¹Department of Botany, Government First Grade College, Vijayanagar, Bengaluru, India
²UGC-Malaviya Mission Teacher Training Centre, Bangalore University, Jnana Bharathi Campus, Bengaluru, India

*Corresponding author:Rathna Kumari BM, Department of Botany, Government First Grade College, Vijayanagar, Bengaluru, India Email: bmrathnakumari@gmail.com

Copyright: ©Rathna Kumari BM, and Nagaraja N. 2024. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Article Information:Submission: 18/04/2024; Accepted: 04/06/2024; Published: 08/06/2024

The soil amendments are known to reduce the toxicity of heavy metals in soil. A pot experiment was conducted to evaluate the effect of four different concentrations (220:117:140, 440:117:140, 220:117:280 and 440:117:280) of Nitrogen, Phosphorous and Potassium (NPK) and their effects on Acacia auriculiformis and Casuarina equisetifolia in phytoremediation of CCA compounds. Results showed that the growth and biomass of the seedlings treated with CCA (1000 mg kg-1 soil) were significantly increased with an increase in NPK concentrations. Similarly, the amounts of Cu, Cr, and As found in the roots of A. auriculiformis at 440:117:2080 mg g-1of NPK were 3.577, 3.070, and 0.094 mg g-1 respectively. Similarly, the quantity of Cu, Cr, and As found in stem and leaves in similar concentrations of NPK were 1.683 and 2.220 mg g-1, 2.600 and 1.913 mg g-1, and 0.077 and 0.085 mg g-1 respectively. Furthermore, the amount of Cu, Cr, and As found in the roots of C. equisetifolia at similar concentrations of NPK were 1.670, 3.320 and 0.240 mg g-1 respectively. Nevertheless, the amount of Cu, Cr, and As found in stem and leaves were 0.350 and 0.398 mg g-1 and 0.110 and 1.530mg g-1, 2.540 and 0.154 mg g-1 at 440:117:2080 mg of NPK respectively. The higher concentrations of NPK significantly (p≤0.05) increased the biochemical parameters of both the test plants. The maximum amount of total carbohydrates, proteins, and chlorophyll content found in A.auriculiformis and C. equisetifolia were 4.06, 1.61 and 0.28 mg g-1, and 3.575, 1.073 and 0.369 mg g-1 at NPK ratio of 220:117:280 mg kg-1 soil respectively. The present study advocates that the higher concentrations of NPK improve soil properties and plant productivity by reducing heavy metal solubility and the NPK can greatly enhance phytoremediation in CCA-contaminated soils using A. auriculiformis and C. equisetifolia tree species.

Keywords:Acacia auriculiformis; Casuarina equisetifolia; CCA; NPK; Biochemical parameters

Heavy metals remain persistent in the soil for a long period by posing a long-term threat to the environment [1,2]. These are biologically magnified and affect soil fertility thereby reducing crop production [3]. Chromated copper arsenate (CCA) is a wood preservative, used in the protection of timber from microbial decay and insect damage [4]. The CCA-treated woods are widely used as construction material resulting in the release of its components viz Cr. Cu and As in to agricultural fields, which initiates concern for food safety [5].

Phytoremediation is an effective technology in mitigating heavy metal contamination of the soil [6]. Plants used in phytoremediation produce relatively high biomass, tolerant to metal toxicity and exhibit high metal absorption capacity [7]. Acacia auriculiformis and Casuarina equisetifolia are moderatesized fast-growing tree species widely distributed in India. They have a shallow root system and produce huge biomass in short periods even in dry conditions [8]. These plant species are potential candidates for the production of huge biomass and are well adapted to even poor soil conditions [9]. Furthermore, these species are found to accumulate huge amounts of metals in their tissues [10]. Since these plant species show better tolerance to heavy metal stress, they are used in the remediation of CCA contaminated sites [11].

The chemical enhanced technology helps the uptake of heavy metals in plants [12]. The immobilization of heavy metals using different soil amendments is one of the most effective ways to remediate contaminated soil [13]. Several chemical amendments have been used for the enhancement of phytoextraction process[14]. The EDTA and DTPA are known to increase biochemical constituents in seedlings of A auriculiformis and C equisetifolia treated with chromated copper arsenate[15]. Nitrogen is constituent of amino acids and is widely applied in nurseries. The optimization of N, P, and K doses for improved seedling growth in Eucalyptus species has been reported [16]. The application of 100 ml NPK stimulated 45% shoot biomass and 26% Cr uptake compared with the non-fertilized cultures [17]. Surprisingly, information on the influence of inorganic fertilizers viz NPK which are required for healthy growth and development of plants in phytoremediation of CCA in tree plant species is meagre. The objective of the study was to investigate the influence of NPK on the growth, biochemical changes and uptake of CCA components viz Cu, Cr and As in the seedlings of A. auriculiformis and C. equisetifolia tree species in pot conditions.

Experiments on the effect of four levels of inorganic fertilizers viz. Nitrogen, Phosphorus, and Potassium (NPK) treated with chromated copper arsenate (1000 mg kg-1 soil) were conducted in the seedlings of Acacia auriculiformis and Casuarina equisetifolia species in Bengaluru, India. The healthy seeds of A. auriculiformis and C. equisetifolia were procured from the Institute of Forest Genetics and Tree Breeding (IFGTB), Coimbatore, India. The seeds were surface sterilized with bavistin (50% WP) followed by a double wash with deionized water. The CCA (1000 mg kg-1 soil) concentration was prepared from commercially available C-type CCA with a proportion of Cr (CrO3-47.5%), Cu (CuO- 18.5%), and As (As2O5- 35%). Four levels of NPK viz 220:117:140 mg, 440:117:140 mg, 220:117:280 mg, and 440:117:280 mg kg-1 soil were prepared and were applied separately.

The healthy seedlings of A. auriculiformis and C. equisetifolia were transplanted from root trainers in plastic pots (2000cc) filled with a sufficient quantity of air-dried soil mixed with compost. All the pots were arranged in the greenhouse in four replications. The potting medium (sand and soil in 2:1, v/v) was prepared, mixed thoroughly with CCA (1000 mg kg-1 soil) and four levels of NPK (220:117:140 mg,440:117:140 mg, 220:117:280 mg,and 440:117:280 mg kg-1 soil). The potting medium consisted of sand and soil (2:1 v/v) without NPK was used as control. Regular management practices, including sufficient irrigation and weed control, were performed during the experiments.

After six months, the shoots (stem and leaves) and roots of the seedlings of each test tree species were harvested and washed thoroughly with deonized water. The seedling growth indices in terms of height were measured with measuring tape and collar diameter was measured with a calliper. The fresh and dry weights of the seedlings were recorded for each sample using electronic balance. The dry weights of the seedlings were measured after oven drying at 65-75o C till the weight becomes constant. The washed plant samples were separated into shoots and roots dried and powdered. One gram of the root and shoot (stem and leaves) were digested in 10 ml acid mixture of 60% HNO3 and 2 mL 30 % H2O2 in a microwave oven drying at 200±50 C for 20 min. Furthermore, the concentrations of Cu, Cr, and As accumulated in roots, stems, and leaves were analyzed using an atomic absorption spectrometer (Shimadzu, AA-6880, Japan).

The amount of total carbohydrates, protein, and chlorophyll content found in the 6-month-old seedlings of A. auriculiformis and C. equisetifolia was estimated by spectrophotometric methods. The leaves separated from each treatment and were cut into fine pieces and then ground with mortar and pestle. 0.5 g of fresh leaf powder from each treatment was homogenized with 5 μL of 80% acetone, incubated overnight at room temperature, and centrifuged at 5000 rpm for 5 min. The supernatant was made up to 5 μL with 80% acetone and the optical densities were measured at 645 and 663 nm wavelengths using UV-VIS Spectrophotometer (Shimadzu, UV-1900, Kyoto, Japan). The amount of total carbohydrates was analyzed by the Anthrone method [18]. Similarly, the total protein contents of the seedlings were determined by Lowry’s method [19]. Furthermore, the amount of total chlorophyll was estimated using Arnon’s method [20] and the results obtained in different treatments were compared with the control samples.

The experimental data on seedling growth, biomass, accumulation of Cu, Cr and As compounds in root, stem and leaves, and changes in total carbohydrates, proteins, and chlorophyll content of A. auriculiformis and C. equisetifolia on exposure to different NPK concentrations treated with CCA (1000 mg kg-1 soil) were statistically analyzed by one-way analysis of variance (ANOVA). The data are presented as mean, standard error and coefficient of deviation (CD) (0.05). Furthermore, different treatments of the study were analyzed statistically for significant differences at p≤0.05 by Duncan’s multiple range test (DMRT).

The growth and biomass of the seedlings of A. auriculiformis and C. equisetifolia treated with four different concentrations of NPK along with CCA (1000 mg kg-1 soil) were varied [Table 1] .

The height and collar diameter of the seedlings were significantly (p≤0.05) increased with an increase in NPK concentrations. The maximum and minimum height and collar diameter of A. auriculiformis recorded were 68.75 and 44 cm and 69.75 cm and 5.38 and 5.10 cm at NPK concentration of 440:117:280 and control respectively. Similarly, the highest and lowest height and collar diameter of C. equisetifolia recorded were 69.75 and 44.25 cm and 4.38 and 3.37 cm at NPK ratio of 440:117:280 and control respectively. Furthermore, the maximum and minimum fresh and dry weights of A. auriculiformis were found to be 27.39 and 16.15 mg and 16.17 and 10.29 g in NPK concentration of 220:117:280 25.15 and control respectively. Similarly, the maximum and minimum fresh and dry weight of C. equisetifolia were found to be 32.82 and 20.23 mg and 13.61 and 8.40 g in NPK concentration of 220:117:280 25.15 and control respectively.

The soil rich in sufficient nutrients are required for the healthy growth and development of plants. However, the soil contaminated with toxic heavy metals may affect the morphological and biochemical parameters of plants. The influence of different concentrations of NPK on the biochemical parameters of the seedlings of A. auriculiformis and C. equisetifolia is presented in [Table 2] . The results showed a gradual increase in total carbohydrate, protein, and chlorophyll content with an increase in NPK concentrations. The minimum and maximum amount of total carbohydrates, proteins and chlorophyll content recorded in the seedlings of A. auriculiformis were 3.04 and 4.06 mg g-1, 0.95 and 1.61mg g-1 and 0.20 and 0.28 mg g-1 in control and 220:117:280 mg kg-1 soil concentrations of NPK respectively. Similarly, the minimum and maximum amount of total carbohydrates, proteins and chlorophyll content recorded in the seedlings of C. equisetifolia were 2.475 and 3.575 mg g-1, 0.826 and 1.073mg g-1 and 0.210 and 0.369 mg g-1 in control and 220:117:280 mg kg-1 soil concentrations of NPK respectively.

Plants respond differently to fertilizers depending on their ability to accumulate heavy metals in shoots and root. Accumulation of CCA in the form of its components viz Cu, Cr, and As amended with different concentrations of NPK were recorded in the root of six months old A. auriculiformis seedlings [Table 3] . The results revealed a significant (p≤0.05) increase in accumulation of CCA compounds with an increase in concentration of NPK in both root and shoot. The highest amounts of Cu, Cr, and As found in the root were 5.027, 4.207, and 0.147 mg g-1 respectively at 220:117:280 NPK level. Similarly, the maximum amount of Cu, Cr, and As recorded in stem and leaves were 2.333, 3.623, and 0.117 mg g-1 and 3.343, 3.650, and 0.134 mg g-1 respectively in the similar concentration of NPK.

The amount of CCA components viz Cu, Cr, and As amended with different concentrations of NPK were recorded in the root, stem, and leaves of the seedlings of C. equisetifolia [Table 4] . The observations showed a significant (p≤0.05) increase in the amount of CCA compounds with an increase in concentration of NPK in both stem and leaves. The maximum amount of Cu, Cr, and As found in the root were 3.650, 4.230, and 0.329 mg g-1 respectively at 220:117:280 NPK level. Similarly, the maximum amount of Cu, Cr, and As recorded in stem and leaves were 0.645, 0.874, and 0.198 mg g-1 and 2.870, 3.870, and 0.242 mg g-1 respectively in the similar concentration of NPK.

The addition chemical amendments to soil contaminated with heavy metals enhance the phytoremediation process, and fertilizers are the most widely used amendments[21]. Reports show that the mobility of metals in the soil can be considerably increased by the addition of chelating agents. A number of natural and synthetic chelating agents are available, although their effectiveness varies with plant and soil types. The results of the study showed an increased growth and biomass of the seedlings treated with NPK in CCA-contaminated soils. It is apparent that fertilizers enhance the plant growth, and heavy metal extraction capacity of plants [22]. The increase in seedling height on application of nitrogen might lead to increased production of photosynthesis [23]. There was an increase in the mean plant biomass and tolerance, on treated with fertilizers in Pb, Cd and

Zn-contaminated soils [24]. The observations of [25] showed that the addition of NPK fertilizers significantly increased the biomass of Leersia hexandra with higher Cu extraction efficiency. Nitrogen plays an important role in plant growth since N is a component of all plant structures such as proteins, enzymes, and chlorophyll. Appropriate fertilization could increase the plant biomass content and may enhance their ability to uptake and tolerate heavy metals [26].

Nutrient solutions were applied to the soil to improve the growth of plants for phytoremediation.Furthermore, nutrient availability is an important factor governing the success of phytoremediation and can be regulated through the addition of fertilizers.Several studies showed that the application of NPK significantly increase heavy metal uptake in plants. Phosphorus contributes to root expansion and plant nutrient uptake [27].The increased accumulation of CCA in the seedlings could be attributed to the increased solubility of CCA compounds in the soil. Urea was found to enhance the Cd phytoaccumulation in Carpobrotus rossii [28]. Furthermore, nitrogen application reduces the production of a large number of reactive oxygen species (ROS) induced by biotic and abiotic stress [29]. Nitrogen fertilizers improve the resistance and tolerance to plants exposed to Cd stress [30]. Similarly, Cd phytoremediation efficiency of Solanum nigrum was found to be improved with the addition of nitrogen fertilizer [31]. The N, P, and K are important components of plant amino acids, proteins, genetic material, and enzymes [32].

It is evident from the studies that, the NPK enhances the biochemical composition of A. auriculiformis and C. equisetifolia seedlings. Results of the study showed increased amount of carbohydrate content, proteins and chlorophyll content with an increase in the concentrations of NPK in the seedlings of test species. The reactive oxygen species (ROS) causes severe changes in protein structure, altering the proteins’ functions, with subsequent effects on metabolic pathways [33]. The results of our study showed an increase in the chlorophyll content in the seedlings exposed to different concentrations of NPK. Accumulation of excess amounts of Cu damages the ultra-structure of the chloroplast, which directs changes in the composition of the thylakoid membrane [34].

The studies reveal that the NPK plays a pivotal role in the enhancement of the plant defense system and alleviation of CCA phytotoxicity by enhancing the plant biomass of the seedlings of tree species. The NPK also improved the accumulation of Cu, Cr, and As in the root, stem, and leaves of Acacia auriculiformis and Casuarina equisetifolia. These plants could be used as phytoaccumulators for remediation of CCA compounds on application of NPK. However, further studies are required on the evaluation of the phytoremediation efficiency of these tree species in field conditions.

The authors are grateful to Bangalore University, Bengaluru, India for extending research facilities for conduct of these studies.