Evaluation of Cu, Zn, Fe, and Mn Concentrations in Water, Soil, and Fruit Samples in Sargodha District, Pakistan

Abstract

This study aimed to assess the concentrations of copper (Cu), zinc (Zn), iron (Fe), and manganese (Mn) in the edible parts of grapefruit and kinnow fruit irrigated with sewage water (SW), tube-well water (TW), and canal water (CW). Preparation of the samples used in the study for metal analysis was carried out via the wet acid digestion method. Atomic absorption spectrometry (AAS) was used for metal determination. According to the results, Cu concentration ranged from 0.152 to 0.754 mg/L in water, 5.254 to 41.659 mg/kg in soil, and 0.128 to 0.864 mg/kg in fruit samples. Zn concentration varied from 0.574 to 2.723 mg/L in water, 17.812 to 112.954 mg/kg in soil, and 2.658 to 42.642 mg/kg in fruit samples. Fe concentration ranged from 0.254 to 1.245 mg/L in water, 10.635 to 48.638 mg/kg in soil, and 1.062 to 7.584 mg/kg in fruit samples. Mn concentration ranged from 0.154 to 0.638 mg/L in water, 51.283 to 183.865 mg/kg in soil, and 0.136 to 1.464 mg/kg in fruit samples. The Pollution Load Index (PLI) indicated that Cu and Mn exceeded a PLI value of one, and Zn had a PLI > 1 only in sewage water-irrigated sites. Bioconcentration Factor (BCF), Enrichment Factor (EF), Daily Intake of Metal (DIM), and Health Risk Index (HRI) values for all metals were within permissible limits, indicating no immediate health risks associated with consuming these fruits.

1. Introduction

Citrus holds the distinction of being the most widely cultivated fruit worldwide, occupying a large area of production. Global citrus production spans approximately 8712 thousand hectares, yielding an impressive 143,755.6 thousand tonnes [1]. Citrus fruits, belonging to the Rutaceae family with 158 genera and 1900 species, are extensively cultivated on a large scale in more than fifty countries, with China, the U.S.A., Brazil, and Mexico accounting for approximately 47% of global citrus production [2]. The citrus group encompasses seven distinct types, namely mandarins (Citrus reticulata), pummelos (Citrus maxima), sweet oranges (Citrus sinensis), grapefruits (Citrus paradisi), lemons (Citrus limon), citrons (Citrus medica), and limes [3].

The kinnow (Citrus reticulata Blanco.) is a high-yielding mandarin hybrid that is widely planted in the Punjab provinces of Pakistan and India. It is a cross between the citrus cultivars “Willow Leaf” (Citrus deliciosa) and “King”, which were initially created by Howard B. Frost at the University of California Citrus Experiment Station [4]. The kinnow is widely cultivated in the Sargodha district of Pakistan and is celebrated for its exceptional taste and tangy flavour. This citrus variety is greatly valued for its abundant vitamin C content and many health benefits, making it a preferred choice among consumers [5]. Similarly, grapefruit (Citrus paradisi) has gained global recognition for its delightful taste and nutritional value [6]. Enriched with compounds such as glucosides, furanocoumarins, and limonoid aglycones, grapefruit has become increasingly popular. The worldwide production of grapefruit totals around 9.504 million tonnes [1].

The environmental problems caused by rapid industrialisation following the Industrial Revolution gave rise to the concept of sustainability [7,8]. Although industrialisation increased production, unregulated waste created pollution that disrupted the natural balance of air, water, soil, noise, and electromagnetic pollution. This is exemplified by nitrogen oxides, sulphur oxides, acidic gases, and heavy metals which cause cumulative pollution within the atmosphere [9]. Furthermore, potentially harmful elements (PHEs), particularly heavy metals, are a major pollution threat to water resources, soil, and living organisms [10]. Climate change has caused significant shifts in weather patterns resulting in a shortage of freshwater. In countries where traditional agricultural practices are still common, limited availability of freshwater has resulted in the usage of domestic wastewater for irrigation purposes. Even though wastewater offers mineral resources for plant growth, continuous use and flood irrigation for crops are inefficient and spread diseases [7]. In developing countries like Pakistan, which have main economic reliance on agriculture, wastewater is widely used due to limited freshwater availability and arid climatic conditions [11]. Irrigation water quality is crucial for crops, as heavy metals (HMs) and other toxic substances can affect soil and plant health, resulting in yield losses and contamination of the food chain. Global freshwater scarcity is prominent, making mixed water solutions valuable. However, heavy metals from wastewater can contaminate soil and food, posing risks to humans [12]. Wastewater irrigation affects soil properties and fertility [7]. The decline in freshwater availability necessitates mixed water usage, but prolonged sewage water application can damage soil and impede nutrient uptake [13].

Heavy metals, characterized by their long half-life, can gradually accumulate in the body’s organs over time, leading to adverse effects [14,15]. The accumulation of heavy metals can result in severe health consequences including renal dysfunction and failure, liver damage, and disruption of essential biochemical processes within the body, contributing to bone deformation, nervous system impairment, and cardiovascular issues [16]. Copper, zinc, iron, and manganese are essential micronutrients for humans and plants. Excessive copper levels can lead to inflammation in brain tissues and various health issues such as acne, cancer, hair loss, migraines, anorexia, and anxiety [17,18]. Zinc is vital for growth and metabolism in humans and plants, playing structural and enzymatic roles in biochemical reactions [19]. Citrus trees are particularly susceptible to zinc deficiency, resulting in decreased growth of immature tissues, smaller leaves with yellowing along the veins, and reduced carbohydrate production [20,21].

Iron is an essential micronutrient for citrus plants, playing a pivotal role in chlorophyll formation, photosynthesis, enzyme activation, and nutrient uptake [22]. It contributes to vibrant fruit colour, strengthens plant defence against pathogens, and enhances overall growth and productivity. However, both iron deficiency and excess can adversely affect citrus plants, emphasizing the importance of maintaining optimal iron levels. Manganese plays a crucial role in plants by supporting enzyme activity, calcium absorption, and hormone biosynthesis [23]. In humans, manganese contributes to vital processes like enzyme biosynthesis, hormone production, blood clotting, and blood sugar regulation, thereby reducing the risk of heart disease and cancer. It also acts as an anti-ageing agent, improves health conditions, and reduces the risk of certain diseases. Overall, copper, zinc, and manganese are crucial micronutrients for both human and plant health, but maintaining the right balance is essential to avoid toxicity or deficiency-related issues [24].

In recent times, the contamination of heavy metals in food has emerged as a significant challenge [25]. The presence of heavy metals in food is a major issue, as they can accumulate in biological systems from polluted water, air, or soil. This issue is exacerbated by the limited availability of fresh water, compelling farmers to resort to alternative water sources such as sewage water, polluted canal water, or industrial wastewater for irrigation needs [26]. The use of wastewater for orchard irrigation poses a risk of heavy metal accumulation in the soil. Consequently, plants grown in such contaminated soil can accumulate these metals in various parts, rendering them unsuitable for human consumption if they contain higher levels of these metals. Moreover, the heavy metals can also extend to their juices, further compromising their safety [27]. Moreover, the presence of these metals in the edible parts of fruits can negatively impact their quality, including taste and aroma. Given that fruits are an essential part of our diet and are widely consumed, it is crucial to enforce strict monitoring measures to ensure food safety and protect consumers from the harmful effects of heavy metal contamination [28]. Elevated concentrations of metals in fruits can also have detrimental economic impacts, affecting a country’s trade [29].

Amid all these concerns, it is essential to address the issue of heavy metal contamination in fruits, particularly in terms of irrigation sources and monitoring practices, to safeguard both public health and the country’s reputation for the export of fruit items. To address these problems, this study aimed to assess the influence of various irrigation sources on the levels of heavy metals, such as copper, zinc, iron, and manganese, in the edible parts of citrus fruits. The objective is to ensure that the consumption of these fruits aligns with the standards set by the World Health Organization (WHO) and the Food and Agriculture Organization (FAO), thus guaranteeing food safety for consumers.

2. Materials and Methods

2.1. Study Area

Sargodha is located in the Punjab province of Pakistan with geographical coordinates of 32°4′56.8776″ N latitude and 72°40′8.8608″ E longitude, covering an area of 5854 km² (Figure 1). Sargodha experiences a hot and humid summer season, and short, dry, and cool winters, with generally clear weather conditions. Sargodha is renowned for its superior quality kinnow, earning it the nickname “California of Pakistan”. The district is also famous for other citrus fruits like orange, lime, grapefruit, and lemon, which are exported to other countries.

2.2. Sample Collection

Three citrus orchard sites with different irrigation regimes (tube-well water, canal water, and sewage water) were selected for sampling. All samples were collected 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 in all three sites (Figure 1).

Soil samples were collected from all three sites to analyse the soil properties in citrus orchards. The sampling involved digging up to a depth of 60 cm using an augur, and each soil sample was a composite mixture of four parts taken from depths of 0–15 cm, 15–30 cm, 30–45 cm, and 45–60 cm. With this sampling method, three replicates were obtained from each of the three different irrigation areas. These samples were carefully collected in air-tight plastic bags and appropriately labelled. Subsequently, the soil samples were transported to the laboratory for further analysis. The water samples were also collected from the same sites selected for soil sampling and were collected in 500 mL plastic bottles, sealed, and stored in an icebox at 5 °C to maintain their integrity during transportation to the laboratory. In the laboratory, water samples were subjected to analysis to determine their electrical conductivity (EC) and other physicochemical properties, following the procedures outlined by Al-Lahham et al. [30]. Before metal analysis, the water samples were filtered and stored in a refrigerator. Furthermore, the physical parameters of both soil and water samples were measured using appropriate laboratory techniques [31].

Fruit samples of Citrus reticulata and Citrus paradisi were collected from orchards located in all three sites. The collection of fruit samples involved selecting three replicates from each site in a randomized manner. The samples were carefully handpicked, placed in labelled envelopes, and transported to the laboratory for further analysis. In the laboratory, fruits were thoroughly washed with water to remove any external contaminants. The edible portions of the fruits from both varieties were then air-dried and subsequently dried in an oven to facilitate the analysis of mineral elements present in the fruit.

2.3. Sample Preparation

Soil samples gathered from three selected sites were initially air-dried for a few days. Subsequently, the dried samples were placed in an oven for 72 h at 72 °C to ensure that there was no moisture left in the samples. The samples were then weighed using a digital balance after removing them from the oven. To prepare the fruit samples, they were subjected to air-drying for several days until they were completely dried. Subsequently, the dried samples were placed in an electric oven at 72 °C for 48 h until no moisture content was left in the samples. The samples were then completely ground into a fine powder and were stored at room temperature in a desiccator [32].

2.4. Digestion of Samples Using Wet Acid Digestion Method

Three replicates of powdered samples weighing 1 g each were accurately measured and placed in crucibles for further analysis. The edible portion of the fruits was utilized for mineral and metal analysis. Both soil and fruit samples were digested using an acid mixture to ensure complete solubilization. The digestion procedure followed the method described by Khan et al. [33]. Initially, 1 mL of water sample or 1 g of powdered soil/fruit sample was treated with 10 mL of concentrated (65%) HNO₃ and left overnight without heating. The mixture was then heated on a hotplate at 70 °C until evaporation occurred. After cooling to room temperature (25 °C), 5 mL of concentrated HClO₄ (70%) was added, and the mixture was heated again at 70 °C until dense white fumes were observed, indicating complete digestion. Heating on a hot plate was employed to expedite dissolution. The solutions were then transferred quantitatively to volumetric flasks, ensuring complete dissolution. The sample was cooled once more to room temperature, and the digested samples were filtered using Whatman filter paper #42. The filtered samples were transferred to 50 mL volumetric flasks and filled up to the mark with deionized water [34]. Deionized water was added drop by drop until the lower meniscus reached the mark on the flask, and thorough mixing was achieved by shaking the flask. The concentration of the solution was verified by checking the meniscus to ensure the concentration of the solution was alike at the bottom which was at the 100 mL mark.

2.5. Standard Preparation and Metal Analysis

Standard solutions were prepared for the analysis of copper, zinc, iron, and manganese in water, soil, and fruit samples using an atomic absorption spectrophotometer. Glassware and equipment were thoroughly washed and dried before use. The samples were weighed using an analytical balance and dissolved in a suitable solvent or water. Analysis of the metals (copper, zinc, iron, and manganese) was performed using atomic absorption spectrophotometry (AAS) with a Perkin-Elmer Corporation instrument (1980 Waltham, MA, USA). Standard solutions were prepared to set particular curves. The operating conditions of the instrument are presented in

Table 1. Operating conditions of AAS for studied metals.

Metal	Wavelength (nm)	Slit Width (mA)	Current of Lamp
Cu	324.7	0.2	3.5
Zn	213.8	0.2	2.0
Fe	248.3	0.2	2.0
Mn	279.6	0.2	2.0

.

2.6. Statistical Analysis

SPSS (Statistical Program for Social Sciences) was used for statistical analysis. One-way ANOVA was performed to calculate the mean values, with significance levels set at 0.001, 0.01, and 0.05. Tukey’s pairwise comparison test was conducted to assess differences between means, and lettering was employed to indicate variance among the means, following the approach described by Steel et al. [35].

2.7. Bioconcentration Factor

The bioconcentration factor is the concentration of metals transferred from soil to edible parts of fruits. It was calculated by the bioconcentration factor (BCF) index using the following formula for its calculation.

$$\mathrm{BCF}=\frac{\mathrm{M}\mathrm{e}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e} \mathrm{i}\mathrm{n} \mathrm{e}\mathrm{d}\mathrm{i}\mathrm{b}\mathrm{l}\mathrm{e} \mathrm{p}\mathrm{a}\mathrm{r}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t}}{\mathrm{M}\mathrm{e}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e} \mathrm{i}\mathrm{n} \mathrm{s}\mathrm{o}\mathrm{i}\mathrm{l}}$$

2.8. Enrichment Factor

The enrichment factor was calculated by the technique and standard values of Ahmad et al. [36]. For this assessment, the following formula is used:

$$\mathrm{E}\mathrm{F}=\frac{(\mathrm{M}\mathrm{e}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e} \mathrm{i}\mathrm{n} \mathrm{f}\mathrm{r}\mathrm{u}\mathrm{i}\mathrm{t}\mathrm{s}/\mathrm{M}\mathrm{e}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e} \mathrm{i}\mathrm{n} \mathrm{s}\mathrm{o}\mathrm{i}\mathrm{l}) \mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}{(\mathrm{M}\mathrm{e}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e} \mathrm{i}\mathrm{n} \mathrm{f}\mathrm{r}\mathrm{u}\mathrm{i}\mathrm{t}\mathrm{s}/\mathrm{M}\mathrm{e}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e} \mathrm{i}\mathrm{n} \mathrm{s}\mathrm{o}\mathrm{i}\mathrm{l}) \mathrm{S}\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{d}\mathrm{a}\mathrm{r}\mathrm{d}}$$

2.9. Daily Intake of Metals

For the calculation of the amount of metal intake by humans through a fruit diet, the DIM index was developed. The DIM values in this study were determined in accordance with Sajjad’s [37] definition:

$$\mathrm{D}\mathrm{I}\mathrm{M}=\frac{\mathrm{C}\times \mathrm{F}\times {\mathrm{D}}_{\mathrm{f}\mathrm{o}\mathrm{o}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}}}{\mathrm{W}}$$

where C represents the concentration of metal in plants, D_{food intake} represents the daily intake of fruits, F shows the conversion factor, and W is the average weight of the human body.

2.10. Pollution Load Index

The pollution load index (PLI) is a measurement tool used to evaluate heavy metal pollution. The PLI is usually compared to a threshold value (often set at one). If the PLI is greater than one, it suggests that pollution levels in the environment exceed acceptable limits, indicating potential environmental stress. Conversely, if the PLI is less than one, it suggests that pollution levels are within acceptable limits. It provides a simplified numerical representation of the overall pollution status by considering multiple pollutants or contaminants simultaneously.

PLI = Metal value in examined soil/Reference value of soil metal

The reference soil values (mg/kg) of Cu (8.39), Fe (56.90), Zn (44.19), and Mn (46.75) were taken according to Ugulu et al. [32].

2.11. Health Risk Index

The HRI assesses the potential risks to human health presented by the consumption of metal-contaminated fruits [38].

$$\mathrm{H}\mathrm{R}\mathrm{I}=\frac{\mathrm{D}\mathrm{I}\mathrm{M}}{{\mathrm{R}}_f\mathrm{D}}$$

Here, DIM is daily intake of the metal and R_fD is the oral reference dose of 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.

3. Results

3.1. Metal Concentrations in Water Samples

The ANOVA table displayed a non-significant effect (p > 0.05) of the sites on Cu in water. The mean quantity of Cu in water used for irrigation purposes was 0.508 ± 0.125 to 0.642 ± 0.029 mg/L at the SW site. The mean concentration of Cu in canal water used for irrigation purposes ranged from 0.239 ± 0.033 to 0.25 ± 0.041 mg/L at the CW site. At the TW site, the mean concentration of Cu in water used for irrigation purposes was 0.209 ± 0.024 to 0.227 ± 0.037 mg/L. The maximum quantity of Cu was present in Citrus paradisi at the SW-irrigated site (

Table 2. Metal concentrations in the water samples (mg/L) and ANOVA results.

Water	Cu					Zn
	Fruit Site	Mean	SE Mean	Min	Max	Mean	SE Mean	Min	Max
C. reticulata	SW	0.508	0.125	0.348	0.754	1.81	0.475	1.124	2.723
	CW	0.239	0.033	0.181	0.294	1.087	0.337	0.621	1.742
	TW	0.209	0.024	0.162	0.238	0.942	0.206	0.574	1.285
Mean Square	0.081 ns					0.649 ns
C. paradisi	SW	0.642	0.029	0.587	0.687	1.878	0.444	1.154	2.684
	CW	0.25	0.041	0.168	0.297	1.045	0.210	0.715	1.435
	TW	0.227	0.037	0.152	0.265	0.94	0.188	0.623	1.273
Mean Square	0.163 ns					0.792 ns
	Fe					Mn
C. reticulata	Fruit site	Mean	SE Mean	Min	Max	Mean	SE Mean	Min	Max
	SW	0.884	0.141	0.646	1.134	0.48	0.064	0.374	0.596
	CW	0.439	0.042	0.385	0.521	0.295	0.017	0.264	0.322
	TW	0.277	0.017	0.254	0.311	0.268	0.033	0.232	0.341
Mean Square	0.297 ns					0.038 *
C. paradisi	SW	0.829	0.209	0.585	1.245	0.538	0.059	0.434	0.638
	CW	0.484	0.049	0.386	0.541	0.291	0.020	0.254	0.321
	TW	0.317	0.023	0.275	0.354	0.214	0.033	0.154	0.268
Mean Square	0.205 ns					0.086 ns

* significant at 0.05 level; ns, non-significant.

).

The ANOVA results displayed a non-significant effect (p > 0.05) of the locations on Zn in water samples. The mean value of Zn fluctuated from 0.942 ± 0.206 to 1.81 ± 0.475 mg/L in Citrus reticulata. The mean concentration of Zn fluctuated from 0.94 ± 0.188 to 1.878 ± 0.444 mg/L in C. paradisi. The maximum amount of Zn was present in SW sites of both varieties. The minimum concentration of Zn was present in tube-well water. The highest amount of Zn was present in water samples from the SW-irrigated site of C. paradisi (Table 2).

The ANOVA results exhibited a non-significant effect (p > 0.05) on the locations of Fe in water. The mean value of Fe fluctuated from 0.277 ± 0.017 to 0.884 ± 0.141 mg/L in water samples of C. reticulata, while the mean level of Fe in water samples fluctuated from 0.317 ± 0.023 to 0.829 ± 0.209 mg/L. The level of Fe was significantly higher in the water of both varieties of citrus at the SW site (Table 2).

The ANOVA results exhibited a significant effect (p < 0.05) for C. reticulata and a non-significant effect (p > 0.05) of sites for Mn in water samples. The minimum amount of Mn in C. reticulata was 0.277 ± 0.033 mg/L at the TW site, while the maximum was 0.48 ± 0.064 mg/L at the SW site. The minimum quantity of Mn was in C. paradisi grown water, which was 0.214 ± 0.033 mg/L at the TW site, while the maximum was 0.538 ± 0.059 mg/L at the SW site planted with C. paradisi. Overall, a higher amount of Mn was present in both citrus varieties at SW sites (Table 2).

3.2. Metal Concentrations in Soil Samples

ANOVA results revealed that “Sites” had significant differences in Cu concentrations (p < 0.001) with regard to the soil samples (

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

Cu			Zn
Source of Variation	DF	Mean Square	Source of Variation	DF	Mean Square
Sites	2	925.858 ***	Sites	2	4228.6 ***
Plants	1	18.942 ns	Plants	1	669.0 ns
Sites × Plants	2	0.273 ns	Sites × Plants	2	170.6 ns
Error	12	28.288	Error	12	228.1
Total	17		Total	17
Fe			Mn
Source of Variation	DF	Mean Square	Source of Variation	DF	Mean Square
Sites	2	983.975 ***	Sites	2	8855.16 ***
Plants	1	2.410 ns	Plants	1	5.38 ns
Sites × Plants	2	3.734 ns	Sites × Plants	2	117.57 ns
Error	12	61.57	Error	12	695.50
Total	17		Total	17

*** significant at 0.001 levels; ns, non-significant.

). At the SW-irrigated site, the mean Cu concentration in soil for both citrus varieties ranged from 33.565 ± 1.559 to 35.452 ± 3.501 mg/kg. At the CW-irrigated site, the mean Cu concentration in soil ranged from 14.607 ± 4.879 to 17.143 ± 2.351 mg/kg. At the TW-irrigated site, the mean Cu concentration in soil ranged from 10.094 ± 1.300 to 11.826 ± 3.295 mg/kg. The highest Cu concentration was observed in C. reticulata grown soil at the SW-irrigated site, while the minimum concentration was found in the soil of TW-irrigated sites of both varieties. Significantly higher concentrations of Cu were present in the SW-irrigated site soil of both citrus varieties (

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

Soil	Cu					Zn
	Fruit Site	Mean	SE Mean	Min	Max	Mean	SE Mean	Min	Max
C. reticulata	SW	35.452	3.501	29.542	41.659	67.882	8.877	54.245	84.547
	CW	17.143	2.351	13.451	21.512	34.129	7.832	18.624	43.812
	TW	11.826	3.295	5.254	15.541	28.7	6.224	17.812	39.368
C. paradisi	SW	33.565	1.559	30.853	36.254	92.128	12.884	68.574	112.954
	CW	14.607	4.879	7.284	23.854	42.473	8.771	31.857	59.874
	TW	10.094	1.300	8.436	12.658	32.689	5.862	25.398	44.285
	Fe					Mn
	Fruit Site	Mean	SE Mean	Min	Max	Mean	SE Mean	Min	Max
C. reticulata	SW	35.378	7.806	20.521	46.961	128.732	25.014	89.354	175.145
	CW	16.557	2.652	12.685	21.632	77.121	5.807	65.861	85.214
	TW	12.931	1.188	10.854	14.968	64.582	7.665	51.283	77.834
C. paradisi	SW	36.448	7.128	23.952	48.638	139.709	24.708	98.417	183.865
	CW	14.691	1.621	11.853	17.469	75.533	7.643	63.412	89.659
	TW	11.532	0.572	10.635	12.595	58.472	1.976	54.921	61.749

).

Similarly, ANOVA results indicated that “Sites” were significantly affected (p < 0.001), while “Plants” and “Sites × Plants” were not significantly affected (p > 0.05) by Zn concentration in soil samples (Table 3). The amount of Zn in soil samples of C. reticulata ranged from 28.7 ± 6.224 to 67.882 ± 8.877 mg/kg, while in C. paradisi, it fluctuated from 32.689 ± 5.862 to 92.128 ± 12.884 mg/kg. Overall, the maximum amount of Zn was present in the soil samples of SW-irrigated sites of both citrus varieties. The highest concentration of Zn was found in the soil of C. paradisi at the SW-irrigated site (Table 4).

For Fe concentration in the soil, a significant difference (p < 0.001) in accumulation at all sites was observed (Table 3). The amount of Fe varied from 12.931 ± 1.188 to 35.378 ± 7.806 mg/kg in C. reticulata soil, while in C. paradisi, it ranged from 11.532 ± 0.572 to 36.448 ± 7.128 mg/kg. The maximum amount of Fe was found in C. paradisi soil irrigated with SW (Table 4).

Additionally, ANOVA results clearly demonstrated that sites had significantly different concentrations (p < 0.001) of Mn in soil samples (Table 3). The concentration of Mn in C. reticulata soil ranged from 64.582 ± 7.665 to 128.732 ± 25.014 mg/kg, while in C. paradisi, it ranged from 58.472 ± 1.976 to 139.709 ± 24.708 mg/kg. The highest Mn values were found in the soil of C. paradisi (139.709 ± 24.708 mg/kg) at the SW-irrigated site (Table 4).

3.3. Metal Concentrations in Fruit Samples

ANOVA results indicated that “Plants” were significantly affected (p < 0.05), while “Sites” and “Sites × Plants” were not significantly affected (p > 0.05) by Cu concentration in fruit samples (

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

Cu			Zn
Source of Variation	DF	Mean Square	Source of Variation	DF	Mean Square
Sites	2	0.004540 ns	Sites	2	80.26 ***
Plants	1	0.268400 *	Plants	1	3352.79 ***
Sites × Plants	2	0.028425 ns	Sites × Plants	2	45.20 **
Error	12	0.038228	Error	12	5.57
Total	17		Total	17
Fe			Mn
Source of Variation	DF	Mean Square	Source of Variation	DF	Mean Square
Sites	2	5.7869 ***	Sites	2	0.735198 ***
Plants	1	44.8373 ***	Plants	1	0.568533 ***
Sites × Plants	2	4.5465 **	Sites × Plants	2	0.340177 ***
Error	12	0.4700	Error	12	0.005673
Total	17		Total	17

*, **^, *** significant at 0.05, 0.01, and 0.001 levels; ns, non-significant.

). The mean Cu concentration ranged from 0.185 ± 0.028 to 0.546 ± 0.176 mg/kg in both fruit varieties. The highest Cu concentration was found in C. paradisi at the CW-irrigated site, while the lowest concentration was observed in C. reticulata at the TW site. Overall, C. paradisi exhibited higher Cu levels compared to C. reticulata (

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

Fruit	Cu					Zn
	Fruit Site	Mean	SE Mean	Min	Max	Mean	SE Mean	Min	Max
C. reticulata	SW	0.344	0.061	0.235	0.448	38.594	2.447	34.187	42.642
	CW	0.200	0.027	0.161	0.253	28.664	1.517	26.114	31.364
	TW	0.185	0.028	0.128	0.214	26.637	1.312	24.124	28.547
C. paradisi	SW	0.431	0.078	0.328	0.584	5.007	0.966	3.854	6.927
	CW	0.546	0.176	0.254	0.864	3.842	0.260	3.385	4.284
	TW	0.484	0.184	0.158	0.795	3.159	0.359	2.658	3.854
	Fe					Mn
	Fruit Site	Mean	SE Mean	Min	Max	Mean	SE Mean	Min	Max
C. reticulata	SW	6.762	0.453	6.021	7.584	0.405	0.033	0.342	0.452
	CW	4.42	0.617	3.254	5.351	0.244	0.028	0.192	0.287
	TW	3.105	0.267	2.752	3.628	0.187	0.014	0.168	0.214
C. paradisi	SW	1.723	0.232	1.341	2.143	1.309	0.095	1.136	1.464
	CW	1.593	0.217	1.162	1.856	0.358	0.015	0.341	0.389
	TW	1.500	0.427	1.062	2.354	0.235	0.005	0.227	0.243

).

ANOVA results showed that Zn concentration varied considerably (p < 0.001, p < 0.01) for “Plants” and “Sites” with regards to Zn concentration in fruit samples (Table 5). The concentration of Zn in C. reticulata fruits ranged from 26.637 ± 1.312 to 38.594 ± 2.447 mg/kg, while in C. paradisi, it ranged from 3.159 ± 0.359 to 5.007 ± 0.966 mg/kg. Significantly higher amounts of Zn were observed in C. reticulata fruits compared to C. paradisi. The highest Zn concentration was found in C. reticulata fruits irrigated with SW (Table 6).

For Fe concentration in fruit samples, ANOVA results revealed that “Plants” and “Sites” were significantly affected (p < 0.001) and “Sites × Plants” were also significantly affected (p < 0.01; Table 5). The mean Fe concentration ranged from 1.500 ± 0.427 to 1.723 ± 0.232 mg/kg in both citrus varieties. The concentration of Fe was significantly higher in C. reticulata fruits compared to C. paradisi. The maximum Fe concentration (6.762 ± 0.453 mg/kg) was found in C. reticulata fruits irrigated with SW (Table 6).

Furthermore, ANOVA results indicated that “Plants”, “Sites”, and “Sites × Plants” were significantly affected (p < 0.001) by Mn concentration in fruit samples (Table 5). The mean Mn concentration in C. reticulata was 0.187 ± 0.014 and 0.405 ± 0.033 mg/kg in TW- and SW-irrigated sites, respectively, whereas the mean Mn concentration in C. paradisi was 0.235 ± 0.005 and 1.309 ± 0.095 mg/kg in TW- and SW-irrigated sites, respectively. The highest mean Mn value was observed in C. reticulata fruit samples (0.405 ± 0.033 mg/kg) at the SW site (Table 6).

3.4. Pollution Load Index

In C. reticulata, the highest PLI for Cu was observed at the SW site (4.225), while the lowest was at the TW site (1.2031) in C. paradisi. For Zn, the SW site showed significantly higher PLI values, with the maximum value observed in C. paradisi (2.0518). The minimum value for Zn was in C. reticulata at the TW site (0.6392). Similarly, the PLI for Mn was highest at the SW sites in both citrus varieties, with C. paradisi exhibiting the maximum PLI (2.9884) and C. reticulata showing a slightly lower value (2.7536) (

Table 7. Pollution Load Index for heavy metals.

Site	Cu		Zn		Fe		Mn
	C. reticulata	C. paradisi	C. reticulata	C. paradisi	C. reticulata	C. paradisi	C. reticulata	C. paradisi
SW	4.2255	4.0006	1.5118	2.0518	0.6218	0.6406	2.7536	2.9884
CW	2.0433	1.7410	0.7601	0.9459	0.2910	0.2582	1.6496	1.6157
TW	1.4095	1.2031	0.6392	0.7280	0.2273	0.2027	1.3814	1.2507

).

3.5. Bioconcentration Factor

The highest BCF was observed in C. paradisi irrigated with TW, while the lowest BCF was found in C. reticulata at the SW-irrigated site. Both fruits exhibited higher BCF values for Zn and Fe. The BCF for Cu in the study varied from 0.0097 to 0.0479. For Zn, the BCF ranged from 0.0543 to 0.9281. C. reticulata showed comparatively higher BCF values for Zn compared to C. paradisi, with the lowest BCF observed in C. paradisi irrigated with SW and the highest in C. reticulata irrigated with TW. In both citrus varieties, the BCF for Mn ranged from 0.0473 to 0.2669. BCF values were comparable in both citrus varieties with respect to Mn (

Table 8. Bioconcentration Factor for heavy metals.

Site	Cu		Zn		Fe		Mn
	C. reticulata	C. paradisi	C. reticulata	C. paradisi	C. reticulata	C. paradisi	C. reticulata	C. paradisi
SW	0.0097	0.0128	0.5685	0.0543	0.1911	0.0473	0.0032	0.0094
CW	0.0117	0.0374	0.8399	0.0905	0.2669	0.1084	0.0032	0.0047
TW	0.0156	0.0479	0.9281	0.0966	0.2401	0.1301	0.0029	0.0040

).

3.6. Enrichment Factor

The EF for Cu in the study ranged from 0.2115 to 0.8046. C. paradisi irrigated with TW exhibited a higher EF, indicating a greater enrichment of Cu. Overall, C. paradisi showed higher EF values compared to C. reticulata, with the lowest EF observed in C. reticulata at the SW site. For Zn, the EF concentrations ranged from 0.0407 to 0.6945 in both citrus varieties. The highest EF was found in C. reticulata (0.6945), while the lowest was observed in C. paradisi (0.0407). In both fruit varieties, the EF values for Mn varied from 0.1345 to 0.7595. The highest EF level was found in the CW-irrigated site grown with C. reticulata, while the lower EF level was observed in C. paradisi at the SW site. Overall, C. reticulata exhibited higher EF values compared to C. paradisi. The EF concentrations for the citrus varieties ranged from 0.4512 to 1.4601. The highest EF was found in C. paradisi (1.4601) irrigated with SW, while the lowest concentration was observed in C. reticulata (0.4512) irrigated with TW. Overall, C. paradisi showed higher EF values than C. reticulata (

Table 9. Enrichment Factor for heavy metals.

Site	Cu		Zn		Fe		Mn
	C. reticulata	C. paradisi	C. reticulata	C. paradisi	C. reticulata	C. paradisi	C. reticulata	C. paradisi
SW	0.1628	0.2155	0.4254	0.0407	0.5438	0.1345	0.4903	1.4601
CW	0.1958	0.6272	0.6285	0.0677	0.7595	0.3085	0.4930	0.7386
TW	0.2625	0.8046	0.6945	0.0723	0.6831	0.3701	0.4512	0.6263

).

3.7. Daily Intake of Metal

The DIM for Cu was highest in C. paradisi at the CW site, with a value of 2.0884 × 10⁻⁵, while the lowest DIM was observed in C. reticulata at the TW site, measuring 7.0762 × 10⁻⁶. DIM values were generally higher in C. paradisi compared to C. reticulata. For Zn, the highest DIM concentration was found in C. reticulata at the SW site, measuring 1.4762 × 10⁻³, while the lowest value was observed in C. paradisi at the TW site, measuring 1.2083 × 10⁻⁴. The sequence of DIM values followed the order SW > CW > TW. In the case of Fe, the upper peak DIM value was 2.5865 × 10⁻⁴ in C. reticulata at the SW site, while the lowest DIM value was found in C. paradisi irrigated with TW, measuring 5.7375 × 10⁻⁵. The sequence of DIM values followed the order SW > CW > TW. Regarding Mn, the maximum DIM concentration was observed in C. paradisi at the SW site, measuring 5.0069 × 10⁻⁵, while the minimum DIM was found in C. reticulata at the TW site, measuring 7.1528 × 10⁻⁶. Overall, DIM values were higher for C. paradisi compared to C. reticulata (

Table 10. Daily intake of heavy metals.

Site	Cu		Zn		Fe		Mn
	C. reticulata	C. paradisi	C. reticulata	C. paradisi	C. reticulata	C. paradisi	C. reticulata	C. paradisi
SW	1.315 × 10−5	1.6486 × 10−5	1.476 × 10−3	1.915 × 10−4	2.586 × 10−4	6.59 × 10−5	1.5491 × 10−5	5.006 × 10−5
CW	7.650 × 10−6	2.0884 × 10−5	1.096 × 10−3	1.469 × 10−4	1.690 × 10−4	6.09 × 10−5	9.3330 × 10−6	1.369 × 10−5
TW	7.076 × 10−6	1.8513 × 10−5	1.018 × 10−3	1.208 × 10−4	1.187 × 10−4	5.73 × 10−5	7.1528 × 10−6	8.988 × 10−6

).

3.8. Health Risk Index

The upper peak value of HRI was found in C. paradisi at the CW site, measuring 5.221 × 10⁻⁴, while the lowest value was observed in C. paradisi at the TW site. Overall, the HRI of C. reticulata was lower than that of C. paradisi. The upper peak HRI value was found in C. reticulata at the SW site, measuring 3.989 × 10⁻³, while the lowest HRI value was observed in C. paradisi at the TW site, measuring 3.265 × 10⁻⁴. The order of HRI values followed the sequence SW > CW > TW. The maximum HRI value was observed in C. reticulata (0.0369) irrigated with SW, while the lowest HRI was observed in C. paradisi (0.0082) irrigated with TW. Overall, higher HRI values were observed in C. reticulata compared to C. paradisi. The order of HRI values was SW > CW > TW for both citrus varieties. The upper peak HRI values were found in C. paradisi at the SW site, measuring 3.576 × 10⁻⁴, while the lowest values were found in C. reticulata at the TW-irrigated location, measuring 5.109 × 10⁻⁵. The order of HRI values for C. paradisi and C. reticulata followed the sequence SW > CW > TW (

Table 11. Health Risk Index for heavy metals.

Site	Cu		Zn		Fe		Mn
	C. reticulata	C. paradisi	C. reticulata	C. paradisi	C. reticulata	C. paradisi	C. reticulata	C. paradisi
SW	3.289 × 10−4	4.121 × 10−4	3.989 × 10−3	5.176 × 10−4	0.0369	0.0094	1.106 × 10−4	3.576 × 10−4
CW	1.912 × 10−4	5.221 × 10−4	2.963 × 10−3	3.971 × 10−4	0.0242	0.0087	6.666 × 10−5	9.781 × 10−5
TW	1.769 × 10−4	4.628 × 10−4	2.753 × 10−3	3.265 × 10−4	0.0170	0.0082	5.109 × 10−5	6.420 × 10−5

).

4. Discussion

The current study was undertaken to gain a deeper understanding of the environmental conditions within citrus orchards, particularly concerning heavy metal contamination. Given the export value of these fruits, it is imperative to monitor the level of heavy metals. Copper is an essential trace element that plays a vital role in the defence against oxidation, respiration, and the maintenance of a healthy central nervous system, bones, and immune system. It also upholds body pigments and prevents anaemia. In humans, a deficiency of copper can lead to skin diseases, growth retardation, and digestive tract disorders [24]. In the present study, the concentration of copper in the irrigation water of both citrus varieties ranged from 0.209 to 0.642 mg/L. Notably, the SW site water exhibited the highest copper concentration in both fruit orchards. This can be attributed to the higher nutrient content and presence of waste materials in wastewater. It is worth mentioning that the concentration of Cu in the water samples of both citrus varieties was below the safe limit of 2 mg/L recommended by the World Health Organization [40]. Our findings slightly exceeded the results reported by Almeelbi et al. [13], who reported Cu concentrations in SW as 0.102 ± 0.021 and in potable water as 0.051 ± 0.004 μg mL⁻¹. However, our results were consistent with those of Khan et al. [41], who reported copper concentrations in groundwater (GW), canal water (CW), and sewage water (SW) as 0.023 ± 0.012, 0.031 ± 0.002, and 0.032 ± 0.004 mg/L, respectively. Similarly, Ahmad et al. [36] described copper concentrations in GW, CW, and SW as 0.01 ± 0.001, 0.03 ± 0.001, and 0.03 ± 0.002 mg/L, respectively. The elevated copper levels in wastewater can be attributed to the presence of higher nutrient and waste content.

Zn is another essential metal which supports the body’s defensive system and is crucial in cell division and growth. Zinc deficiency can result in various symptoms such as skin sores, slow growth, and delayed wound healing, which can be alleviated by zinc supplements and zinc-rich foods [42]. Adequate amount of zinc intake, recommended by the Institute of Medicine [43], ranges from 8 to 13 mg/day. Excessive uptake of Zn causes vascular shock, tachycardia, nausea, and pancreatic diseases [42]. The concentration of Zn in the water samples ranged from 0.94 to 1.878 mg/L, which is below the acceptable limit of 5 mg/L suggested by USEPA [39]. Our findings aligned with those of Almeelbi et al. [13] who reported Zn in SW as 0.191 ± 0.011 and in potable water as 0.021 ± 0.001 μg/mL. Yadav et al. [44] also reported Zn concentrations of 0.17 mg/L in the sewage waters of Haryana, India. Similarly, Khan et al. [41] reported Zn concentrations in GW, CW, and SW as 0.613, 0.637, and 0.646 mg/L, respectively. In another study by Ahmad et al. [36], Zn concentrations in GW, CW, and SW were 0.49 ± 0.03, 0.61 ± 0.04, and 0.62 ± 0.04 mg/L, respectively. The elevated Zn levels in SW can be attributed to the discharge of various types of waste into this water source.

Iron (Fe) is an important mineral that serves several roles in humans. It is involved in ATP generation, synthesis of myoglobin, haemoglobin, and certain enzymes, as well as neurotransmitters and collagen. In plants, Fe is required for the formation of chlorophyll [45]. In the present study, the concentration of iron in the water samples ranged from 0.277 to 0.884 mg/L. These concentrations were significantly below the maximum permissible limit of 5 mg/L, as indicated by Griffiths et al. [46]. Our findings were consistent with those of Almeelbi et al. [13], who reported iron concentrations in SW as 2.362 ± 0.131 and in potable water as 0.532 ± 0.006 μg mL⁻¹. Additionally, Abdel-Shafy and El-Khateeb [47] reported mean Fe concentrations in primary treated sewage water, groundwater, and canal water as 0.53, 0.24, and 0.14 mg/L, respectively. Similar results were reported by Khan et al. [41] and Ahmad et al. [36] regarding Fe concentrations in different water sources.

Manganese (Mn) is another essential mineral that plays a vital role in enzyme function, sex hormones, blood clotting factors, and connective tissues [23]. It also helps in maintaining blood sugar levels and reduces the risk of cancer and heart disease [24]. It is known for its antioxidant properties, which contribute to delaying ageing and improving overall health conditions [48,49]. In the present study, the concentration of manganese in all the samples ranged from 0.214 to 0.538 mg/L, which is below the maximum permitted value of 0.4 mg/L recommended by the WHO [40]. The permissible limit for daily manganese intake is 2.3 mg/day according to the WHO [40]. The higher manganese concentration in SW can be attributed to contamination from various sources.

In our study, the concentration of Cu in soil samples ranged from 10.094 to 35.452 mg/kg, which was below the maximum permissible limit of 50 ppm reported by Adagunodo et al. [50]. These findings were consistent with the results of Almeelbi et al. [13], who reported a Cu concentration of 145.81b ± 11.79 μg/g in soil irrigated with sewage water (SW) and 17.54a ± 3.02 μg/g in soil irrigated with potable water. However, our results differed from the findings of Khan et al. [41], who reported lower Cu concentrations in soil samples irrigated with GW, CW, and SW, ranging from 2.364 to 3.838 mg/kg. Ahmad et al. [36] also reported Cu concentrations in soil irrigated with domestic wastewater ranging from 3.14 ± 0.175 to 3.49 ± 0.205 mg/kg, and in soil irrigated with GW, CW, and SW ranging from 2.79 ± 0.16 to 4.13 ± 0.19 mg/kg. These variations in Cu concentrations can be attributed to the different sources of irrigation water and the presence of soluble salts, which can affect soil pH and electrical conductivity [49].

Regarding zinc (Zn) concentrations in soil, our study found concentrations ranging from 28.7 to 92.128 ppm, which were below the maximum acceptable limit of 250 mg/kg reported by Adagunodo et al. [50]. These results were lower than those reported by Almeelbi et al. [13], who found a Zn concentration of 371.24b ± 25.9 μg/g in soil irrigated with SW and 45.87 ± 6.28 μg/g in soil irrigated with potable water. Shah et al. [51] reported mean Zn concentrations in citrus orchard soil ranging from 0.24 to 10.10 μg/g. Khan et al. [41] reported Zn concentrations in soil irrigated with GW, CW, and SW ranging from 1.683 to 3.307 mg/kg. Ahmad et al. [36] reported Zn concentrations in soil irrigated with wastewater ranging from 6.71 ± 0.52 to 9.96 ± 0.41 mg/kg. The variations in Zn concentrations can be attributed to different agricultural practices and the presence of contaminants in the irrigation water.

Iron (Fe) concentrations in soil were significantly higher in samples irrigated with SW, ranging from 11.532 to 36.488 mg/kg. However, these concentrations were far below the maximum allowed limit of 50,000 mg/kg reported by Chiroma et al. [52]. Almeelbi et al. [13] reported a Fe concentration of 20.99 ± 1.98 μg/g in soil irrigated with SW and 0.278 ± 0.12 μg/g in soil irrigated with potable water. Khan et al. [41] reported Fe concentrations in soil irrigated with GW, CW, and SW ranging from 34.43 to 44.55 mg/kg. Ahmad et al. [36] also reported Fe concentrations in soil irrigated with wastewater ranging from 42.9 ± 0.64 to 48.4 ± 1.42 mg/kg. The higher Fe concentrations in soil irrigated with SW can be attributed to the presence of effluents and contaminants in sewage water.

The concentration of manganese (Mn) in soil ranged from 58.472 to 139.709 mg/kg, which was considerably lower than the maximum permissible value of 2000 ppm reported by Chiroma et al. [52]. Our results differed from those of Shah et al. [51], who reported mean Mn concentrations in citrus orchard soil ranging from 0.14 to 40.72 μg/g. Ahmad et al. [36] reported Mn concentrations in soil irrigated with wastewater ranging from 18.9 ± 1.26 to 34.8 ± 1.27 mg/kg. The availability of Mn in the soil can be influenced by soil pH, with alkaline conditions reducing the uptake of Mn and other metals by plants [51].

In citrus fruit samples, the concentration of Cu ranged from 0.185 to 0.546 mg/kg. These concentrations were below the maximum allowed limit of 0.5 mg/kg as described by JEFCA [53], except for C. paradisi, which exceeded the limit. Ghani et al. [24] reported copper concentrations ranging from 2.036 to 2.71 mg/kg in different tehsils of the Sargodha district. Almeelbi et al. [13] found a copper concentration of 0.156b ± 0.009 μg/g in citrus fruit samples irrigated with SW and 0.056 ± 0.004 μg/g at the potable water site. Other studies have reported varying Cu concentrations in citrus fruit, ranging from 5.91 ppm [54] to 22.73 ± 0.17 mg/kg [55] in C. paradisi and from 0.28 ± 0.02 to 1.80 ± 0.22 mg/kg in C. reticulata [56]. Ateshan et al. [57] reported the presence of various heavy metals in oranges, including copper at a concentration of 4.81 mg/kg.

The amount of Zn was higher in C. reticulata than C. paradise in the current study. In C. reticulata, the concentration of Zn ranged from 26.637 to 38.594 mg/kg, while in C. paradisi, it ranged from 3.159 to 5.007 mg/kg. These concentrations of Zn in fruit samples were significantly far below the maximum tolerable limit of 100 mg/kg reported by Chiroma et al. [52]. Our results were in agreement with Paul and Shaha [58] who estimated the concentration of Zn in citrus fruits ranging from 0.12 to 0.48 mg/100 g, while grapefruit, pomelo, and orange had 0.14, 0.48, and 0.15 mg/100 g mean Zn levels. Our results also differed for Zn mean level in C. reticulata in a study by Rasool et al. [55], who reported Zn concentrations of 1.87 ± 0.12 mg/kg in C. paradisi and 1.80 ± 0.05 mg/kg in C. reticulata in Sargodha. Furthermore, Almeelbi et al. [13] also reported different Zn concentrations in citrus fruit samples irrigated with SW (0.321 ± 0.021 μg/g) and TW (0.032 ± 0.001 μg/g). However, Brima and Mohamed [56] reported Zn concentrations of 0.78 ± 0.36 mg/kg in C. reticulata and 7.17 ± 2.93 mg/kg in C. paradisi in their fruit juice. Ghani et al. [24] stated that the concentration of Zn fluctuated from 26.463 ± 1.57 to 31.726 ± 1.31 mg/kg in different tehsils of the Sargodha district with the highest amount reported in tehsil Silanwali. Similar results were reported by Mbong et al. [59], who found Zn concentrations of 16.4 and 24.4 ppm in rural and urban orchards, respectively. Galal et al. [60] reported that the mean concentration of Zn in the pulp of navel oranges was 6.82 mg/100 g in the SW-irrigated site and 1.52 mg/100 g for the pulp of navel oranges irrigated with the fresh water. Despite the high concentration of Zn in soil and water, only a small fraction was present in the fruit, due to the limited translocation of Zn to the plants and, consequently, to the fruits.

The amount of Fe in the fruit pulp of C. reticulata was higher than C. paradisi. The concentration of Fe ranged from 1.5 to 6.762 mg/kg in the fruit samples, which was below the maximum acceptable limit of 20 mg/kg [51]. These findings were consistent with the results of Paul and Shaha [58], who reported Fe concentrations of 0.51 and 0.37 mg/100 g in grapefruit and orange, respectively. Rasool et al. [55] reported Fe concentrations of 3.72 ± 0.18 mg/kg in C. paradisi and 2.68 ± 0.16 mg/kg in C. reticulata in Sargodha, which differed from our results. Our results were similar to the findings of Soceanu et al. [61] who reported that the concentration of Fe in orange pulp was 1.135 mg/kg while in grapefruit pulp it was 1.096 mg/kg. Abdel-Shafy and El-Khateeb [47] reported that the mean values of metals iron (Fe), manganese (Mn), zinc (Zn), copper (Cu), chromium (Cr), nickel (Ni), lead (Pb), and cadmium (Cd), in the juice of C. sinensis (variety Washington Navel) irrigated with primary treated sewage water was 23.25, 10.09, 6.50, 4.50, 0.45, 0.300, 0.25, and 0.200 mg/kg, respectively, of the dry weight. The results of another study indicated that the concentration of Fe was recorded as 4.044 ± 0.531 at sewage-water-irrigated location, while at the potable water site, it was notably lower at 0.241 ± 0.006 μg g⁻¹ [13]. The mean values of metals like iron, manganese, zinc, lead, copper, chromium, nickel, and cadmium in the juice of C. sinensis (variety Laring) irrigated with primary treated sewage water were 26.34, 14.3, 7.5, 5.4, 0.3, 0.4, 0.32, and 0.35 mg/kg, respectively, of the dry weight [47].

The concentration of Mn in the fruit samples ranged between 0.187 and 0.405 mg/kg. The concentrations of Mn in fruit samples irrigated with SW exceeded the permissible limit of 0.3 ppm [53], while those irrigated with TW remained below the limit. Rasool et al. [55] had similar results for Mn in C. paradisi (0.52 ± 0.05 mg/kg) and C. reticulata (0.40 ± 0.04 mg/kg) in Sargodha. Brima and Mohamed [56] reported that the concentration of Mn in C. reticulata and C. paradisi was 0.33 ± 0.09 and 4.57 ± 0.6 mg/kg, respectively, in the fruit juice of the two varieties. Ghani et al. [24] stated that the quantity of Mn varied from 0.051 ± 0.01 to 0.163 ± 0.05 mg/kg in different tehsils of the Sargodha district; the highest concentration of Mn was present in tehsil Silanwali in the Sargodha district. Soceanu et al. [61] reported similar findings of Mn mean level in orange pulp (0.1075 mg/kg) and grapefruit pulp (0.2355 mg/kg). Galal et al. [60] stated that the mean concentration of Mn in the pulp of navel oranges was 1.85 mg/100 g at the SW-irrigated site and was not detected in the pulp of navel oranges irrigated with fresh water. All the current study values are also higher than the maximum allowable limit of 0.30 mg/kg devised by WHO and FAO. Despite the presence of Mn in the soil and water samples, a much smaller quantity of Mn was present in the fruits’ edible parts, as plants mainly accumulate these metals in the roots and translocate only a small fraction to their other parts.

The PLI value for Cu was greater than one in all the samples which showed that they could cause health hazards. The value of PLI was higher for SW than CW and TW. The bioconcentration factor was less than one which shows that a very small amount of Cu was accumulated in the edible part of the plant. The value of EF was less than one. DIM for Cu for all the fruit samples was very low. No health hazard was indicated due to the consumption of these citrus fruits with respect to Cu. HRI for all the fruit samples was less than one which suggests that there was no health effect posed by the consumption of these fruits [62].

For Zn, PLI was greater than one for SW while for CW and TW it was less than one. The bioconcentration factor for all the samples was below one which indicated that no health risk was posed. EF value was higher for C. reticulata than C. paradise and was less than one. DIM for all citrus fruit samples was below one, signifying safe consumption of these fruits as only a small amount of metals had accumulated in the citrus fruit. The HRI values remained significantly below one, indicating that the consumption of the studied fruit poses no health hazards [62].

The PLI values for Fe were less than one, showing minimal health risks. BCF values were less than one and there are no health risks associated with the consumption of eatables having PLI > 1 for the metal in question [62]. EF values were comparatively higher for C. reticulata than C. paradisi. EF values were less than one and they showed no significant effect. The values of DIM and HRI for all the samples were very low and the consumption of these fruits could cause no harm.

PLI values for Mn were greater than one for all the sites. There were potential problems of pollution due to the higher concentration of Mn. The bioconcentration values were, contrarily, less than one. EF values were higher in C. paradisi than C. reticulata. EF and HRI values were also less than one. DIM by the consumption of citrus fruits was negligible.

5. Conclusions

The various irrigation sources investigated in this study did not appear to significantly influence the mineral content of the fruits. It is noteworthy, however, that PLI for Mn exceeded one, indicating a potential pollution concern. On the other hand, the PLI for iron (Fe) was below one, suggesting a lower risk of pollution associated with Fe. The BCF, EF, DIM, and HRI values for all metals were found to be below one, which is within the permissible limits. Therefore, the consumption of these fruits does not pose any health risks, as the levels of all studied metals in the fruit varieties were below the maximum permissible limits recommended by reputable organizations such as the WHO and FAO.

The study results showed that heavy metal accumulation was higher as a result of sewage water application for all samples than the other irrigation waters. For this reason, it is important to emphasize the need for controlled monitoring of these metals, especially considering the long-term application of sewage water for irrigation. Prolonged use of sewage water may alter soil properties and potentially lead to health hazards. Therefore, continuous monitoring and appropriate measures should be implemented to ensure the safety and quality of the fruits.