Different Sources of Irrigation Water Affect Heavy Metals Accumulation in Soils and Subsequently on Physiological Determinants and Physico-Chemical Properties of Guava Fruits

Guava is a highly nutritious fruit and widely grown in tropical and sub-tropical regions of the world. Guava orchards are frequently irrigated with contaminated water in different regions of the world. In the present study, fruits of two guava cultivars, i.e., Gola and Surahi were collected from orchards grown in peri-urban areas of Multan, Pakistan, which were irrigated with different sources of irrigation i.e., canal water, tubewell water and sewage water. The soil (at four different layers), water, and fruit (at harvesting) samples were collected from the orchards for their physical, chemical, and heavy metals analysis. The results revealed that sewage water contained more amount of copper, zinc and nickel as compared to tubewell and canal water. The soil irrigated with sewage water also contained the highest copper, nickel and zinc contents, while tubewell water irrigated soils contained the lowest copper, nickel and zinc contents. As regards different soil layers, the top soil layer (0–15 cm) contained the maximum copper, nickel and zinc contents. In contrast, the minimum copper, nickel and zinc contents were estimated in the lower most soil layer (46–60 cm). The different physiological parameters of guava leaves i.e., photosynthesis (A net ), stomatal conductance (gs), transpiration (E), leaf chlorophyll uorescence in light (Fv'/Fm') and in dark-acclimated (Fv/Fm) and non-photochemical quenching in dark-acclimated (NPQ) were decreased with sewage water irrigation. Further, the sources of irrigation water signicantly altered the different physical and chemical properties of the fruits. It is concluded that sewage water improved the physico-chemical properties of fruits but these also had high amounts of different heavy metals. Further, the soil irrigated with sewage water also had high contents of different heavy metals.


Introduction
Generally, fruits and vegetables can replenish humans with an adequate amount of minerals and vitamins and are recommended by health organizations as an essential part of daily food intake. Fruits provide not only essential components of the human's diet but also other bioactive compounds such as natural antioxidants (Nicoli et al. 1999). However, agricultural commodities grown in suboptimal conditions or with substandard inputs can malnourish human with toxic quantities of these minerals. Intake of excessive levels of toxic metals causes gastrointestinal, pancreas and urinary bladder cancer. Cr in high concentrations causes skin rashes, stomach upset and ulcer, respiratory problems, weakened immune systems, kidney and liver damage, alteration of genetic material and lungs cancer (Avena 1979).
The concentration of Pb if found in maximum limits affects the nervous system, bones, liver, pancreas, teeth, gums and blood function (Abbas et al. 2010). The high concentration of Cd may cause severe tubular growth, kidney damage, cancer, diarrhea and incurable vomiting (Abbas et al. 2010). Ni toxicity causes lungs and blood cancer (Agarwal 2002). Increased level of copper in food can cause hepatic and kidney damage, hemolytic anemia and methemoglobinemia (Chugh et al. 1975). Even high nitrate (NO 3 − ) accumulation in plants is also harmful to humans, particularly to children (Ikemoto et al. 2002).
Developing countries like Pakistan is facing different environmental challenges because of the increase in urbanization. Growing population not only catalyzes the growth of cities, industry and transportation, it also disturbs the ecosystems, especially in peri-urban areas where major sources of contamination are air pollution, untreated wastewater and city and industry drains ushing into canals or rivers. As costeffective mass production of food is necessary to meet the demands of the growing economy and population, cheaply available untreated wastewater and canal water are frequently used for irrigation.
Besides, fertilizers containing As and Cd as an enrichment have added to the soil contamination. In Pakistan, polluted water is used for drinking and irrigation purposes because of shortage of water.
Approximately 80% of the diseases were due to unsafe drinking water in Pakistan and Azad and Jammu Kashmir (Javaid et al. 2008).
For the production of quality fruits, irrigation and fertilizer application have signi cant effects. Irrigation in arid and semi-arid conditions is required in a regular and adequate amount for commercial production of fruit crops. The need is usually met through canal, tube well, and sewage wastewater. The use of wastewater is an economical option but risky as it is loaded with salts, toxic metals and disease-causing microorganisms. Many farmers are irrigating their crops with sewage or such polluted water. Agricultural commodities are contaminated with heavy metals and causing several human health hazards. A major cause of heavy metal accumulation in soils near urban areas is irrigation with sewage water (Singh et al. 2004; Sharma et al. 2006Sharma et al. , 2007. Moreover, fruits and vegetables were also polluted with the uncontrolled application of different fertilizers and other agrochemicals (Oliver 1997 (Ang and Ng 2000). The highest accumulation of heavy metals was recorded in urban areas of orchards because of irrigated water is mostly contaminated (Farooq et al. 2008). Several methods were adopted for the treatment of wastewater treatment i.e. coagulation, ltration, ion exchange and adsorption (Priya et al. 2009). Further, food authorities were working on the control of sewage water applications to crops.
Guava (Psidium guajava L.) belongs to Myrtaceae family and is successfully cultivated in tropical and sub-tropical regions of the world (Gupta et al. 2011). It is a highly nutritious fruit crop after mango and citrus because of different vitamins (Mehmood et al. 2018). Guava is commercially cultivated in the Sindh and Punjab provinces of Pakistan with excellent fruit quality (Menzel et al. 1985). Guava is a rich source of vitamins i.e., A, B, C & K, minerals, different sugars, oxalic acid and lactic acid. Further, higher antioxidants properties were also recorded in guava fruit crops (Gull et al. 2012). However, the use of sewage water for irrigation purposes is widespread in guava orchards. Fruits orchard irrigated with sewage water may accumulate high amount of heavy metals in edible parts of fruit trees. Little research work on the application of sewage water is available in different fruit trees. In a view, concentrations of these metals in tree fruits are very low even when grown on contaminated soils. Othman  Therefore, in the current study, we determine the effect of different sources of irrigation water i.e., canal, tubewell and sewage on different heavy metals accumulation such as copper, nickel, zinc and cadmium in soil. Moreover, the effects of accumulation of these heavy metals in soil on physiological attributes of leaves and physico-chemical properties of the fruit of two guava cultivars were studied.

Plant materials
The experiment was conducted in the peri-urban areas of Multan, Pakistan. The preliminary survey was conducted to identify the different orchards irrigated with different sources of irrigation water, i.e. canal water, tubewell water and sewage water. The fruits of two guava cultivars, i.e. Gola and Surahi were collected at physical maturity from these orchards for further physical and biochemical analysis. These physical and biochemical analysis of fruits were conducted in the Postgraduate lab of the Department of Horticulture. The study was designed according to randomized complete block design (RCBD) with two factors (cultivar and irrigation sources) and three replications.

Heavy metals estimation in soil
For heavy metal estimation, soil samples were collected from guava orchards irrigated with canal water, tubewell water and sewage water at four different depths i.e. S1 = 0-15 cm, 16-30 cm, 31-45 cm and 46-60 cm by using an auger apparatus. All samples were brought in the laboratory and dried at room temperature without sunlight for three days. After that, the samples were dried in the oven for two days at 70 °C. After drying, soil samples were saved for further analysis. Heavy metals estimation in soil samples was made through diethylene triamine penta acetic acid (DTPA). For analysis, 20 g soil sample was added in 40 mL of DTPA (7.3 pH) and reading was noted through an Atomic absorption spectrophotometer (Thermo Scienti c 3000 series, USA). The reaction solution contained 1.96 g DPTA, 1.47 g calcium chloride and 12.65 mL tri-ethanol amine).

Heavy metals estimation in water
Water samples were collected from the above mentioned irrigation sources. Water samples were then ltered with Whatman lter paper No. 2. Atomic absorption spectrophotometer (Thermo Scienti c 3000 series, USA) was used for the estimation of heavy metal in water samples.

Heavy metals estimation in fruits
The fruits were collected from the orchards and dried at 65°C in an oven until their weight became constant. The dried fruits were then ground into a ne powder. Then, 0.5 g dried fruit samples were added in nitric acid and perchloric acid with the ratio of 3:1 and kept for overnight. After that, samples were kept at 200°C until colourless solutions obtained. The samples were then diluted by adding 25 mL of distilled water (Farooq et al. 2008). After digestion Cu, Zn, Cd, and Ni were determined using an Atomic absorption spectrophotometer (Thermo Scienti c 3000 series, USA).

Physiological attributes of plant leaves
Different leaf physiological parameters, A net , gs and E were measured with an infrared gas analyzer (LCi-SD, BioScienti c Ltd. UK). The Fv'/Fm', Fv/Fm and NPQ in leaves were measured with a chlorophyll uorometer (FluorPen FP-100, Photon Systems Instruments, Czech Republic). Different photosynthetic variables were measured between 9 am -11 am. In contrast, for dark-acclimated photosynthetic variables leaves were covered for one hour before taking the measurements.

Fruit physical attributes
The physical parameters of fruits, i.e., fresh and dry fruit weights were measured by using a digital weighing balance. The fruit diameter (equatorial and polar) was determined by using a digital vernier calliper.

Fruit biochemical attributes
Page 6/21 The fresh fruit pulp was used for the determination of different biochemical parameters. The total soluble solids (TSS) was measured using hand refractometer, pH and electrical conductivity (EC) were estimated through pH and EC meters, titratable acidity (TA) and vitamin C content were measured using a titration method (Ruck, 1963). Total phenolic content (TPC), antioxidant activity (AA) and antioxidant capacity (AC) were measured according to Anjum et al. (2018). Total carotenoid content was measured according to a method of Lee and Castle (2001) with little modi cations. The absorbance of the sample for carotenoid was measured using a spectrophotometer (UV-1900 UV/Vis) at wavelength 450 nm. Fresh fruit pulp was used for determining total avonoids in guava fruits according to a method as described earlier (Kaushik et al. 2013).

Statistical analysis
The analysis of variance was performed by using Statistix 8.1 (Tallahassee Florida, USA). At a 5% probability level, the least signi cant difference test was used for the comparison of means (Gomez and Gomez 1984).

Gas exchange and chlorophyll uorescence
Different physiological parameters were signi cantly affected by different sources of irrigation water. The A net , gs and E in leaves of both guava cultivars were high in tube well water irrigated orchards compared to sewage and canal water irrigated ones and the difference was signi cant. The low values of physiological attributes, as mentioned above, were observed in sewage water irrigated orchards (Fig. 1).
The Fv/Fm and Fv´/Fm´ were decreased in leaves of both guava cultivars irrigated with the sewage water.
In contrast, the NPQ was increased in leaves of both guava cultivars irrigated with the sewage water. The Fv/Fm and Fv´/Fm´ were high in tubewell and canal water irrigated orchards. In contrast, the NPQ was low in tubewell and canal water irrigated orchards (Fig. 1).

Heavy metals content in water, soil and fruit
The different sources of irrigation water i.e. canal water, tubewell water and sewage water contained the signi cantly different amount of heavy metals ( Table 1). The sewage water contained the highest amounts of different heavy metals i.e. Cu (0.08 ppm), Ni (0.40 ppm) and Zn (0.55 ppm), while the tubewell water contained the lowest amounts i.e. Cu (0.00 ppm), Ni (0.07 ppm) and Zn content (0.16 ppm).   (Table 3). Moreover, the heavy metal accumulation was not affected by the cultivars studied and the difference was non-signi cant. The cadmium was not detected in, all different sources of irrigation water, all layers of soils and fruits of both cultivars.

Effect of heavy metals on physical and organoleptic properties of guava fruits
Different physical properties of fruits like fresh and dry fruit weights and polar and equatorial diameters were not signi cantly affected by the different sources of irrigation water. However, the value of different physical properties was a bit high in fruits irrigated with sewage water (Table 4). For different organoleptic properties, avor, eating quality and texture were signi cantly better in fruits irrigated with sewage water when compared to other sources of irrigation. Further, the aroma was signi cantly better in fruit irrigated with canal water and shelf life was signi cantly better in fruits irrigated with tubewell water (Table 5).

Effect of heavy metals on biochemical properties of guava fruits
The different biochemical properties of fruits were signi cantly affected by the different sources of irrigation water. The pH (3.78), EC (3.63), titratable acidity (1.17 %), ascorbic acid content (80.58 mg/ 100 mL), total phenolics (511.32 µg GAE/mL), antioxidant activity (98.20 %) and total avonoids (274.01 QE/ 100 g) were high in fruits irrigated with sewage water (Table 6). In contrast, the values of the abovementioned attributes were lower in fruits irrigated with tubewell water. The values of TSS (11.00 Brix) and total carotenoids (6.67 µg/mL) were higher in fruits irrigated with canal water and lower in fruits irrigated with tubewell water.

Discussion
The guava orchards in periphery of Multan city are mainly grown under sewage water. The orchards grown with sewage water showed the excess amount of heavy metals in soil and also the accumulation of heavy metals in fruits. Heavy metals are very much injurious to human health. In this study, we explored the physiological attributes of trees and fruit analysis and heavy metal detection of guava orchards irrigated with canal water, sewage water and tubewell water. The photosynthetic rate was strictly restricted by the presence of toxic metals in plants. These metals signi cantly affected the biosynthesis of chlorophyll, photochemical reactions and enzymes of the Calvin cycle (Chugh and Sawhney 1999). In our ndings, the trees irrigated with sewage water showed the maximum amount of heavy metals, as a result, the guava plants showed a decrease in photosynthetic rate, transpiration rate, and stomatal conductance (Fig. 1). Our results are in line with the ndings of previous researchers that photosynthetic variables in plants decreased In our ndings, the plants exposed to sewage water showed less Fv/Fm and Fv´/Fm´ than tubewell water and canal water, which depicts that decrease in these variables showed the stress. The decline in Fv/Fm and Fv´/Fm´ of PSII indicates the photoinhibition caused by excess photon ux density when plants are exposed to environmental stress (Björkman and Demming 1987). Moreover, the increase in NPQ was observed in the plants irrigated with the sewage water. The increase in NPQ is mainly due to dissipation of damaging excess energy, which may also compete with photochemical quenching and reduce photosynthetic electron transport, as evidenced by the reduction in Fv´/Fm´ (Chaves et al. 2009).
These days, with the growth of the global economy and changing climate, water and soil contamination by different heavy metal has increased, which results in deterioration of the global ecosystem. The different sources of irrigation water can have different levels of minerals and heavy metals content. The heavy metals increasingly pollute the agricultural soil through different sources of irrigation water; hence, heavy metal toxicity is in owing into the food chain, which affects the food quality and safety for humans. Our results also suggested that heavy metals accumulation in soil were different with different sources of irrigation water. Sewage water contained the highest heavy metals content than tubewell and

Conclusion
Guava is a highly nutritious fruit crop with higher minerals and vitamins. Wastewater use is going to increase with time. The use of sewage water is also affecting plant health by disturbing its physiological attributes, and increase accumulation of toxic metals in fruits, which poses many threats to human health. Sewage water irrigation improved the quality of guava cultivars, which seems to be attractive to the farmers. However, at the same time, sewage water contaminated the soils, leaves and fruits with heavy metals in comparison to canal and tubewell water. Zinc and cadmium were not detected in any water, soil, leaf and fruit samples in the studied areas. Copper and nickel were high in soils, leaves and fruits of sewage water irrigated orchards. Canal and tubewell water irrigated orchards accumulated low metals contents in soils, leaves and fruits.