**1. Introduction**

A 1–3 ◦C increase in global warming is predicted by the 2050s [1]; the warming in the South Asian region is expected to be higher (2.2–3.3 ◦C) [2,3], with surface warming as high as 4.2 ◦C predicted to be in the northern regions of Pakistan [4] under the RCP8.5 emission scenario. Subsequently, enhanced glacier melts [5,6], soil surface drying, and water table lowering [7] are broadly expected, which may result in an acute shortage of surface- and/or groundwater supply for irrigating crops. Therefore, Pakistan, whose economy is ~21% agriculture driven, would be one of the regions most severely affected by climate change [6]. Some of these impacts are already being observed. That is why some of the suburban crops are irrigated using dilutes of city wastewater [8,9]. Likewise, approximately 20 million hectares of vegetable or cereal crops grown in a total of 50 countries are also being supplied with substandard waters, including ~80% untreated or partially treated wastewater of household or industrial nature [10,11] to cope with the issue of food security [12,13]. However, given the use of untreated wastewater for growing vegetables or cereal crops for human or animal consumption, human and soil health are at risk.

Wastewaters potentially contain a large variety of pollutants [9], including, but not limited to, unknown chemicals (organic, inorganic or biological nature) and/or salts, metals

**Citation:** Ahmad, I.; Malik, S.A.; Saeed, S.; Rehman, A.-u.; Munir, T.M. Phytoextraction of Heavy Metals by Various Vegetable Crops Cultivated on Different Textured Soils Irrigated with City Wastewater. *Soil Syst.* **2021**, *5*, 35. https://doi.org/10.3390/ soilsystems5020035

Academic Editors: Matteo Spagnuolo, Paola Adamo and Giovanni Garau

Received: 24 May 2021 Accepted: 17 June 2021 Published: 18 June 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and metalloids, pathogens and hosts, residual drugs and pesticides, endocrine-disrupting chemicals, or active ingredients of human care products. On one hand, these pollutants can impair soil and environmental health [14]; on the other hand, they can be taken up by the growing field crops resulting in buildups of toxic levels of heavy metals in the vegetable biomass. The toxic crops (especially vegetables) when consumed by humans may put their health at risk. Therefore, urban and farming communities' environments and human and animal health are at risk, which indirectly poses an even greater risk of global food security.

A heavy metal is defined as a chemical element of 500% higher specific gravity than that of normal water [15]. Continuous use of heavy metals containing wastewater for vegetable crop irrigation results in heavy metal accumulation in soil [16] and subsequent transfer to vegetable plants above the safe limits [17,18]. The plant accumulation concentration divided by soil accumulation concentration is called the vegetable transfer factor (VTF) [19], which shows the vegetable accumulation rate concerning soil accumulation concentration [20]. The soil–plant transfer of heavy metals is largely dependent on the plant species and is evaluated using the soil–plant TF [21]. The transfer factor is further controlled by several factors: plant age and species, crop variety, heavy metal concentration and its physical and chemical properties, and duration of effect [22].

Cd toxicity, even at low levels, has been attributed to its bioaccumulation and long half-life of ~30 years [8,23,24]. Cd also is known for its high mobility across the soil–water– plant–environment continuum [25]. A few major toxic plant effects include, but are not limited to, leaf chlorosis, stunted growth, and limited uptake of essential nutrients and protein synthesis [24,26]. Cr toxicity also reduces crop yield via impaired leaf and root hair growth, reduced enzymatic dynamics, and mutagenesis [27]. Toxic soil–water—plant concentrations are reported to impair overall plant growth and reproduction [28]. Excessive amounts of soil Zn together with soil Cu may decrease overall plant growth but increase TF, and ingestion of these higher TF vegetables result in acute depression symptoms in humans [29]. Excess levels of Cu alone in human blood showed acute stomachache and subsequent liver damage in many patients [30].

While the accumulation of heavy metals by wastewater-irrigated crops has been studied over several soil types, these investigations were made using one crop and one soil type at a time. Thus, various crops have not been compared for their comparative VTFs using the same or different soil types. Phytoremediation of the contaminated soils irrigated with wastewaters is an environmentally friendly and green technique [31,32] to remediate the soil–water–air continuum and quantify the translocation of these heavy metals by calculating the VTF.

Thus, an evaluation of the comparative phytoextraction efficiencies of various crops grown on the same and different soil types and irrigated with several wastewater types, or comparison of the VTFs, remained largely unexplored in Pakistan. Therefore, our unique study aims at identifying and assessing the potential sources of contamination to the soil, water, or plant, and evaluating the comparative VTFs of various crops grown on the same and different textured soils irrigated with a variety of wastewaters. The conclusions of this study will be helpful to sugges<sup>t</sup> the necessary mitigation measures and inform policy development.

We hypothesize that the comparative VTF evaluation of various crops grown on the same or different textured soils irrigated with a variety of wastewaters will differ. Therefore, the present study was carried out to quantify the heavy metal (Zn, Cu, Mn, Fe, Cd, Cr, Ni, and Pb) accumulation in four vegetables (spinach, brinjal, lettuce, and cauliflower) grown on the same and different soil types irrigated with different wastewaters, and to evaluate the suitability of the wastewater used for growing these vegetables.

The specific objectives of the present study are (1) to quantify the contents of heavy metals in two different soil types and their irrigation wastewater samples, both collected from six different study sites of the Multan suburban area; and (2) to monitor the comparative accumulation of heavy metals between the edible portions of the vegetables grown on the same and different textured soils irrigated with wastewaters, by quantifying

the vegetable transfer factor. The VTF will also be related to the soil concentrations of heavy metals.

#### **2. Materials and Methods**

*2.1. Study Sites*

To determine the vegetable transfer factors (VTF) in the effluent-irrigated vegetable crops, a total of six study sites were chosen in the vicinity of the WASA disposal stations within the suburban area of Multan city. The sites had several open and covered drainage channels that fed the vegetable crops. Each site was around one acre in size. We divided the sites (6) into two major soil texture types: sandy loam (3) and clay loam (3). Each of the texture types was irrigated with three types of water: normal, waste (sewage), and normal + waste. The brinjal (*Solanum melogena* L.) and spinach (*Spinacia oleacea* L.) crops were sown during January 2016 on a quarter of each site, randomly, while the cauliflower (*Brassica oleracea* L.) and lettuce (*Lactuca sativa* L.) were also sown on the rest of the quarters of each site during September 2015. Details of the study sites, their textural classes, the irrigation water types, and the heavy metal concentrations of soil and irrigation waters are shown in Table 1.

**Table 1.** Chemistry (EC and pH) and concentrations of the heavy metals in the study soils and their irrigation wastewaters, sampled during 2015–2016 from six major vegetable production areas in the Multan region of Pakistan †.


† Each value is a mean of four sample months. \* Permissible limits for liquid municipal and industrial effluents in Pakistan. \*\* Threshold levels of trace elements in irrigation water. ECs and pHs, and ECiw and pHiw denote electrical conductivity and pH of the soil and irrigation water, respectively. After four months, the study sites (soils) were significantly different in chemistry and heavy metal concentrations (ECs: *p* = 0.001; pHs: *p* < 0.001; Zn: 0.036; Cu: *p* = 0.002; Fe: *p* < 0.001; Mn: *p* < 0.001; Cd: *p* = 0.006; Cr: *p* = 0.003; Ni: *p* = 0.005; Pb: *p* < 0.001). The variable buildup of concentrations that was observed may be due to sewage water irrigation during the experimental period. All respective irrigation waters were also significantly different except for Cr (ECs: *p* = 0.019; pHs: *p* < 0.001; Zn: 0.031; Cu: *p* = 0.005; Fe: *p* = 0.012; Mn: *p* = 0.003; Cd: *p* = 0.004; Cr: *p* = 0.058; Ni: *p* = 0.002; Pb: *p* = 0.013).

## *2.2. Sampling and Analysis*

A total of 4 composite surface soil (0–20 cm) samples were randomly collected monthly from each of the six sites receiving wastewater regularly for irrigation, (4 samples \* 6 sites \* 4 months = 96 samples). However, the soil samples from the brinjal and spinach crop sites were collected during January–April 2015, compared to the cauliflower and lettuce crop site soil samples collected during September–December 2016. The soil samples were

air-dried, crushed and sieved to <2 mm, and stored at room temperature before analyses of the physicochemical properties and heavy metal concentrations. Soil samples were analyzed for textural class, saturation paste electrical conductivity (ECs), and saturation paste pH (pHs) following methods described by the US Salinity Laboratory Staff following Richards [33]. Textural class of only the first batch (month) of soil samples was analyzed. To quantify the water-soluble soil Zn, Cu, Fe, Mn, Cd, Cr, Ni, and Pb concentrations, 10 g of dry soil was extracted with 50 mL deionized water following Zia et al. [34].

Irrigation wastewater samples were also collected monthly during the soil sampling campaigns. Four replicate polyethylene bottles (acid washed) of 500 mL each were filled with wastewater one by one at an interval of 10 s from an open channel flowing to the study site, for all sites. Each of the collected wastewater samples was acidified immediately with 1 mL of concentrated HCl to avoid microbial degradation of the heavy metals. The samples were placed in a cooler and transported to a soil- and water-testing laboratory in Multan. Within a week, 50 mL of the sample was digested with 10 mL of concentrated HNO3 at 80 ◦C until the solution turned clear [35]. The clear solution was then filtered through a Whatman ™ 42 filter, diluted back to 50 mL using distilled water, and stored for analysis.

Edible parts of the harvested vegetables were thoroughly washed sequentially with 1% HCl and deionized water (to clean/remove any dust material), air-dried in shade for 24 h, and then oven-dried at 70 ◦C until a constant weight. The dried matter was ground to a powder form and then sieved to <1 mm. One gram of the powder was digested with a mixture of HNO3 and HClO4 in a 2:1 ratio, respectively. The clear digest was filtered and diluted to 50 mL using deionized water and stored for analysis.

Plant total and soil and wastewater soluble Zn, Cu, Fe, Mn, Cd, Cr, Ni, and Pb concentrations were measured from the stored extracts using an Atomic Absorption Spectrophotometer (Model AAS Vario 6, Analytik Jena AG, Jena, Germany).

Transfer factors of the vegetables were calculated by dividing the vegetable total heavy metal concentration with the soil water-soluble heavy metal concentration [20], to interpret comparative bioaccumulation of heavy metals by the experimental vegetables grown on the same or different textures soils irrigated with various wastewater sources in Multan.
