Heavy metals uptake by the global economic crop (Pisum sativum L.) grown in contaminated soils and its associated health risks

The aim of the present investigation was to determine the concentration of heavy metals in the different organs of Pisum sativum L. (garden pea) grown in contaminated soils in comparison to nonpolluted soils in the South Cairo and Giza provinces, Egypt, and their effect on consumers’ health. To collect soil and plant samples from two nonpolluted and two polluted farms, five quadrats, each of 1 m2, were collected per each farm and used for growth measurement and chemical analysis. The daily intake of metals (DIM) and its associated health risks (health risk index (HRI) were also assessed. The investigated heavy metals were cadmium (Cd), arsenic (As), chromium (Cr), copper (Cu), nickel (Ni), iron (Fe), manganese (Mn), zinc (Zn), silver (Ag), cobalt (Co) and vanadium (V). Significant differences in soil heavy metals, except As, between nonpolluted and polluted sites were recorded. Fresh and dry phytomass, photosynthetic pigments, fruit production, and organic and inorganic nutrients were reduced in the polluted sites, where there was a high concentration of heavy metals in the fruit. The bioaccumulation factor for all studied heavy metals exceeded 1 in the polluted sites and only Pb, Cu and Mn exceeded 1 in the nonpolluted sites. Except for Fe, the DIM of the studied heavy metals in both sites did not exceed 1 in either children or adults. However, the HRI of Pb, Cd, Fe, and Mn in the polluted plants and Pb in the nonpolluted ones exceeded 1, indicating significant potential health risks to consumers. The authors recommend not to eat garden peas grown in the polluted sites, and farmers should carefully grow heavy metals non-accumulating food crops or non-edible plants for other purposes such as animal forages.


Introduction
Vegetables are edible plants that store reserve food materials in their roots, stem, leaves, and/or fruits, where essential dietary elements, including iron, calcium, vitamins, protein, and a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 cultivated as a main food crop for Egyptians and for exportation [25], consequently this crop must be produced with high quality. Thus, the results of this study are worldwide relevant. According to the authors' knowledge, so far, no studies have been carried out in Egypt on P. sativum grown at polluted sites in comparison with the reference (nonpolluted) sites. Therefore, the aim of the present investigation was to determine the accumulation potential of heavy metals in the different organs (root, leaf, and fruit) of P. sativum cultivated in polluted soils in comparison to nonpolluted soils in the South Cairo Province, Egypt, and to assess the associated impact of these accumulated heavy metals on the health of the public consumers.

Sampling sites
Plant sampling was carried out through two farms in nonpolluted (29˚44 0 38.82@ N and 311 53.00@ E) and two others in polluted (29˚44 0 45.47@ N and 31˚17 0 46.56@ E) sites, located in the South Cairo and Giza Provinces, respectively, during the winter season of 2017. The owner of the farms gave us the permission to conduct the study on these sites. Each farm has an area of approximately three acres. Nonpolluted farms received clean water from the Nile River tributaries, while polluted farms received industrial waste (National Cement Company, Egyptian Iron and Steel, Bricks factories, and Helwan Fertilizer Company) and municipal discharge. These wastes mainly constitute hydrocarbons, nutrients, and heavy metals. The agricultural lands of the study sites, which extends along the River Nile, were characterized by Alluvial soils. The prevailing climate of the study area showed that the mean annual rainfall was 1.67-2.13 mm/year, while the mean annual temperature was 21.08˚C, and the annual mean relative humidity was 52.68-56.08%.

Plant sampling and analysis
At each farm, 5 quadrats (each of 1 m 2 ) were randomly selected to represent the growth of P. sativum. From each quadrat, individual pea plants were harvested, separated into roots, shoots (stems + leaves), and fruits, weighed to determine the fresh weight, packed in polyethylene bags, and transferred to the laboratory. Plant samples were washed twice with tap water and then with distilled water, followed by oven-drying at 105˚C until they reached a constant weight, to determine the dry weight (kg/acre) and the plant production [26], and then ground into powder using a metal-free plastic mill for nutrient analysis. For pigment analysis, three pea leaves from each quadrat in the nonpolluted and polluted sites were collected and mixed to make three composite samples. Chlorophyll and carotenoids were extracted from the definite fresh weight of leaves (approximately 2 g) in 50% (v/v) acetone in complete darkness and kept overnight at 4˚C, then taken and measured using a spectrophotometer against a blank of aqueous acetone at three wavelengths (453, 644, and 663 nm). The concentration of each pigment fraction in mg/l was calculated using the following equations [27]: The values were then conveyed as mg/g fresh weight. Total soluble carbohydrates were estimated by the anthrone-sulfuric acid method [28], while the total soluble proteins were measured spectrophotometrically by the Bio-Rad protein assay [29]. Heavy metals and nutrient (N, P, and K) concentrations of the different plant organs were extracted using the mixed acid digestion method. A ground sample of 1 g was digested in 20 mL of a tri-acid mixture of HNO 3 :H 2 SO 4 :HClO 4 (5:1:1, v/v/v) until a transparent color appeared, then the digested plant was filtered and diluted with double-distilled water to 25 mL [30]. Twelve heavy metals (Ag, As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, V, and Zn) and K were determined by atomic absorption spectrometry (Shimadzu AA-6300; Shimadzu Co. Ltd., Kyoto, Japan), while total P was determined using a spectrophotometer via the ammonium molybdate method. Total N was determined using a CHN Elemental Analyzer (Yanako CHN Corder MT-5, Yanaco Apparatus Development Laboratory Co. Ltd., Kyoto, Japan). All procedures are outlined in the study of Allen [26]. Atomic absorption spectrometry was calibrated by standard solutions, which contain known concentrations of each element. Standard solutions were prepared by diluting available high-purity stock solutions (BDH) [26].

Soil sampling and analysis
Three composite soil samples were collected from the profiles of 0-20 cm from each farm. Soil water extracts (1:5, w/v) were prepared to determine the soil salinity (μS/cm) using an electrical conductivity meter (Corning Model 311, Corning Incorporated, New York, USA), and soil reaction using a pH meter (ICM Model B-213, ICM, Hillsboro, USA). N, P, K, and the 12 heavy metals were extracted and determined with the methods used for plant samples.

Quality assurance and quality control
A certified reference material (SRM 1573a, tomato leaves) was used to verify the accuracy of the heavy metal determinations. This reference material was digested and analyzed using the same methods applied to the P. sativum samples. Heavy metal digestions and measurements were performed in triplicate. Accuracy was determined by comparing the measured concentration with the certified value, and the result was expressed as a percentage. The recovery rates ranged from 95 to 104% for SRM 1573a. The detection limits of heavy metals (in μg/l) were as follows: 3.0 for Ag, 5.0 for Fe; 1.5 for As, Cu, Mn and Zn; 15.0 for Pb and Cd; 9.0 for Co; 3.0 for Cr, 2.0 for V and 6.0 for Ni. The detection limits for all heavy metals were established on a 95% confidence level (3 standard deviations).

Data analysis
The soil pollution by each metal was assessed using the pollution load index (PLI), calculated as follows [31]: PLI ¼ concentration of heavy metal in polluted soils=concentration of heavy metal in nonpolluted soils The significance of the variation in soil and plant variables between the nonpolluted and polluted sites was tested using a paired-samples t-test. Before performing two-way analysis of variance (ANOVA-2), the data were tested for their normality of distribution and homogeneity of variance, and when necessary, the data were log-transformed. ANOVA-2 was used to assess the significant variation in the nutrients and heavy metals in the different plant organs between the nonpolluted and polluted sites. Duncan's multiple range test at p < 0.05 was used to identify significant differences between means. Statistical analyses were carried out using SPSS software [32]. The bioaccumulation factor (BF), measuring the plant's ability to accumulate a specific metal in relation to its concentration in the soil, was calculated as follows: BF = Croot/Csoil, where C root and C soil are the concentrations of heavy metals in the roots and soil. The translocation factor (TF), which assesses the relative translocation of heavy metals from the roots to shoots of a plant, was calculated as TF = Cshoot/Croot, where C shoot and C root are the concentrations of heavy metals in the plant's shoots and roots [9].
A health risk assessment of any pollutant requires an estimation of the level of exposure by noticing methods of exposure to the target organisms. The daily intake of metals (DIM) was measured as the average consumption of polluted plants for both adults and children [33]. DIM = (Cmetal × Cfactor × Dfood intake)/Baverage weight, where C metal is the metal concentration in the plant (mg/kg), C factor is a conversion factor, D food intake is the daily intake of vegetable, and B average weight is the Egyptian average body weight. The conversion factor (0.085) was used to convert fresh weight to dry weight [34]. The average daily intake of metal for children and adults is 0.345 and 0.232 kg/person/day, while their average body weights are 32.7 and 55.9 kg, respectively [35]. Moreover, the health risk index (HRI) for the local inhabitants consuming contaminated plants was calculated as the ratio of the assessed crop exposure and the reference oral dose [36]. An HRI value greater than 1 is a danger for human health [37] and may cause a health risk for the consumers.

Soil analysis
Significant differences for all soil variables (except As) were recognized between the nonpolluted and polluted sites ( Table 1). The polluted soils had a higher pH (7.6), salinity (5.8 μS/cm), and heavy metal concentrations, but lower contents of N (49.7 mg/kg), P (6.9 mg/kg), and K (35.4 mg/kg) than the nonpolluted soils. The pollution load indexes (PLI) of the investigated

Phytomass
A significant difference was detected for the fresh and dry phytomass, as well as fruit production, between nonpolluted and polluted sites (Fig 1). In the polluted soils, the fresh phytomass of P. sativum was greatly reduced from 5223 to 1654 kg/acre, while the dry phytomass was decreased from 404 to 126 kg/acre. In addition, the fruit production was reduced by 85.2% under pollution stress.

Plant analysis
The analysis of P. sativum leaves indicated a significant reduction in chlorophyll a and a nonsignificant reduction in chlorophyll b and carotenoids in the polluted sites (Fig 2). Chlorophyll a was reduced by 43.8%, while chlorophyll b and carotenoids were reduced by 12.5% and 33.3%, respectively. The nutrient contents of the above-and below-ground parts of P. sativum showed a significant reduction under pollution stress (

Heavy metals in the fruits
There was a high accumulation of heavy metals in the fruits of P. sativum plants cultivated in the polluted farms ( Table 3). The concentration of most of the heavy metals in the fruit tissues

Heavy metal bioaccumulation and translocation
The bioaccumulation factor (BF) of P. sativum for the studied heavy metals was greater than 1 in the polluted sites, while the translocation factor (TF) did not exceed 1 in either the nonpolluted or polluted sites ( Table 4). It was found that Cd had the highest BF (242.9) in the polluted Table 3. Heavy metal concentrations (mean ± standard deviation) in the fruits of Pisum sativum grown in nonpolluted and polluted soils. Difference = ((Cp-Cn)/ Cp) × 100, where Cp and Cn are the metal concentrations in the polluted and nonpolluted soils, respectively.

Daily intake of metals (DIM)
The DIM of heavy metals (except Fe and Cd) in the nonpolluted and polluted sites did not exceed 1 in either children or adults (

Assessment of health risks
The health risk index (HRI) indicated that Pb in the nonpolluted sites and Pb, Cd, Fe, and Mn in the polluted sites have the greatest potential to cause health risks to public consumers, and the HRI of these metals for children and adults exceeded unity ( Table 6). In the polluted sites, the HRI of Cd (60 and 70) was the highest, followed by that of Pb (60 and 70), Mn (2.1 and 2.9), and Fe (1.8 and 1.9) for both adults and children. In contrast, the HRI of Pb in the nonpolluted sites was 5 for both adults and children.

Discussion
Plants cultivated in heavy-metal-polluted soils, resulting from the increase in anthropogenic and geologic activities, show reduced growth due to changes in their biochemical and physiological activities [43]. In the present study, a remarkable decrease in N, P, and K in the soil and plants (roots and shoots) of the polluted sites was recorded. In most agricultural conditions, the availability of usable nitrogen is a common limiting factor of high growth, as nitrogen is important for vegetative growth and assimilation of amino acids, and it is a constituent of chlorophyll. In addition, a suitable amount of P plays an important role in plants yielding more fruit and having healthier shoots and root systems, and plants with sufficient P may mature much faster than those without phosphorus. An inadequate supply can cause green and purple discoloration, wilting, and small flowers and fruit. K helps plants use water efficiently, preventing many diseases and heat damage, and aids the cycle of nutrients through the leaves, roots, and stems [44]. In this study, the concentration of heavy metals was higher in polluted soils than in nonpolluted soils, which coincides with the results of Galal [6], and Shehata and Galal [9]. Based on the Environmental Quality Standards set by the US Environmental Protection Agency [37], the polluted and nonpolluted soils in the present study were within the safe level. This could be due to the incessant removal of heavy metals by the crops cultivated in these sites and the leaching of heavy metals into the soil's deeper layer [14]. The values of all of the metals determined were above the tolerable limits recommended by the World Health Organization [38], except for Cr (0.4 mg/kg), Cu (16.3 mg/kg), and Ni (0.6 mg/kg), which were below the standard maxima of 30, 0.27−100.0, and 5.0 mg/kg, respectively.
According to Borah and Devi [22], heavy metals affect the growth performance of P. sativum by decreasing its biomass, yield, and photosynthetic pigment (chlorophyll a and b) in plants grown in wastewater-irrigated soil. In our study, a reduction in the fresh and dry phytomass and productivity of P. sativum cultivated in the polluted soils was recorded. In addition, the contents of chlorophyll a and b were also reduced, which may have led to a decrease in P. sativum productivity. According to Mansur and Garba [45], heavy metal concentrations affect soil fertility, as they reduce the nutrient (e.g., K and P) availability by forming complexes (e.g., lead phosphate and copper phosphate) at a certain pH. These complexes cannot be absorbed/ taken up by plants [46]. The reduction in plant nutrients such as N, P, and K under the effect of pollution may lead to a reduction in the economic yield and phytomass of almost all vegetable crops [6].
Various abiotic stresses decrease the chlorophyll content in plants [47]. In our study, the reduction of chlorophyll a and b in P. sativum cultivated in the polluted soils may have been the result of high concentrations of heavy metals, which inhibits chlorophyll synthesis and destroys the chloroplasts [48]. Moreover, P. sativum was found to accumulate higher concentrations of Cd, Cr, Ni, and Pb. Horler et al. [49] studied, in detail, the leaf pigments of P. sativum plants and showed that chlorophyll a and b decreased under the stress of Cd, Cu, Pb, or Zn. A similar result was obtained by Gubrelay et al. [50] in barley. Heavy metals may inhibit or promote the synthesis of some proteins [51] with a general trend of decline in the overall content. Our study reported that proteins and carbohydrates declined in the tissues of P. sativum cultivated in polluted soils. This result coincides with the study of Galal [6], who reported a decrease in soluble protein content under heavy metal stress in Cucurbita pepo. The reduction in protein content may have been caused by increasing protein degradation as a result of the increased activity of protease enzyme [52], which increases under stress conditions. Gubrelay et al. [50] reported a decrease in carbohydrate content in Cd-treated Oryza sativa and attributed this to chlorophyll biosynthesis inhibition. Ahmed et al. [53] reported that carbohydrates can be inhibited if the Cd concentration is more than 5 mg/kg in P. sativum tissues. In the present study, the cadmium concentration in the P. sativum tissues grown in the polluted soils exceeded 100 mg/kg.
The accumulation of metals in vegetables depends on many factors, such as the type of vegetable (leafy, tuber, or fruit), the concentration of metals in the soil, soil organic carbon, and pH. Soil pH plays a significant role in heavy metal investigations, where a low pH enhances the bioavailability of metals [54]. P. sativum cultivated in polluted soils, with a low pH value, accumulated high concentrations of heavy metals in our study. Nanda and Araham [55] reported that plants accumulating heavy metals exceeding 1000 mg/kg in their tissues were considered to be hyperaccumulators; therefore, P. sativum is considered a hyperaccumulator for Cd and Fe.
Vegetables absorb metals from contaminated soil and from the particulate matter deposited on their parts from the ambient air [1]. The BF of the studied heavy metals was greater than 1, and this may be a result of the high accumulation power of P. sativum in relation to these metals [6]. Similar results were reported by Shehata and Galal [9] in Egypt, and by Ali and Al-Qahtani [15] and Eid et al. [3] in Saudi Arabia, for some vegetable crops. Chen et al. [56] reported that legumes are more likely to accumulate Cr, while leafy vegetables accumulate higher concentrations of Cd and Pb. This is in agreement with our finding that Cd had the highest BF (442.9). In contrast, the TF of the studied heavy metals did not exceed unity in either the nonpolluted or the polluted sites. Therefore, these heavy metals are suitable elements for phytostabilization, which decreases the mobility of metals and their leaching into groundwater; and hence decreases the metal bioavailability and risk of entry into the food chain [9].
Although there are several pathways for human exposure to heavy metals, eating contaminated food is one of the major pathways [57]. The DIM for the assessed heavy metals (except Fe) in the polluted and nonpolluted sites was less than 1 in both children and adults, suggesting that the health risk of single heavy metal exposure through the food chain is generally low [57]. However, Horiguchi et al. [58] reported that the amount of ingested of heavy metals is unequal to the absorbed pollutant amount in reality, because the heavy metals ingested may be excreted, with the remainder accumulating in body tissues and affecting human health. According to US-EPA [37], an HRI of >1.0 is considered dangerous for human health. Washington et al. [59] showed that human exposure to Cd, Cu, Pb, Zn, and Cr through the food chain is at safe levels because the HRI for heavy metals in vegetables is <1.0. However, the present study indicated that the HRI of Pb, Cd, Fe, and Mn in the polluted sites and Pb in nonpolluted sites was >1.0 by many folds. This will threaten the health of the local population in Egypt, where P. sativum constitutes a high proportion of the diet. Cadmium is not essential for biological function in humans. The kidney is the main human organ impacted by cadmium exposure in both the general population and in those who are occupationally exposed, while copper is currently categorized by the US-EPA as a Group D carcinogen and can cause the destruction of red blood cells, possibly resulting in anemia [60]. Radwan and Salama [5] reported that Pb, Cd, Cu, and Zn concentrations in fruit and leafy vegetables in the Egyptian markets exceed the permissible limits of heavy metals in the food given by the WHO/FAO [40]. In addition, Hare [61] mentioned that "nonessential" elements such as Cd and Pb, even at low concentrations, are toxic for humans. Therefore, the consumption of P. sativum poses a high risk to human health, and the cultivation of such an important vegetable in farms exposed to pollution is not desirable.

Conclusions
According to the Environmental Quality Standards set by the US-EPA, the polluted and nonpolluted soils were in the normal range, and this may be due to the incessant removal of heavy metals by crops cultivated in these sites and the leaching of heavy metals into the soil's deeper layer. The values of all of the metals (except Cr, Cu, and Ni) were above the tolerable limits recommended by the World Health Organization. The BF of the studied heavy metals was greater than 1, while the TF did not exceed unity in either the nonpolluted or polluted sites. Therefore, these heavy metals are suitable elements for phytostabilization, which decreases the mobility of metals and their leaching into groundwater, and hence decreases the metal bioavailability and its risk of entering into the food chain. The DIM for the assessed heavy metals (except Fe) in the polluted and nonpolluted sites was less than 1 in both children and adults, suggesting that the health risk of single heavy metal exposure through the food chain is generally low. The present study indicated that the HRI of Pb, Cd, Fe, and Mn in the polluted site and Pb in nonpolluted site was >1.0 by many folds. This will threaten the health of the local and global populations, where P. sativum constitutes a high proportion of the diet worldwide.