on their distance range from road i.e. group-I (0-10 m distance), group-II (10-40 m distance), group-III (40-80 m distance), and group-IV (80-120 m distance). Five replicates were taken for each plant at each range. Similarly, ve soils samples were randomly collected from each range. Plants (Spinacia oleracea, Allium cepa, Rumex dentatus, and Trachyspermum ammi) were selected for the present investigation based on their importance as vegetables.

Acid digestion of soil and plant samples
Collected soil samples were ground and sieved (2 mm) to remove large particles. Parameters such as electrical conductivity and pH were calculated using a pH meter (Model CON.3173) and electrical conductivity meter (Model CON 5). For acid digestion, 0.25 g from each soil sample was added in acid solution (5 ml nitric acid and 1 ml sulfuric acid) in digestion tubes and placed overnight in a fume hood. The next day, each soil sample in acid solution was heated until a clear aliquot was obtained. The aliquot was ltered and the total volume was raised to 50 mL with distilled water. Each solution was stored in plastic bottles until analyzed for heavy metals.
Collected plant samples were cut into parts (roots, stem, and leaves) and then kept in paper envelopes.
The samples were dried in the oven at 80 °C for 48 h and then crushed into a powdered form using a commercial blender. Each sample was digested by the method of Allen (1974). A 0.25gm of the sample was taken in the conical ask and then a mixture of acids (5 ml nitric acid and 1 ml sulfuric acid) was added to it and boiled on a hotplate until digested completely. After digestion, each sample was cooled, ltered and then the nal volume is raised to 50 mL by adding distilled water. The solutions were stored in plastic bottles for metal analysis. The metal analysis of both soil and plants samples was carried out at Central Resource Laboratory, Peshawar using atomic absorption spectrophotometer (AA2407, USA).

Health risk assessment
Bioconcentration factor (BCF) It is the ratio of heavy metal concentration in the edible part of the plant to heavy metal concentration in a soil sample (Sharma et al. 2018). BCF was calculated using the following formula: where C plant is heavy metal content in the edible part of plant and C soil is heavy metal content in respective soil. The value of BCF greater than 1 indicates that the plant is a potential accumulator of the metal being considered for analysis.

Estimated daily intake (EDI)
The estimated daily intake of the metals was determined based on their mean concentration in each Target hazard quotient (THQ) The target hazard quotient (THQ) values were estimated to assess non-carcinogenic human health risk from the consumption of vegetables contaminated by heavy metals. The THQ values were calculated using the following equation as described by Chen et al., (2011).
Where EDI is the estimated daily metal intake of the population in mg/day/kg body weight and RfD is the The cancer risk posed to human health due to the ingestion of individual possibly carcinogenic metals was estimated using the following equation as described by Sharma et al. (2018). Then, the target cancer risk (TCR) resulting from heavy metals (Pb and Ni) ingestion, which may promote carcinogenic effect depending on the exposure dose, was calculated using the following equation as described by Kamunda et al. (2016).
where CR represents cancer risk over a lifetime by individual heavy metal ingestion, EDI is the estimated daily metal intake of the population in mg/day/kg body weight, CPS o is the oral cancer slope factor in (mg/kg/day)-1 and n is the number of heavy metals considered for cancer risk calculation. The CPS o values used for Pb and Ni were 0.0085 and 1.7, respectively. It has been pointed out that the slope factor converts the estimated daily intake of the metal averaged over a lifetime of the exposure directly to the incremental risk of an individual developing cancer (Kamunda et al. 2016).

Statistical analysis
The mean value of the data was calculated and then it was subjected to analysis of variance (ANOVA) and correlations between different parameters were established using statistical software SPSS 16 and MS Excel 2010.

Results
Physicochemical properties of soil Different physicochemical properties of soils collected at different distances from the roadside are presented in Table 1. Almost all the properties varied with distance from the roadside. A decreasing trend in soil pH while an increasing trend in electrical conductivity (EC) was noted with increasing distance from the roadside. Similarly, the concentrations of lead (Pb) and nickel (Ni) in the soil decreased as the distance from the road increased. Concentrations of Pb and Ni were signi cantly higher at 0-10 meters from the road compared to the other distances. The highest concentrations of Pb and Ni recorded at 0-10 m from the road were 60.6 and 35.0. The concentration of Pb in the soil was under the normal limit (85 mg kg -1 ) but that of the Ni was at the edge of the permissible limit (35 mg kg -1 ) by WHO, 2001. The soil was loamy in texture, which is suitable for agricultural activities purposes because it has constituents more or less the same amount required for plants growth.
Lead and nickel concentrations in Rumex dentatus L.
As clear from Figure 1 From the data presented in Figure 3, the bioaccumulation of Pb and Ni in Spinacia oleracea L. was decreased as the distance from the road was increased and vice versa. The highest accumulation of both Pb and Ni was in the root portion followed by leaves, and stems. The Pb concentration in all the parts of Spinacia oleracea L. was above the permissible limits (0.3 mg kg -1 ) by WHO/FAO, 2001 up to 80 m distance from the road. At 80-120 m distance, the Pb concentration was above the permissible limits only in the root of Spinacia oleracea L. In the case of Ni, a similar decreasing trend in Ni concentration with increasing distance was recorded in different parts of Spinacia oleracea L. The concentration was above the permissible limits in root, leaves, and stem portions of Spinacia oleracea L. up to 10 m distance from the road. At 10-40 distance, the Ni concentration was above the permissible limit in only the root portion of the Spinacia oleracea L. For the rest of the plants after 40-120 m, the concentration of Ni was below the permissible limits as given by WHO, FAO, 2001.
Lead and nickel concentrations in Allium cepa L.
A decreasing trend with increasing distance from the roadside regarding the concentrations of Ni and Pb in different parts of Allium cepa L.was recorded ( Figure 4). As clear from the data, the root portion accumulated the maximum amount of Pb, followed by stem and leaves. In the case of Ni, the maximum accumulation was observed in the case of the stem, followed by stem and leaves. Regarding the permissible limit of Pb concentration, only stem and leaves portion at 80-120 away from the roadside were below the permissible concentration of Pb while all the others were above the permissible limit of Pb i.e. 0.3 mg kg -1 . In the case of Ni, the concentration was above the permissible limit (1.5 mg kg -1 ) in root, stem, and leaves at 0-10 m away from the road while only root and stem portions had Ni concentrations above the permissible limits.

Lead and nickel bioaccumulation
As clear from the data presented in Table 2, the maximum bioaccumulation of Pb was recorded in Rumex dentatus L., followed by Trachyspermum ammi (L.) Sprague ex Turrill, Spinacia oleracea L., and Allium cepa L. The shoot and root portion of Rumex dentatus L. bioaccumulated 46.2 and 29.4 mg kg -1 DW.

Correlation between Ni and Pb concentration in soil and plant samples
A positive strong correlation (R 2 = 0.984) was found between Ni and Pb concentration in the soil as given in Figure 5. Similarly, a positive correlation existed between the concentration of Ni and Pb in all the studied plants as shown in Figure 6. The correlation was found to be highly signi cant in Trachyspermum ammi (L.) Sprague ex Turrill (R 2 = 0.969), Spinacia olerace (R 2 = 0.988), Allium cepa (R 2 = 0.959) and Rumex dentatus (R 2 = 0.906).

Bioconcentration factor
As clear from the data presented in Table 2, the bioconcentration factor (BCF) decreased with increasing distance from the road in all the species studied. The maximum value of BCF (0.7629) was observed in Rumex dentatus for the Pb, followed by 0.4949 in Trachyspermum ammi for Ni. The values of BCFs in Spinacia oleracea, Allium cepa, and Rumex dentatus were more for Pb as compared to that observed for Ni in the same species. The minimum BCF value (0.0312) was observed in the case of Trachyspermum ammi at 80-120 m away from the road for Pb. A decreasing trend in BCF values was observed in all the studied species as the distance from the road was increased.

Health risk assessment
After calculation of BCF and TF, the data on heavy metals concentration in different plant species studied was analyzed regarding health risks associated. It was found that the values of estimated daily intake (EDI), target hazard quotient (THQ), hazard index (HI), cancer risk (CR), and target cancer risk (TCR) were decreased as the distance of sampling site from the road was increased. In general, the values of EDI and THQ were higher in the case of Pb as compared to those observed for Ni. The maximum EDI (0. . Similar is the case with nickel, which also occurs naturally in petroleum products. Nickel in soils can be found in several different forms: adsorbed or complex with organic cation surfaces or on inorganic cation exchange surfaces, inorganic crystalline minerals, water-soluble, chelated metal complexes, or free ion in soil solution. Lead does not seem to be of big concern outside urban areas but may ultimately become a problem due to decreased pH of soil caused by limited use of soil liming in agriculture and mobilization because of increased acid rain (Bai et al. 2011).
Plants grown in contaminated soils are exposed to the contaminants. Since plants have the natural ability to absorb dissolved substances from the soil solution and in doing so, plants also absorb toxic metals present in the soil. Comparison among lead concentrations in different plant species demonstrated signi cant variations with distances from roads. Pb concentration in plants occurred in the order of Rumex dentatus > Spinacia oleracea > Trachyspermum ammi > Allium cepa. Differences in Pb concentration in plants depend on several factors such as developmental stage, genetic makeup, transpiration coe cient, plant roots system in soil, plant growth rate, and nutrients needed by plants (Cenkci et al. 2010). Roots of plants absorbed the highest Pb concentration as compared to the other plant parts. This is because roots are more exposed to the pollutants in the soil as compared to the other parts of the plant. Many plants have been reported to retain up to 95% of Pb in their root portions and a little concentration is transported to aerial parts of a plant (Shahid et al., 2011). This restricted transport of Pb to the aerial parts of the plants might be due to the immobilization of Pb on plant cell walls by the negatively charged pectin (Arias et al. 2010). Despite this, some of the Pb moves through intercellular spaces present between plant cells and translocation with water to endodermis (Wang et al. 2007;Shahid et al. 2011). Pb is either stored in a plasma membrane attached with pectin, crystallized in intercellular spaces, or stored in outer root cortical cells (Yang et al. 2007). Plants collected near the roadside were more exposed to lead as compared to the plants away In the case of THQ, values noted for Pb and Ni were greater than unity, which shows that there is potential risk associated with the consumption of all the tested vegetables up to 0-10 m away from the road. Moreover, the consumption of Rumex dentatus was not safe up to 40 m away from the road. All the other species were safe to use after 10 m away from the road except Rumex dentatus, which was safe to utilize after 40 m away from the road. A similar trend in HI was observed regarding the usage of different vegetables tested in the present study as observed in the case of THQ. In the case of TCR, all the values were greater than 1 × 10 -4 , which shows that there is a risk of cancer with the consumption of all the tested vegetables. Based on these indices, it was found that the consumption of the tested vegetables was very dangerous and may lead to higher risks of cancer.

Conclusions
The results con rmed the presence of Pb and Ni in selected soils and plant samples. The concentration of Pb in Rumex dentatus, Trachyspermum ammi, Spinacia oleracea, and Allium cepa was above the safe limits of WHO within 80 m range from the road. The results demonstrated that plants collected beyond the 80 m range contained Pb concentration below the WHO safe limit. While the plant was found safe in the case of Ni beyond the 10 m range where the concentration of Ni in plant tissues was found below the WHO safe limit. Moreover, the consumption of studied vegetables grown along the roadside was not safe based on the values of different indices. In the case of target hazard quotient (THQ), the values noted for Pb and Ni were greater than unity, which shows that there is potential risk associated with the consumption of all the tested vegetables up to 0-10 m away from the road. In the case of target cancer risk (TCR), all the values were greater than 1 × 10 -4 , which shows that there is a risk of cancer from Ni with the consumption of vegetables to the people in the study area. Based on these results, it is highly recommended to have strict regulatory control on the cultivation of these vegetables along the roadside in the study area.

Declarations
Funding This research did not receive any speci c grant from funding agencies in the public, commercial, or notfor-pro t sectors.    Correlation between Pb and Ni in soil.