Accumulation and translocation of eight trace metals by the different tissues of Abelmoschus esculentus Moench. irrigated with untreated wastewater

Due to water scarcity, the use of wastewater to irrigate crops is on the rise all over the world, including in Egypt (particularly untreated wastewater). The purpose of this study is to see if irrigation with untreated industrial wastewater in natural fields can cause Abelmoschus esculentus Moench. (okra plant) to accumulate and translocate eight trace metals (lead: Pb, cadmium: Cd, chromium: Cr, copper: Cu, iron: Fe, manganese: Mn, nickel: Ni, and zinc: Zn) in its different tissues. It was extended to look at the effects of wastewater irrigation on the farmed okra plants’ growth characteristics, nutrients, colors, and organic content. Two studied sites at South of Cairo have been investigated: the first site (29°42'31.17" N and 31°15'11.56" E) represented by five cultivated fields irrigated with Nile water (control) and the second site (29°42'37.87" N and 31°17'14.53" E) fields irrigated with effluent received untreated industrial wastewater. Three composite soil and irrigated water samples were collected from each site. Because of wastewater irrigation, soil and plant nutrients (nitrogen, potassium, and phosphorus) decreased significantly (at P < 0.01), whereas trace metals increased significantly (at P < 0.01 and P < 0.05) for soil and plant samples irrigated with untreated wastewater. Due to irrigation with untreated wastewater, there was also a significant decrease in okra growth metrics (at P < 0.05) and leaves photosynthetic pigments: chlorophyll a and b, and carotenoids (at P < 0.01 and P < 0.05). In the plant’s fruits (edible section) watered with wastewater, iron was the most abundant metal. Besides, Cd, Cu, Fe, Mn, Ni, and Zn concentrations were also in the phytotoxic range (42.57, 140.67, 2756.67, 1293.33, 1326.67, and 877.83 mg kg-1, respectively). All trace elements examined accumulate in the roots of wastewater irrigated okra (Bioaccumulation factor > 1). Okra plants, on the other hand, did not have an accumulated trace metals strategy in their shoots since the translocation factor was less than one. Because of substantial trace metal accumulation in their edible sections, the scientists advised against eating okra plants grown in fields watered with untreated wastewater.


Introduction
Food production continues to rise in response to rising population demands, posing a significant challenge for the agricultural community to raise food output by more than 70%. (Kannan and Anandhi 2020). Organic food production necessitates the availability of sustainable resources such as water and electricity. Due to water scarcity, untreated wastewater is commonly used to irrigate crops around the world, particularly in developing countries (Malakar et al. 2019). Untreated wastewater was the primary source of contaminated food crops, soil, and freshwater. Using contaminated industrial, municipal, or agricultural wastewater in crop irrigation alters soil quality and increases trace metals concentration in soil and crops (Malakar et al. 2019).
The sewage water and industrial wastes are drained to agricultural areas and used to irrigate a variety of vegetable crops. These industrial sewage effluents are a major source of nutrients and organic matter, but they also contain a variety of trace metals (e.g., chromium: Cr, cobalt: Co, lead: Pb, Cu, Cd, Fe, Mn, Ni, and Zn), which accumulate in the soil and are then transferred to plants growing in these polluted soils (Balkhair and Ashraf 2016;Nzediegwu et al. 2020). Because different crop plants have different capacities to remove and accumulate trace metals in their tissue parts (Slima and Ahmed 2020), continuous irrigation with wastewater raises trace metal concentrations in soil to toxic levels, which then enter the food chain by accumulating in crop tissues (Ahmed and Slima 2018). The buildup of trace metals in plants can damage the quality of vegetable crops cultivated in polluted soils, as well as have negative health consequences for the surrounding population (Dan et al. 2017;Khan et al. 2019). Trace metals enter the food system by crops growing in polluted soils uptaking and collecting these metals (Christou et al. 20).
Vegetables are a source of carbs, proteins, vitamins, minerals, and fibers, and they are an important part of human nutrition. Okra, also known as lady's finger (Abelmoschus esculentus Moench. ), is a Malvaceae fruit crop planted for its delicious immature fruits (Petropoulos et al. 2017). Okra is a valuable vegetable crop grown in tropical and subtropical climates, with origins in North Asia and East Africa. Okra seeds are used for oil production and as a non-caffeinated coffee substitute (Petropoulos et al. 2017); they contain a high fiber content that aids in blood sugar stabilization (Ngoc et al. 2008); they treat digestive disorders and colon health (Messing et al. 2014); they promote healthy pregnancy (Zaharuddin et al. 2014); they improve heart health and control body cholesterol level; they improve heart health and control body (Gemede et al. 2015). Okra is grown in Egypt for its immature fruits, which are used as human food. The total cultivated area is roughly 17510 acres, with an average annual production of 88.8 tonnes (Ministry of Agriculture and Land Reclamation 2019).
Heavy metal accumulation in plants is influenced by plant species and plant efficacy for metal absorption, as measured by transfer factors (bioaccumulation factor: BF and translocation factor: TF) (Eltaher et al. 2019). The potential of heavy metals to bioaccumulate in subsurface organs serves as a self-protective mechanism for plant shoots against dangerous poisonous trace metals (Ahmed et al. 2021). The accumulation of trace metals in edible sections of plants was caused by crop growing in polluted soil, posing serious health concerns to humans and animals (Shehata and Galal 2020). Because untreated industrial wastewater is used to irrigate crops in Egypt, the current study aims to assess the okra plant's ability to accumulate and translocate eight trace metals (Pb, Cd, Cu, Cr, Fe, Mn, Ni, and Zn) in various tissues as a result of irrigation with untreated industrial wastewater in natural fields. The okra plant is grown as Egypt's principal food crop and for export; as a result, it must be produced to a high standard. As a result, the findings of this study are applicable all over the world. To the best of the authors' knowledge, no investigations on okra plants growing in polluted areas vs reference (non-polluted) sites have been conducted in Egypt.

Soil sampling and analysis
The soil samples that support the growth of okra were collected from two sites in south Cairo in summer 2018. The first from El-Attef city (29°42'31.17" N and 31°15'11.56" E) as control (fields irrigated with Nile water) and the second from Ekhsas Gharb city (29°42'37.87" N and 31°17'14.53" E) as treatment (fields irrigated with effluent receives untreated industrial wastewater). Each site's soil samples were taken from the surface layer at a depth of 0-50 cm, air-dried, and sieved to remove pebbles and plant debris using a 2 mm sieve. According to Brower and Zar (1984), a soil water extract (1:5 w/v) was prepared for determining soil pH using a pH meter (Model 9107 BNORION type) and electrical conductivity (EC) as an indicator for soil salinity using an electrical conductivity meter (60 Sensor Operating Instruction Corning) according to Rowell (1994). To determine the soil nutrient content, the soil was digested using the acid digestion method, where one-gram soil sample was digested in 20 mL tri-acid mixture of HNO 3 /H 2 SO 4 /HClO 4 (5:1:1, v/v/v) for 8 hours at 80 °C, digestion was continued until the solution became clear, then the transparent digests were filtered using a 0.45 m pore size cellulose nitrate membrane filter paper (Mill (Pb, Cd, Cr, Cu, Fe, Mn, Ni, and Zn). The total soluble nitrogen (N) was determined according to Piper (1947) using Kjeldahl method and the molybdenum blue method was used to determine P content in soil samples using a spectrophotometer (UNICO Vis Model 1200, USA) at 660 nm in case of N and 700 nm in case of P. At the same time, the potassium (K) content was measured using a Flame Photometer (CORNING M410). All mentioned procedures were according to Allen (1989). The concentration of the investigated trace metals was determined according to APHA (1999) using Atomic Absorption Spectrophotometer (a Perkin-Elmer 3100).

Water sampling and analysis
Three composite water samples were taken from the surface of the Nile River (control) and a wastewater discharge that absorbs untreated industrial wastes from pottery and faience clay brick companies and flows into various irrigation canals. At the end of the growth season, water samples were collected in plastic bottles and stored in the refrigerator until evaluated. To evaluate the content of several heavy metals such as Pb, Cd, Cr, Cu, Fe, Mn, Ni, and Zn, water samples were acidified immediately after collection with nitric acid (1 mL HNO 3 /L) (APHA 1999). A dual pH/electrical conductivity meter (60) was used to assess water pH and total dissolved salts (TDS) according to Rowell (1994). Chemical and biological oxygen demand (COD and BOD, respectively) were used to assess the relative oxygen requirements for water samples. COD was measured using the titrimetric analyses method as reported by Pitwell (1983), while BOD was measured using the 5day biochemical oxygen demand method as given by Delzer and Mckenzie (2003), utilizing a BOD incubator (Presto's biochemical oxygen demand incubator "Prima series"). BOD is a metric for measuring organic pollution in water that looks at how quickly microorganisms in the water use dissolved oxygen (DO) as they digest organic contaminants. DO is measured in each sample at the start and end of a 5-day period in a dark incubator at 20°C. The difference in DO is the BOD result. Results are reported in mg (O 2 )/L.

Okra cultivation
Okra seeds were surface sterilized for 20 min with 3.5% sodium hypochloride and then washed several times with distilled water. For a higher yield, the seeds were sown in rows at a depth of ½ to 1 inch, with a spacing of 6 inches between each seed and 24 to 36 inches between rows. Drip irrigation was used to irrigate the okra plants because it delivers consistent moisture throughout the growth season. Plants in the early growth stage of the okra crop required 2.4 L/day of water, while plants in the fruiting stage required 7.6 L/day.

Sampling of okra plants
Under natural settings, samples of okra plants were taken from five fields (each about one acre) that were evenly dispersed in both study sites: El-Attef city as control and Ekhsas Gharb city as treatment. Ten quadrats of 4 m 2 were chosen at random from each planted field to sample okra plants.
The first was irrigated with water from the Nile River, while the second was watered with wastewater from the effluent, which receives untreated industrial wastes. At the end of the growing season, the plants were harvested and several growth indicators were measured (number of individuals per m2, stem length, root length, number of leaves per individual). We took samples and analyzed them. The collected samples were divided into root, shoot and fruits (edible part) for determining fresh weights of leaves and stems per m 2 , dry weights for leaves and stems per m 2 , fresh and dry biomass and productivity kg per acre. Dry weight for leaves and stems was carried out by keeping samples at 40 0 C for 3 days, according to Allen et al. (1986).

Plant analyses
The plant samples were separated into roots, shoots and fruits, then air-dried and grounded using the electrical mill to determine the concentration of different heavy metals (Pb, Cd, Cr, Cu, Fe, Mn, Ni, and Zn) and plant nutrients (N, P, and K), proteins and carbohydrates. One gram from each plant sample (control and polluted samples) was digested using the acid-mixture digestion method (Lu 2000). Ten mL of conc. HNO 3 (69%) and 0.5 mL hydrofluoric acid (40%) were added to 1 g of dry plant sample in a closed Teflon vessel at a temperature of 130 °C for 24 h. Digestion continues until the solution was clear. The digested samples were then filtered using 0.45 μm cellulose nitrate membrane filter paper (Millipore). The volume was diluted to 50 mL using distilled water. The concentration of different heavy metals in plant samples was measured using atomic absorption spectrophotometer (DW-AA320N), while nutrients (N, P and K) were determined as mentioned in section 2.1. Total soluble proteins and carbohydrates were measured spectrophotometrically (UNICO Vis Model 1200, USA) using Bio-Rad protein assay and the anthrone-sulfuric acid methods, respectively (Lowry et al. 1951 andUmbriet et al. 1959, respectively).
To determine leaf pigments (chlorophyll a, b, and carotenoids), 2 g of fresh okra plant leaves were extracted with about 20 mL of 50% acetone (v/v) in complete darkness (as light causes chlorophyll degradation) and stored at 4 °C overnight, after which the samples were measured spectrophotometrically (UNICO Vis Model 1200, USA) according to Metzner et al. (1965). Chlorophyll a = 10.3 E 663 -0.918 E 644 , Chlorophyll b = 19.7 E 644 -3.87 E 663 , and Carotenoids = 4.2 E 453 -(0.0264 chl.a + 0.426 chl.b). E is the absorbance at a specific wavelength (nm).

Data analyses
A paired-sample t-test was used to examine the differences between the studied variables for soil, water, and plant tissues in the studied sites. The data were checked for normality and variance homogeneity. Using SPSS software (version 23), the significance of changes in trace metals in different plant organs was assessed using one-way analyses of variance (ANOVA) and Duncan's test in mean multiple comparisons (SPSS 2006). The plant's ability to store a specific metal in relation to its soil concentration is referred to BF. It is calculated as follows: BF = C root / C soil , where C root and C soil represent the heavy metal concentration in the root and soil respectively. The relative translocation of metal from root to shoot of the plant is referred to TF and it is calculated as follows: TF = C shoot / C root , where C shoot and C root represent the trace metal concentration in the plant shoot and root, respectively (Galal et al. 2021a). The pollution load index (PLI) for soil irrigated with wastewater was calculated as follows: PLI = Cp / Cn, where Cp and Cn represent the trace metal concentrations in the soil irrigated with untreated industrial wastewater and soil irrigated with Nile water, respectively (Slima and Ahmed 2020).

Soil and water properties
Irrigating with untreated industrial effluent has a considerable negative impact on soil chemical characteristics ( Table 1). The TDS and trace metals concentrations in soil irrigated with untreated wastewater were significantly raised (P < 0.01 and P < 0.05, respectively), whereas the pH was dramatically decreased (P < 0.01). At the same time, the use of untreated wastewater in irrigation resulted in a substantial drop (P < 0.01) in soil nutrient concentrations (N, P, and K). Soil irrigated with Nile water had a neutral pH (pH = 7) and EC (1.84 μS cm -1 ), whereas soil irrigated with wastewater had a more acidic pH (pH = 5.78) and was more salinized (EC = 5.70 μS cm -1 ). Except for Fe, Mn, and Zn, which surpassed WHO's tolerable limits (WHO 1996), heavy metal concentrations remained within the permissible limits (Table 1). Furthermore, the PLI revealed that soil irrigated with untreated industrial effluent included high levels of trace metals; Zn had the highest PLI value (21), followed by Pb (16.92),and Fe (15.46).
When compared to Nile water (control), industrial wastewater was somewhat acidic (pH = 6.89), salinized (TDS = 2339 μS cm -1 ) and had high BOD and COD (312 and 639 mg L -1 , respectively). With the exception of Cr and Cu, which were within the range of permissible levels for untreated wastewater, all trace metals examined were considerably higher (P < 0.001 and P < 0.01) and were over the bearable limits (Table 2).

Plant analyses
Photosynthetic pigments Plants irrigated with untreated industrial wastewater had significantly (P < 0.05 and P < 0.01) lower levels of photosynthetic pigments (chlorophyll a and b) than control plants (Fig. 2). Carotenoids also  Fig. 3. In okra roots, the concentration of carbohydrates and proteins declined by 37.9 % and 33.3 %, respectively. At the same time, the concentration of carbohydrates and proteins declined by 26.8 % and 37.8 % in shoot tissues of okra plants irrigated with untreated wastewater. There is a significant increase (P < 0.01) in the concentration of eight studied trace metals for okra root and shoot tissues irrigated with untreated industrial wastewater compared to control (  (Table 4). Besides, fruits of okra plants irrigated with untreated industrial wastewater recorded a highly significant increase (P < 0.001) for all studied trace metals concentrations compared with control   (Table 5). The results showed that Fe (2756.67 mg kg -1 ) was the highest metal accumulated in the plant's fruits irrigated with untreated industrial wastewater, followed by Ni, Mn, Zn, Pb, Cu, Cd, and Cr.

Trace metal bioaccumulation and translocation
Metal BF values were generally higher than one in okra plants irrigated with untreated industrial wastewater, but TF values were less than one (

Soil and water properties
The availability, mobility, and transportation of heavy metals in the soil are influenced by soil pH and TDS levels, and the mobility of heavy metals rises with increasing TDS (Salman et al. 2019) and decreasing soil pH (Nzediegwu et al. 2020). When watered with untreated industrial wastewater, soil pH and TDS increased, indicating that the soil became acidic (pH = 5.78) and more saline (TDS = 5.7 μS cm -1 ). Because crops absorb and retain trace metals from polluted soil in their edible sections, increased metal mobility is detrimental for agricultural fields (Ahmed and Slima 2018;Shehata and Galal 2020;Slima and Ahmed 2020;Galal et al. 2021b, c).
Increasing the quantity of trace metals in soil influences the activity (growth and metabolism) of soil microbial populations, which might alter plant nutrition availability (Xie et al. 2016). The present study found that irrigation with wastewater reduced soil nutrient concentrations in soil polluted with trace metals. According to Derome and Lindroos (1998), heavy metals may limit mineral nutrient pools in soils by impeding mineralization processes. It is worth noting that dangerous trace metal concentrations were found to be many times higher than those found in soil irrigated with unpolluted water, exceeding FAO/WHO (2001) permitted limits. According to WHO (1996) and the Environmental Protection Agency (US-EPA 2006), the trace metals concentration in wastewater is above the safe limits, while Nile water may have a low concentration of heavy metals below the instrument's detection limit.

Growth parameters
Most of the development metrics of okra plants irrigated with untreated industrial wastewater were significantly reduced (P < 0.01 and P < 0.05). Heavy metal buildup in plant tissues resulted in physiological and biochemical alterations (Singh and Singh 1981;Fisher et al. 1981). According to Batool et al. (2014), heavy metals have an impact on morphological and physiological characteristics, as well as a reduction in plant growth indices. In the present study, all plant growth measurements including the number of individuals per m 2 , stem length, root length, number of leaves per individual, leaves fresh weight, stem fresh weight, leaves dry weight and stem dry weight showed a significant decrease under pollution stress. This result is in agreement with Mami et al. (2011), who reported a reduction in the growth parameters of tomato plant irrigated with water containing a high concentration of trace metals especially Fe, Pb and Cu. Likewise, Galal et al. (2021a) found a reduction in the growth parameters of Cyperus alopecuroides as a result of the high concentration of trace metals present in irrigation water. Similar results were reported on rice plants by Islam et al. (2006), who recorded a reduction in plant height and the number of tillers per pot and shoot dry matter weights due to irrigation with contaminated water. Many studies, such as Farahat et al. (2017) on wheat and maize, Ahmed and Slima (2018) on Corchorus olitorius L., Galal et al. (2018) on cabbage, Slima and Ahmed (2020) on Pisum sativum L., Shehata and Galal (2020) on cucumber, and Galal et al. (2021b) on Ricinus communis L., have reported reduced plant growth parameters due to irrigation with untreated wastewater. In this study, okra plants irrigated with untreated industrial effluent had lower fresh and dry biomass and production than okra plants irrigated with Nile water (control). This decline could be related to ecophysiological factors such as water availability or other negative factors such as trace metal pollution (Galal and Shehata 2016). According to Ahmed and Slima (2018), Corchorus olitorius irrigated with untreated industrial wastewater had worse plant development metrics due to less water and nutrient uptake, or because root protein synthesis was inhibited. Galal et al. (2021a) found that heavy metals in high concentrations reduced the biomass and productivity of Cyperus alopecuroides when irrigated with contaminated wastewater. Galal et al. (2021c) found that irrigation with untreated wastewater reduced the production of Pisum sativum. Batool et al. (2014) mentioned that Ni and Cd when present in high concentrations caused a decrease in the productivity and biomass of plants.

Plant analysis
Photosynthetic pigments Okra leaves showed a significant reduction in photosynthetic pigments (chlorophyll a and b) when irrigated with untreated industrial wastewater compared to control; this could be due to inhibition of enzyme activity, which would then inhibit metabolic processes (Zengin and Munzuroglu 2005;Shakya et al. 2008), particularly enzymes involved in chlorophyll biosynthesis (Żurek et al., 2014). Similar results were found in spinach (Saini et al. 2014), strawberry (Muradoglu et al. 2015), Corchorus olitorius (Ahmed and Slima 2018), and Pisum sativum (Slima and Ahmed 2020). Trace metals in wastewater used in agriculture irrigation reduce the sensitivity of the plant's chloroplast machine ( Nutrients and heavy metals Because of irrigation with untreated industrial wastewater, the concentrations of N, P, and K in different plant organs (root and shoot) have decreased significantly in the current study. Begum et al. (2011) reported on the effect of employing textile industrial effluent in rice crop irrigation and the loss in nutrients, which they attributed to the presence of high levels of Pb in irrigation water. Also, according to Osawa and Tajuke (1990), the decrease in the percentage of N was caused by an increase in the value of Cu, which had an antagonistic effect on N content. In this study, dirty water used to irrigate okra contained levels of Pb and Cu above the allowed limit, resulting in a decrease in okra N content. Trace metals also lowered nutrient absorption and harmed legume plants' ability to fix nitrogen (Singh and Kalamdhad 2011). The presence of large quantities of Pb, Zn, and Cu in the wastewater, which had an antagonistic effect with P and K, may have caused the crop's P and K content to fall (Muchrimsyah and Mercado 1990). The result of the present study agreed with Shukry (2001), Begum et al. (2011), Ahmed and Slima (2018) and Slima and Ahmed (2020).
In the current investigation, photosynthetic suppression resulted in a decrease in carbohydrate and protein concentrations, which matched the findings of Galal (2016) on Cucurbita pepo L. and Farahat et al. (2017) on maize and wheat irrigated with wastewater. Protein breakdown by increasing the activity of protease enzymes may result in lower protein content in plant tissues as a result of the presence of the plant under stress conditions (Palma et al. 2002).
The amounts of all trace metals tested in okra roots and shoots irrigated with untreated industrial effluent are significantly higher than in control, implying that the okra plant is a hyperaccumulator for all heavy metals detected in the study except Cr. All estimated heavy metals (except Cr) were discovered in excess of allowed levels and above the phytotoxic range in various plant tissues (root, shoot and fruits). All of these findings are consistent with Ahmed and Slima (2018) findings on Corchorus olitorius irrigated with wastewater, as well as Slima and Ahmed (2020) findings on pea plants.

Trace metal bioaccumulation and translocation
The mobility of trace metals from soil to plant and from plant root to plant shoot is calculated by calculating BF and TF, which is dependent on the nature of the plant (Slima and Ahmed 2020). Plants that have a BF value more than one prefer to store metals in their roots rather than move them to their shoot tissues, whereas plants that have a TF value greater than one prefer to transport and store metals in their shoot tissues (Ahmed et al. 2021). The difference in BF and TF values, trace metals accessibility, interactions between physicochemical characteristics and the plant cultivated in these polluted soils, and the plant uptake efficiency of trace metals are all influenced by the trace metals-binding capacity of roots (Slima and Ahmed 2020). Despite the high levels of examined trace metals in okra plant shoots and fruits (edible component) watered with untreated industrial wastewater, the current study found that okra plants acquire trace metals in their root tissues rather than their shoot tissues. Chelating metals may cause trace metal accumulation in root tissue rather than shoot tissue by creating phytochelatins or metallothioneins metal complexes at the intracellular and intercellular levels (Emamverdian et al. 2015). Because most metal ions are insoluble and unable to flow through the vascular system on their own, they are blocked in apoplastic and symplastic compartments after producing carbonate, sulfate, or phosphate precipitates in root tissues (Thakur et al. 2016).

Conclusion
This research aims to raise awareness about the dangers of utilizing untreated industrial effluent to irrigate okra plants. According to the findings, irrigation with untreated wastewater causes significant soil pollution and changes the physicochemical parameters of the soil. Using wastewater in the irrigation of the okra plant, on the other hand, harmed it (causing a decline in different growth parameters, declining NPK nutrition, and photosynthetic pigments) and led to heavy metals (Pb, Cd, Cr, Cu, Fe, Mn, Ni, and Zn) accumulation in its various plant parts (especially edible fruits) that exceeded the tolerable limits. Due to its capacity to accumulate heavy metals in its various organs, especially edible parts, the current study recommended that soil and plant pollution be monitored regularly and that okra plants not be grown in heavy metals polluted soil or soil irrigated with toxic metals contaminated water, as these toxic metals enter the food chain and cause many health harmful effects to consumers.