Typha Latifolia L. Grown in Carrying Petroleum Secondary E uent Supply Nutrients Enhance for Biomass Growth and Phytoremediation of Heavy Metals and TPH


 Phytoremediation is an innovative tool which can be used for the treatment of industrial and agricultural wastewater. Typha latifolia L. (T. latifolia L) is an aquatic plant which grows on petroleum secondary effluent (PSE) containing metals like cadmium (Cd), cobalt (Co), manganese (Mn) and TPH (total petroleum hydrocarbon). The growth performance in biomass, nutrient concentrations and heavy metals in parts of the T. latifolia L. The reason for the accumulation of Cd, Co and Mn in T. latifolia L. can be explained as a tolerance strategy due to its transfer index (TI) which is higher than 2.9. The enrichment coefficients of the metals present in the root compared to stem of T. latifolia L. were higher than 3.31 to 2.56 for Cd, 5.35 to 3.55 Co. But, for Mn were found to be lower 1.98 than 3.51 at 75%. Similarly, the enrichment coefficients of all the metals, except for Co, in roots of T. latifolia L. were higher than 5.36. (TI) for Co (2.95) and Mn (2.55) which is absolutely better as compared to the enrichment coefficients of Cd (2.35) and TPH (3.45) in PSE. Thus, there is a possibility that PSE could be a source of important nutrients.


Introduction
Industrial wastewater contains various metals, hydrocarbons and inorganic compounds which are a threat to soil and water systems. With the onset of the industrial revolution, metal and nutrient pollution has intensi ed swiftly which has posed a major environmental and health risk to the ecosystem (Ahmad, 2018;. As industrial pollution may cause major environmental and human health problems, we need to turn our attention to the effective and affordable tools required for the remediation of pollution from soil and water (Akpor et al. 2014; Kaumari and Tripathi, 2015). Heavy metals (HMs) and total petroleum hydrocarbons (TPHs) present in soil and water coming from the industrial processes, enters the food chain and ultimately the biosphere (Foshtomi et al. 2019; Al-Thani and Yaseen, 2020).
Furthermore, the heavy metals leach into the soil surrounding them which also includes the agricultural elds and passed to food chain (Arivoli et al. 2015). Considering the Hazardous consequences of toxicity due to metal contamination, feverish efforts have been made to phytoremediate metals from the biosphere, soil and water system (Ahmad et al. 2010;Brankovic et al. 2015).
Typha latifolia L. and certain species like parthenium, rye grass, brassica have been identi ed to have potential to sequester of heavy metals in maximum amounts and feasibility of phytosequesteration of heavy metals from soil ; Ahmad and Ahmad, 2014;Hammami et al. 2018). Typha latifolia L. plant have shown characteristics to faster growth of biomass with the sources available metals like Fe, Ca, P and nutrients from e uents, an under unfavorable condition to propagate growth rates compared to other hyperaccumulator plant for heavy metals (Banerjee et al. 2019; Afzal et al., 2019). Due to this characteristics, ecologically sustains and recommended as a suitable for phytoremediation of heavy metals and total hydrocarbon such as Co, Mn, Ni, and Cd and had high biomass (Chayapan et al. 2015). The ecological engineered way for bioaccumulation, enrichment factor and transfer of HMs and TPH by plant biomass from contaminated sediments (Ali and Chaudhury, 2016).
The micronutrients to change the physiological and molecular mechanisms responsible for metal hyperaccumulation and tolerance immunity in plant have been studied widely (Adeyeye, 2005;Truu et al. 2015).
Mechanism of pollution accumulation, it must be strongly emphasized that phytoremediations e ciency not only depends on plant factors such as metal tolerance, metal transfer, enrichment coe cient, biotransformation, metal accumulation, and so on (Yadav et al. 2018). It also depends on soil factors such as metal mobility and crucially, soil metals with wastes amendment and phytoavailability (Klomjek, 2016;Muthusaravanan et al. 2018;Zhaoet al. 2020). After all, rather than total concentrations, a major factor governing the phytotoxicity of metals in soil is their bioavailability (Pandey, Ahmad et al. 2020). Several aquatic and terrestrial plants is identi ed as a species able to phytoextraction of metals, nutrients and petroleum haydrocarbon from a multiply contaminated soil (Mustapha and Lense, 2018), take up and accumulate into its above-ground parts stem for the metals such as Cd, Co, Cu, Ni, Mn, Pb and TPH (Galal et al. 2017). In eearlier studies it has been reported that T. latifolia L has good tolerance and potential to withstand with higher levels of heavy metals and petroleum hydrocarbon (Samuel et  The aim of this study to investigation the effect of petroleum secondary e uent concentrations on T. latifolia L biomass and the phytoremediation of metals. Four dosage concentrations of PSE (25,50,75 and 100%) were tested in the vertical wetland. Analyses of T. latifolia L bionss growth, metals accumulation, transfer index, enrichment coe cients and nutrients role. Biomass growth and distribution of nutrients of chemical precipitates were monitored during the experimental periods

Physico-chemical parameters
Petroleum secondary e uent (PSE) was collected from Sur re nery, Oman. The petroleum is totally under the operation using extraction and re ne petrol from the crude oil production. The levels of various physicochemical and heavy metals (Co, Mn and Cd) and TPH determined in PSE are shown in Table 1 and 2. The lake water collected from Sur lake and analyzed the properties of pH 7.9, total dissolved solids 145 mgL -1 , total hardness 240 mgL -1 , calcium hardness 106 mgL -1 , dissolved oxygen 3.6 mgL -1 , chloride ion 83 mgL -1 , alkalinity 110 mgL -1 , Na 25, K 6 mgL -1 . The soil collected from university garden and had the properties of pH 7.4, electrical conductivity-1.13 dsm -1, total nitrogen (%) 0.09, total phosphorus (%) 0.78, organic carbon (%) 0.49 and Zn 23, Fe 5100, Mg 150, Ni 130 µg/g dry weight (DW), Pb and Hg non detectable.

Experimental design and performance
Typha latifolia L. were collected from Sur petroleum re nery, Oman. The plants were grown and the plant was raised in 75"/50" plastic rectangular tubs wetlands. After raised the plants, in four tubs having 25 plants were irrigated on per day with different concentrations of petroleum secondary e uent (PSE). For the treatment application, the different concentrations ratio was applied as 25% petroleum secondary e uent (PSE) (25% PSE + 75% lake water), 50% PSE (50% PSE + 50% lake water), 75% PSE (75% PSE + 25% lake water) and 100% PSE (100% PSE) serial order of required dilution, of the petroleum secondary e uent. Three samples were taken for each treatment. Plant grown in lake water served as control. The T. latifolia L. from each set of tub working volume is 25 l was placed under natural conditions as in open environment. The roots and stem from each plant were detached and washed repeatedly using tap water to remove unwanted debris and blotted. Fresh biomass contents were also recorded. For heavy metals (HMs) and TPH analysis in water and in plant parts, dried 1.0 g plant samples were ground in a grinder and digested in HNO 3 :HClO 4 (3:1, v/v) at 80 0 C. Metals (Co, Mn and Cd) were estimated by metals concentrations in the plant samples which were determined using Perkin Elmer Corporation Atomic Absorption Spectrophotometry (Perkin Elmer, AAS 1500) was used for calibration and quality assurance for each analytical batch. The detection limits of Co, Mn and Cd were 0.5, 1.0 and 0.01 µg/l, respectively. Replicate (n=3) analyses were conducted to assess the precision of the analytical techniques. Triplicate analysis for each metals varied by no more than 5%. I conducted the estimation biomass growth after 60 days of experimental periods plants were collected. The study of root, stem length, fresh and dry weight of root and stems. For the estimation of dry biomass kept at 85 o C for three days for weighted biomass.

Analysis of protein and carbohydrate
Plant samples for the estimation of carbohydrate (Murphy, 1958) and protein according to Ahmad et al (2019) 1 g of fresh plant tissues was crushed in 3 ml of potassium phosphate buffer (50 mM, pH =7.0) and centrifuged for 20 min. Supernatant (0.1 ml) was added to test tube and diluted with 1 ml of distilled water. From 5 ml of reagent C (Reagent A comprised of sodium carbonate (2%) in sodium hydroxide (0.1 N), Reagent B comprised of copper sulphate (0.5%) in potassium sodium tartarate (1%). Reagent C consists of 100 ml of reagent A and 2 ml of reagent B) was poured into the test tube and incubated at room temperature for 10 min. After this 0.5 ml of Folin-Ciocalteu reagent was added to the reaction mixture in the test tube followed by an incubation of 30 min at 30 o C at dark condition. To determine the protein and carbohydrate content, BSA (bovine serum albumin) standard curve was used, absorbance was analyses at 660 nm The concentration of protein and carbohydrate was expressed as mg/g fresh weight.
Bioaccumulation of HMs and TPH The bioaccumulation, translocation and phytoextraction of metals and TPH by the T. latifolia L. was assessed at the level in roots and stem. The method of total metals and TPH analysis in different plant parts were prepared as an according to methods APHA (1998) using atomic absorption spectrophotometry (Perkin Elmer, AAS 1500). Plant mass was being analyzed for transfer index (TI) including stem concent. Stem biomass include root concent. Root biomass divided by stem and root biomass multiplies days of growth Enrichment coe cient factor (ECF) The plant was analysed the enrichment coe cient factor (ECF) for stem and roots from soil values greater than or equal to 1.5 (Ef ≥ 1) expresses that it is a hyperaccumulator and decent for phytoremediation practice. It also indicates that the plant has a high ability to accumulate and tolerate a higher concentration of heavy metals and TPH in its petroleum secondary e uent. The enrichment factor (Ef) for various heavy metals accumulated in the tissue of T. latifolia L. after phytoremediation was calculated using Eq. 1.
where Cf and Ci are the mean metal and TPH concentration in the e uent sample and concentration of value in the tissue of the plant, respectively. The plants materials and water were collected and statistically analyzed. The treatments were carried out in triplicates and the data furnished in gures and tables are mean ± SE (Standarderror of the mean of three replicates).
Statistical analyses of samples for two-way analysis of variance (ANOVA) was done on all the data to con rm the variability of data and validity of results. Differences among means were determined by the analyses of variance. The SPSS (Statistic Program for Social Sciences) statistical program package (Release 12.0) was used for statistical analyses of data. Different letters (a-strongly signi cant and blower signi cant) mean signi cant differences between treatment and control at a part of plant, error bars indicate the standard error of individual measurements.

Results And Discussion
The physico-chemical, metals, heavy metals (HMs), and total petroleum hydrocarbon (TPH) properties of petroleum secondary e uent (PSE) are shown in Table 1 and 2. From the Fig. 1, was found to be PSE doses exposures. Bio-accumulation of HMs and TPH in parts of the plant at 75% was highest compared to 100% doses of PSE represented in Fig. 1. The bioaccumulation of TPH compared with metal was found higher and showed that the T. latifolia L Co> Mn> Cd, (Fig. 2), has the maximum 49 cm root and 105 cm shoot, the plant exhibited better growth at 75% of PSE. It's may be due to availability of macro nutrients and calcium, magnesium and iron in cellular level with the improvement of structure of chloroplast showed growth of biomass at lower concentration of Cd and Co. As a result, the roots were unable to deliver the nutrients to shoot, for which the growth of the plants were affected (Fig. 2).

Variation of HMs and TPH in plants
The accumulation of the HMs and TPH reduction was TPH>Co>Mn>Cd from petroleum secondary e uent (PSE) (Fig. 2). The levels of TPH and HMs accumulated in root and stem were signi cantly different in different parts of plant at different of PSE concentrations. However, the TPH and HMs concentrations in lake water to soil lower than the parts of the plant (Fig. 2). The TPH and HMs bioaccumulation reached up to 97.3% for TPH, 97.5% for Co, 91% for Mn, and 42.7% for Cd (Fig. 2). The accumulation of TPH and HMs was observed in this experiment found higher compared to other studies The results of this experiment showed that T. latifolia L plant have the biomass of dry weight was 45g at 25%, 78g at 50%, 97g at 75% and 23.9 g at 100%, so that at 75% dosing PSE showed highest biomass compared with lower and 100% PSE (Fig. 3). The plants biomass was observed in Fig. 3, biomass growth totally depends on PSE concentration at 75%, biomass 97 g/Kg highest than lower and higher concentration of PSE compared with control. Thus, the biomass development of the plant was signi cant at 25 and 75% dosing as compared to the biomass of at 100% PSE, as a result biomass growth increased constantly on all the amendments except at 100% PSE dosing (Fig. 3).

Transfer index for TPH and HMs
Transfer index factor can be used to estimate a plant's potential for phytoremediation purpose in T. latifolia are shown in Table 3. The bioaccumulation of TPH and HMs rate depend on PSE doses and biomass. The transfer index (TI) increases with increase in the concentration of TPH and HMs but the bio-concentration in plant decreases with the application of higher concentration of TPH and HMs ( Table  3). The maximum value of TI, was observed 1.9 at 75% dosing. Bioaccumulation of TPH and HMs in biomass to transfer via transportation index (TI) can be considered as an effective tool for identi cation of hyper accumulator species. The uptake of metals from soil to plants and then translocation those to aerial parts allow to determine the capabilities of individual species to accumulate the metals and therefore to recognized as a potential hyper accumulator (Rana and Maiti, 2018; Ahmad et al. 2020). The TIs changed between 0.65 to 2.95 for HMs and for TPH between 1.15 to 3.45 from 25-75% dosing of PSE, whereas at decrease at 100% compared to control ( Table 3). The mean TI for Cd, Co and Mn in T. latifolia L. was higher than 2.56 at 75% dosing of PSE, but mean TI for higher dosing metals were generally lower due to toxicity and did not effectively transfer heavy metals from root to plant body ( Table 3). The TPH for TI at 75% dosing was 3.45 compared to higher dosing lower transfer due to availability with plants parts (Foshtomi et al. 2019;Ahmad et al. 2020). The plant parts having ability was in the order of TPH>Co>Cd>Mn. The values of TI in different dosing of PSE indicated that different metal has different phytotoxic effect on T. latifolia (Table 3). Ahmad et al. (2010) and Ahmad and Ahmad (2014) observed that TI higher than 2.0 were determined in metal hyper accumulator plants whereas TI was lower than 1.5 in metal accumulator plants. In this study TI higher than 2.0 indicates an e cient ability to transport metal from soil to root to stem, most highly effective remediate metals due to e cient metal transporter systems through its biomass (Ariolo et al. 2015; Klomjek, 2016).

Enrichment coe cient for the root and stem
The plant showed bio-concentration of TPH and HMs indicates the e ciency of the plants to eliminate the TPH and HMs metals from the soil to plant. The enrichment coe cients roots (ECR) in the roots of T. latifolia L. were higher for 5.10>4.05 TPH, 3.31>2.56 Cd, 5.36>3.55 Co at 75% of dosing PSE compared to 100% found lower due to bioavailability toxicity (Table 3). This characteristics of plant means that the roots of T. latifolia L. showed highest capacity for TPH>CO>Cd Mn.. However, the bioaccumulation of TPH and HMs by the root of T. latifolia L. was higher due to ECR more than 3.0 except for Mn (Table 3). Enrichment coe cients are a very important factor, which indicate phytoremediation of a given species  Table 3). The TPH and HMs concentrations in root>shoot were generally higher than that in soil (Table 3). Researcher supported our ndings to this situation indicated a special ability of T. latifolia L to with stand bioaccumulate, transfer of TPH and HMs from PSE soil and well grown of biomass their root and stem ( (Fig. 4). However, the contents of Fe, Mg, Ca and other important metals for plant growth, at 50-75% of PSE remained higher than those at 25, 100%, indicating that iron and calcium precipitate and the stem chloroplast adheres to the plant (Fig. 4). The results of this analysis supports the conclusion that stem of T. latifolia L containing metal elements; Ca and Fe and macronutrients like protein, carbohydrate can be observed at 50-75% dose but not at 25,100% of PSE dose (Fig. 4). Due to the concentration of Fe and Ca at the dosing of 50 and 75%, they form hematite (Fe2O3) and calcium pyrophosphate (Ca2P2O7). Along with (Fe2O3) and (Ca2P2O7), (Ca2Fe2O5) and (Fe3(PO4)2(OH)3) were also found in the plant at 75%., (Rout and Sahoo, Gao and Cutright, 2019). After 60 days of cultivation of T. latifolia L, the contents of P, Fe and Ca in the plant at 100% was lower than that at 75% dose of PSE (Fig. 4). Taking into consideration, the contents of macro and micronutrients increased rapidly at 50 to 75% but not at 25, 100%, this implies that the growth of biomass of T. latifolia L increases at 50 to 75% dosing of PSE ( The amount of TPH, HMs and metals nutrients transferred from the soil to the plant biomass and the remaining masses of chemical precipitations are depicted in Fig. 5 and Table 4. Therefore, it can be understood that the weight ratio of TPH and HMs precipitations in soil is 11.6 µg/g with the biomass growth of plant 109 g/Kg in 75% compared with the biomass 37.5 g/Kg and 56.7µg/g at 100% dosing of PSE (Fig. 5). As shown in Fig. 5, the contents of HMs and TPH in soil of range (11.6 µg/g) were relatively stable in 75%, while the contents of TPH, HMs in soil were shown to increase as the dosing of PSE range increased from 25 and 100%. These results prove that 75% of PSE per gm of biomass of T. latifolia L removed 17.98 TPH, 6.37 Co, 11.99 Mn and 3.1 Cd mg/g DW respectively. The amounts of Co and Mn removed from soil were observed highest than that utilized by the plant in both control and treatment (Galal et

Role of metals and micronutrient for HMs and TPH toxicity removal
In Table 4, experiment was performed on the system in order to determine the TPH and HMs' removal pathways. The dosing of PSE 75%, the remained contents in water of protein-carbohydrate and Ca, Fe, Mg and P in water decreased signi cantly due to mass balance to maximum amount of metals and microonutrient transfer in T. latifolia L. for biomass growth. The signi cant decrease the contents of Ca 11.35, P 1.71, Fe 5.76, Mg 2.45 and protein 6.92, carbohydrate 5.65 in water was observed at 75% dosing of PSE. The per g mass growth using metals and micronutrient range increased from <P to Fe>Ca>Mg and Carbohydrate>Protein. These results clearly con rm that growth plant biomass utilized more 97.

Conclusions
The experiment for phytoremediation T. latifolia L in PSE having TPH and HMs contaminated water-soil produced tremendous amounts of biomass, which is directly proportional to uptake of TPH and HMs. The optimal PSE dosage was 75% for TI, ECR and ECR formation and stability as well as removals of removed 17.98 TPH, 6.37 Co, 11.99 Mn and 3. Availability of data and materials Self-laboratory work and original data generated in the course of the research Tables   Table 1. Physico-chemical properties of petroleum secondary e uent and values are means of three replicates ± SD