Trace Metals in Vegetables and Associated Health Risks in Industrial Areas of Savar, Bangladesh

Background. The occurrence of high levels of trace metals in foodstuffs represents a significant threat to human health. Vegetables grown in metal-contaminated soil or irrigated with wastewater can accumulate metals and bioaccumulate in the food chain affecting animals and humans. Objectives. The present study aimed to measure the levels of lead (Pb), cadmium (Cd), chromium (Cr) and cobalt (Co) in common vegetables grown in the industrial areas of Savar, Bangladesh, and to determine their potential health risks. Methods. Five vegetables species: jute (Corchorus capsularis), red amaranth (Amaranthus gangeticus), okra (Abelmoschus esculentus), zucchini (Luffa aegyptiaca) and stem amaranth (Amaranthus viridis) were sampled randomly from agricultural fields across each study site. Vegetable samples were digested in a microwave digestion system (Berghof Microwave MWS-2, Germany). Metal concentrations were determined using an atomic absorption spectrophotometer (AA-7000, Shimadzu Corporation, Japan). Results. The range of Pb, Cd, Cr and Co in analyzed vegetables was 0.643–3.362, 0.041–0.049, 1.681–2.431 and 1.612–2.492 mg/kg, respectively. The target hazard quotient (THQ) of Pb in zucchini and stem amaranth and the THQs of Cr in all analyzed vegetables was greater than one. The target carcinogenic risk (TCR) of Pb and Cd for all analyzed vegetables was in the unacceptable range. In all vegetable samples, lead content was detected to be higher than the maximum permissible limits. The THQ values indicate the possibility of non-carcinogenic health risk through consumption of these vegetables. In addition, the TCR values of Pb and Cd indicate a lifetime carcinogenic health risks to consumers. Conclusions. Consumption of vegetables grown in this area may pose long-term health risks. Competing Interests. The authors declare no competing financial interests.


Introduction
Trace metals are widely distributed in the environment. In addition to their vital role in plant growth and/or human nutrition, some metals, such as arsenic (As), lead (Pb), cadmium (Cd), cobalt (Co), nickel (Ni), chromium (Cr), mercury (Hg) etc., cause adverse effects to human health even at low concentrations. 1 The occurrence of metals in food can have geogenic or anthropogenic origins. 2 Evidence suggests that anthropogenic activities such as wastewater irrigation, solid waste disposal, mining, smelting, sewage sludge applications, fertilizers, fungicides and industrial activities contribute significantly to the deposition of these metals in cultivable land. [3][4][5] Crops and vegetables grown in contaminated soil or irrigated with wastewater could take up metals and accumulate them in their edible parts. 6 Human can be exposed to metals through various pathways. One major exposure pathway is through consumption/diet. 4 Vegetables constitute an important part of the human diet. Therefore, it is reasonable to hypothesize that vegetables contaminated with metals pose a potential health risk to consumers, in addition to their ability to fulfill nutritional requirements.
Some metals like copper (Cu), zinc (Zn), selenium (Se), etc. are essential for maintaining the physiological functions and biochemical processes in humans. 7 However, metals like As, Pb, Cd, Co, Cr, Hg are considered potentially toxic because these metals cause adverse effects to human health when ingested over a long period. 8,9 Moreover, metals like As, Pb and Cd have shown carcinogenic effects. 10 Previous studies have reported the concentration of metals in different foodstuffs. 11- 15 Vegetables can uptake metals from polluted soil and water, which depends largely on soil composition, water quality, metal Research solubility, and absorption ability. 6, 11 The exceedingly higher concentrations of metals in vegetables pose serious health risks to humans through the food chain. 1, 16 Over the past decades, Bangladesh has undergone structural transformation from agriculture to industrialization. Studies show that vegetables growing near industrial zones display higher concentration of metals. 11, 16 Savar upazila is an important industrial zone in Bangladesh. Most of its industries are located at the bank of the Turag River. Several studies have reported on concentrations of metals in this river's water. 17,18 Moreover, the area is exposed to different degrees of industrial pollution, especially from tannery waste and textile dyeing. The vegetables growing in this area may become contaminated by metals through water irrigation or soil absorption. Previous studies revealed metals like As, Cd, Pb, Hg, Cr, manganese (Mn), Ni, Cu, and Zn are significant vegetable contaminants in Bangladesh. 11,12, 19 However, the potential health risk associated with the consumption of vegetables in the study area of Bangladesh has not been fully investigated. Therefore, the present study aimed to measure the levels of Pb, Cd, Cr and Co in commonly consumed vegetables grown in industrial areas of Savar upazila, Bangladesh, and to determine associated potential health risks. In the present study, health risks from exposure to Pb, Cd, Cr and Co through the consumption of vegetables were evaluated using different indices, including estimated daily intake of metals (EDI), target hazard quotient (THQ), hazard index (HI) and target carcinogenic risk (TCR). The THQ and HI evaluate potential noncarcinogenic risk, while TCR evaluates carcinogenic risk that can occur due to consumption of these vegetables.

Methods
The samples were collected from Fulbaria, Katlapur, and the Dhaka Export Processing Zone of Savar upazila (Figure 1). The area of Savar upazila is 280.13 km 2 and located at 23.8583° N 90.2667° E. It is situated about 25 km to the northwest of Dhaka city (the capital city of Bangladesh). Savar is an important industrial region of Bangladesh and is highly vulnerable to environmental pollution. The area is home to a variety of industries such as garments, tanneries, pharmaceuticals, packaging, dyeing, battery manufacturing, textile, and different food processing industries which produce large volumes of effluents that may contain heavy metals. After production, these industries discharge untreated wastes randomly into nearby rivers and canals. The disposed wastes are then mixed with soils, which are continuously being polluted by toxic elements in the area.

Sample collection and preparation
Five commonly consumed and seasonally available vegetables species, i.e. jute (Corchorus capsularis), red amaranth (Amaranthus gangeticus), okra (Abelmoschus esculentus), zucchini (Luffa aegyptiaca) and stem amaranth (Amaranthus viridis) were collected randomly from agricultural fields across each site between May and August in 2019 ( Table 1). The samples were collected by hand using vinyl gloves, carefully packed into polyethylene bags, and then brought to the laboratory for analysis. The analysis was done at the Wazed Miah Science Research Center, Jahangirnagar University. Only the edible parts of the collected vegetable samples were used. The samples were washed with distilled water to remove foreign materials. The samples were partially dried at room temperature to remove moisture and then cut into small pieces with a knife of stainless steel. The samples were then ovendried at 80°C for 72 hours. The oven dried samples were powdered using a pestle and mortar and sieved through a mesh of 2 mm.

Metal analysis
Analytical reagent grade chemicals and deionized water were used to prepare all solutions. A total of 0.5 g of each powdered sample was treated with 65% nitric acid and 30% hydrochloric acid in the volume ratio of 5:1 in a closed Teflon vessel and then digested in a microwave digestion system (Berghof Microwave MWS-2, Germany). Samples were cooled at room temperature and filtered using Whatman filter paper. The final volume was made up to 50 ml using double distilled water. Metal concentrations were determined using an atomic absorption spectrophotometer (AA-7000, Shimadzu Corporation, Japan). Standard solution (1000 mg/l) of different metals viz. Pb, Cd, Cr and Co were procured from Agilent Technologies. A standard curve was prepared using various concentrations made from standard solution. Digested samples were then analyzed for the metal content. All test batches were evaluated using an internal quality approach and validated if they satisfied the defined internal quality controls. Blank samples were scrutinized after every three samples for the purpose of ensuring that obtained results were within the correct range. The levels of metals were calculated based on dry weight and all the examinations were replicated in triplicate.

Estimated daily intake of metals
The EDI of metals via consumption of foods depends on the metal concentrations in foods, daily food consumption, as well as body weight. In the present study, the EDI of the metals of interest (Pb, Cd, Cr and Co) were evaluated with Equation 1. 20

EDI = ((FIR × C)/BW) × 10 -3
In Equation 1, FIR is the daily vegetable consumption rate (g/ person/day), C is the estimated metal concentration in vegetable samples (mg/kg fresh weight (fw)), BW is the average body weight and 10 -3 is the conversion factor (Table 2).

Target hazard quotient
The THQ can be defined as the ratio of exposure to a toxic substance and the level at which no adverse health effects are expected. A THQ value less than one implies no non-carcinogenic health risk from lifetime exposure to the substance, while if the THQ is higher than one, the toxicant may produce an adverse non-carcinogenic effect. The equation used for estimating the THQ is as follows. 20

THQ = ((EF × ED × FIR × C)/ (RFD × BW × AT)) × 10 -3
In Equation 2, EF is the exposure frequency (365 days/year), ED is the exposure duration, FIR is the daily vegetable consumption rate (g/ person/day), C is the estimated metal concentration in vegetable samples (mg/kg fw), RFD is the oral reference dose (mg/kg/day), BW is the average body weight, AT is the average exposure time for non-carcinogenic effects and 10 -3 is the conversion factor. The main exposure factors that have been taken into account to carry out the risk assessment calculations are listed in Table 2.

Hazard index
The HI is the sum of the THQs of individual metals in each food item. The HI assumes that exposure to multiple metals results in additive effects. Even if the individual THQs for the metals in the food item are lower than one individually, adverse health effects may be posed by the cumulative effect of consumption. A hazard index greater than one indicates significant non-carcinogenic risks. 25 The following equation was used for HI evaluation. 20

Target carcinogenic risk
The TCR is the possibility of an individual lifetime health risk of developing cancer upon exposure to carcinogens. A TCR value between 10 -6 and 10 -4 indicates an increased risk of developing cancer. A value of 10 -4 is the upper limit of the range and a value above this range indicates lifetime cancer risk to populations who consumed contaminated foods. 26 Lead and Cd are considered carcinogenic elements. 10 The carcinogenic potential of these metals was calculated using the following equation: 20

TCR = ((EF × ED × FIR × C × CSF)/ (BW × AT)) × 10 -3
In Equation 4, EF is the exposure frequency (365 days/year), ED is the exposure duration, FIR is the daily vegetable consumption rate (g/person/day), C is the estimated metal concentration in vegetable samples (mg/kg fw), CSF is the oral carcinogenic slope factor (mg/kg/ day), BW is the average body weight, AT is the average exposure time for non-carcinogenic effects and 10 -3 is the conversion factor. The oral carcinogenic slope factors for Pb and Cd are presented in Table 2.

Data analysis
All the calculations were performed using Microsoft Office Excel 2019. Descriptive statistics such as mean, SD, and ranges for the target parameters were calculated. Tables were used to describe the calculations.
All results are reported as fresh weight (fw). Of these analyzed vegetable species, okra had the lowest mean concentration of Pb at 0.643 mg/kg and zucchini had the highest mean concentration of Pb at 3.362 mg/kg. The lowest Cd content was 0.041 mg/ kg detected in zucchini, while the

Non-carcinogenic risk
The THQs for non-carcinogenic risk of the tested metals from consumption of vegetables for children and adults are presented in  Figure 2. In the present study, the HI for all analyzed vegetable samples was greater than one.

Carcinogenic risk
The carcinogenic risks of Pb and Cd were evaluated based on the TCR from consumption of vegetables and are presented in Table 6. In this study, the TCR values of Pb ranged from 1.8E-05 in okra to 9.3E-05 in zucchini and 5.1E-04 in zucchini to 5.9E-04 in okra for Cd for children (Table 6). For adults, the TCR values of Pb ranged from 1.2E-05 in okra to 6.1E-05 in zucchini and 3.4E-04 in zucchini to 3.9E-04 in okra for Cd.

Discussion
The present study aimed to measure the levels of Pb, Cd, Cr and Co in commonly consumed vegetables grown in the industrial areas of Savar, Bangladesh, and to determine their potential health risks. The average levels of metals occurred in the following declining order in all vegetable samples: Co > Cr > Pb > Cd (Table 3). A great variety of metal concentrations were observed among different vegetable species. The accumulation of these metals were attributed to the differential absorption capacities of vegetables for different metals. 6,13 Lead, a highly toxic metal, is absorbed in inorganic forms through ingestion of food and water and inhalation. Lead is known to induce kidney damage, increase blood pressure and cardiovascular disease risk for adults and hinder cognitive development in children. 30 In the present study, Chromium is an essential trace mineral that humans need in very small quantities. Chronic exposure to high levels of Cr leads to the development of gastrointestinal and central nervous system cancers. 35 The results of this study showed that stem amaranth samples had the lowest mean concentration of Cr at 1.681 mg/kg and red amaranth samples had the highest mean concentration of Cr

Figure 2 -Hazard index for metals in analyzed vegetables
Research at 2.431 mg/kg. Chromium contents in all studied vegetables (except red amaranth) were found to be below the standard limit (2.3 mg/kg). 27 In the literature, Cr concentrations of 1.171-3.835 mg/kg have been reported in vegetables in Savar areas, and 2.28-11.84 mg/kg in vegetables of the Dhaka Export Processing Zone areas (also in Savar). 11, 31 Previous Bangladeshi studies found Cr concentrations in vegetables of 2.10 mg/kg to 33.16 mg/ kg and 0.30 mg/kg to 1.11 mg/kg. 12, 19 The occurrence of Cr in vegetables may be attributed to soil uptake. The main sources of Cr in soils are the application of chemical fertilizers and pesticides, wastewater irrigation and open dumping of industrial wastes in the environment. 36 Cobalt is another essential trace mineral required by humans for proper functioning of bodily systems. However, when ingested beyond permissible limits over a long period of time, Co can lead to human neurological, cardiovascular and endocrine deficits. 37 In the present study, the Co content was found to be the lowest in zucchini (1.612 mg/kg) and highest in red amaranth (2.492 mg/kg). Mean Co concentrations in all the vegetable samples were below the maximum permissible limit (50 mg/ kg). 27 The EDIs of Pb, Cd and Cr from individual vegetable species were below the maximum tolerable daily intakes recommended by international regulatory bodies; indicating that these vegetable species may not pose health risks to consumers. However, combined metal EDIs might pose a risk to consumers.
In the present study, the noncarcinogenic risk from consuming vegetables in the study area was evaluated for adults and children. The THQ for non-carcinogenic risk of Pb in zucchini and stem amaranth, and the THQs of Cr in all analyzed vegetables were greater than one, indicating a non-carcinogenic health risk through consumption of vegetables. These findings indicate that Pb and Cr were the major components contributing to the potential health risk via consumption of the analyzed vegetables. Therefore, consumers are at high risk with respect to Cr which can pose non-carcinogenic risks to adults and children. The HI assesses multiple effects of metals. The potential risk could be multiplied when considering all the metals together. In the present study, the HI for all analyzed vegetable samples was greater than one, indicating potential non-carcinogenic health risks from consuming analyzed vegetables for both children and adults. Consequently, consumption of these vegetables is considered unsafe and their consumption on regular basis is not recommended. Therefore, consumers (both children and adults) are at high risk with respect to these metals which can cause noncarcinogenic effects.
The present study also evaluated the carcinogenic risk from Pb and Cd for adults and children from consuming vegetables, as Pb and Cd show carcinogenic effects. 10 The TCRs for carcinogenic risk of Pb for all analyzed vegetables were in the unacceptable range (