water Biogeochemical Behavior of Lead and Nickel as Inﬂuenced by Phosphatic Fertilizer Applied to Rice ( Oryza sativa L.) Cultivars Grown under City Efﬂuent Irrigation

: The hydrology of arid to semi-arid regions is substantially different from that in humid regions due to scarce hydrological data distributions with dry climates and strong evapo-transpirative conditions. In the perspective of global water shortage, food security for all of the living beings has become a matter of great concern. Efﬁcient use of water resources both in urban and rural environments and application of non-conventional water resources for irrigation are becoming increasingly important. In order to sustain crop production, the re-use of treated wastewater for irrigation of crops could be a good option. A pot experiment was set up to evaluate effects of different doses of di-ammonium phosphate (DAP) fertilizer on lead (Pb) and nickel (Ni) phytoavailability by two cultivars of rice irrigated with city efﬂuent. Experiment was conducted in a completely randomized design (CRD) each with three replications. The results showed effective immobilization of Pb with applied phosphatic fertilizer. Among all of the tested treatments, the most effective treatment to reduce phyto-availability of Pb was T 4 (248 kg P ha − 1 ) due to antagonistic interaction. While Ni showed inconsistent behavior with both synergistic and antagonistic interaction (biphasic) to applied phosphorus (P) rates. Data regarding various growth parameters such as plant height, number of tillers, shoot and root dry weights, straw and grain yields, and physiological attributes such as total chlorophyll contents, photosynthetic and transpiration rates showed signiﬁcant ( p ≤ 0.05) responses to P application. An increasing trend was revealed in determined parameters with increased P application rates, with the exception of decreased plant height. The conclusion of the present remedial approach indicated that addition of P can signiﬁcantly reduce the risks of rice grown under city efﬂuent irrigation containing high Pb and Ni contents.


Introduction
Pakistan lies in an arid and semi-arid region of the world and agricultural irrigation is prominently dependent upon Indus River system. Over 16 million hectares of land receive Therefore, the present work aims at effective use of P fertilizer in the immobilization/remediation of Pb and Ni growing coarse and fine/basmati rice cultivars irrigated with city effluent. Phyto-availability of Pb and Ni and their possible interactions, accumulation, and distribution patterns in the different tissues of rice plants are also investigated.

Soil Sampling and Its Characterization
A pot experiment was set up in the wire house at the Institute of Soil and Environment Sciences (ISES), University of Agriculture Faisalabad (UAF), Pakistan. Bulk soil was collected from an experimental area of the institute and was air dried, finely grounded with a wooden roller to pass through a sieve (2 mm), homogenized and stored in plastic containers for further physical and chemical analysis following Iqbal et al. [25].
A hydrometer method [26] with sodium hexametaphosphate [(NaPO 3 ) 6 ] as the dispersion agent was employed for particle size analysis. Using UDSA textural triangle, the class of soil texture was revealed.
Total metals in soil were extracted by a method described by Amacher [27] using a flame atomic absorption spectrometer (FAAS; Model Thermo S-Series, Model Thermo S-Series, Thermo Electron Corporation, Cambridge, UK). A sample of 1 g of air-dried soil was taken in a 50 mL conical flask with the addition of 10mL concentrated HNO 3 and was placed overnight. Next morning, this mixture was heated at 200 • C and cooled. After addition of 1 mL of HNO 3 and 4 mL of HClO 4 sample was again heated at 200 • C until fumes if HClO 4 appeared. Sample was cooled after fumes of HClO 4 appeared and heated again to 70 • C for one hour after adding 5 mL of 1:10 HCl. The sample was cooled again and 1% HCl was added to make a final volume of 50 mL. The sample was filtered through a Whatman filter paper no. 42 to get the filtrate for heavy metal analysis. The filtrate was extracted from saturated soil paste by applying negative pressure using a pressure pump. To avoid precipitation of salts during storage, sodium hexametaphosphate (0.1%) at the rate of one drop per 25 mL extract was added.
The AB-DTPA extraction method [31] was followed to determine plant available Pb and Ni by using FAAS. The determined important initial physical and chemical characters of the soil are presented in Table 1.

Characteristics of Faisalabad City Effluent
Thirty samples of raw city effluent were collected from Nallah from Faisalabad metropolitan with the help of plastic bottle attached to a long wooden stick. Concentrated HCl (5 mL) was inserted to each water sample to maintain low pH to avoid any type of Pb and Ni precipitation. Afterwards, Pb and Ni were determined in the laboratory using FAAS [7,10]; the obtained data are presented in Table 2. The electrical conductivity of the city effluent was 2.9 to 6.5 dS m −1 . Minimum and maximum concentrations of the Pb in city effluent were 0.015 and 1.137 mg L −1 , respectively, averaging at 0.08 mg L −1 in collected city effluent. The Ni concentration was relatively low in city effluent as compared with Pb. Minimum and maximum concentrations of the Ni were 0.022 and 0.073 mg L −1 , respectively, whereas average concentration of Ni was found 0.0437 mg L −1 in city effluent.

Rice Nursery and Its Transplantation
Seeds of two cultivars of rice (Shaheen Basmati and KS-282) were taken from Rice Research Institute Kala Shah Kaku, Shiekhupura, Pakistan. Healthy seeds were chosen from both cultivars and were grown in polythene lined trays containing washed sand. Sand was washed with tap and distilled water, twice each and with 1% HCl solution. Nursery was irrigated with distilled water. Then, four weeks old rice seedlings were transplanted at three seedlings per hill and four hills were maintained per pot.

Experimental Setup and Design
Glazed pots were filled with 10 kg of soil. Five treatments viz. control (T 0 ) phosphorus at 62 kg ha −1 (T 1 ), phosphorus at 124 kg ha −1 (T 2 ), phosphorus at 186 kg ha −1 (T 3 ), and with phosphorus at 248 kg ha −1 (T 4 ) were set up and arranged in completely randomized design (CRD). Three replicates were used for each treatment to increase experimental accuracy (Table 3). Rice crop was fertilized at 120: 62 kg ha −1 of N: K as Urea, and K 2 SO 4 (SOP), respectively. While P as DAP was applied at four different rates as mentioned in Table 3, whole of P 2 O 5 and K 2 O was added at transplanting whereas urea was applied thrice in the same dose: 1/3rd at transplanting, 1/3rd after 25 days of transplanting and 1/3rd after 45 days of transplanting. Effluent samples were taken at each irrigation for analysis of Ni and Pb concentrations using FAAS.

Plant Sampling and Analysis
Randomly selected flag leaves in pots were used to measure transpiration rates, photosynthesis rates and total chlorophyll contents of both the rice cultivars. Leaf total chlorophyll content index in terms of Special Products Analysis Division (SPAD, a division of Minolta) values were determined, from leaf tip to leaf base via a handheld SPAD-502 meter (Minolta, Osaka, Japan). The photosynthetic and transpiration rates were measured using a portable infrared gas analyzer (IRGA, LCA-4, Analytical Development Company, Hoddesdon, England).
The rice crop was harvested at harvest maturity. Plants were dug out carefully from the pots. Roots were gently washed using tap water to remove attached soil particles and were then washed with distilled water. The uprooted plants were divided into roots, shoots, and paddy. Root and shoot samples were blotted in filter paper sheets, collected in separate paper bags, air dried and oven dried up to a constant weight at 65 • C. Oven-dried root and shoot were finely grinded with stainless steel Willey grinding machine and stored for analysis. Rice paddy were separated manually and weighed. Root and shoot dry weights of rice were also recorded. Soil samples from each pot after harvest were kept for further Pb and Ni analysis, and rice samples were also used for metal determination [32].
From this dried plant material, one gram was taken and digested by addition of 5 mL of concentrated HNO 3 and 5 mL of HClO 4 in a conical flask and was kept for overnight. The very next morning, 5 mL of concentrated HNO 3 was added again for digestion a hot plate until the solution became clear. Following digestion, the resultant material was cooled before being diluted to 25 mL by adding distilled water and was stored in air-tight bottles and was kept for analysis of Pb and Ni via FAAS.
The Pb or Ni uptake (mg pot −1 ) by root or shoot or paddy was computed by Pb or Ni concentration (mg kg −1 ) in root or shoot or paddy × root or shoot or paddy yield (g pot −1 )/1000 [7,10].

Statistical Analysis
All the obtained data were subject to analysis of variance (ANOVA) at a significance level of p ≤ 0.05. The LSD test was used to differentiate between applied treatment effects [33] using M-STAT Version 1.10 computer based software package.

Results and Discussion
City effluent is a prosperous basis of plant nutrients and organic matter as well as contains heavy metals which when enter into food chain cause severe health problems. Phosphorus application may be one option for immobilizing heavy metals including Pb and Ni. In this section, under city effluent irrigation, rice growth, physiological responses and metals uptake by different rice plant parts as affected by varying P application rates have been presented and discussed.

Rice Growth and Yield Responses
Addition of P along with city effluent irrigation brought a gradual increase in rice growth and yield attributes. Rice showed a positive response to increased P rates. Coarse rice cultivar (KS-282) produced more biomass as compared to fine cultivar (Shaheen Basmati).

Number of Tillers per Hill and Plant Height
Effects of P application were found to be significant (p ≤ 0.05, Table 4) on the number of tillers per hill ( Figure 1a) and plant height of rice (Figure 1b), irrigated with city effluent. The number of tillers increased significantly compared to the control. Maximum number of tillers was recorded with treatment T 4 in both cultivars, but fine Shaheen basmati cultivar produced more tillers, and its plant height was higher as compared with number of tillers and plant height recorded in coarse KS-282 rice cultivar. But statistically, both cultivars remained non-significant in producing number of tillers at control treatment. The treatments T 2 , T 3 , and T 4 differ significantly from each other and from control, but T 1 remained at par with control. The treatment order for number of tillers per hill remained T 4 ≥ T 3 > T 2 > T 1 ≥ T 0 . Maximum total number of tillers of rice with T 4 can be attributed to sufficient P concentration to support normal plant growth in the soil [34]. Maximum plant height (169.6 cm) and (164.0 cm) was recorded in Shaheen basmati and KS-282, respectively, with T 1 treatment while minimum with T 0 treatment (control). Mean plant height of cultivars remained in the order of T 1 > T 2 > T 3 > T 4 > T 0 . Crop yield is the product of yield components, of which total number of tillers is the most important component. A higher number of tillers reflects good crop stand [18]. Decrease in plant height with increase in P rates may be due to increased plant and soil metal owing to fertilizer application [35,36] which in turn decreased plant physiological processes. Brennan and Bolland [37] also observed increased Cd concentration of canola and wheat from P fertilization in a soil. In normal and salty Pb-anxious soils, Iqbal et al. [16] demonstrated that rice growth and yield was improved through phosphate amendments by forming Pb pyromorphite, thus reducing Pb bioavailability [38]. In another study, it Crop yield is the product of yield components, of which total number of tillers is the most important component. A higher number of tillers reflects good crop stand [18]. Decrease in plant height with increase in P rates may be due to increased plant and soil metal owing to fertilizer application [35,36] which in turn decreased plant physiological processes. Brennan and Bolland [37] also observed increased Cd concentration of canola and wheat from P fertilization in a soil. In normal and salty Pb-anxious soils, Iqbal et al. [16] demonstrated that rice growth and yield was improved through phosphate amendments by forming Pb pyromorphite, thus reducing Pb bioavailability [38]. In another study, it was also evident that applied DAP increased the development of Pb pyromorphite, resultantly; growth was increased due to the abridged Pb availability to plants [39].

Rice Root and Shoot Dry Weight
The root dry weight (RDW, Figure 1c) and shoot dry weight (SDW, Figure 1d) increased significantly (p ≤ 0.05, Table 4) with increasing P rates in both rice cultivars as affected by city effluent irrigation. The maximum RDW and SDW were obtained with treatment T 4 and minimum with control treatment. In fine Shaheen basmati cultivar, the highest SDW (94.4 g pot −1 ) was observed in T 4 treatment which was 33.1% increased relative to the control treatment. Shoot dry weight increased by 4.8, 14.3, 27.9 and 33.1% with increasing P rates (T 1 , T 2 , T 3 , T 4 , respectively) compared to control. In coarse cultivar, the increase in SDW was 3.9, 11.5, 16.3 and 25% over control with T 1 , T 2 , T 3 , T 4 , respectively. Likewise, in case of KS-282, the increase in RDW was 6.1, 17.3, 30.7, and 41.4% while in Shaheen basmati it was 9.8, 21.8, 33.3, and 41% relative to the control treatment (T 0 ). The two rice cultivars behaved differently for SDW and RDW; as fine Shaheen basmati cultivar showed more increase in SDW in comparison with coarse KS-282 cultivar. Mean SDW and RDW of both cultivars were found in the order of T 4 > T 3 >T 2 > T 1 > T 0 . Coarse cultivar produced more SDW compared with the fine cultivar.
In agreement with the present study, comparable results were reported by Jamali et al. [40] that coarse cultivar produced more SDW as compared to fine cultivar irrigated with city effluent. Overall KS-282 produced significantly more relative to Shaheen basmati. Increase in RDW and SDW with increasing applied P rates owing to highly soluble phosphate resource was seen to enhance the potential for pyromorphite formation [41,42] thereby promoting root development and photosynthesis, and extensive root and shoots growth with increased P rates [43]. To explore the relative toxicity or sensitivity of plants to toxic metal, the growth of root was a greater intended feature [44,45].
In agreement with present study, Shaheen basmati produced the highest root and shoot weights followed by KS-282 under Cd [44] and Pb stress [46]. Iqbal et al. [16] established that increasing application rates Ca and P based amendments steadily increased plant height, biomass of straw and paddy yield in normal and salty Pb-anxious soils. The positive influence of P based amendments on growth and yield of plants can be documented either due to improved nutrition of P and additional energy, otherwise, the impact of P to lessen toxicity of metal; or amalgamation of both mechanisms [38].

Paddy Yield and 1000 Grain Weight
Effects of applied P treatments and cultivars on rice paddy yield ( Figure 1e) and 1000 grain weight (Figure 1f) were found significant (p ≤ 0.05, Table 4) under city effluent irrigation. Paddy yield and 1000 grain weight increased significantly with increasing P rates, being found maximum with treatment T 4 . In KS-282, the increase in paddy yield was 3.7, 5.6, 11.1, and 22.2% compared to control. In fine cultivar the increase was 7.2, 11.6, 14.3, and 19.7% with its respective control. Total paddy yield increased gradually with increasing P rates and the highest paddy yield (32.8 g per pot) was recorded with treatment T 4 .
Coarse KS-282 cultivar produced a little higher 1000 grain weight at 248 kg P ha −1 as compared with fine Shaheen basmati. Maximum 1000 grain weight was recorded with T4 while minimum was with T 0 in both cultivars. In coarse cultivar the increase was 4.2, 14.2, 19.8, and 28.4% compared with that of control while in fine cultivar the increase was 5.5, 12.5, 15.2, and 21% as compared with the control. However, statistically both cultivars remained non-significant. The treatment's effectiveness to produce mean paddy yield and 1000 grain weight of both cultivars was observed in decreasing order of T 4 > T 3 >T 2 > T 1 > T 0 .
These results correlate with the findings of Jamali et al., [40] who found maximum wheat yield with the application of domestic sewage sludge at normal rates of nitrogen (N), P and potassium (K). City effluent contains organic matter, but increase or decrease in crop yield depends upon the nature of organic matter present in the effluent. If the organic material consists of easily decomposable fractions with low C: N ratio, it may have beneficial effects on crops yield, while organic matter with wider C: N ratio may immobilize N present in the soil or applied through fertilizer and accordingly may result in yield reduction [47]. Application of P enhances crop yield by promoting root development, crop maturity, water use efficiency, photosynthesis, N fixation, sugar translocation and plant disease resistance [43]. Iqbal et al. [16] also found that the applied DAP increased the straw dry matter, plant height and paddy yield in normal and salty Pb-anxious soils.
The increase in grain yields can be due to the beneficial effect of applied P on the metabolism of plants, however, extent of increase depended on plant species and their cultivars [45]. Patra et al. [48] further illustrated that plant species and crop genotypes demonstrate disparity in tolerance or sensitivity to metal bearing on metacentric or diploid chromosomes number and total length of diploid complement. Iqbal et al. [46] also derived that Shaheen basmati had shown better growth, yield and physiological functions in comparison with KS-282 due to genetic differences under diverse levels of applied Pb in normal and salty anxious soils

Rice Physiological Responses
In this study, under city effluent irrigation, physiological parameters such as total chlorophyll contents, photosynthesis rate and transpiration rate of both cultivars increased significantly (p ≤ 0.05, Table 4) with increasing P rates. The response of both cultivars for total chlorophyll contents ( Figure 2a) and photosynthetic rate (Figure 2b) statistically differed significantly from each other while their response for transpiration rate (Figure 2c) remained non-significant. Coarse and fine cultivars produced the highest total chlorophyll contents 36.8 and 36.4, respectively, with T 4 treatment. Mean increase in total chlorophyll contents was found in the order of T 0 > T 1 > T 2 > T 3 > T 4 .
The reason is the immobilization of heavy metals by P which in turn enhanced the chlorophyll contents. Similar results were reported in rice by Huang et al. [49]. They also reported that coarse rice cultivar produced more chlorophyll contents as compared to fine cultivar.
The increase in transpiration rate in KS-282 was 15.7, 34.1, 40.8, and 46.5% over its respective control treatment. However, in Shaheen Basmati the increase was 12.6, 22.6, 36.4 and 47% as compared with control. The reason may be that city effluent used for irrigation contains higher amount of plant nutrients and organic matter which increased plant growth that ultimately led to increased physiochemical processes of plants. The response of cultivars regarding transpiration rate remained non-significant. Mean increase in transpiration rate was remained in the order of T 0 > T 1 > T 2 ≥T 3 ≥ T 4 . organic material consists of easily decomposable fractions with low C: N ratio, it may have beneficial effects on crops yield, while organic matter with wider C: N ratio may immobilize N present in the soil or applied through fertilizer and accordingly may result in yield reduction [47]. Application of P enhances crop yield by promoting root development, crop maturity, water use efficiency, photosynthesis, N fixation, sugar translocation and plant disease resistance [43]. Iqbal et al. [16] also found that the applied DAP increased the straw dry matter, plant height and paddy yield in normal and salty Pb-anxious soils.
The increase in grain yields can be due to the beneficial effect of applied P on the metabolism of plants, however, extent of increase depended on plant species and their cultivars [45]. Patra et al. [48] further illustrated that plant species and crop genotypes demonstrate disparity in tolerance or sensitivity to metal bearing on metacentric or diploid chromosomes number and total length of diploid complement. Iqbal et al. [46] also derived that Shaheen basmati had shown better growth, yield and physiological functions in comparison with KS-282 due to genetic differences under diverse levels of applied Pb in normal and salty anxious soils

Rice Physiological Responses
In this study, under city effluent irrigation, physiological parameters such as total chlorophyll contents, photosynthesis rate and transpiration rate of both cultivars increased significantly (p ≤ 0.05, Table 4) with increasing P rates. The response of both cultivars for total chlorophyll contents ( Figure 2a) and photosynthetic rate (Figure 2b) statistically differed significantly from each other while their response for transpiration rate (Figure 2c) remained non-significant. Coarse and fine cultivars produced the highest total chlorophyll contents 36.8 and 36.4, respectively, with T4 treatment. Mean increase in total chlorophyll contents was found in the order of T0 > T1 > T2 > T3 > T4.
The reason is the immobilization of heavy metals by P which in turn enhanced the chlorophyll contents. Similar results were reported in rice by Huang et al. [49]. They also reported that coarse rice cultivar produced more chlorophyll contents as compared to fine cultivar.
The increase in transpiration rate in KS-282 was 15.7, 34.1, 40.8, and 46.5% over its respective control treatment. However, in Shaheen Basmati the increase was 12.6, 22.6, 36.4 and 47% as compared with control. The reason may be that city effluent used for irrigation contains higher amount of plant nutrients and organic matter which increased plant growth that ultimately led to increased physiochemical processes of plants. The response of cultivars regarding transpiration rate remained non-significant. Mean increase in transpiration rate was remained in the order of T0 > T1 > T2 ≥T3 ≥ T4. Deviations in chlorophyll contents of crop plants due to abiotic or biotic disturbances are interrelated to illustrate symptoms of plant disorders and photosynthetic capability [45,50]. With escalating DAP rates, the lethal impacts of Pb on physiological and gas exchange rates were steadily diminish can be ascribed to lessen Pb concentration in tissues of rice [16]. The halt of Pb noxious was persuaded by P fertilizer via dropping Pb solubility owing to configuration of pyromorphite in soils [51], whereas augmented application rate of DAP gradually lessens the Pb accumulation in rice [16].

Concentration of Pb in Root, Shoot and Paddy
The results showed Pb concentrations (mg kg −1 DW) decreased significantly (p ≤ 0.05, Table 4) in root (Figure 3a), shoot ( Figure 3b) and paddy (Figure 3c) at increasing P rates in both the rice cultivars under city effluent irrigation. Lead concentration was the highest with treatment T0 and the lowest with treatment T4 in both cultivars. Deviations in chlorophyll contents of crop plants due to abiotic or biotic disturbances are interrelated to illustrate symptoms of plant disorders and photosynthetic capability [45,50]. With escalating DAP rates, the lethal impacts of Pb on physiological and gas exchange rates were steadily diminish can be ascribed to lessen Pb concentration in tissues of rice [16]. The halt of Pb noxious was persuaded by P fertilizer via dropping Pb solubility owing to configuration of pyromorphite in soils [51], whereas augmented application rate of DAP gradually lessens the Pb accumulation in rice [16].

Concentration of Pb in Root, Shoot and Paddy
The results showed Pb concentrations (mg kg −1 DW) decreased significantly (p ≤ 0.05, Table 4  The highest Pb concentration in shoot of KS-282 was 3.9 mg kg −1 DW, while in Shaheen basmati it was 3.8 mg kg −1 DW with treatment T 0 . Furthermore, its concentration reduced to 2.8 mg kg −1 DW in KS-282 and 2.6 mg kg −1 in Shaheen basmati with treatment T 4 . In shoot, the decrease in Pb concentration in coarse cultivar was 27.4, 23.3, 15.6, and 9.7% as compared with its control treatment. While in fine cultivar the decrease was 30.4, 26.1, 15.4, and 7.4% with its respective control treatment. Lead concentration in paddy was found the lowest with T 4 both in case of coarse (1.30 mg kg −1 DW) and fine (1.03 mg kg −1 DW) cultivars. The decrease in Pb concentration in paddy in KS-282 was 31.0, 26.1, 15.1, and 5.9% with respect to control. While in of Shaheen basmati the decrease was 42.6, 39.7, 32.7, and 17.3% in paddy Pb concentration from its respective control. Significantly more Pb concentration in paddy was accumulated in KS-282. The less Pb accumulation in Shaheen basmati might be due to the fact that it immobilized Pb more effectively.
The response of both cultivars regarding immobilization of Pb remained significantly different from each other and Shaheen basmati immobilized Pb more effectively as compared with coarse cultivar. Mean decrease in Pb concentration in roots, shoots, and paddy were found in the order of T 0 > T 1 > T 2 > T 3 ≥ T 4 .
In present study, the low concentration of Pb in plant seems due to its low bioavailability, its retention by root surface and low mobility in soil owing to formation of Pb phosphate and other lead complexes in soil [52]. Zaragüeta et al. [53] also explored influence of longterm addition of sewage sludge to a calcareous soil on its total and bioavailable content of trace elements including Pb and Ni, and their transmittance to the tested barley and sunflower crops. Kartas et al. [54] assessed metal accretion in wheat irrigated by wastewater and reported that most of heavy metals (Cr, Mn, Pb, Zn, Ni and Cu) were retained in roots and their concentration was 4.2, 3.6, 42.8, 5.5, 1.7 and 40.1 mg kg −1 , respectively. Abbas et al. [55] also reported that sufficient quantities of trace elements (Zn, Cu, Fe, Mn) accumulated in rice straw and roots. Concentration of Pb in shoots and paddy was lower than that of Ni concentration. It was generally higher with the application of city effluent indicating antagonistic interaction of Ni with Pb absorption. It is reported that Ni had the enhancing effect on Pb in wheat straw [56]. The potential of other ions in also important on the stabilization of Pb (e.g., sulphate can reduce the solubility of Pb-phosphate) [57]. Reduced Pb uptake by Shaheen basmati can either be due to chelate secretion and/or its deposition of Pb in cell wall components [58].
Similar trends in results were also described by Iqbal et al. [59], Basta et al. [60], Cao et al. [61] and Bolan et al. [52] that Pb translocated to upper parts in very small amount as P immobilized Pb in soil due to phosphate induced Pb adsorption or precipitation with solution P as metal phosphates. Other reason for low Pb concentration in paddy could be the Pb accumulation mostly on cell walls of roots and permit restricted quantity of Pb for translocation to shoots, resultantly, less loading of grains with Pb [49]. Plants such as barley and rice did not absorb much Pb or transport Pb to grains [62].

Concentration of Ni in Root, Shoot and Paddy
The concentration of Ni in root (Figure 3d) of both cultivars remained non-significant (p ≤ 0.05, Table 4) as these accumulated statistically the same concentration of Ni (i.e., 23.9 and 23.3 mg kg −1 , respectively). Effects of treatments were found statistically significant. Mean Ni concentration was decreased in roots in the order of T 2 ≥ T 3 >T 0 ≥ T 4 > T 1 . Treatment T 2 exhibited maximum decrease in Ni concentration. Among rice plant parts, Ni was mostly retained in roots.
The present results were in agreement with Wang et al. [63], who recognized significant variations for Ni accumulation among 72 rice varieties. The Ni concentration in rice shoot was found to be certainly associated with its translocation, however, not with Ni concentration in roots (due to diverse Ni-transport capabilities of rice varieties rather than the restricting Ni in roots). Andreeva et al. [64] reported that distribution of Ni in plants is dependent on the plant's developmental stages and most of Ni accumulation was found in roots of oat (Avena satia L.) at booting and tillering stages.
In the present study, the response of both rice cultivars remained inconsistent for Ni in root and shoot against the applied P treatments. The Ni showed both synergistic and antagonistic interaction with P application. Nickel concentration in shoot (Figure 3e) of KS-282 increased with T 1 but it decreased with T 2 , T 3 , and T 4 .
Statistically Shaheen basmati accumulated more Ni (14.7 mg kg −1 DW) as compared with KS-282 (14 mg kg −1 DW). The effect of treatments was statistically non-significant. The effectiveness of treatments for decreasing Ni concentration in shoot was in the order of T 0 > T 2 ≥ T 3 > T 1 ≥ T 4 . Overall, Ni accumulation was statistically more with control while the other treatments remained at par with each other.
Recognition of genetic variability among cultivars to take up essential and/or toxic substances in plant tissue is measured as satisfactory criterion [46,65]. A maximum permissible level of Pb in rice grain is 0.2 mg kg −1 [66]. Marschner [12] concluded that increasing P concentrations in growth environment can decrease the concentrations of Mn, Zn and Ni in typical soil. The present study results are in disparity with these reports since the maximum level of P we used in experiment was 248 kg ha −1 while some scientists have used P @ 2300 mg P kg −1 soil for immobilizing metals. Cao et al. [67] and Basta et al. [60] reported phosphate to be effectual for immobility of Pb than for Zn, Ni, Cu, and Cd. This may be the reason that Ni concentration in paddy, shoots and roots was more than Pb since Ni was not effectively immobilized due to its interactive effect with Pb under phosphate applications.
The Ni concentration in paddy (Figure 3f) of both coarse and fine cultivars had nonsignificant (p ≤ 0.05, Table 4) response to P application. Application of treatments i.e., T 0 , T 1 and T 2 had similar response, whereas, treatments T 3 and T 4 were significantly different from the each other for Ni concentration in paddy. Statistically both cultivars remained non-significant as they accumulated almost similar concentration of Ni (2.7 mg kg −1 and 2.74 mg kg −1 ). While the interactive effects of treatment and cultivar also remained non-significant.
Khoshgoftarmanesh and Kalbasi [68] established that with garbage leachate Ni < 1 mg L −1 applied at 600 t ha −1 , the paddy Ni concentration is found not greater than 4 mg kg −1 . Kashem and Singh [36] observed that solubility of Ni decreased by two to five times in P amended soils due to formation of metal complexes. Less concentration of Ni in grains than that in shoot indicates some physiological barrier which restricts the transport of Ni to grains [69].

Post Harvest Soil Pb and Ni Concentration
Lead was significantly (p ≤ 0.05, Table 4) immobilized in post rice soil with increasing P application rates (Figure 3g). Lead was effectively immobilized with T 3 (36.3%) which was 3.52 mg Pb kg −1 soil and with T 4 (39.3%) which was 3.3 mg kg −1 soil. Increasing P rates increased immobilization of Pb in soil under both cultivars, but both cultivars remained statistically non-significant. Mean increase in Pb immobilization in soil was in the order of T 0 > T 1 ≥ T 2 ≥ T 3 ≥ T 4 . Maximum Pb was immobilized with T 4 which was 39.4 % of the control.
Phosphorus treatments have been shown to exhibit effectiveness in chemical immobilization of Pb [60]. Application of DAP significantly reduced (92%) the bioavailability of Pb [39]. Lead in soil was immobilized mainly as Pb phosphate as reported by Bolan et al. [52], Cao et al. [61]. Other mechanism of immobilization includes direct metal absorption and phosphate anion induced metal absorption. Another reason can be that when superphosphate (SSP) are added in soil, monocalcium phosphate (MCP) dissolves and forms soluble dicalcium phosphate (DCP) and releases phosphoric acid in the vicinity of fertilizer granules. Phosphoric acid can dissociate into hydrogen ions (H + ) and phosphate. The protons can cause a reduction in pH around fertilizer granules down to very low rates and this lower pH leads to increased dynamics of Pb minerals and subsequent release of Pb [52].
The effect of treatments on Ni immobilization in post rice soil (Figure 3h) was found non-significant (Table 4). Statistically all of the applied P treatments had shown same trends in results. There was no immobilization of Ni with increasing P rates. Cultivars had non-significant effect with each other. The interactive effects of treatments and cultivars were also non-significant.
The present results can be due to the fact that P was effective in remediating Pb, Zn, Cu, and Cd (and to a very little extent Ni) [60]. Another reason might be the lower rates of P used in our experiment. Mcgowen et al. [70] evaluated the performance of DAP as chemical amendment and reported that among different concentrations applied, 2300 mg P kg =1 was most valuable for immobilization Pb, Zn, and Cd. Iqbal et al. [16] established that increasing application rates of DAP gradually decreased AB-DTPA extractable Pb in post-rice soil in normal and salty noxious soil conditions. Equivalent tendency was noted for AB-DTPA Pb extractability in post-wheat soil under normal and salty soil environment [71].

Accumulation Pattern of Pb and Ni in Root, Shoot and Paddy
Similar behavior for Pb uptake by KS-282 and Shaheen basmati was observed, with applied P treatments receiving city effluent irrigation. The KS-282 showed maximum uptake (0.58 mg pot −1 ) in roots (Figure 4a) with control treatment (T 0 ) which decreased significantly with increasing P rates and the lowest uptake (0.43 mg pot −1 ) was recorded with T 4 treatment (248 kg P ha −1 ). Similarly, maximum Pb uptake (0.35 mg pot −1 ) in shoots was seen in T 0 and decreased to the lowest value of (0.25 mg pot −1 ) with T 0 treatment. Furthermore, maximum uptake of Pb by paddy was 0.10 mg pot −1 with T 0 decreasing to 0.04 mg pot −1 with T 4 .
non-significant effect with each other. The interactive effects of treatments and cultivars were also non-significant.
The present results can be due to the fact that P was effective in remediating Pb, Zn, Cu, and Cd (and to a very little extent Ni) [60]. Another reason might be the lower rates of P used in our experiment. Mcgowen et al. [70] evaluated the performance of DAP as chemical amendment and reported that among different concentrations applied, 2300 mg P kg =1 was most valuable for immobilization Pb, Zn, and Cd. Iqbal et al. [16] established that increasing application rates of DAP gradually decreased AB-DTPA extractable Pb in post-rice soil in normal and salty noxious soil conditions. Equivalent tendency was noted for AB-DTPA Pb extractability in post-wheat soil under normal and salty soil environment [71].

Accumulation Pattern of Pb and Ni in Root, Shoot and Paddy
Similar behavior for Pb uptake by KS-282 and Shaheen basmati was observed, with applied P treatments receiving city effluent irrigation. The KS-282 showed maximum uptake (0.58 mg pot −1 ) in roots (Figure 4a) with control treatment (T0) which decreased significantly with increasing P rates and the lowest uptake (0.43 mg pot −1 ) was recorded with T4 treatment (248 kg P ha −1 ). Similarly, maximum Pb uptake (0.35 mg pot −1 ) in shoots was seen in T0 and decreased to the lowest value of (0.25 mg pot −1 ) with T0 treatment. Furthermore, maximum uptake of Pb by paddy was 0.10 mg pot −1 with T0 decreasing to 0.04 mg pot −1 with T4.  T0  T1  T2  T3  T4  T0  T1  T2  T3  T4 KS-282 Shaheen basmati (b) Ni uptake (mg pot -1 ) by rice In Shaheen basmati, maximum uptake (0.39 mg pot −1 ) was recorded by roots with T 0 which decreased with increasing P rates to the lowest value of 0.26 mg pot −1 . Similarly, Pb uptake by shoots was maximum (0.27 mg pot −1 ) with T 0 and minimum (0.16 mg pot −1 ) uptake was observed with T 4 treatment. The lowest uptake of Pb (0.03 mg pot −1 ) by paddy was recorded with treatment T 4 (248 kg P ha −1 ). Results showed the Pb concentration in different parts of Shaheen basmati and KS-282 was in the subsequent sequence: as roots > shoots > paddy.
In agreement with the present study, the Pb uptake by rice shoots and paddy of Pb grown under Pb-noxious soils were notably higher than normal and salty soils. Iqbal et al. [16] estab-lished that the DAP was graded second effectual amen dement among tested amendments and its application lessened Pb concentration in rice shoot and paddy under normal and salty Pb noxious soils.
The Ni uptake by plant root, shoot and paddy was also significantly affected by P treatment rates receiving city effluent irrigation. The maximum (1.30 mg pot −1 ) recorded Ni uptake by shoots of rice cultivar KS-282 was with T 4 (248 kg P ha −1 ) treatment and its minimum uptake (1.12 mg pot −1 ) was with T 0 treatment (Figure 4b). Whereas, maximum (1.16 mg pot −1 ) Ni uptake by root was with T 3 and the lowest (0.75 mg pot −1 ) was observed in case control (T 0 ). Moreover, maximum Ni uptake in paddy was (0.09 mg pot −1 ) with T 4 treatment in both cultivars. In Shaheen Basmati, maximum Ni uptake in shoots was 1.46 mg pot −1 with T 4 (248 kg P ha −1 ) and minimum (1.08 mg pot −1 ) was recorded with T 0 treatment. Likewise, maximum Ni uptake by roots was (1.09 mg pot −1 ) with T 4 and minimum uptake (0.69 mg pot −1 ) was observed with control treatment. The both cultivars remained inconsistent to applied P rates and that most of Ni was accumulated in shoots followed by roots and paddy.
Aslam et al. [72] explained that based on heavy metals tolerance responses and mechanisms, the cereal crops can be categorized into three classes: (i) metal excluders, (ii) metal indicators, and (iii) metal accumulators. The metal 'excluders' use avoidance policy towards the accumulation and translocation of metal in their shoots. The metal 'indicators' accumulate toxic metals systematically in aerial parts or shoots. The metal 'accumulators' related to absorbance, translocation and ultimately these accumulate toxic ions, at levels higher than those recognized in soil medium, in diverse parts of their shoot tissues. In procession with present pot study, Murtaza et al. [24] revealed that N and P fertilization had significantly influenced the difference tissues concentration of Cd, Cu, and Zn in wheat and maize crops. Undue Ni in plants restrain photosynthestic system due to disturbed electron transport chain and assimilatory CO 2 [73], openings and closings of stomata and conductance mechanisms [74]. Nevertheless, the accumulations of Ni in edible ingredients pollute the foodstuff succession, hence menacing health of human at risk [75]. Aziz et al. [76] investigated that the noxious impacts of Ni on rice growth, physiology, and Ni concentration in rice and illustrated the ameliorative functions of Ca on Ni toxicity to plants. Nickel is reported to induce the deficiency of Fe and Zn, and hampers the uptake of Cd, Co, Cr, and Pb [10].

Conclusions
In present study, the addition of P has been evaluated in soil irrigated by city effluent, and the bioavailability of Pb and Ni under their interactions in the presence of P in two cultivars of rice has been investigated. Lead was effectively immobilized in soil, showed antagonistic interaction with P and its translocation to upper plant parts was significantly decreased with applied P rates. The Ni showed both synergistic and antagonistic interaction (inconsistent behavior) to P applications. The Ni showed more bioavailability and most of it was accumulated in the shoots and roots of rice, while Pb was mostly retained in the roots. The increased Pb immobilization in soil was found in the order of T 0 > T 1 ≥ T 2 ≥ T 3 ≥ T 4 . The addition of P at higher rates also promoted rice growth parameters significantly with the exception of decreased plant height, while the physiological functions of both the rice cultivars were increased with increased P rates. Increasing rate of P along with steady level of NK appears to be an effective and viable option for immobilization of Pb in effluent irrigation. This remedial approach can considerably reduce the risks of growing rice with city effluent containing high Pb and Ni. There is need to carry out more detailed work to assess and establish a sound basis for the suitability and economic feasibility of P for the immobilization of Pb and Ni receiving city effluent for irrigation on farmer's field and further need to be verified with other rice cultivars and crop species.