Heavy Metals in Vegetables: Screening Health Risks of Irrigation with Wastewater in Peri-Urban Areas of Bhakkar, Pakistan

One of the key concerns in public health is food security in the food sector. Due to the large amounts of potentially hazardous metals in wastewater, this practice may pose serious environmental and health risks to neighboring residents. In this study, the health effects of heavy metals in vegetables irrigated with wastewater were studied. The findings indicated a massive accumulation of heavy metals in wastewater-irrigated soil and vegetables collected from Bhakkar, Pakistan. The current study looked at the effects of wastewater irrigation on metal buildup in the soil–plant continuum and the health hazards that come with it (Cd, Co, Ni, Mn, Pb, and Fe). Heavy metal concentrations in vegetables cultivated on soil irrigated with untreated wastewater were not significantly lower (p ≥ 0.05) than in vegetables grown on wastewater-irrigated soil and were below the World Health Organization’s recommended limits. A considerable amount of the selected hazardous metals was also swallowed by adults and children who consumed these vegetables, according to the research. On soil that had received wastewater irrigation, Ni and Mn were substantially different at p ≥ 0.001 levels. Pb, Ni, and Cd had health risk scores higher than the ones in all ingested vegetables, while Mn had a health risk score greater than the ones in turnips, carrots, and lettuce. The results also showed that both adults and children who consumed these vegetables absorbed a significant amount of the chosen toxic metals. Pb and Cd were shown to be the most dangerous chemical compounds to human health, and everyday consumption of agricultural plants irrigated with wastewater may pose a health risk, according to the health risk criteria.


Introduction
More than 200 illnesses, from cancer to diarrhea, are brought on by contaminated food that contains dangerous bacteria, viruses, parasites, or chemicals. Because of the estimated 600 million people who get unwell from consuming contaminated food and the 420,000 fatalities globally, 33 million disability-adjusted life years are lost each year. (DALYs). Children under the age of five bear 40% of the weight of food-borne illnesses, which results in 125,000 yearly fatalities. Diarrheal diseases, which yearly infect 550 million people and cause 230,000 deaths, are the most common illnesses caused by consuming contaminated food [1]. Macro and micronutrients are responsible for providing materials that are required for growth. Some heavy metals are classified as xenobiotics since they play no role in body nutrition and can potentially be detrimental in small doses. The presence of inappropriate absorption causes minor symptoms, but their high concentration still makes them hazardous [12]. Due to rising urbanization and industrialization, human metal inputs outweigh natural sources. Heavy metals are found in groundwater, surface water, and soils from a variety of sources, including industrial wastes, air deposition from congested cities, and various home wastes [13].
Heavy metals such as cadmium, chromium, and nickel may be present in soil as a result of parent materials during soil formation [14]. Soil serves as a foundation for all living things on the planet. The most significant aspect is that soil serves as a substrate for plant development, recycling nutrients, and additional resources. Heavy metals (HMs) in contaminated rivers and wastewater are absorbed by the soil, causing negative effects on vegetable development. Roots absorb wastewater and nutrients in a solution as they develop in the soil [15]. Heavy metals bind to soil aquatic and soil elements, which are absorbed by plant roots and stored in vegetables [16]. Some plants can absorb significant quantities of metals from the soil. One of them is leafy vegetables, where research revealed that a high degree of soil pollution constituted a possible threat to vegetables growing nearby [17]. These problems have compelled researchers to evaluate the accumulation of heavy metals in numerous fruits and vegetables, such as mangoes and mushrooms, which are significant agricultural goods found in enormous quantities in local East and Southeast Asia markets [18,19]. High levels of untreated wastewater are used in agricultural areas in the Punjab region of Pakistan's Bhakkar. Contamination of the land and crops, as well as health issues for the local people, pose the greatest concern. As a result, it is vital to analyze the potential health and environmental consequences of using untreated wastewater for crop irrigation in Pakistan. In Bhakkar District, as in other Pakistani cities, wastewater is routinely used for vegetable irrigation with no prior treatment. As a result, the current study's objectives were to evaluate (i) HM concentrations in municipal wastewater, (ii) potential HM accumulation in the soil-plant continuum, and (iii) potential health dangers to residents from consuming wastewater-irrigated food crops.

Zone Description
Bhakkar is a Punjab district well-known for its agricultural products. The present research was carried out in Pakistan's Punjab Province and Bhakkar District. Bhakkar City is also the administrative center of Bhakkar Tehsil, one of the four tehsils in the district. Within the Bhakkar Tehsil, there are three union councils that make up the city of Bhakkar [20]. Industrial study zones were selected and separated into three sampling zones, i.e., zone-DK (Darya Khan), zone-BK (Bhakkar), and zone-SM (Sarai Mahajir) as shown in Figure 1. The three zones were located nearly 10 km away from each other.

Sample Collection
The sampling locations were divided into three groups. A random sample was taken. Soil samples were collected at a 35 cm depth. Vegetable samples at various stages of development were gathered, and characteristics including pH, TDS, and electrical conduc-

Sample Collection
The sampling locations were divided into three groups. A random sample was taken. Soil samples were collected at a 35 cm depth. Vegetable samples at various stages of development were gathered, and characteristics including pH, TDS, and electrical conductivity (EC) were measured.

Water Sampling
Seventy-two water samples (twenty-four from each zone) were collected at once for the determination of heavy metal concentration and calculation of physical parameters. Highdensity polyethylene bottles and all glassware were cleansed in 3% nitric acid. De-ionized water was used to wash the objects first. Wastewater samples were collected in 250 mL plastic bottles from various sites and analyzed in a laboratory. Twenty-five milliliters of each sample were moved to a sterile 50 mL container. In a beaker, 5 mL of nitric acid was calculated. It was then processed at 90 • C until it became visible. Whatman no 42filter paper was used to strain the digestion contents into a measuring flask. Filtrate was created using deionized water and was kept at 5 • C. The pH (pH-7110), EC (HANNA edge EC), and TDS were measured. Heavy metal concentrations (Co, Fe, Mn, Pb, Cd, and Ni) were measured and calibrated using an atomic absorption spectrophotometer. For quality assurance, a non-polluted ionized water sample was utilized as a blank. The analyses of different samples were carried out three times using atomic absorption spectroscopy [21].

Soil Sampling
Three sub-zones within each zone were selected for soil sampling. At a depth of around 30 cm, plants from soil samples were used. Before being pulverized using a pestle, all soil samples were dried in the open air. The mesh size was raised to 1 mm to separate any unwanted pieces, and the samples were kept in plastic bags at 3 • C awaiting testing. The method also includes the preparation of tri-acid solutions: nitric acid at 5%, perchloric acid at 1%, and sulfuric acid at 1%. They were held in the digestion compartment until the evaporation ended. We sifted the digested samples after cooling them. The samples were then placed in a 50 mL measuring beaker and filled the rest of the way with deionized water. Samples were tested for parameters such as pH, EC, and organic matter (OM). Heavy metal concentrations (Co, Fe, Mn, Pb, Cd, and Ni) were measured and calibrated using an atomic absorption spectrophotometer.

Vegetable Sampling
A total of one hundred and eight vegetable samples from various families were collected in labeled polythene bags from the designated zones where soil samples were gathered. Fresh vegetables such as cauliflower and cabbage were collected at random from several Bhakkar localities. The samples were evaluated in a laboratory. After being cleaned with tap water, they were rinsed with water. The edible parts of the vegetables were cut into small pieces and dehydrated in the oven at room temperature. They were broken into well-defined particles with a porcelain mortar and pestle and kept in an airtight polythene bag. The ratio of solution 5:1:1, a tri-acidic solution, and a heated plate were also used to digest the powder of the vegetable samples. The digested samples were filtered using Whatman 42-filter paper into a 20 mL measuring flask, and the filtrate was warmed up with deionized water before being maintained at 5 • C and monitored with an atomic absorption spectrophotometer.

Analysis of Heavy Metals
A questionnaire was used to gather information on people's weight, family size, ages, vegetable consumption, and vegetable source to perform a nutritional survey and evaluate the risk of consuming wastewater-irrigated vegetables. A total of 150 healthy persons were chosen at random from the population of Punjab, Pakistan Bhakkar area. Three vegetable products were included in the dietary questionnaire, and each was measured in kilograms per person per day using the one-week recall technique. For several vegetables, data on intake frequency and amount were collected. Heavy metal concentrations were measured using an atomic absorption spectrophotometer.

Quality Control
The chemicals used in this experiment were of the highest quality. The water research facility and analytical laboratory at the Institute of Chemical Sciences at Gomal University in D.I. Khan, Pakistan, provided the deionized water for the solutions. All samples were evaluated in triplicate for quality assurance, and blanks and standards were run after each batch of samples.

Data Analysis
Data were evaluated by using Statistical package for the social sciences (SPSS) software.

Heavy Metal Transfer Factor
Heavy metal transmission and accumulation from soil to crops is a complicated process. The ratio of heavy metal concentration in flora to heavy metal content in soil was calculated using the formula below: HMTF = C veg /C soil Metals in vegetables are represented by C veg , whereas metals in soil are represented by C soil [21,22].

Risk Assessment
The hazard quotient (HQ) is a relationship between the computed dose and the reference dose that is used to quantify the risk of metal contamination in vegetables for human health (R f D). If the ratio is <1, the population is safe. However, if the figure surpasses or equals one, the population is in serious danger [23].
The HQ was calculated using the formula below: [W plant ] = dry weight (mg·dL −1 ) of ingested vegetables, dry weight (mg·dL −1 ) of consumed vegetables.

Daily Dietary Index (DDI)
The following formula was used to compute the daily dietary index: where X = vegetables with heavy metals, Y = vegetables' dry weight, Z = vegetable consumption daily, and B = the average weight of the users.

Daily Intake of Metals (DIM)
The DIM was calculated using the equation DIM = C metal × C factor × D food intake /B average weight where C metal = metal concentration in vegetables (mg·kg −1 ), C factor = conversion coefficient (0.085 for fresh vegetable weight to dry weight), D food intake = daily consumption of vegetables, and B = BMI (body mass index) [24].

Health Risk Index (HRI)
The HRI was computed using the DIM and a reference oral dose as follows: Health Risk Index = DIM = R f D: If the HRI value is <1, the population exposed is considered safe [25].

Physicochemical Parameters and Concentration Level of Heavy Metals in Wastewater
The physicochemical parameters of the wastewater samples collected randomly from the three zones are shown in Table 1. pH levels in the three zones (DK, BK, and SM) of wastewater varied from 3.20 to 5.09, 3.21 to 4.80, and 3.00 to 5.00, respectively. The WHO's acceptable pH limit for water is 6.5-8.5. Because waste-tainted water contains more CaCO 3 , NaCl, and Na 2 SO 4 than wastewater, the pH of wastewater was slightly higher than that of fresh water. The EC of wastewater ranged from 72.0 to 90.0 µS/cm, 65.0 to 87.0 µSc/m, and 75.0 to 97.0 µS/cm, respectively. The EC of wastewater was below the WHO's limits (1400 µS/cm total dissolved solids (TDS) in wastewater ranged from 43.0 to 97.0 (mg/L), 25.3 to 39 (mg/L) and 35.0 to 80.0 (mg/L), respectively). The TDS of wastewater-contaminated water were below the WHO's limit of 1000 mg/L. Therefore, fresh water's EC and TDS are acceptable for irrigation while waste-polluted water is not acceptable for irrigation. A study conducted in Kangal, Andhra Pradesh, found that the pH and EC values in the water and soil were identical [26]. In waste-contaminated water, heavy metals Cd, Co, Fe, Mn, Ni, and Pb had ranges of 0.47-0.87, 0.40-0.60, 0.61-0.95, 0.38-0.87, 0.03-0.85, 0.14-0. 19 .98-20.50, 0.20-0.50, and 6.78-9.26 (mg/L), respectively, as shown in Table 1. In wastewater, all heavy metals except Co were over the WHO's permitted levels. Heavy metals in wastewater from the three zones were in the following order: DK zone, Pb > Cd > Ni > Mn > Fe > Co; BK zone, Ni > Cd > Pb > Mn > Fe > Co; and in the SM zone, the order of Cd > Pb > Ni > Mn > Fe > Co differed from that of the fresh water order Fe > Mn > Co > Ni > Cd > Pb.
ANOVA analysis showed that Ni, Cd, Pb, Mn, Fe, and Co concentrations in the present study were not significantly different at p ≥ 0.05 levels in wastewater, as shown in Table 2. In waste-contaminated water, the concentrations of heavy metals Cr, Cd, Ni, and Mn ranged from 3.8 to 7.32, 1.80 to 18.20, 0.27 to 0.64, 0.21 to 1.29, and 0.64 to 4.88 mg·L −1 , whereas fresh water values ranged from 1.32 to 4.12, 0.27 to 1.67, 0.14 to 0.44, 0.05 to 0.20, and 0.12 to 0.72 mg·L −1 , respectively [27]. These heavy metal concentrations were substantially greater than the results of a previous study: Cd (0.09) mg·L −1 , Ni (0.06) mg·L −1 , and Pb (0.03) mg·L −1 in wastewater from Varanasi, India, were found at lower levels of concentration [28]. The concentrations of heavy metals Cd, Ni, Pb, and Cr were similarly lower than those reported in [29]. The wastewater from Peshawar's region (Bara River and Warsak Canal) is not suitable for irrigation due to a high concentration of heavy metals [30].

Physicochemical Analysis and Heavy Metal Content in Soil
The physicochemical parameters of wastewater-irrigated soil samples collected randomly from the three zones are as follows: The pH of wastewater-irrigated soil ranged from 4.90 to 5.09, 3.29 to 5.91, and 3.80 to 5.50; the wastewater-irrigated soil pH was within the range as required by the WHO. Because waste-polluted water contains a high amount of calcium, magnesium, and bicarbonates, the pH of wastewater-irrigated soil was slightly higher than that of fresh water. The EC of wastewater-irrigated soil ranged from 172.0 to 285.0 µS/cm, 180.0 to 250.0 µSc/m, and 175.0 to 189.0 µS/cm. The EC of wastewater was below the WHO's limits (1400 µS/cm). The EC of wastewater-irrigated soil was not acceptable for irrigation [31]. The amounts of organic matter (OM) in wastewater-irrigated soil were 1.00 to 1.20, 0.60 to 1.10, and 0.30 to 0.50, respectively. Khan   ANOVA analysis showed that Ni and Mn were significantly different at p ≥ 0.001, and Cd, Pb, Co, and Fe were non-significantly different at p ≥ 0.05 levels in wastewater-irrigated soil (Table 4). In this research, irrigation with heavy-metal-contaminated wastewater is primarily responsible for soil pollution. In some studies, except for Cd, which exceeded the permissible limit, in contrast to the current research work, the results obtained were in agreement with the present findings, where the minimum and maximum values for Pb, Cr, Cd, Ni, and Mn ranged from 18.33 to 66.78, 31.65 to 61.65, 7.13 to 11.13, 30.05 to 64.30, and 11.50 to 90.00 and were all within the safe limits of the current research work [27]. In another study, with the exception of Pb, which was above the WHO's limits, the results obtained, in contrast to the current results, showed that the experiential minimum and maximum values for Pb, Cr, Cd, Cu, Zn, and Ni ranged from 34

Heavy Metal Content of Vegetables
Heavy metals Co, Fe, Pb, Mn, Cd, and Ni in wastewater-irrigated vegetables from zone-DK ranged from 1.09 to 13.0, 3.0 to 150, 0.42 to 500, 6.89 to 85.0, 0.67 to 7.0, and 6.62 to 67.90 mg·kg −1 , and those from zone-BK wastewater-irrigated soil ranged from 1.09 to 14.89, 4.0 to 150.0, 0.49 to 500, 7.89 to 85.0, 0.71 to 7.90, and 7.98 to 67 mg·kg −1 . Heavy metals Co, Pb, Fe, Mn, Cd, and Ni in wastewater-irrigated vegetables from zone-SM ranged from 1.01 to 14.8, 4.0 to 150, 1.78 to 500, 8.90 to 85.0, 0.80 to 8.0, and 4.29 to 67.90 mg·kg −1 , respectively. Heavy metal elements Pb, Cd, and Ni were found to be above the WHO's permissible limit when comparing wastewater-irrigated crops to wastewater-irrigated vegetables, having the order of Cd > Pb > Fe > Co > Mn > Ni, Ni > Pb > Mn > Fe > Co, and Pb > Ni> Fe > Mn > Co > Cd, respectively. As shown in Figure 2, Pb, Ni, and Cd concentrations were higher in all nine vegetables Spinacia oleracea, Brassica oleracea var. capitata, Brassica oleracea var. botrytis Brassica rapa subsp. Rapa, Raphanus sativus, Colocasia esculenta, Benincasa fistulosa, Daucus carota subsp. sativus, Lactuca sativa, and Daucus carota subsp. sativus, Brassica rapa subsp. sativus, Brassica rapa subsp. sativus Spinacia oleracea, and Benincasa fistulosa, with Ni having greater concentrations than the other heavy metals. Cd levels were also greater in crops watered with wastewater. In the current research, it was discovered that all crops irrigated with wastewater contained higher levels of the metals under investigation than vegetables irrigated with tube well water. All crops irrigated with wastewater contained heavy metals such as Cd, Pb, and Ni in excess of the WHO's permitted limit, showing that after the crops were irrigated with wastewater, a buildup in the vegetables took place. Plants cultivated on wastewater-irrigated soils contained heavy metals in excess of the WHO's allowed levels, suggesting a serious health risk to consumers, according to a prior study [34].

Heavy Metal Transfer Factor
To investigate the human HRI associated with vegetables grown on wastewaterirrigated soil, it is essential to analyze the heavy metal transfer factor. Pb, Cd, Co, Ni,  Table 5. The HMTF trend for heavy metals in all three zones in various vegetables was Cd > Ni > Co > Zn > Cr > Pb > Mn > Fe. Cd, Co, Ni, and Pb had a greater HMTF than the other metals in all wastewater-irrigated crops. The high absorption ratio of heavy metals in wastewater-treated vegetables is attributable to the direct absorption of heavy metals from wastewater, according to the ANOVA study. The current study found higher HMTF values for all metals except Zn and Cr, ranging from 0.04 to 0.11 (Pb), 0.12 to 0.29 (Cr), 0.51 to 1.47 (Cd), 0.32 to 0.51 (Co), 0.36 to 0.57 (Ni), and 0.21 to 0.41 (Zn) mg·kg −1 , compared to those reported by Khan et al. [35], which could be due to differences in soil with increasing total metal concentrations in soils. Vegetables have also been shown to have an inverse association.

Heavy Metal Transfer Factor
To investigate the human HRI associated with vegetables grown on wastewater-irrigated soil, it is essential to analyze the heavy metal transfer factor. Pb, Cd, Co, Ni, Fe, and Mn concentrations in zone-DK wastewater varied from 0.016 to 0.085, 0.039 to 0.183, 0.058 to 0.532, 0.696 to 3.478, 0.379 to 0.859, 0.294 to 0.656, and 0.247 to 0.924. Water from zone-BK contained varying amounts of Pb, Cr, Cd, Co, Zn, Ni, Fe, and Mn, with values ranging
In nations such as Pakistan, where irrigated wastewater consumption is unregulated, assessing health risks via the food chain is critical. Human contact with heavy metals through food is one of the most common routes, along with air, water, and soil [37]. Similarly, the results achieved for Pb and Cd had HRI values greater than 1, implying that these metals are dangerous to human health even at extremely low doses [27]. The current study's findings for Pb, Cd, and Ni, which are the most dangerous to human health, were consistent with previous findings [36].

Heavy Metal Correlation Analysis
In wastewater, the physical parameters EC and TDS showed a strong relation and correlation (r = 1). There was a significant correlation at p ≤ 0.05 between Co, Cd, Fe (r = 0.141), Mn (r = 0.425), and Pb (r = 0.249). A high correlation at p ≤ 0.05 between Co, Fe, Mn (r = 0.516), Pb (r = 0.631), Cd, and Ni was found in wastewater-irrigated soil in [38].

Conclusions
The measured wastewater parameters indicated a wide range of fluctuation. Pb, Cd, Co, Ni, Fe, and Mn amounts in wastewater, as well as Pb, Cd, and Ni concentrations in vegetables, were found to be above the WHO's acceptable limit. In the three tested zones, the transfer factor for four heavy elements, namely Cd, Co, Ni, and Zn, was higher. The observed parameters for wastewater showed a significant range of variation. The EC, TDS, and heavy metals Pb, Cd, Co, Ni, Fe, and Mn in wastewater and Pb, Cd, Co, Fe, Mn, and Ni concentrations in vegetables were below the WHO's permissible limit. The DIM value for adults and children consuming vegetables from the three zones (DK, BK, SM) for three heavy metals (Pb, Mn, Ni) was above the tolerable daily intake rate. The HRI of Pb and Cd was >1 for all the studied vegetables, and the HRI for Ni was >1 in three vegetables, viz. S. oleracea, B. fistulosa, and L. sativa. The studies provide a detailed insight into the present scenario of vegetable contamination and human health risk estimations. To minimize heavy metal buildup in vegetables and, eventually, lower the chronic health risk to the population that consumes vegetables, it is urgently necessary to rigorously monitor the wastewater irrigation system in the research region.

Conflicts of Interest:
The authors declare no conflict of interest.