The physico-chemical, metals, heavy metals (HMs), and total petroleum hydrocarbon (TPH) properties of petroleum secondary effluent (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). Similar results were observed by others researchers (Peris et al. 2017; Ali et al. 2020). The inhibition of the root growth is a primary symptoms of heavy metal toxicity which can be taken as a measure for root stress tolerance (Ali at al. 2018; Ahmad et al. 2020).
Impact of dosing petroleum secondary effluent
The applied concentration of PSE 25-50% did not show any significant accumulation of HMs and TPH in plant parts. However, HMs and TPH accumulations rate of stem> root also depends on the doses of PSE. The highest amount of TPH was transferred in stem 894 and root 477 µg/g DW was found at 75% doses of PSE. However, a much lower accumulation of TPH was observed at 25-50% or higher concentrations i.e.100% of PSE. The accumulation of HMs in plant from PSE were Mn, Co and Cd, (638, 653, 186 μg/g) in root, (458, 768, 198 μg/g) in stem and (368, 463, 86 μg/g) in leaves T. latifolia L and final remained (precipitate) heavy metal concentration in the water are presented in (Fig. 2) respectively. The lowest accumulation of metal Mn and Cd, due to the initial concentration of metals in PSE was lowest compared to TPH and Co. However, the mass of the plants average accumulation for TPH and HMs from PSE to root to stem indicated by the depend on biomass enrichment coefficient and transfer index of metals (Ali et al. 2019; Anudeep et al. 2020).
Variation of HMs and TPH in plants
The accumulation of the HMs and TPH reduction was TPH>Co>Mn>Cd from petroleum secondary effluent (PSE) (Fig. 2). The levels of TPH and HMs accumulated in root and stem were significantly 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 bio-accumulation 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 (Ahmad et al. 2019; Jaskulaki et al. 2020). In our study, TPH and HMs accumulation in the root and stem of T. latifolia L increased along with biomass with the increasing dosing of PSE. Similar observations by other researcher were also found (Galal and Shehata, 2015; Ahmad et al. 2019).
Impact on growth and yields of biomass
The high concentration of TPH and HMs in T. latifolia L tissue could decrease the growth, biomass (Waheed et al. 2019; Ahmad et al. 2020). The reduction in biomass of plants at 100% might be due to toxicity of TPH and HMs, which corroborates our previous results using varying concentrations of HMs on growth of rye grass plants (Ahmad et al. 2019). The growth and development of roots were affected at very high concentration of PSE available on T. latifolia L. As a result, the roots were unable to deliver the nutrients to shoot, for which the growth of the biomass plants were affected (Rehman et al., 2019). Similar findings were estimated by related researchers (Ahmad and Ahmad, 2014; Jampsari and Saeng-Nigam, 2019: Ahmad et al. 2019). The inhibition of the biomass is a primary symptom of TPH and HMs toxicity which can be taken as a measure for plant stress tolerance (Ahmad et al. 2010; Newete and Byrne, 2016). The result showed that the biomass of plant was able to phytoremediate the metals petroleum secondary effluent (Ahmad et al. 2019; Ali et 2019). 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 significant 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 identification 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 efficient ability to transport metal from soil to root to stem, most highly effective remediate metals due to efficient metal transporter systems through its biomass (Ariolo et al. 2015; Klomjek, 2016).
Enrichment coefficient for the root and stem
The plant showed bio-concentration of TPH and HMs indicates the efficiency of the plants to eliminate the TPH and HMs metals from the soil to plant. The enrichment coefficients 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 coefficients are a very important factor, which indicate phytoremediation of a given species (Ahmad et al. 2011; Kumari and Tripathi, 2015; Hammami et al. 2018; Ali et al. 2018). (Table 3). The TPH and HMs concentrations in root>shoot were generally higher than that in soil (Table 3). Researcher supported our findings to this situation indicated a special ability of T. latifolia L to with stand bio-accumulate, transfer of TPH and HMs from PSE soil and well grown of biomass their root and stem (Li et al 2018; Outa et al. 2020; Steliga and Daluk, 2020).
Tolerance strategy of plants and chemical precipitates in biomass-metals, HMs and TPH
Fig. 4 and 5 shows the protein and carbohydrate content in T. latifolia L at 75% dose of petroleum secondary effluent (PSE) stabilized at around 87.5 and 73.216.4 mg/Kg DW. The content of Fe, P, Mg and Ca in the plant at 75% dosage is 98.5, 0.7, 2.5 and 23.6 mg/Kg respectively. After cultivating for 45 days, it is evident that the metal content in the plant decreased to 90% (At 100% concentration of PSE) (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 (Fig. 4) (Mustapha and Lens, 2018; Bokhari et al. 2019).
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 al. 2017: Ahmad et al. 2019; Ali et al. 2020)).
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 significantly due to mass balance to maximum amount of metals and microonutrient transfer in T. latifolia L. for biomass growth. The significant 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 confirm that growth plant biomass utilized more 97.35% of TPH and HMs, had low content of chemical precipitations (Ahmad et al. 2011; Adeyeye, 2005; Ahmad and Ahmad, 2014; Jampsari and Saeng-Ngam, 2019) or release in water (Table 4). The plants used for the phytoremediation of with stand long exposure of these TPH and HMs might bring about accumulation and increase the content inside the plants. However, the fact that TPH and HMs accumulated in plants from PSE, despite the presence of TPH and HMs in water- soil, their concentrations were relatively very low and fulfilled the conditions for the discharge of treated effluent (Rana and Maiti, 2018; Truu et al. 2015; Gao and Cutright, 2019; Ahmad et al. 2019; Ahmad et al. 2020; Hejna et al. 2020).