The Effects of Irrigation with Diverse Wastewater Sources on Heavy Metal Accumulation in Kinnow and Grapefruit Samples and Health Risks from Consumption

: This study aimed to assess the concentrations of Pb, Cr, Cd, and Ni in the edible parts of grapefruit and kinnow fruit irrigated with sewage water, tube-well water, and canal water. Preparation of the samples used in the study for metal analysis was carried out by the wet acid digestion method. Atomic absorption spectrometry was used for metal determination. All of the studied metals were more abundant in water, soil, and fruit samples from sites irrigated with sewage water than from those irrigated with tube-well and canal water. The concentrations of Pb were established in the range of 0.047 to 0.175 mg/L in water, 12.652 to 47.863 mg/kg in soil, and 0.023 to 0.124 mg/kg in fruit samples of both varieties. The range of Cr varied from 0.107 to 0.741 mg/L in water, from 1.121 to 5.835 mg/kg in soil, and from 0.054 to 0.784 mg/kg in fruit samples of both varieties. The range of Cd varied from 0.004 to 0.028 mg/L in water, from 1.258 to 4.246 mg/kg in soil, and from 0.012 to 0.131 mg/kg in fruit samples of both varieties. The range of Ni varied from 0.384 to 1.815 mg/L in water, from 5.367 to 18.658 mg/kg in soil, and from 0.028 to 1.475 mg/kg in fruit samples of both varieties. The pollution load index indicated that Pb exceeded a value of 1, while Cd and Ni exceeded 1 only in sewage-water-irrigated sites. The bioconcentration factor, enrichment factor, daily intake of metal, and health risk index values for all metals were below 1, within permissible limits, indicating no immediate health risks associated with consuming these fruits.


Introduction
Citrus is a genus of flowering trees and shrubs belonging to the family Rutaceae, native to subtropical and tropical regions of Asia, Australia, and the Americas [1].Citrus species are known for their diverse range of fruits, including oranges, lemons, limes, grapefruits, and tangerines.The kinnow and grapefruit are two of the most popular citrus fruit species that are widely cultivated and consumed worldwide.Kinnow is a hybrid cultivar that was made with a cross between Citrus nobilis and Citrus deliciosa [2].It was first produced by a researcher named H.B. Frost working at the Citrus Research Centre, USA [3].Kinnow, a readily available and inexpensive crop, contains significant amounts of antioxidant, anticancer, and antimicrobial compounds in both its juice and its peel [4].Grapefruit is a subtropical citrus cultivar that is renowned for its taste and nutritional value, and it contains bioactive compounds with antioxidant, anticancer, and nutraceutical properties, including vitamin A and lycopene [5].
Irrigation water quality is very important for plant growth, as it can affect soil properties and, ultimately, impact mineral uptake in the plants [6].In water-stressed countries like Pakistan, where a significant portion of the population is dependent on agriculture Water 2023, 15, 3480 2 of 18 for their livelihood, the use of wastewater (e.g., sewage water, polluted canal water, or industrial wastewater) for irrigation is becoming increasingly common due to the depletion of freshwater supplies [7].However, wastewater can contain increased levels of salts and organic compounds that can contribute to increased electrical conductivity in the soil and promote crop productivity [8,9].Studies have shown that the use of reclaimed wastewater can lead to increased productivity and improved physiology in citrus crops [10].
The flood irrigation method, commonly used in citrus orchards, is sub-economical due to water loss and the potential introduction of disease to healthy trees [11].Citrus does not use a lot of water in its production, but still, proper irrigation is necessary for high-quality fruit.Grapefruit also requires adequate water for its normal growth, as does mandarin, with an average water requirement ranging from 900 to 1200 mm depending on environmental conditions and soil characteristics [12].When using wastewater for the irrigation of citrus fruit trees, strict monitoring of heavy metal and salinity levels is necessary [13].Mixing wastewater with fresh water can be a practical approach to mitigate the risks of high boron concentration and salinity in the irrigation of citrus fields [14].
Heavy metals (HMs) pose serious health risks to humans and affect food chains.Some HMs, like Zn, Cu, Fe, Ni, and Mn, are beneficial micronutrients in lower quantities, but their toxicity causes harm to several important metabolic processes and reduces the antioxidant qualities of fruits at higher levels [15][16][17].Heavy metals are much more abundant in wastewater than in groundwater, and the use of wastewater for irrigation results in an increase in HMs in soil, plants, and animals [18][19][20].Reactive oxygen species generated in response to heavy metals can harm plant growth and cause metal toxicity, which can interfere with reproductive growth [21].The buildup of heavy metals in the liver and kidneys can lead to serious illnesses, including kidney problems, nervous disorders, bone diseases, and cardiovascular diseases [22,23].
Accumulation of HMs in edible plant parts or fruits may render them unsuitable for human consumption, impacting the fruit's taste and aroma [24].Consequently, plants grown in contaminated soil accumulate heavy metals in various organs, posing a health risk to humans if consumed.Given the importance of fruits in our diet and the risk posed by heavy metal contamination, strict monitoring of heavy metals in plants and their products is necessary [25].It is essential to ensure that heavy metals are checked in products and kept within permissible limits to maintain human health and safety.
Lead (Pb) and copper (Cu) are the main pollutants among all heavy metals that contaminate the soil, and Pb is particularly toxic to both food items and humans [26].Heavy metals like Pb have adverse effects on plants' growth, physiological and biochemical processes, water potential, and molecular activities.Exposure to lead can cause miscarriage, anemia, brain injury, and learning disabilities in children [27].Similarly, excessive amounts of chromium (Cr) in food can lead to skin rashes, stomach upset, liver and kidney damage, and lung cancer.Cr is significantly accumulated in the citrus pulp, and in small quantities it can be crucial for the maintenance of human health, while its deficiency can lead to diabetes [28].Cadmium (Cd) in soil inhibits productivity and decreases important plant processes, such as photosynthesis, mitosis, and the absorption of water.Pb and Cd may cause toxicity symptoms like dark green leaves, older leaves wilting, stunted foliage growth, and short brown roots.Nickel (Ni) is very important for the active sites of many important metalloenzymes.High concentrations of Ni can cause deficiencies in zinc or iron, malfunctioning of many enzymes, and mutations above the permissible limits, potentially leading to cancer [29].
Because of all of this, it is crucial to address the problem of heavy metal contamination in fruits, especially in terms of irrigation sources and monitoring techniques, in order to protect both the reputation of fruit exports and the health of the general population.This study aims to evaluate the impacts of various irrigation sources on the levels of HMs (Pb, Cr, Cd, and Ni) in the edible sections of fruits in order to resolve these problems.The goal is to make sure that the contents of these fruits comply with the guidelines established by the Food and Agriculture Organization (FAO) and the World Health Organization (WHO), thus ensuring consumer food safety.

Study Area
Sargodha, a city in Pakistan's Punjab Province, is situated at 32 • 4 56.8776N latitude and 72 • 40 8.8608 E longitude and has a total area of 5854 km 2 (Figure 1).Summers in Sargodha are hot and muggy, whereas winters there are brief, dry, and chilly, with often clear skies.Sargodha has earned the moniker "California of Pakistan" because of its reputation for producing very high-quality kinnow.Additionally, the region is also known for various citrus fruits that are exported to other nations, including oranges, limes, grapefruit, and lemons.
Because of all of this, it is crucial to address the problem of heavy metal contamination in fruits, especially in terms of irrigation sources and monitoring techniques, in order to protect both the reputation of fruit exports and the health of the general population.This study aims to evaluate the impacts of various irrigation sources on the levels of HMs (Pb, Cr, Cd, and Ni) in the edible sections of fruits in order to resolve these problems.The goal is to make sure that the contents of these fruits comply with the guidelines established by the Food and Agriculture Organization (FAO) and the World Health Organization (WHO), thus ensuring consumer food safety.

Study Area
Sargodha, a city in Pakistan's Punjab Province, is situated at 32° 4′ 56.8776″ N latitude and 72° 40′ 8.8608″ E longitude and has a total area of 5854 km 2 (Figure 1).Summers in Sargodha are hot and muggy, whereas winters there are brief, dry, and chilly, with often clear skies.Sargodha has earned the moniker "California of Pakistan" because of its reputation for producing very high-quality kinnow.Additionally, the region is also known for various citrus fruits that are exported to other nations, including oranges, limes, grapefruit, and lemons.

Sample Collection
The samples of water, soil, and fruits were collected from three different locations randomly.All samples were taken from Chak No. 39 N.B (Site-SW, irrigated with sewage water), Chak No. 37 N.B (Site-CW, irrigated with canal water), and Chak No. 67 N.B (Site-TW, irrigated with tube-well water).Fruit samples of Kinnow (Citrus reticulata) and grapefruit (Citrus paradisi) were collected from orchards located at all three sites (Figure 1).
Soil samples were taken from all three orchards to examine the soil properties in citrus orchards.Each soil sample was a composite combination of four components obtained from depths of 0-15 cm, 15-30 cm, 30-45 cm, and 45-60 cm.The sampling required digging to a depth of 60 cm using an auger.These samples were meticulously collected and labelled before being placed in airtight plastic bags.The soil samples were then taken to the laboratory for detailed examination.The sites chosen for soil sampling were also used for the collection of water samples.These water samples were collected in 500 mL plastic bottles, capped, and stored in an ice box at 5 °C to preserve their integrity on the way to

Sample Collection
The samples of water, soil, and fruits were collected from three different locations randomly.All samples were taken from Chak No. 39 N.B (Site-SW, irrigated with sewage water), Chak No. 37 N.B (Site-CW, irrigated with canal water), and Chak No. 67 N.B (Site-TW, irrigated with tube-well water).Fruit samples of Kinnow (Citrus reticulata) and grapefruit (Citrus paradisi) were collected from orchards located at all three sites (Figure 1).
Soil samples were taken from all three orchards to examine the soil properties in citrus orchards.Each soil sample was a composite combination of four components obtained from depths of 0-15 cm, 15-30 cm, 30-45 cm, and 45-60 cm.The sampling required digging to a depth of 60 cm using an auger.These samples were meticulously collected and labelled before being placed in airtight plastic bags.The soil samples were then taken to the laboratory for detailed examination.The sites chosen for soil sampling were also used for the collection of water samples.These water samples were collected in 500 mL plastic bottles, capped, and stored in an ice box at 5 • C to preserve their integrity on the way to the laboratory.Following the steps described by Al-Lahham et al. [30], the water samples were analyzed in the lab to assess their electrical conductivity (EC) and other physicochemical parameters.The water samples were filtered and kept in a refrigerator prior to metal analysis.Additionally, using the proper laboratory procedures, the physical properties of the soil and water samples were measured [31].
Citrus reticulata and Citrus paradisi fruit samples were gathered from orchards situated at each of the three sites.Three replicates from each site were randomly chosen for the fruit sample collection.The samples were hand-selected, packaged in marked envelopes, and delivered to the lab for additional examination.The fruits were properly cleansed in water in the lab to get rid of any contaminants from the outside.To make it easier to analyze the Water 2023, 15, 3480 4 of 18 mineral components found in the fruits, the edible parts of both types of fruit were then air-dried and baked in an oven.

Sample Preparation
Soil samples were taken from three different places and dried in the air for a few days.The dried samples were then heated to 72 • C in an oven for 72 h.The samples were dried up completely to remove any remaining moisture.These samples were then taken out of the oven and weighed using a digital balance.The materials were digested using the wet acid digestion procedure in order to conduct further analyses.Additionally, water samples were collected in 500 mL polypropylene bottles from three chosen sites.These samples were correctly packaged and kept in a refrigerator.Three specific locations provided samples of the fruits' edible parts.The samples were dried completely by air-drying for a few days.The dried samples were then heated in an electric oven at 72 • C for 48 h to remove any remaining moisture.After being thoroughly crushed into a fine powder, the samples were kept in a desiccator at room temperature.The wet acid digestion procedure was used to digest the materials [32].

Digestion of Samples by the Wet Acid Digestion Method
For further investigation, three replicate powdered samples were precisely weighed at 1 g each and put into crucibles.The fruits' edible parts were used for the examination of minerals and metals.To guarantee full solubilization, acid mixtures were used to digest samples of both soil and fruit.The digestion process adhered to the methodology outlined by Khan et al. [33].Initially, 10 mL of concentrated (65%) HNO 3 was added to 1 mL of water sample or 1 g of powdered soil/fruit sample, and the mixture was allowed to sit undisturbed for an entire night.Following that, the mixture was heated on a hot plate at 70 • C until evaporation took place.After the mixture had cooled to a normal temperature (25 • C), 5 mL of concentrated HClO 4 (70%) was added.The mixture was then heated once more at 70 • C until dense white vapors were seen, indicating full digestion.The sample was cooled down to room temperature once more, and Whatman filter paper #42 was used to filter the digested samples.The filtered samples were then transferred to 50 mL volumetric flasks and fully topped up with 0.1 N HNO 3 [34].

Standard Preparation and Metal Analysis
An atomic absorption spectrophotometer was used to generate standard solutions for the examination of Pb, Cr, Cd, and Ni in water, soil, and fruit samples.Before usage, the glassware and equipment were carefully cleaned and dried.The materials were weighed using an analytical balance before being dissolved in either water or a suitable solvent.The dissolution process was sped up by heating on a hot plate.Then, to ensure thorough dissolution, the solutions were quantitatively transferred to volumetric flasks.After shaking the flask to ensure thorough mixing, deionized water was gradually added until the lower meniscus met the mark on the flask.The meniscus was examined to confirm the solution's concentration and that it was the same at the bottom, where the 100 mL mark was located.Using atomic absorption spectroscopy (AAS), the metals (Pb, Cr, Cd, and Ni) were analyzed.Specific curves were established using standard solutions.Table 1 lists the instrument's operating conditions.

Statistical Analysis
The statistical program SPSS (Statistical Program for Social Sciences) was utilized.The mean values were calculated using one-way ANOVA, with significance levels of 0.001, 0.01, and 0.05.Following the method outlined by Steel et al. [35], Tukey's pairwise comparison test was used to evaluate differences between means, and lettering was used to highlight similarities between means.

Bioconcentration Factor
The bioconcentration factor is the metal concentration that is transmitted from the soil to the plant's edible parts.It was computed using the following formula by the bioconcentration factor (BCF) index:

BCF =
Metal value in edible part of plant Metal value in soil

Enrichment Factor
The method and reference values used by Ahmad et al. [36] were utilized to compute the enrichment factor.This evaluation used the following formula: EF = (Metal value in fruits/Metal value in soil) Sample (Metal value in fruits/Metal value in soil) Standard

Daily Intake of Metals
The DIM index was created to calculate the quantities of metals consumed by individuals through a plant-based diet.Sajjad's [37] definition was used to derive the DIM values in this study.

DIM =
C × F × D food intake W where C represents the concentration of a metal in plants, D food intake represents the daily intake of fruits, F is the conversion factor, and W is the average weight of the human body.

Health Risk Index
The HRI evaluates the possible dangers to human health posed by consuming fruits that are contaminated by metals [38].
where DIM is the daily intake of the metal, and R f D is the oral reference dose of the metal.The RfD values for Cu, Fe, Ni, Pb, Zn, and Mn, as reported by the USEPA [39], are 0.04, 0.7, 0.3, and 0.04 mg/kg/day, respectively.

Metal Concentrations in Water Samples
The results from ANOVA showed a significant effect (p < 0.01) of site on Pb in water.The mean concentration of Pb in water varied from 0.062 ± 0.012 mg/L at the TW site to 0.131 ± 0.024 mg/L at the SW site in C. reticulata.The mean concentration of Pb in water varied from 0.075 ± 0.006 mg/L at the TW site to 0.128 ± 0.027 mg/L at the SW site in C. paradisi.The highest quantity of Pb was seen in water samples of C. reticulata (Table 2).The ANOVA showed a non-significant effect (0.05 < p) of location on Cr in water.The mean concentration of Cr in water used for irrigation purposes was 0.524 ± 0.057 to 0.586 ± 0.095 mg/L at the SW site.The mean concentration of Cr in water used for irrigation purposes was 0.255 ± 0.032 to 0.259 ± 0.029 mg/L at the CW site.At the TW site, the mean concentration of Cr in water used for irrigation purposes was 0.18 ± 0.037 to 0.21 ± 0.019 mg/L.The maximum value of Cr was present in C. reticulata at the SW-irrigated site (Table 2).
The ANOVA displayed a significant effect (0.001 > p) of site on Cd in water samples.The mean quantity of Cd varied from 0.007 ± 0.001 to 0.023 ± 0.004 mg/L in C. reticulata.The mean amount of Cd varied from 0.008 ± 0.001 to 0.022 ± 0.003 mg/L in C. paradisi.The maximum quantity of Cd was present at the SW site of C. reticulata.The minimum quantity of Cd was present at the TW site of C. reticulata.The highest quantity of Cd was present in the water of SW-irrigated sites (Table 2).
The ANOVA results exhibited a non-significant effect (0.05 < p) of site on Ni in water.The mean quantity of Ni fluctuated from 0.569 ± 0.049 to 1.394 ± 0.211 mg/L in water samples of C. reticulata, while the mean quantity of Ni in water samples of C. paradisi was different, ranging from 0.54 ± 0.119 to 1.38 ± 0.175 mg/L.The levels of Ni were significantly higher in the water of both varieties of citrus at the SW site (Table 2).

Metal Concentrations in Soil Samples
The outcomes from ANOVA of the data displayed that sites had a significantly effect (0.001 > p) while plants and sites × plants had non-significant effects (0.05 < p) on the Pb concentration in soil samples (Table 3).The quantity of Pb in the soil irrigated with SW was significantly greater than at the CW and TW sites.At the SW site, the mean concentration of Pb was 37.2 ± 5.716 mg/kg in C. reticulata and 37.188 ± 7.582 mg/kg in C. paradisi.At the CW site, the mean quantity of Pb was 19.044 ± 1.927 mg/kg in C. reticulata and 18.778 ± 1.558 mg/kg in C. paradisi.At the TW site, the mean concentration of Pb was 16.883 ± 2.129 mg/kg in C. reticulata and 17.486 ± 0.877 mg/kg in C. paradisi.The maximum concentration of Pb was present in the soil of C. reticulata at the SW-irrigated site (Table 4).The outcomes from ANOVA of the data revealed that sites had a significant effect (0.01 > p) while plants and sites × plants had non-significant effects (0.05 < p) on the Cr concentration in soil samples (Table 3).At the SW-irrigated site, the mean values of Cr in the soil used for the cultivation of the two citrus varieties ranged from 4.463 ± 0.528 to 4.73 ± 0.629 mg/kg.For the CW-irrigated site, the mean value of Cr in the soil was 2.646 ± 0.629 to 3.21 ± 0.413 mg/kg.For the TW-irrigated site, the mean quantity of Cr in the soil was 2.151 ± 0.723 to 2.639 ± 0.340 mg/kg.The largest amount of Cr was present in C. reticulata at the SW-irrigated site.The minimum concentration of Cr was present at the TW-irrigated site of C. reticulata, while significantly higher concentrations of Cr were present at the SW-irrigated sites of both varieties (Table 4).
The results from ANOVA of the data exhibited that sites had significant effects (0.001 > p) while plants and sites × plants had non-significant effects (0.05 < p) on the Cd concentration in soil samples (Table 3).The quantity of Cd in soil samples of C. reticulata ranged from 1.665 ± 0.302 to 3.606 ± 0.283 mg/kg.The quantity of Cd in soil samples of C. paradisi varied from 1.808 ± 0.274 to 3.684 ± 0.292 mg/kg.Overall, the maximum concentration of Cd was present at the SW-irrigated sites of both varieties.The largest amount of Cd was present in the soil of C. paradisi at the SW-irrigated site (Table 4).
The results from ANOVA of the data displayed that sites had significant effects (0.01 > p) while plants and sites × plants had non-significant effects (0.05 < p) on the Ni concentration in soil samples (Table 3).The amount of Ni was significantly higher in the soil from the SW-irrigated site.The concentration of Ni varied from 8.177 ± 1.804 to 15.4 ± 2.171 mg/kg in C. reticulata, while in C. paradisi it varied from 8.642 ± 1.068 to 15.296 ± 1.582 mg/kg.The maximum amount of Ni was present in C. reticulata soil irrigated with SW (Table 4).

Metal Concentrations in Fruit Samples
The results from ANOVA of the data exhibited that sites had a significant effect (0.001 > p) while plants and sites × plants had non-significant effects (0.05 < p) on the Pb concentration in fruit samples (Table 5).The mean value of Pb at the TW site for both varieties ranged from 0.031 ± 0.003 to 0.037 ± 0.012 mg/kg.The mean value of Pb at the CW site for both varieties ranged from 0.038 ± 0.005 to 0.049 ± 0.013 mg/kg.The mean value of Pb at the SW site for both varieties ranged from 0.095 ± 0.015 to 0.081 ± 0.008 mg/kg.The maximum amount of Pb (0.095 ± 0.015 mg/kg) was present in C. reticulata at the SW site (Table 6).The results from ANOVA of the data displayed that sites (0.05 > p) and plants had significant effect (0.001 > p) while sites × plants had non-significant effects (0.05 < p) on the Cr concentration in fruit samples (Table 5).The mean values of Cr varied from 0.137 ± 0.068 to 0.72 ± 0.049 mg/kg in both of the fruit varieties.The maximum concentration of Cr was present at the SW-irrigated site of C. paradisi.The minimum concentration of Cr was present in C. reticulata at the TW site.Overall, significantly higher amounts of Cr were present in C. paradisi samples than in C. reticulata samples (Table 6).
The outcomes from ANOVA of the data revealed that plants and sites had significant effects (0.001 > p) as did sites × plants (0.05 > p), on the Cd concentration in fruit samples (Table 5).The concentration of Cd in C. reticulata fruit varied from 0.049 ± 0.008 to 0.113 ± 0.014 mg/kg, while in C. paradisi it varied from 0.015 ± 0.002 to 0.033 ± 0.004 mg/kg.Overall, a significantly higher concentration of Cd was present in the fruit of C. reticulata than in those of C. paradisi.The maximum concentration of Cd was present in the fruit of C. reticulata irrigated with SW (Table 6).
The outcomes from ANOVA of the data revealed that sites (0.05 > p) and Plants had significant effects (0.001 > p) while sites × plants had non-significant effects (0.05 < p) on the Ni concentration in fruit samples (Table 5).The mean value of Ni varied from 0.033 ± 0.003 to 1.273 ± 0.122 mg/kg in both citrus varieties.The concentration of Ni was significantly higher in fruit samples of C. paradisi than in those of C. reticulata.The maximum concentration of Ni (1.273 ± 0.122 mg/kg) was found in SW-irrigated C. paradisi (Table 6).

Pollution Load Index
The maximum PLI of Pb was 4.5644 and 4.5629 in C. reticulata and C. paradisi at the SW site, respectively.The minimum PLI of Pb was 2.0715 and 2.1455 in C. reticulata and C. paradisi at the TW site, respectively.The sequence of PLI in both citrus fruit varieties was SW > CW > TW (Table 7).The maximum PLI of Cr was observed in C. reticulata at the SW site, i.e., 0.5215, while the minimum PLI of Cr was in C. reticulata at the TW-irrigated site, i.e., 0.2372.The sequence of PLI in C. reticulata and C. paradisi was SW > CW > TW (Table 7).
The PLI of Cd was significantly high at the SW-irrigated sites of both varieties.In both varieties, it ranged from 0.5946 to 1.3157.The maximum PLI was observed in C. paradisi irrigated with SW, i.e., 1.3157.The minimum PLI (0.5946) was observed at the TW-irrigated site in C. reticulata.The sequence of PLI in C. reticulata and C. paradisi was SW > CW > TW (Table 7).
The PLI for Ni was significantly higher at the SW-irrigated sites of both fruit varieties.The maximum PLI was observed at the SW site of C. reticulata (1.6998), while the minimum was observed at the TW site of C. reticulata (0.9025).The sequence of PLI in both fruit varieties was SW > CW > TW (Table 7).

Bioconcentration Factor
The BCF for Pb for both citrus varieties varied from 0.0018 to 0.0026.The highest BCF was found at the CW-irrigated site of C. paradisi, while the lowest BCF was observed at the TW-irrigated site of C. reticulata (Table 8).The range of bioconcentration factor for Cr in the present study was 0.0637-0.1842.The bioconcentration factor of Cr was highest in C. paradisi irrigated with TW.The lowest BCF was observed in C. reticulata at the TW-irrigated site.Overall, C. paradisi showed a higher BCF than C. reticulata (Table 8).
The BCF for Cd fluctuated from 0.0083 to 0.0313 in the current study.A significantly higher BCF was present in C. reticulata than in C. paradisi.The lowest concentration of BCF was present in C. paradisi irrigated with TW, while the highest was in C. reticulata irrigated with SW (Table 8).
In the current study, the BCF for Ni ranged from 0.0040 to 0.1033 in both citrus varieties.The bioconcentration factor of Ni was highest at the TW-irrigated site for C. paradisi, while it was lowest in C. reticulata irrigated with TW.Overall, higher BCF was observed in C. paradisi than in C. reticulata (Table 8).

Enrichment Factor
The EF concentration for Pb fluctuated from 0.1496 to 0.2127.The peak EF level was observed in C. paradisi watered with CW, while the lowest level of EF was found in C. reticulata irrigated with TW (Table 9).The concentration of EF for Cr ranged from 0.2512 to 0.7262.A higher EF was found in C. paradisi irrigated with TW.Overall, the EF of Cr was higher in C. paradisi than in C. reticulata.The minimum EF was observed in C. reticulata at the TW site (Table 9).
The concentration of EF for Cd varied from 0.1236 to 0.4669 in both citrus varieties.The largest EF was seen in C. reticulata (0.4669) at the SW site, while the lowest was present in C. paradisi (0.1236) at the TW site (Table 9).
The EF of Ni varied from 0.0183 to 0.4681 in both fruit varieties.The highest EF level was found at the TW-irrigated site for C. paradisi, while the lowest EF level was found in C. reticulata at the TW site.Overall, higher EF was observed in C. paradisi than in C. reticulata (Table 9).

Daily Intake of Metals
The DIM of Pb was 3.6338 × 10 −6 and 3.0982 × 10 −6 at the SW site in C. reticulata and C. paradisi, respectively.The DIM of Pb was 1.4535 × 10 −6 and 1.8742 × 10 −6 at the CW site in C. reticulata and C. paradisi, respectively.The DIM of Pb was 1.1858 × 10 −6 and 1.4152 × 10 −6 at the TW site in C. reticulata and C. paradisi, respectively.The sequence of DIM in C. reticulata and C. paradisi was SW > CW > TW (Table 10).The DIM of Cr was greatest in C. reticulata at the TW site, i.e., 5.2402 × 10 −6 , while the minimum DIM was present in C. paradisi at the SW site, i.e., 2.7540 × 10 −5 .The DIM of C. reticulata was less than the DIM of C. paradisi (Table 10).
The DIM of Cd showed an upper peak concentration in C. reticulata at the SW site (4.3222 × 10 −6 ), while the lowest value was observed in C. paradisi at the TW site (5.7375 × 10 −7 ).The sequence of DIM was SW > CW > TW (Table 10).
The DIM of Ni indicated an upper peak value of 4.8692 × 10 −5 in C. paradisi at the SW site, while the lowest value of DIM was found in C. reticulata irrigated with TW (1.2622 × 10 −6 ).The sequence of DIM was SW > CW > TW (Table 10).

Health Risk Index
The HRI values of both citrus varieties ranged between 3.3879 × 10 −4 and 1.0382 × 10 −3 .The maximum HRI value was observed in C. reticulata irrigated with SW (1.0382 × 10 −3 ), while the minimum HRI was observed at the TW-irrigated site of C. reticulata.In C. reticulata and C. paradisi, SW > CW > TW was the order of HRI (Table 11).The upper peak value of the HRI for Cr (0.0918) was found in C. paradisi at the SW site, while the lowest value of HRI was found in C. reticulata at the TW site.Overall, the HRI of C. reticulata was lower than the HRI of C. paradisi (Table 11).
The upper peak value of HRI for Cd (0.0086) was found in C. reticulata at the SW site, while the lowest HRI (0.0012) was found in C. paradisi at the TW site.The order of HRI in C. reticulata and C. paradisi was SW > CW > TW (Table 11).
The maximum HRI value for Ni was observed in C. paradisi irrigated with SW (0.0024), while the lowest HRI was observed in C. reticulata irrigated with TW (6.3112 × 10 −5 ).Overall, a higher HRI was observed in C. paradisi than in C. reticulata.The order of HRI was SW > CW > TW in both citrus varieties (Table 11).

Discussion
The aim of the current study was to gain more knowledge about the environmental conditions in citrus orchards, particularly with regard to heavy metal pollution.It is crucial to keep an eye on the amounts of heavy metals, given the export value of these fruits.Lead has adverse effects on health.Higher concentrations of lead can cause cancer, mental retardation, brain problems, and behavioral disorders [40].The quantity of Pb varied from 0.062 to 0.128 mg/L.The quantity of Pb was higher at the SW sites.The concentration of Pb in all of the samples was more than the maximum tolerable limit of 0.065 mg/L [41].Khan et al. [42] presented similar results, where the concentration of Pb in GW, CW, and SW was 0.233 + 0.014, 0.268 + 0.007, and 0.337 + 0.017 mg/L, respectively.Khan et al. [43] reported that the quantity of Pb in GW, CW, and SW was 0.214 ± 0.017, 0.245 ± 0.016, and 0.301 ± 0.008 mg/L, respectively.Our results are comparable with the results of Ahmad et al. [36], who reported that the concentration of Pb in GW, CW, and SW was 0.27 ± 0.01, 0.28 ± 0.01, and 0.29 ± 0.001 mg/L, respectively.Different sources of contamination increased the concentration of Pb in the sewage water.
Cr is an essential element for the complete metabolism of carbohydrates, which are essential for the brain and body.It is involved in the metabolism of glucose and insulin functioning.Its deficiency may cause diabetes and many other malfunctions [44].The recommended dosage for men varies between 14 and 35 µg/day [45].The amount of Cr in water samples ranged from 0.18 to 0.586 mg/L.The amount of Cr in the water of C. reticulata at the SW site was above the maximum permissible value of 0.55 mg/L [41].Perveen et al. [46] described Cr concentrations in sewage water in Peshawar ranging from 0.01 to 0.10 mg/L, with a mean quantity of 0.03 mg/L across various localities.Wastewater has higher concentrations due to contamination from various sources.
The results exhibited that the quantity of Cd in water samples varied from 0.007 to 0.023 mg/L.The concentration of Cd in SW was higher than the maximum permissible limit of 0.1 mg/L [41].Our findings were consistent with those of Almeelbi et al. [18], who reported that the quantity of Cd in citrus fruits irrigated with SW was 0.036b ± 0.002 while in potable water it was 0.014 ± 0.003 µg mL −1 .A similar study by Perveen et al. [46] showed that the Cd levels in sewage water in Peshawar ranged from 0.06 to 1.43 mg/L in various localities.
Ihesinachi and Eresiya [47] reported that nickel (Ni) is present in the environment only at very low levels.This metal is vital in small doses; however, it can be hazardous when the maximum tolerable limits are exceeded.In our study, the results revealed that the quantity of Ni for the water samples ranged between 0.54 and 1.394 mg/L.A higher concentration of Ni was present in SW.The amount of Ni in the water samples was less than the maximum permissible limit of 1.40 mg/L [41].Khan et al. [43] reported similar findings, where the concentration of Ni in GW, CW, and SW was 0.091 ± 0.012, 0.108 ± 0.010, and 0.154 ± 0.007 mg/L, respectively.Almeelbi et al. [18] reported that the quantity of Ni in SW was 0.112 ± 0.015 while in potable water it was 0.045 ±0.006 µg mL −1 .Ahmad et al. [36] reported that the amount of Ni in GW, CW, and WW was 0.04 ± 0.01, 0.06 ± 0.01, and 0.07 ± 0.01 mg/L, respectively.The presence of a high amount of Ni in the SW was due to contamination from different sources.
The results indicated that the amount of Pb was between 16.883 and 37.188 mg/kg.The concentration of Pb in soil irrigated with SW was higher than the mean quantity of Pb in all soil samples and was much less than the maximum permissible limit of 200 mg/kg [48].Mbong et al. [49] reported similar findings, where soil samples from urban and rural Citrus reticulata orchards contained the following: Zn, 26.3 and 14.3; Pb, 16.2 and 10.7; Mn, 54.4 and 52.8; Fe, 1014.0 and 143.2; Cd, 4.3 and 3.6; and Cr, 13.8 and 11.7 ppm, respectively.Ahmad et al. [36] described similar values of Pb in soil irrigated with GW, CW, and SW (24.13 ± 2.71, 27.25 ± 1.78, and 30.74 ± 0.81 mg/kg, respectively).Khan et al. [42] stated that the concentrations of Pb in GW-, CW-, and SW-irrigated soil were 28.17, 30.43, and 36.95mg/kg, respectively.Sebastian et al. [50] reported that the concentrations of Cd and Pb in the soil of C. sinensis orchards along the roadside were 0.157 ± 0.014 and 0.568 ± 0.088 mg/kg, respectively.
In this research, the concentration of Cr in soil ranged from 2.151 to 4.73 mg/kg.A higher quantity of Cr was present in soil samples irrigated with SW.The amount of Cr in all of the soil samples was far less than the maximum allowable limit of 200 mg/kg [48].Our findings were consistent with those of Ahmad et al. [36], who reported that the quantity of Cr in soil irrigated with wastewater ranged from 0.07 ± 0.01 to 0.09 ± 0.04 mg/kg.Our results are dissimilar to the results reported by Ahmad et al. [36], who reported that the amount of Cr in soil irrigated with domestic wastewater ranged from 0.063 ± 0.002 to 0.076 ± 0.003 mg/kg in soil.
The results showed that the concentration of Cd in soil varied from 1.665 to 3.684 mg/kg.The quantity of Cd in all soil samples was less than the maximum allowable limit of 10 ppm [48].Almeelbi et al. [18] stated that the concentration of Cd in citrus fruits irrigated with SW was 6.14 b ±1.12, while in potable water it was 1.91 ± 0.15 µg g −1 .Elbagermi et al. [51] reported that the concentrations of Co, Ni, Cd, and Pb in oranges were 0.763, 1.099, 0.030, and 0.200 mg/kg, respectively.A similar study by Ahmad et al. [36] reported that the quantity of Cd in soil irrigated with wastewater ranged from 10.9 ± 0.54 to 15.4 ± 0.29 mg/kg.Ogunkunle et al. [52] reported that the heavy metal concentrations in soils were 2.27 ppm for Cu, 0.25 ppm for Pb, and 3.9 ppm for Cd.Sewage water had greater amounts of Cd due to contamination from various sources.
In our research, the amount of Ni in soil ranged from 8.177 to 15.4 mg/kg.The quantity of Ni in all soil samples was less than the highest permissible limit of 100 mg/kg [48].Khan et al. [42] reported that the quantities of Ni in GW-, CW-, and SW-irrigated soil were 6.368 ± 0.127, 7.136 ± 0.079, and 8.245 ± 0.203 mg/kg, respectively.Our findings were different from the readings of Almeelbi et al. [18], who found that the concentration of Ni in fruits irrigated with SW was 76.12 ± 11.91, while in potable water it was 18.21 ± 3.27 µg g −1 .Ahmad et al. [36] reported that the concentrations of Ni in soil irrigated with GW, CW, and WW were 26.25 ± 1.74, 31.54 ± 1.72, and 33.23 ± 2.68 ppm, respectively.
The amount of Pb in fruit samples ranged from 0.031 to 0.095 mg/kg.A higher amount of Pb was present in fruits irrigated with SW.Only a small amount of Pb was present in the citrus fruit.The concentration of Pb was far below the maximum allowable limit of 0.1 mg/kg [53].Thus, there were no health risks posed by the consumption of these fruits.Our results were dissimilar from the findings of Brima and Mohamed [54], who reported that the concentrations of Pb in the fruit juice of C. reticulata and C. paradisi were 0.29 ± 0.16 and 0.10 ± 0.004 mg/kg, respectively.Elbagermi et al. [51] described similar mean con-centrations of the metals Pb and Cd, with values of 0.20 ± 0.04 and 0.03 ± 0.02 mg/kg, respectively, in orange fruit samples.Rahim et al. [55] reported that the mean concentrations of Pb and Cd in oranges were 0.0392 ± 0.0071 and 0.0106 ± 0.0003 mg/kg, respectively.Despite the presence of Pb in the soil and water samples, only a small quantity of Pb was present in the fruits' edible parts.This is because plants mainly accumulate these metals in the roots and translocate only a small fraction to other parts of the plants.
The amount of Cr in the fruit samples ranged between 0.137 and 0.72 mg/kg.A higher concentration of Cr was present in C. paradisi than in C. reticulata.The amount of Cr in the fruit samples was far below the maximum allowable limit of 2.3 mg/kg [51].Our results were consistent with the findings of Brima and Mohamed [54], who reported that the concentrations of Cr in the fruit juice of C. reticulata and C. paradisi were 0.20 ± 0.08 and 0.77 ± 0.05 mg/kg, respectively.Ghani et al. [26] showed that the concentration of Cr fluctuated from 0.031 to 0.342 mg/kg in different tehsils of the Sargodha district.The highest concentration of Cr was present in the tehsil of Kotmomin.A similar study by Rasool et al. [56] reported that the Cr content in C. paradisi was 0.08 ± 0.01 mg/kg in Sargodha, while in C. reticulata it was 0.22 ± 0.03 mg/kg in Sargodha.These findings show that heavy metal accumulation in citrus fruits grown in the Sargodha region generally have approximate values, but in some studies very high metal values were also found.For example, Rashid et al. [57] stated that high metal values in orange samples may be caused by the use of inorganic fungicides that contain toxic compounds containing heavy metals.
Our results showed that the quantity of Cd ranged between 0.015 and 0.113 mg/kg.The quantity of Cd in fruit samples of C. reticulata irrigated with SW was above the maximum permissible limit of 0.10 mg/kg [53].The concentrations of Cd in the fruits of C. paradisi were far below the maximum permissible limit.Almeelbi et al. [18] reported that the concentration of Cd in orange fruit samples irrigated with SW was 0.104 ± 0.008, while at the potable water site it was 0.034 ± 0.003 mg/kg.A similar study by Brima and Mohamed [54] reported that the concentrations of Cd in the fruit juice of C. reticulata and C. paradisi were 0.06 ± 0.07 and 0.01 ± 0.0002 mg/kg, respectively.A significantly higher concentration was reported by Sebastian et al. [50], who showed that the concentrations of Cd and Pb in the fruit of C. reticulata orchards along the roadside were 0.45 ± 0.04 and 1.34 ± 0.09 mg/kg, respectively.Although Cd was present in the samples of water and soil, the fruit samples had small concentrations of Cd due to limited translocation.
The quantity of Ni in fruit samples ranged from 0.033 to 1.273 mg/kg.Larger amounts of Ni were present in C. paradisi as compared with C. reticulata.The concentration of Ni in all fruit samples was less than the maximum allowable limit of 2 mg/kg [40].Our findings were similar to the results of Brima and Mohamed [54], who reported that the concentrations of Ni in the fruit juice of C. reticulata and C. paradisi were 0.08 ± 0.02 and 0.87 ± 0.26 mg/kg, respectively.A similar study by Almeelbi et al. [18] showed that orange fruit samples irrigated with SW had a Ni concentration of 0.112 ± 0.011, while at the potable water site the concentration was 0.061 ± 0.006 mg/kg.Similar research by Galal et al. [58] reported that the average value of Ni in the pulp of navel oranges from the SW-irrigated site was 0.275 mg/kg, while it was not detected in the pulp of navel oranges irrigated with fresh water.Elbagermi et al. [51] reported that the mean concentration of Ni was 1.099 ± 0.368 mg/kg in orange fruit samples.Rahim et al. [55] reported that the mean amount of Ni in oranges was 0.1608 ± 0.0043 mg/kg.Ni was only present in the fruit samples in negligible amounts, due to the limited translocation of Ni in the edible portion of the fruits.
Rashid et al. [57] conducted a study on the presence of potentially hazardous metals in oranges collected from eleven farms in Sargodha, Punjab, Pakistan.As a result of the study, the heavy metal intervals detected in the sample oranges were as follows: 0.0745-1.36 for Pb, 0.0092-1.351for Cd, 0.71-2.0978for Ni, and 0.001-1.572for Cr.The heavy metal values in the samples taken from different orange groves were higher than the values found in kinnow and grapefruit samples in this study.Rashid et al. [57] stated that high metal values in orange samples may be caused by the use of inorganic fungicides that contain toxic compounds containing Hg, Pb, Cd, and Cr.Atta et al. [59] conducted a study in Sargodha to assess the impact of air pollution on the growth of Citrus limon (lemon) and Citrus sinensis (kinu), as well as the accumulation of heavy metals in their leaves within the industrial areas of the tehsil Shahpur Sadar.The metal values obtained from the above study for lemon and kinu samples were higher than the values obtained in this study.The fact that the sampling points were selected from the roadsides and industrial areas in the aforementioned study may be one of the reasons for this situation.
The PLI values for Pb at all of the sites were greater than 1, which shows that there is risk associated with the contamination of Pb.The highest values of PLI were observed at the SW site.The bioconcentration factor values were less than 1, which shows no risk associated [60][61][62][63][64].The EF values were less than 1.Only a small fraction of Pb was accumulated in the fruits.The DIM was negligible.There were no health risks posed by the consumption of these citrus fruits.The HRI values were much less than 1, which shows that consumption of these citrus fruits is safe.The values of PLI for Cr were less than 1 for all of the samples studied.There was no pollution due to the Cr concentration.A higher BCF was present in C. paradisi than in C. reticulata.The values of BCF were less than 1.The EF of C. paradisi was greater than that of C. reticulata.The values of EF were less than 1.The DIM values were very small, which indicates that there were no health effects associated with the consumption of these citrus fruits.The HRI values were less than 1 for all of the studied fruits.
The PLI values for Cd were greater than 1 for the SW site.There were hazards of Cd pollution due to the irrigation with the SW.The BCF values were less than 1.The EF values were higher for C. reticulata than for C. paradisi.The EF values were less than 1 for all of the samples.The DIM of Cd was negligible.The HRI values of Cd were far less than 1, so there was no health risk due to the use of these citrus varieties.The PLI values for Ni were greater than 1 for the SW and CW sites.It was evident that Ni could cause pollution.The BCF value was less than 1 for all of the sites.The EF values were higher for C. paradisi than for C. reticulata.The EF values were less than 1.The DIM values were very small.The HRI values were less than 1, so there were no health risks associated with Ni toxicity posed by the consumption of these citrus fruits.

Conclusions
The different sources of irrigation used did not have a significant impact on the mineral contents of the fruits.The pollution load index for Pb was more than 1, but for Cr it was less than 1.The PLI for Cd was more than 1 only at sewage water sites, while for Ni the PLI was more than 1 at the sewage and canal water sites.The BCF, EF, DIM, and HRI for all metals were less than 1 and within the permissible limits.There were no health risks associated with the consumption of these fruits, as the concentrations of all metals in both fruit varieties were less than the maximum permissible limits recommended by the WHO and FAO.Still, there is a need for controlled monitoring of these metals, as a long-term application of sewage water may change the soil properties and cause health hazards.

Table 1 .
Operating conditions of AAS.

Table 2 .
Metal concentrations in the water samples (mg/L), and ANOVA results.
Notes: * significant at the 0.01 level; ** significant at the 0.001 level; ns, non-significant.

Table 3 .
Analysis of variance in the data for metal values in soil.

Table 4 .
Metal concentrations in the soil samples (mg/kg).

Table 5 .
Analysis of variance in the data for metal values in fruits.

Table 6 .
Metal concentrations in the fruit samples (mg/kg).

Table 7 .
Pollution load index for heavy metals.

Table 8 .
Bioconcentration factor for heavy metals.

Table 9 .
Enrichment factor for heavy metals.

Table 10 .
Daily intake of heavy metals.

Table 11 .
Health risk index for heavy metals.