Heavy metals immobilization and bioavailability in multi-metal contaminated soil under ryegrass cultivation as affected by zno and mno2 nanoparticle-modified biochar

ABSTRACT Pollution by heavy metals (HMs) has become a global problem for agriculture and the environment. In this study, the effects of pristine biochar and biochar modified with manganese

dioxide (BC@MnO2) and zinc oxide (BC@ZnO) nanoparticles on the immobilization and bioavailability of Pb, Cd, Zn, and Ni in soil under ryegrass (_Lolium perenne_ L.) cultivation were

investigated. The results of SEM–EDX, FTIR, and XRD showed that ZnO and MnO2 nanoparticles were successfully loaded onto biochar. The results showed that BC, BC@MnO2 and BC@ZnO treatments

significantly increased shoots and roots dry weight of ryegrass compared to the control. The maximum dry weight of root and shoot (1.365 g pot−1 and 4.163 g pot−1, respectively) was reached

at 1% BC@MnO2. The HMs uptake by ryegrass roots and shoots decreased significantly after addition of amendments. The lowest Pb, Cd, Zn and Ni uptake in the plant shoot (13.176, 24.92,

32.407, and 53.88 µg pot−1, respectively) was obtained in the 1% BC@MnO2 treatment. Modified biochar was more successful in reducing HMs uptake by ryegrass and improving plant growth than

pristine biochar and can therefore be used as an efficient and cost effective amendment for the remediation of HMs contaminated soils. The lowest HMs translocation (TF) and bioconcentration

factors were related to the 1% BC@MnO2 treatment. Therefore, BC@MnO2 was the most successful treatment for HMs immobilization in soil. Also, a comparison of the TF values of plant showed

that ryegrass had a good ability to accumulate all studied HMs in its roots, and it is a suitable plant for HMs phytostabilization. SIMILAR CONTENT BEING VIEWED BY OTHERS EXTRACTION OF HEAVY

METALS FROM COPPER TAILINGS BY RYEGRASS (_LOLIUM PERENNE_ L.) WITH THE ASSISTANCE OF DEGRADABLE CHELATING AGENTS Article Open access 01 April 2024 ACIDIFIED BIOCHAR IMPROVES LEAD TOLERANCE

AND ENHANCES MORPHOLOGICAL AND BIOCHEMICAL ATTRIBUTES OF MINT IN SALINE SOIL Article Open access 30 May 2023 CHEMICAL FORMS OF CADMIUM IN SOIL AND ITS DISTRIBUTION IN FRENCH MARIGOLD

SUB-CELLS IN RESPONSE TO CHELATOR GLDA Article Open access 20 October 2022 INTRODUCTION Heavy metal (HMs) pollution caused by industrialization and economic development has become a major

environmental, agricultural, and public health problem worldwide1,2. Heavy metal are non-degradable and stable in nature, and at concentrations of less than 1 ppm, they are considered toxic

to plants and animals3,4,5,6. Moreover, due to their high mobility, these metals pose a major threat to human and animal health as they accumulate in water bodies and agricultural soils and

enter the food chain7,8,9,10. Therefore, in order to clean and restore soil health and eliminate or limit the bioavailability of HMs to plants and humans, it is essential to use

environmentally sound and economically viable technologies11,12,13. To date, various methods have been used to remediate soils contaminated with HMs, including electrokinetic remediation,

bioremediation, soil washing, and chemical precipitation14,15,16,17, but the application of these technologies is limited due to their high cost, environmental contamination from the

generation of secondary chemicals, and damage to soil structure15,16,18,19. The Environmental Protection Agency (EPA) has identified soil immobilization as the most effective, economical,

and environmentally safe technique for remediation of HM-contaminated soils on a broad scale14,20,21. Immobilization using ion exchange reactions changes the adsorption, complexation, and

precipitation of HMs from active to stable phases and reduces the bioavailability of HMs in soil22,23,24,25. In situ immobilization of HMs requires careful consideration of the choice of

immobilization substance26. Many substances such as montmorillonite, limestone, compost, metal oxides, and biochar have been used to immobilize HMs in the soil7,27,28. Biochar is a

carbon-rich adsorbent produced from the pyrolysis of various raw materials in the absence or limited presence of oxygen29,30,31. Due to its high porosity, active functional groups, and high

CEC, biochar has been considered an ideal amendment for the immobilization of HMs in24,32,33. Therefore, biochar can reduce the availability, mobility and leaching of HMs in soil, and

limiting their bioavailability for the plants15,30. Biochar immobilizes HMs in soil through various mechanisms such as complexation, cation exchange, electrostatic interactions, reduction

and precipitation30. Biochar also protects the plant from HMs by increasing antioxidant activity31. However, unmodified biochar has a limited capacity to immobilize and adsorb HM in

soil23,24,34,35. Therefore, it is important to modify biochar to improve its ability to immobilize HMs in soil. Various physical, chemical, and biological techniques have been used to

improve the properties of biochar, e.g. specific surface area, porosity, number of adsorption sites on the surface, and number of surface functional groups36,37,38. Recently, the

modification of biochar with nanomaterials, especially nanometals, has attracted attention due to their high efficiency, low toxicity and environmental compatibility39,40,41. Nanomaterials

are particles ranging in size from 1 to 100 nm42,43. In the last decade, nanomaterials have been used extensively for the removal of HMs from water and soil due to their tiny size, strong

reactivity, and high surface activity44,45,46. However, these materials tend to aggregate and deactivate due to their high surface energy, which limits their ability to remove

contaminants33,41,44,45,46,47. To overcome these problems, biochar can serve as a carrier for metal oxide nanoparticles due to its porosity and high ion exchange capacity48,49. The composite

of biochar and metal nanoparticles eliminates their deficiencies and creates a material that combines the advantages of both properties50,51. The functional surface groups, the specific

surface area, and the cation exchange capacity increase by modifying biochar with metal nanoparticles. This increase leads to improvement of ion exchange, complexation and thus the

stabilization of HMs36,49,52. Among metal nanoparticles, zinc oxide nanoparticles have attracted attention due to their low toxicity, compatibility, high binding energy, low production cost

and biodegradability compared to other nanoparticles44,53,54. They are also widely used for the removal of HMs from water and soil39. In addition, manganese oxide nanoparticles have been

shown to be well suited for stabilization of HMs due to their high specific surface area, high affinity for HMs, abundance of hydroxyl functional groups on their surface, stability in a wide

pH range, and high negative surface charges55,56,57,58. Numerous studies have demonstrated the effect of biochar modified with various nanoparticles on the immobilization of HMs in

soil37,59,60,61,62. However, studies on the effect of biochar modified with ZnO and MnO2 nanoparticles on HMs immobilization in soil under plant cultivation is still limited and need to be

explored in detail. Ryegrass (_Lolium perenne_ L.) was selected as the study plant in our research due to its fast growth, high biomass, high potential for accumulation of metals, and high

tolerance to these metals63,64,65. In this study, we tested the hypothesis that MnO2 and ZnO nanoparticle-modified biochars outperforms the pristine biochar in terms of their ability to

immobilize HMs, reduce their bioavailability, and improve plant development. Therefore, the objectives of this study include: (i) Synthesis of modified biochar with MnO2 and ZnO

nanoparticles and comparison their morphology, physicochemical properties (ii) Evaluation and comparison of the efficiency of pristine biochar (BC), biochar modified with ZnO nanoparticles

(BC@ZnO), and biochar modified with MnO2 nanoparticles (BC@MnO2) in term of immobilization of Pb, Cd, Ni and Zn in multi-metal contaminated soils and (iii) Comparing the effects of pristine

and ZnO and MnO2 nanoparticle modified biochar on the HMs uptake and ryegrass (_Lolium perenne_ L.) growth. RESULTS CHARACTERISTICS OF THE BC, BC@MNO2 AND BC@ZNO FT-IR ANALYSIS FTIR analysis

was carried out to investigate the functional groups of BC, BC@MnO2, and BC@ZnO (Fig. 1). The broad band 3300–3940 cm−1 represents the strong and broad O–H stretching of alkyne66,67. The

peaks at 2700–3000 cm−1 corresponded to the C–H stretching vibration68,69. The existence of the C ≡ C triple or C = C = C double bonds of alkene was confirmed by the peaks at 1905 and 2112

cm−170. The peak at 1584 cm−1 is attributed to the stretching vibration of C = C71. The peak at 1373 cm−1 was the characteristic peak of –C–H of the methyl group72. The peaks at 1116 cm−1

are assigned to C–O–C stretching vibrations in ether or saturated chain anhydrides73,74. The peak at 1034 cm−1 is assigned to C–O stretching75. The peaks at 729–880 cm−1 were characteristic

of the bending vibration of aromatic C–H groups76. The peak at 617 cm−1 is attributed to the bending vibration of the O–H group77. The peak at 511 cm−1 is associated with the Mn–O stretching

vibration, shows that the MnO2 nanoparticles were successfully loaded onto the surface of the biochar66,73. Peaks at 646, 596, 516 and 448 cm−1 confirmed the presence of a Zn–O stretching

vibration77,78,79. XRD ANALYSIS To determine the crystal structure and phase composition of BC, BC@MnO2, and BC@ZnO, the X-ray diffraction (XRD) pattern was used (Fig. 2). The peaks of 26.6°

in BC, 27.3° in BC@ZnO and 23° in BC@MnO2 were attributed to graphite, indicating that a stable and regular graphite structure was formed during the pyrolysis process80. The peak at 40.6°

in BC was assigned to quartz or sylvite81,82. The peaks at 50.2° and 58.72° in BC and BC@ZnO and the peaks at 28.4° and 73.7° in BC, corresponded to sylvite83,84,85. The peaks at 29.4° and

30.9° in BC were attributed to calcite74. The peak at 66.4° in BC was assigned to quartz74. The peaks at 26.3°, 32.93°, 34.8°, 37.5°, 41.2° and 60.2° reflect the crystal planes of the

hexagonal structure of wurtzite ZnO recorded in the card (JCPDS card No. 36–1451)78,86,87,88. The 21.4° and 24.5° peaks in BC@ZnO are also indicative of zinc silicate78,86,87,88,89. The XRD

pattern of BC@ZnO is consistent with the results of other related studies90,91,92,93. The 36.7° peak in BC@MnO2 was assigned to amorphous MnO273,94. Compared to the patterns of BC and

BC@ZnO, the XRD pattern of BC@MnO2 revealed weaker peaks. These weak peaks were probably caused by the charge of the amorphous MnO2, which had an impact on the XRD patterns of other

crystals66. In general, the XRD of MnO2-loaded biochar did not show many peaks for phase identification. This could be due to the unfavorable effect of the high reaction rate between Mn(II)

and Mn(VII), in the case of MnO2 synthesized by the co-precipitation method73,95. According to the results of previous studies, amorphous materials have effective adsorption properties due

to their high specific surface area and the large number of active sites on their surfaces73,96. These results are consistent with the observations of this study, which showed that BC@MnO2

plays a more effective role in the surface adsorption of heavy metals compared to other adsorbents. SEM–EDX AND BET ANALYSIS The surface morphology and elemental composition of BC, BC@MnO2

and BC@ZnO were investigated using SEM–EDX analysis (Fig. 3). According to Fig. 3a, the biochar contains honeycomb and porous structures, which are probably due to the removal of volatile

substances during the pyrolysis process. Figure 3b shows that the surface of biochar is uniformly covered with fine and dense particles, indicating the deposition of MnO2 nanoparticles.

Similar results were obtained by20,66,97. The SEM of the BC@ZnO nanocomposite showed spherical and white particulates of ZnO in the form of clusters on the biochar surface. These spherical

particles are probably due to the irregular growth of the ZnO crystals98. These results have also been obtained by other researchers88,93,99,100,101. In general, the surface of the biochar

modified with ZnO and MnO2 nanoparticles was irregular, non-uniform and more uneven compared to pristine biochar confirming the successful loading of ZnO and MnO2 nanoparticles onto the

biochar. Previous research has shown that a rough surface leads to an increase in the specific surface area and increase in the positive charge thereby increasing the adsorption of

pollutant20,99,100,102,103. EDX analysis of the biochar showed the presence of C, O, Ca, Mg, K, Si and Cl elements. The presence of Mn and Zn and the significant increase in O content in

BC@MnO2 and BC@ZnO confirmed the successful loading of the biochar surface with MnO2 and ZnO nanoparticles. Therefore, according to the results of XRD, FTIR and SEM–EDX analyses, it is

likely that the ZnO and MnO2 nanoparticles were successfully loaded onto the BC. The BET analysis showed that the addition of MnO2 and ZnO nanoparticles increased the specific surface area

of pristine biochar from 60.52 m2 g-1 to 430.71 and 341.2 m2 g-1, respectively. This increase is most likely the result of the deposition of ZnO and MnO2 nanoparticles on the surface of

biochar, which led to an increase in surface roughness and consequently an increase in specific surface area82,104. It can be concluded that biochar modified with nanoparticles has

appropriate active sites for the adsorption of HMs, making it a high-potential adsorbent. In similar studies, Liang et al. (2017) and Wang et al. (2020), reported that loading biochar with

MnO2 and ZnO nanoparticles led to a significant increase in specific surface area of nanocomposites66,88. According to the results, biochar modified with MnO2 nanoparticles can absorb heavy

metals more effectively compared to other adsorbents. This superiority can be attributed to the amorphous structure, more exchangeable cations and a higher specific surface area133. EFFECT

OF THE ADSORBENTS ON RYEGRASS GROWTH To investigate the effects of BC, BC@ZnO, and BC@MnO2 at doses of 0.5 and 1% on the growth of ryegrass under Cd, Pb, Zn, and Ni stress, the dry weight of

the roots and shoots of ryegrass was evaluated (Fig. 4). Compared to the controls, the application of BC, BC@MnO2, and ZnO significantly (_p_ < 0.05) increased the dry weight of the

ryegrass shoots and roots. The application of biochar at 0.5% and 1% increased the dry weight of ryegrass shoots and roots by about 50.83–61% and 60.56–49.17%, respectively, compared to the

control treatment. The addition of BC@ZnO and BC@MnO2 at 0.5% and 1% also increased the dry weight of the aerial parts by 78–81.9% and 84.28–85.83%, respectively, compared to the control.

The addition of BC@MnO2 and BC@ZnO at 0.5% and 1% increased the root dry weight by 78.11–79.2% and 81–82.13%, respectively, compared to the control treatment. The dry weight of ryegrass

roots and shoots in biochar treatments modified with ZnO and MnO2 nanoparticles differed significantly (_p_ < 0.05) compared to the pristine biochar treatment. Moreover, the addition of

BC@MnO2 to the soil significantly increased the dry weight of the root and shoot biomass of ryegrass compared to the BC@ZnO treatments (_p_ < 0.05). The type and dose of adsorbent also

had a significant effect on the dry weight of roots and shoots. Increasing the dose from 0.5 to 1% significantly increased the dry weight of the roots and shoots. The results from Fig. 4

show that the BC@MnO2 treatment resulted in the highest dry weight of roots and shoots, while the control treatment gave the lowest. BC@MnO2 was found to be more effective than BC@ZnO in

increasing the dry weight of roots and shoots. In addition, a positive correlation was found between the shoot and root dry weights of the ryegrass plants in all treatments. After increasing

the adsorbent dosage from 0.5 to 1%, the dry weight of the roots and shoots increased. This increase is probably the result of increased HMs immobilization in the soil and improved nutrient

supply at higher doses. In agreement with previous studies, a linear relationship was found between the amount of adsorbent used and the dry weight of the root and shoot of plant11,105,106.

EFFECTS OF BC, BC@MNO2, AND BC@ZNO ON THE UPTAKE OF NI, ZN, PB, AND CD BY RYEGRASS SHOOTS AND ROOTS Table 1 shows data on HM uptake by ryegrass shoots treated with BC, BC@MnO2, and BC@ZnO.

Compared to the control treatment, the addition of pristine biochar and biochar modified with nanoparticles significantly reduced (_p_ < 0.05) the uptake of Pb, Ni, and Cd in the ryegrass

shoot. The application of 0.5% and 1% BC, BC@ZnO, and BC@MnO2 reduced Pb uptake in the shoots by 22.29–47.31%, 67.55–74.79%, and 83.82–89.33%, respectively, compared to the control.

Application of BC, BC@ZnO, and BC@MnO2 at two levels of 0.5% and 1% reduced uptake of Cd in shoot parts by 18.97–45.11%, 75.29–78.37%, and 78.73–82.83%, respectively, compared to control.

Table 1 also shows that the application of 0.5 and 1% BC, BC@ZnO and BC@MnO2 decreased the Ni uptake in the shoots by 11.55–32.58%, 52.76–61.67%, and 68.44–71.88%, respectively, compared to

the control. Although Zn uptake in shoots was significantly (_p_ < 0.05) decreased by different doses of BC (17.86–40.20%) and BC@MnO2 (74.98–79.85%) compared to control, BC@ZnO

significantly (_p_ < 0.05) increased Zn uptake in shoots (4.91–16.61%). Compared to application of BC treatments, the application of BC@MnO2 and BC@ZnO treatments resulted in a greater

reduction in the uptake of HMs in the shoots, with the exception of ZnO nanoparticle-modified biochar, which increased the uptake of Zn in the shoots. In addition, BC@MnO2 was more effective

than BC@ZnO in reducing HMs uptake by the shoots. In addition to the type of adsorbent, the amount of adsorbent also affected the uptake of HMs by the shoots of ryegrass. When the rates of

BC, BC@ZnO, and BC@MnO2 increased from 0.5 to 1%, the Pb, Ni and Cd uptake in shoots decreased. Zn uptake in shoots increased by increasing the amount of BC@ZnO from 0.5 to 1%. Also, BC@MnO2

was more effective than BC@ZnO in decreasing the uptake of HMs by the shoots. According to our results, the lowest uptake of HMs by ryegrass shoots was observed in 1% of BC@MnO2 treatment.

Moreoveer, all adsorbents showed a strong propensity to uptake Pb than other HMs. Table 2 shows the effect of BC, BC@MnO2, and BC@ZnO at 0.5 and 1% on the uptake of HMs by ryegrass roots.

Compared to the control treatment, the addition of BC, BC@MnO2, and BC@ZnO significantly reduced the uptake of Ni, Pb, and Cd by plant roots (_p_ < 0.05). Compared to the control, Zn

uptake was significantly reduced (_p_ < 0.05) by the BC and BC@MnO2 treatments but increased by BC@ZnO. With the exception of the BC@ZnO treatment, which increased the Zn uptake, the

application of biochar modified with nanoparticles decreased HMs uptake in roots more than pristine biochar. Moreover, the dosage of the adsorbents proved to be influential in reducing the

HMs uptake by the ryegrass roots. When the amount of BC was increased from 0.5 to 1%, uptake of Pb, Cd, Zn, and Ni in root decreased by 16.74, 16.74, 15.71, and 22.19%, respectively.

Similarly, increasing the BC@MnO2 dosage from 0.5 to 1% decreased the root uptake of Cd, Pb, Zn, and Ni by 16.46%, 25.2%, 20.2%, and 11.18%, respectively. . The absorption of Cd, Ni, and Pb

in the roots decreased by 16.27%, 19.7%, and 16.15%, respectively, when in the BC@ZnO dose was increased, while Zn uptake increased by 12.79%. As the results show, the roots of ryegrass had

higher concentrations of HMs compared to the shoots. In general, the tendency for uptake HMs by BC and BC@MnO2 was as Pb > Cd > Zn > Ni and for BC@ZnO as Pb > Cd > Ni > Zn

in the roots and shoots of ryegrass; in reality, the plant absorbs these heavy metals to a lesser extent. The lower uptake of heavy metals by the plant leads to a higher immobilization of

heavy metals in the soil, and thus to lower availability for the plant. BIO-CONCENTRATION (BCF) AND TRANSLOCATION FACTORS (TF) To better understand the effect of different amendments on the

uptake and translocation of heavy metals in ryegrass, the bioaccumulation factor (BCF) and translocation factor (TF) were calculated and presented in Figs. 5 and 6. According to the results

shown in Fig. 5, the addition of all adsorbents to the soil, significantly decreased (_p_ < 0.05) the TF for all HMs compared to the control treatment. Therefore, addition of 0.5% and 1%

biochar led to reductions in TF values for Pb, Cd, Zn, and Ni by 13.75–18.75%, 6.17–7.53%, 6.90–12.64%, and 7.63–8.87%, respectively, compared to the control. The effect of BC treatment in

reducing the TF of HMs compared to the control was as follows: Pb > Zn > Ni > Cd. Also, addition of BC@ZnO at two levels of 0.5% and 1% decreased the TF values for Pb, Cd, Zn and Ni

compared to the control treatment from 45 to 52.50%, 27.78–37.90%, 20.69–32.18%, and 16.23–28.06% respectively,. Compared to the control, BC@ZnO treatment reduced the TF of HMs in the

following order: Pb > Cd > Zn > Ni. Similarly, the addition of BC@MnO2 at 0.5 and 1% decreased the TF values for Pb, Cd, Zn, and Ni from 53.75 to 61.25%, 50.49–54.44%, 49.43–50.57%,

and 33.13–35.93%, respectively, compared to the control treatment. The effect of biochar treatment on reduction of TF values compared to the control were as follows: Pb > Cd > Zn >

 Ni. Increasing the amount of adsorbent led to a further reduction in TF. As can be seen in Fig. 5, the TF values of HMs in the nanoparticle-modified biochar treatments were significantly

lower (_p_ < 0.05) than those in the pristine biochar treatment. In addition, BC@MnO2 reduced the TF of HMs in ryegrass better than BC@ZnO. The treatment with 1% BC@MnO2 had the lowest TF

of HMs, while the control treatment had the highest TF. In general, according to the obtained results, the TF of HMs in ryegrass was as follows: Ni > Zn > Cd > Pb, indicating

greater transfer of Ni from the roots to the shoots. Similar to TF, the addition of adsorbent reduced the BCF of HMs compared to the control, and this decrease became greater when the

adsorbent dose was increased. According to the results, the control treatment and the treatment with 1% BC@MnO2 had the highest and lowest BCF values for HMs, respectively. Thus, the BCF

values of Pb, Cd, Zn, and Ni decreased by 96.98, 95.29, 95.08, and 94.35%, respectively in the treatment of 1% BC@MnO2 compared to the control. The BCF in the biochar modified with

nanoparticles was significantly lower compared to the treatment of pristine BC. In general, the BCF in ryegrass in all treatments was as follows: Ni > Zn > Cd > Pb. DISCUSSION

EFFECT OF BC, BC@MNO2 AND BC@ZNO ON PLANT BIOMASS Abiotic stress, such as heavy metals, can lead to physiological changes in plants, including a decrease in plant biomass107,108,109. The

lower dry weight of roots and shoots observed in the control treatment compared to the other treatments may be attributed to the high concentration of HMs in the plants (Fig. 4). This

results is consistent with previous studies108,109. Shoot and root dry weight increased significantly in comparison to the control treatment when biochar and modified biochar were added to

metal-contaminated soils. This might be due to the fact that the addition of biochar and modified biochar stabilize of HMs in the soil and limit their bioavailability to the plants, and

reduce the uptake of metals leading to an improvement in root and shoot growth of the plant. Similar results were obtained by Ali et al. (2019)110 and Shahbaz et al. (2018)111. They reported

that the application of biochar prevents the transfer of HMs in the plant and increases the dry weight and grain yield of wheat, sunflower, and maize. In addition, biochar increases plant

growth by providing macro and micronutrients. In addition, biochar improves the physicochemical properties of the soil, which produces the optimal conditions for better plant growth. These

conclusions have been supported by numerous reports.112,113,114,115. Research by Wang et al. (2021) also aligns with these results and shows that the presence of various biochars improves

nutrient availability116 and HMs immobilization117, resulting in increased ryegrass biomass18. The results of the present study have obviously shown that loading of biochar with ZnO and MnO2

nanoparticles plays a role in increasing the dry weight of ryegrass roots and shoots (Fig. 4). The increase in the dry weight of roots and shoots in modified biochar treatments may be due

to the dual effect of biochar and nanoparticles on the plants. Indeed, the application of biochar immobilizes the HMs in the soil and nanoparticles increase the zinc and manganese content in

the plant. These results are in line with published studies showing that NPs promoted the growth of the plants when exposed to metal stress53,118,119. These results are consistent with

other findings108,109. In one study, Kareen et al. (2023) observed that incorporation of biochar with zinc oxide nanoparticles resulted in improved alfalfa growth in Cd-contaminated soil.

They attributed this result to the competition between nutrients and heavy metals at the root surface and to the immobilization of Cd in the soil109. Lin et al. (2017) and Yu et al. (2017)

reported that the application of MnO2 modified biochar in an arsenic-contaminated soil significantly increased the grain weight and dry weight of rice roots and shoots compared to pristine

biochar120,121. In addition, the BC@MnO2 treatment performed better compared to other treatments in terms of increasing the dry biomass of ryegrass roots and shoots. This is most likely due

to the fact that BC@MnO2, due to its higher specific surface area, amorphous structure, and greater number of exchangeable cations, reduced the bioavailability of HMs in the soil more

effectively than other treatments thus minimizing their impact on ryegrass development. EFFECT OF BC, BC@MNO2 AND BC@ZNO ON PLANT HMS CONCENTRATION Various organic treatments have been

employed to reduce the bioavailability and toxicity of HMs in plants and soils,122,123. As reported by Azim-Zadeh et al. (2014), organic amendments can alter soil properties, ultimately

affecting the bioavailability of HMs124. In this study, the presence of HMs in ryegrass shoots and roots was used as an indicator of the HMs bioavailability in soils (Tables 1 and 2). In the

present study, addition of BC, BC@MnO2 and BC@ZnO significantly decreased the uptake of HMs in the roots and shoots and as the dose was increased, the reduction effect increased. This might

be due to the fact that biochar immobilizes HMs in soil and reduces their bioavailability. Previous research has shown that oxygen-containing functional groups on biochar surfaces

facilitate the immobilization of metal ions through the formation of precipitates and complexes27,125,126. The interaction with the mineral and crystal lattices on the BC surface also allows

the HM ions to form to form insoluble inorganic compounds (such as metal phosphate, metal carbonate, or metal silicate)127,128. EDX analysis revealed (Fig. 3) that the presence of divalent

cations such as Ca (II) and Mg (II) in biochar can replace HM ions on biochar surfaces129. In fact, it can be argued that biochar decreased the bioavailability of HMs by immobilizing HMs in

the soil through the mechanisms of ion exchange, complexation, and precipitation, which in turn reduced uptake by ryegrass roots and shoots. The silica in wheat straw biochar can also lead

to a reduction in bioavailability in the soil, a reduction in adsorption by the plant, and a reduction in the transfer of HMs from the roots to the shoots130. Similar results have also been

reported by other researchers. Based on the research of Wang et al. (2018), biochar significantly reduced the solubility of HMs in soil131. Awad et al. (2020) reported that the HM contents

of soil and plant tissues were significantly reduced after biochar applications132. The results showed that loading MnO2 and ZnO nanoparticles on the biochar led to a more noticeable

decrease in the plant's absorption of HMs, suggesting that BC@ZnO and BC@MnO2 have a greater ability to immobilize HMs than pristine biochar. BC@NP composites have better

physicochemical properties compared to pristine biochar due to the combination of both constituent particles. Therefore, the substantial reduction in HMs uptake by ryegrass with

nanoparticle-modified biochar application may be attributed to the combined effects of biochar and nanoparticles on soil and plant surfaces. In addition to the benefits of biochar in

reducing the bioavailability of HMs, nanoparticles can also reduce the adverse effects of HMs on plant growth and productivity27. Also, according to the results of BET analysis, biochar

modified with nanoparticles had a higher specific surface area compared to pristine biochar. This suggests that these treatments have been more effective in immobilizing HMs133. According to

previous research, cation channels of Ca2+, Zn2+, Mn2+, and Fe2+ are the pathways through which Cd, Pb, Ni, and other divalent heavy metals enter plant cells134,135. When BC@MnO2 and BC@ZnO

are applied, Mn2+ and Zn2+ levels increase, which can compete with HMs in the transport pathway and obstruct their transfer through plant membranes. In similar reports,2,136,137 the

addition of MnSO4 and ZnSO4 amendments could greatly reduce the buildup of HMs by increasing the amount of accessible Mn and Zn content. In the study of Wang et al. (2019) the capacities of

biochar derived from tea branches and Fe–Mn-modified biochar (MnFe2O4-biochar) to immobilize Sb and Cd in contaminated soils and reduce the bioavailability of Sb and Cd in _Lolium

multiflorum_ Lam were assessed. Their results demonstrated that MnFe2O4-biochar application significantly reduced extractable Sb and Cd concentrations, transformed exchangeable Sb and Cd

into less accessible forms, and decreased the accumulation of Sb and Cd in plants138. Furthermore, Suleiman et al. (2020) investigated the biochar with ZnO nanoparticles treatment on

sunflowers grown in wastewater contaminated with HMs. They reported that biochar and ZnO significantly reduced the availability of HMs and decreased their uptake by sunflower compared to

pristine biochar27. As shown in Tables 1 and 2, the BC@ZnO treatment increased Zn2+ uptake by the ryegrass roots and shoots. This is probably due to dissolving of the ZnO nanoparticles in

soil and releasing ionic Zn, and increasing its bioavailability and uptake by plants. Similar results were obtained by Hossein et al. (2018), Shafqat Ali et al. (2019), and Rizwan et al.

(2019). They reported that ZnO nanoparticles increased the bioavailability of Zn2+ and increased its uptake by different plant organs118,139,140. The data in Tables 1 and 2 show that there

was a significant difference between BC@ZnO and BC@MnO2 treatments in reducing the uptake of Pb, Ni, and Zn for the roots and shoots of plants, but no significant difference was observed for

Cd. Previous research has demonstrated that ZnO nanoparticles decrease Cd levels in a variety of plant species44,141,142. Due to the antagonistic effects of both metals, it is possible to

link this decrease in Cd uptake, at least in part, to the rise in Zn levels143. Zn and Cd compete with each other because both are transported to the root surface plasma membrane by a common

carrier144. Since the adsorption of Zn is easier than that of Cd, Cd phytoaccumulation is reduced when Zn is present145. Numerous studies have shown that plants with higher Zn levels

absorbed less Cd22,141,146,147. According to our results, the amount of Pb uptake by the plant was lower than that of other metals. This demonstrates that Pb is more adsorbed and immobilized

onto the adsorption sites than Cd, Ni, and Zn. Zn, Pb, Cd, and Ni are cations with similar valency, therefore, they are able to compete for the same sites and functional groups on the

adsorbent’s surfaces. According to Houben et al. (2013), Pb may have a stronger affinity for carboxylic and phenolic functional groups that are present on the surface of BC148. Similar

findings were observed by Jiang et al. (2020), Norini et al. (2019), and Namgay et al. (2010), who showed that biochar immobilizes Pb more strongly than other cationic HMs and removes it

from the reach of plants149,150,151. Therefore, according to the results of our study, it can be stated that BC, BC@MnO2, and BC@ZnO can effectively decrease the accumulation of HMs in

ryegrass, consequently reducing phytotoxicity. Also, biochar modified with nanoparticles were more effective than pristine biochar in reducing the uptake of HMs by ryegrass. EFFECT OF BC,

BC@MNO2 AND BC@ZNO ON BIO-CONCENTRATION (BCF) AND TRANSLOCATION FACTORS (TF) OF HM IN RYEGRASS TISSUES The two most important metrics for assessing the potential risks of metal ions for

plant growth in contaminated soils are bioconcentration (BCF) and translocation factors (TF). TF indicates the transport of HMs from the roots to the shoots of ryegrass. The TF value (Fig. 

5) of all HMs was less than 1, indicating that the HMs mainly accumulate in the roots. A low TF value also indicates a lower transport of HMs from roots to the shoots. Thus, all adsorbents

were successful in preventing the HMs transport from the roots to the shoots. Compared to pristine biochar, nanoparticle-modified biochar was more effective in reducing the transfer of HMs.

The results presented in Fig. 6 indicated that the BCF values of all HMs except Pb (in the control treatment) were greater than 1 and with the addition of adsorbents reached below 1.

Therefore, the results confirm that in this study, soil amendment with BC, BC@MnO2, and BC@ZnO was effective in reducing the uptake of HMs by ryegrass. In addition, the modified adsorbents

were found to be more effective in reducing the bioavailability of HMs. Similar findings to this study were reported by others18,149,150,151,152,153. The results of this study show that the

adsorbents are able to efficiently immobilize HMs in the soil, reduce their bioavailability to the plant and, on the other hand, prevent their translocation to the aerial parts of the plant.

As a result, ryegrass is less dangerous for primary consumers. Zhang et al. (2016) reported that high BCF and TF values indicate higher HMs concentrations in soil, higher uptake by plants

and higher translocation to aboveground organs, resulting in increased risk to consumers154. These findings suggest that ryegrass has the ability to bind HMs in its roots. For this reason,

ryegrass is considered as a suitable phyto-stabilizing plant155. According to our results, the BCF and TF values of Pb were lower than those of the other HMs in all treatments, indicating

less uptake by the plant roots and less transfer to the aerial parts. This result supports the findings on metal uptake by plants. CONCLUSION The main purposes of this study were to

synthesis modified biochar with MnO2 and ZnO nanoparticles and evaluate and compare their efficiency to remediate HMs contaminated soil and reduce their bioavailability, and improve plant

growth. Results showed that ZnO and MnO2 nanoparticles were successfully loaded onto biochar (BC). The addition of BC, BC@MnO2 and BC@ZnO immobilized Pb, Zn, Cd and Ni in soil, reduced their

uptake by the ryegrass and improved the dry weight of the ryegrass, but BC@MnO2 and BC@ZnO were more effective than unmodified BC in immobilizing HMs in the soil and reduced their uptake by

ryegrass. In addition, the efficiency of immobilization and the reduction of HMs uptake by the plant were higher in treatments with 1% adsorbents than with 0.5%. Overall, our results showed

that the application of 1% BC@MnO2 was better than other treatments in increasing the ryegrass dry weight, improving immobilization and reducing the availability of HMs to ryegrass. The

results showed that adsorbents had a stronger affinity for Pb than the other HMs. It can be concluded that each effectiveness of treatment depends not only on its dose and type but also on

the type of HMs. Results of present study showed that biochar modified with MnO2 and ZnO nanoparticles had more benefit effect on soils remediation compared to pristine biochar, and they can

be used as efficient and low-cost amendments to remediate HMs-contaminated soils and improve plant growth. Also, a comparison of the TF in ryegrass showed that it had a good ability to

accumulate all studied HMs in its roots; therefore, it is a suitable plant for HMs phytostabilization. METHODS MATERIALS AND CHEMICALS All chemicals, lead nitrate (Pb(NO3)2), Zinc nitrate

(Zn(NO3)2), Cadmium nitrate (Cd(NO3)2), Nickel nitrate (Ni(NO3)2), potassium permanganate (KMnO4), Hydrogen Peroxide (H2O2), Sodium hydroxide (NaOH), nitric acid (HNO3), Zinc sulfate

heptahydrate (ZnSO4.7H2O), and potassium hydroxide (KOH) were provided by Merck company (Germany) and used without any further purification. PREPARATION OF BIOCHAR Wheat straw, selected as

the raw material for preparing biochar and obtained from the animal husbandry station of Shiraz University Faculty of Agriculture. Wheat straw was first washed several times with deionized

water to remove impurities, dried at 60°C for 48 h, and then ground. The crushed samples were pyrolyzed at 500°C in an electric furnace with limited oxygen for three hours130. The produced

biochar (BC) was passed through a 2-mm sieve and stored in plastic containers. PREPARATION OF BIOCHAR-LOADED WITH MNO_2_ NANOPARTICLES Biochar modified with MnO2 nanoparticles prepared

according to the Zhang et al. (2020) and Liang et al. (2017) methods. Briefly, 15 g of BC and 3.16 g of KMnO4 (the manganese source) were combined with 150 mL of deionized water and stirred

at 25 °C for 30 min. While stirring, 40 ml of 30% H2O2 was added to the mixture dropwise. After adding 1 M HNO3 and NaOH to bring the pH of 7.0, the mixture was stirred for another 30 min

and allowed to stand at room temperature for three hours. After filtering, the mixture was repeatedly purified with distilled water and dried at 105 °C for 12 h66,156. PREPARATION OF

BIOCHAR-LOADED WITH ZNO NANOPARTICLES Biochar modified with ZnO nanoparticles was synthesized by the precipitation method. 0.15 M KOH and 0.1 M ZnSO4.7H2O solutions were prepared in

distilled water. 100 mL of 0.1 M ZnSO4 solution and 1.0 g of BC were well mixed for 10 min. Then 100 mL of KOH solution was added to the mixture while being vigorously stirred continuously

to ensure homogeneity. The mixture was then left to stand for 1 h and then filtered using a Whatman filter. Finally to remove the moisture, the filtered nanocomposite (BC@ZnO) was oven dried

at 100°C for 30 h100. ADSORBENT CHARACTERIZATION Scanning electron microscopy (SEM) (TESCAN-Vega3, Czechia) with scattered X-ray spectroscopy (EDX) was used to study the morphology and

composition of the adsorbent’s elements. The surface functional groups and the crystal structures of metal minerals of the adsorbents before and after HMs adsorption was characterized by

Fourier transform infrared spectroscopy (FTIR) (Tensor II, Bruker, Germany) and X-ray diffractometer (XRD) (Rigaku Ultima IV, Japan) in the 2θ range of 20° to 80°, respectively. The specific

surface area of the adsorbents was investigated by Brunauer, Emmett,—Teller (BET) (Belsorp mini II, Microtrac Bel Corp, Japan). SOIL SAMPLING The studied soil sample was collected from a

depth of 0–30 cm from the Shiraz University Faculty of Agriculture, Bajgah, Fars Province, Iran. The collected soil was air-dried, ground, and passed through a 2 mm sieve. Some

physicochemical properties of the studied soil such as particle size by hydrometric method157, pH using a pH meter in saturated paste158, electrical conductivity (EC) in the extraction using

an electrical conductivity meter159, cation exchange capacity (CEC) by the replacing cations with NaOAc160, organic matter (OM) by the Walkley–Black method161. DTPA extraction was used to

evaluate the available form of iron (Fe), manganese (Mn), zinc (Zn), copper (Cu), lead (Pb), cadmium (Cd) and nickel (Ni),162 and their concentration was determined by the Shimadzu AA-670G

atomic absorption spectroscopy(Table 3). SOIL CONTAMINATION AND INCUBATION The soil was divided into four equal sections, and 100 mg kg-1 of Zn, Cd, Ni, and Pb (as a nitrate source) were

added to each section. The contaminated soil was carefully mixed and incubated for one month at 25°C in a greenhouse condition. Soil samples were irrigated with distilled water during the

incubation period, and the soil moisture content was kept at field capacity by adding distilled water163. To evaluate the effect of biochar and biochar modified with ZnO and MnO2

nanoparticles on the immobilization of Pb, Ni, Cd, and Zn and their bioavailability for ryegrass, a completely randomized design with three replications was conducted under greenhouse

conditions. The treatments consisted of: HMs polluted soil (Control) (CK), pristine biochar (0.5% (BC-0.5%), 1% (BC-1%)), ZnO NPs-modified biochar (0.5% ([email protected]%), 1% (BC@ZnO-1%)) and

MnO2 NPs-modified biochar (0.5% ([email protected]%), 1% (BC@MnO2-1%)). Adsorbents were added to the contaminated soil, and then they were completely mixed with the soil and incubated for two

weeks at a 25 °C temperature and the field capacity moisture. POT EXPERIMENTS AND HMS MEASURMENTS IN PLANT Ryegrass seeds were selected for cultivation and sterilized in a 1% (_V/V)_ NaOCl

solution for five minutes. Then, it was washed several times with deionized water and soaked overnight in deionized water. The intubated soils with adsorbents were transferred to pots and

100 perennial ryegrass seeds were planted in each pot138.The greenhouse temperature was set steady during the day and night at 25 ± 5 °C. Soil moisture was kept at field capacity by adding

distilled water during the growth period (without any leaching). After 7 weeks aerial parts of the plant were harvested and the roots were separated from the soils. To remove any dust

particles, the collected shoots were first properly rinsed with tap water and then with distilled water. the roots were washed with tap water, EDTA, and distilled water. A dilute EDTA

solution was used to remove HMs that may be present on the surface of the roots. The aerial parts and roots were completely dried at 60 °C for 72 h. After weighting, the dried samples were

ground. The concentrations of Cd, Ni, Pb and Zn in the samples were measured by atomic absorption spectrophotometry (AA-6800, Shimadzu) after wet digestion. Equations 1 and 2 were used to

determine the HMs uptake by the root and shoot 163: $${\text{Root uptake }}\left( {\mu {\text{g pot}}^{{ - { 1}}} } \right) = {\text{root concentration }}\left( {\mu {\text{g g}}^{{ - { 1}}}

} \right) \, \times {\text{ root dry matter }}\left( {{\text{g pot}}^{{ - { 1}}} } \right)$$ (1) $${\text{Shoot uptake }}\left( {\mu {\text{g pot}}^{{ - { 1}}} } \right) \, = {\text{ shoot

concentration }}\left( {\mu {\text{g g}}^{{ - { 1}}} } \right) \, \times {\text{ shoot dry matter }}\left( {{\text{g pot}}^{{ - { 1}}} } \right)$$ (2) BIO CONCENTRATION AND TRANSLOCATION

FACTORS The effectiveness of biochar and biochar modified with MnO2 and ZnO nanoparticles to adsorb HMs and minimize their absorption by plant roots and shoots was determined by transport

factor (TF) and bio-concentration factor (BCF) indexes18. $${\text{TF}}=\frac{\mathrm{Concentration\,of\,metal\,in\,shoots }(\mathrm{mg kg}-1)}{\mathrm{Concentration\,of\,metal\,in\,roots

}(\mathrm{mg kg}-1)}$$ (3) $${\text{BCF}}=\frac{Concentration\,of\,metal\,in\,roots\,and\,shoots (mg kg-1)}{concentration\,of\,metal\,in\,soil (mg kg-1)}$$ (4) STATISTICAL ANALYSIS Data were

analyzed using SAS 9.4 software. Differences between treatments were determined following Duncan’s Multiple Range Test (DMRT), (_P_ ≤ 0.05). Probability levels of 1% and 5% (_P_ ≤ 0.01 or

0.05) were selected to test the significance of the differences. Figures were drawn using Excel 2018 software. ETHICAL APPROVAL No ethical issues were violated in this study. CONSENT TO

PUBLISH All the authors gave their consent to publishing this manuscript in your journal. DATA AVAILABILITY All data generated or analyzed during this study is included in this article.

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_Ecotoxicol. Environ. Saf._ 207, 111315 (2021). Article  CAS  PubMed  Google Scholar  Download references AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Soil Science, School of

Agriculture, Shiraz University, Shiraz, Iran Mahboobeh Varnaseri Ghandali, Sedigheh Safarzadeh & Reza Ghasemi-Fasaei * Faculty of Advanced Technology, Shiraz University, Shiraz, Iran

Sedigheh Zeinali Authors * Mahboobeh Varnaseri Ghandali View author publications You can also search for this author inPubMed Google Scholar * Sedigheh Safarzadeh View author publications

You can also search for this author inPubMed Google Scholar * Reza Ghasemi-Fasaei View author publications You can also search for this author inPubMed Google Scholar * Sedigheh Zeinali View

author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS M.V.G.: Carried out the experiment and analyzed the data. Wrote the manuscript. Discussed the

results and contributed to the final manuscript. S.S.: Conceived of the presented idea. Supervised the project (supervisor). Edited and reviewed the manuscript. Contributed to the final

version of the manuscript. And discussed the results and contributed to the final manuscript. Revised the manuscript. R.G.-F. and S.Z.: Helped review the project (advisor). All authors read

and approved the final manuscript. CORRESPONDING AUTHOR Correspondence to Sedigheh Safarzadeh. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL

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S., Ghasemi-Fasaei, R. _et al._ Heavy metals immobilization and bioavailability in multi-metal contaminated soil under ryegrass cultivation as affected by ZnO and MnO2 nanoparticle-modified

biochar. _Sci Rep_ 14, 10684 (2024). https://doi.org/10.1038/s41598-024-61270-5 Download citation * Received: 08 December 2023 * Accepted: 03 May 2024 * Published: 09 May 2024 * DOI:

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