Enhancing Soil Remediation of Copper-Contaminated Soil through Washing with a Soluble Humic Substance and Chemical Reductant

: The bioavailability and mobility of copper (Cu) in soil play a crucial role in its toxicity and impact on soil organisms. Humic substances, with their abundant functional groups and unique pore structure, have demonstrated the ability to effectively mitigate the toxic effects of heavy metals in soil. This study explores the potential of a soluble humic substance (HS) derived from leonardite for Cu removal from contaminated soils. The effects of various washing conditions, such as concentration and washing cycles, on removal efﬁciency were assessed. The results showed that a single washing with HS solution achieved an optimal removal efﬁciency of 37.5% for Cu in soil, with a subsequent reuse achieving a removal efﬁciency of over 30.5%. To further enhance Cu removal efﬁciency, a two-step soil washing approach using a chemical reductant NH 2 OH · HCl coupled with an HS solution (NH 2 OH · HCl + HS) was employed, resulting in an increased removal efﬁciency to 53.0%. Furthermore, this approach signiﬁcantly reduced the plant availability and bioaccessibility of Cu by 13.6% and 11.4%, respectively. Compared to a single washing with NH 2 OH · HCl, both HS and NH 2 OH · HCl + HS increased the soil pH and organic matter content. These ﬁndings suggest that the two-step soil-washing approach using NH 2 OH · HCl + HS effectively removed Cu from polluted soil. This study demonstrates the potential of humic substances as environmentally friendly materials for remediating heavy metal-polluted soil, promoting green and sustainable applications in soil remediation practices.


Introduction
Due to rapid urbanization and industrial transformation, industrial enterprises in China have relocated, resulting in numerous abandoned industrial and mining sites [1,2].Many of these sites have undergone significant changes, with most of the structures being demolished.Efforts are now underway to repurpose these sites as valuable social amenities, maximizing land utilization and revitalizing surrounding communities.However, because of the long-term and extensive production mode, these areas have severe ecological and environmental problems, exposing historical legacy issues and causing heavy metal pollution in the soil.This pollution not only threatens the safety of human health but also poses potential risks to the groundwater.Copper (Cu) is a commonly detected heavy metal in contaminated industrial sites.Although it is an essential trace element of the human body, exposure to high concentrations of Cu-contaminated soil may pose a significant risk to human health [1].Thus, removing Cu from the soil is crucial to ensure the environmental safety and ecological function of the soil, which is of great significance for sustainable site development [2].
Agronomy 2023, 13, 1754 2 of 13 However, it is crucial to carefully consider the choice of washing agent to minimize potential damage to soil structure and the ecological environment.While strong acids and alkalis may have higher soil washing efficiency, the use of high concentrations of inorganic acids can result in the loss of essential minerals such as calcium (Ca), magnesium (Mg), and phosphorus (P) from the soil.This can lead to the destruction of soil properties and structure, ultimately affecting soil microorganisms and fertility [3].Lower pH conditions increase the contents of dissolved Fe, Al, and Mn in the soil and their toxicity to crops.Chelating agents, although effective for soil washing, often have low biodegradability and can pose reactivation risks.Their use can negatively impact soil structure, result in the loss of soil nutrients, disrupt microbial ecology, and reduce soil productivity [4,5].Therefore, the selection of washing agents is critical to the success of soil washing technology.
Humic substances are effective biosurfactants for removing heavy metals from soil [6].They contain functional groups such as carboxylic, hydroxyl, phenolic hydroxyl, quinone, and aliphatic chains, which aid in chelating heavy metals.The primary interaction between humic substances and heavy metals occurs through the formation of complexes with carboxylic and phenolic hydroxyl groups [7,8].Moreover, humic substances selectively adsorb heavy metals through electrostatic interactions.Kulikowska et al. demonstrated that HS exhibited a higher affinity for binding Cd and Cu compared to Pb, Ni, and Zn [9].Previous studies have successfully used humic substances derived from coal deposits, agricultural wastes, and composted biomass to remediate soil contaminated with heavy metals [10,11].These substances effectively removed cationic metals such as Pb, Zn, Cu, Ni, and Hg due to their ability to form complexes and engage in ion exchange processes [12,13].However, it is important to note that humic substances stabilizing soil heavy metals can potentially dissolve and release heavy metals under certain environmental conditions, such as pH changes.This can lead to the co-migration of the humic substance solution and heavy metals.Therefore, a comprehensive evaluation of heavy metal mobility and effectiveness in soil remediation is necessary to clarify the repair effect on heavy metals.
A single chemical-reducing agent can efficiently remove exchangeable and carbonatebound heavy metals.However, heavy metals immobilized through this method are susceptible to environmental influences.Studies have shown that even after washing with inorganic acids, significant amounts of metal ions remain attached to iron oxides, which may lead to their release due to environmental changes that damage the iron oxide structure [14,15].A two-step soil-washing method was proposed in our previous study to address this issue [16].In the first step, the soil was treated with NH 2 OH•HCl to dissolve the amorphous iron oxide and temporarily release the loosely attached heavy metals.This destruction of the soil structure was followed by the second stage, where the soil was treated with a soluble humic substance (HS) extracted from leonardite to capture and remove the free metal ions rapidly.The two-step washing strategy can effectively remove cadmium and arsenic from artificially simulated, highly contaminated soil while addressing the adverse effects of chemical reducing agents, such as instability and soil acidification.Moreover, incorporating HS can reduce remediation costs and mitigate soil nutrient loss.
This study investigated the impact of NH 2 OH•HCl-enhanced HS washing on Cu-contaminated soil.Specifically, this study aimed to (1) compare the effectiveness of Cu removal using NH 2 OH•HCl alone, HS solution alone, and NH 2 OH•HCl + HS washing; (2) assess the distribution and stability of Cu under different treatment conditions; and (3) investigate the bioavailable fraction of Cu, including its plant availability and bioaccessibility as well as enzyme activity after soil washing.By comparing and analyzing the changes in Cu distribution and bioavailability in the soil after different washing processes, the study aimed to demonstrated the potential of NH 2 OH•HCl-enhanced Hs washing in effectively removing Cu from polluted soil while minimizing risks to the soil ecological environment.

Characterization of the Studied Soil
The soil used in this study was collected from a relocated industrial site in Suzhou City, Jiangsu Province, China.Before use, the soil samples were air-dried, ground, and passed through a 2 mm nylon sieve.The pH of the soil was measured by stirring a 1:5 (w:v) water suspension of the soil sample for 5 min.The potassium dichromate external heating oxidation method determined the soil organic matter (SOM) content.The cation exchange capacity (CEC) of soil samples were analyzed according to standard methods [17].Total Cu content in the soil was extracted by complete dissolution with HF-HClO 4 -HNO 3 and analyzed using inductively coupled plasma mass spectrometry (ICP-MS) with an Elan DCR II instrument from PerkinElmer Co., Waltham, MA, USA.The total concentration of Cu in the soil was 1205 mg•kg −1 .The proportion of Cu forms in the soil were carbonate-bound fraction (CB) (69.2%) > organic matter-bound fraction (OM) (12.8%) > residual fractions (RS) (10.9%) > Fe-Mn oxide-bound fraction (OX) (4.2%) > exchangeable fraction (2.9%).Additional basic soil properties are listed in Table 1.

Preparation of Soluble Humic Substance
The soluble humic substance (HS) was extracted from leonardite from Xinjiang Uyghur Autonomous Region, China.The leonardite powder was mixed with a 0.1 M KOH solution in a 2 L glass beaker at a 1:40 (g:mL) ratio.The mixture was stirred for 2 h at room temperature (25 ± 2 • C) and thereafter centrifuged at 3000× g for 10 min.In the end, the supernatant was oven-dried at 60 • C to obtain the HS.Ultrapure water was added to the HS and prepared in a 10 g•L −1 HS solution.

Evaluation of Cu Removal Efficiency using Different Agents in Batch Experiments
Batch experiments were performed in triplicate at room temperature to investigate the efficacy of NH 2 OH•HCl and HS in removing Cu from the soil.Although high concentrations of chemical reductant may have better leaching effects, lower concentrations of NH 2 OH•HCl may also have sound leaching effects, so a comparison evaluation is needed.The impact of chemical reductants on the physicochemical properties of soil was assessed with two NH 2 OH•HCl concentration gradients (0.008 and 0.2 M) in this experiment for comparison.The HS solution (10 g•L −1 ) prepared in Section 2.2 was acidified by adding 1.0 M HCl to achieve a pH of 7.01 ± 0.05 before the experiment.The contaminated soil was washed with NH 2 OH•HCl and HS solutions separately at a solid-liquid ratio of 1:5 and stirred with a two-paddle agitator at a speed of 400 rpm for 2 h at room temperature (25 ± 2 • C).After shaking, the suspensions were centrifuged at 3000 rpm for 10 min.The soil in the tubes was rinsed twice with deionized water to remove any residual washing agents.Soil residue in the tubes was then digested using HClO 4 -HNO 3 -HF to determine the concentration of Cu in the soil residue.The removal rates of Cu for the two agents were then calculated.
Three recycling cycles were performed to evaluate the reusability of the washing agents.After each soil type's first round of washing, the NH 2 OH•HCl and HS supernatants were decanted and collected.For the second and third rounds of washing, the same NH 2 OH•HCl and HS solutions were used, and the contaminated soils were washed under the same conditions.The resulting mixtures were then centrifuged, and the supernatant was collected for the next round of washing.The third round of washing was performed under the same conditions as the second round.The washed soil was rinsed twice with deionized water and stirred for 5 min to remove any remaining washing solution.Finally, the cleaned soil samples were air-dried, ground, and acid-digested to analyze their Cu contents.

Two-Step Soil Washing by Chemical Reductant and HS Solution
For the two-step washing, the first step was conducted using a pre-acidified hydroxylamine hydrochloride solution with a concentration of 0.008 M NH 2 OH•HCl, followed by a second step using a 10 g•L −1 HS solution.This design aimed to investigate the combined effect of NH 2 OH•HCl and HS on Cu removal from polluted soil.The soil washing conditions were set as aforementioned.The washed soil was rinsed, air-dried, and ground for further analysis.

Chemical Faction of Cu in the Soil after Soil Washing
The modified Tessier procedure [18] was used to determine the chemical faction of Cu in the soil.It involved determining five fractions of different mobility, including soluble and exchangeable (EX), carbonate-bound (CB), Fe-Mn oxide-bound (OX), organic matter-bound (OM), and residual fractions (RS).The reduced partition index (I R ) was then established based on heavy metal fractionation [19].The I R index provides information on changes in the strength of heavy metal binding in soil by reflecting the share of HMs in the fractions and the stability of individual heavy metals in the soil [20], as follows: where i is the index number of the sequential extraction step, for the weakest to the strongest (k) (in the Tessier procedure, k = 5), and F i is the percentage content of the concerned metal in fraction i.

Plant Availability and Bioaccessibility of Cu and Soil Enzymatic Analyses
Soil urease activity was determined using the sodium hypochlorite-sodium phenate colorimetry assay with urea as the substrate.Soil catalase activity was determined using the permanganometric assay with hydrogen peroxide as the substrate, which was expressed as mL.
The plant bioavailability of residual Cu was evaluated using a diethylenetriaminepentaacetic acid (DTPA) extraction method [18].The DTPA extraction solution contained 0.005 M DTPA, 0.01 M CaCl 2 , and 0.1 M triethanolamine, and the pH was adjusted to 7.3.The soil samples were mixed with the DTPA solution at a solid-to-liquid ratio of 1:2 (w:v) and shaken at 180 rpm for 2 h.The Cu concentrations in the filtered extracts were analyzed using ICP-MS.
The bioaccessibility of Cu in the soil to humans and the associated health risks were evaluated using the unified bioaccessibility method (UBM), which includes in vitro tests of simulated stomach and intestine compartments [21].The UBM-G test was conducted by mixing each treated soil sample (0.6 g) with simulated gastric fluid (13.5 mL) and adjusting the pH of the mixture to 1.1 ± 0.2 with concentrated HCl.The tubes were then shaken at 37 • C for 1 h.The UBM-I test was conducted by adding soil (0.6 g) to centrifuge tubes containing the same simulated gastric fluid (13.5 mL).After shaking at 37 • C for 1 h, intestinal fluid (pH 8.0 ± 0.2) and bile (pH 6.3 ± 0.2) were added to the tubes and shaken at 37 • C for 4 h.The supernatant was then filtered through 0.45 µm and acidified for Cu analysis using ICP-MS.
Soil enzymatic analyses were conducted to determine the potential effects of Cu contamination on soil microbial activity.The enzymatic activities of urease and catalase were measured using standard methods.Urease activity was determined using the phenol-hypochlorite colorimetric method, and catalase activity was measured using the permanganometric method [22,23].

Data Analysis
All experimental data are presented as the mean values of triplicate repetitions.Statistical analysis was conducted using SPSS Statistics 26 software.

Cu Removal Efficiency by Soil Washing
The soluble humic substance (HS) can act as both a complexing agent and an adsorbent for heavy metals, leading to various reaction forms such as coordination, adsorption, precipitation, and redox, which ultimately affects the migration and transformation of heavy metals in soil [24,25].Our previous studies indicated that the HS extracted from leonardite are rich in functional groups such as carboxyl and phenolic hydroxyl, which have a specific binding ability and can form complexes with heavy metals in the soil, washing them out and reducing the accumulation and toxic effects of heavy metals in the soil [16].In our study, a single washing with leonardite-derived HS can reduce the total Cu content from 1205 to 452 mg•kg −1 and remove 37.5% of Cu from heavily contaminated soil, as presented in Figure 1.In comparison to previous studies, such as the work conducted by Yang et al., they used a synthetic humic-like acid containing a high COOH content to remediate heavy metal-contaminated agricultural soil.In their research, the total Cu content in the soil was 302 mg•kg −1 , and their single washing approach resulted in the removal of 30.6% of Cu through complexation and adsorption mechanisms [10].These findings demonstrate the potential of HS as an effective washing agent for removing heavy metals from the soil, particularly with high concentrations of heavy metals.

Data Analysis
All experimental data are presented as the mean values of triplicate repetitio tistical analysis was conducted using SPSS Statistics 26 software.

Cu Removal Efficiency by Soil Washing
The soluble humic substance (HS) can act as both a complexing agent and an bent for heavy metals, leading to various reaction forms such as coordination, adso precipitation, and redox, which ultimately affects the migration and transforma heavy metals in soil [24,25].Our previous studies indicated that the HS extracte leonardite are rich in functional groups such as carboxyl and phenolic hydroxyl, have a specific binding ability and can form complexes with heavy metals in th washing them out and reducing the accumulation and toxic effects of heavy metal soil [16].In our study, a single washing with leonardite-derived HS can reduce th Cu content from 1205 to 452 mg•kg −1 and remove 37.5% of Cu from heavily contam soil, as presented in Figure 1.In comparison to previous studies, such as the wo ducted by Yang et al., they used a synthetic humic-like acid containing a high COO tent to remediate heavy metal-contaminated agricultural soil.In their research, th Cu content in the soil was 302 mg•kg −1 , and their single washing approach resulted removal of 30.6% of Cu through complexation and adsorption mechanisms [10] findings demonstrate the potential of HS as an effective washing agent for rem heavy metals from the soil, particularly with high concentrations of heavy metals.As shown in Figure 1, using 0.2 M NH2OHHCl alone effectively extracted C the soil, with a removal rate of 50.4%, which can be attributed to the strong acid As shown in Figure 1, using 0.2 M NH 2 OH•HCl alone effectively extracted Cu from the soil, with a removal rate of 50.4%, which can be attributed to the strong acidity and presence of hydroxyl groups of NH 2 OH•HCl.The -OH groups can form a complex with Cu, enhancing its mobility and allowing it to transfer from the soil to the solution and be washed out for removal [26].However, in practical engineering applications, the choice of reducing agent concentration should consider the cost and feasibility of the actual operation.Using high-concentration chemical reagents in low-permeability, multi-phase, and heterogeneous soil media can destroy soil structure and physical and chemical properties due to the long-term residue of chemical reagents.It can be seen from Table S1 that the addition of 0.2 M NH 2 OH•HCl lowered the soil pH from 7.36 to 6.30, and there is a risk of soil acidification.
Previous studies have shown that some emerging biosurfactants and biodegradable chelators can achieve comparable removal efficiency, but the costs of these washing agents are usually relatively high [27,28].Furthermore, using high-concentration chemical reagents can also cause problems such as nutrient loss [15,29,30].The Cu removal rate decreases to 33.8% when the NH 2 OH•HCl concentration is reduced to 0.008 M, which is comparable to the first washing efficiency of HS.The combination of NH 2 OH•HCl + HS works synergistically to take advantage of both compounds.Compared to using HS or NH 2 OH•HCl alone, the use of NH 2 OH•HCl + HS leaching significantly enhances the removal of Cu in the soil (53.0%).The resulting total Cu content in the soil is 566 mg•kg −1 .This study showed high simultaneous Cu removal efficiency in the soil in the two-step washing treatment, which could be attributed to the fact that NH 2 OH•HCl destroys the chemical structure of the soil, followed by HS increasing the soil organic matter content (Table S1) and strengthening the complexation and adsorption of Cu [9,16].
Figure 1 demonstrates the effect of washing cycles on Cu removal at an NH 2 OH•HCl concentration of 0.008 M/0.2 M and an HS solution concentration of 10 g•L −1 .The results show that even after the second reuse of the reducing agent, 17.7% to 30.0% of Cu can still be removed.

Cu Distribution and Stability in the Soil after Washing
Soil washing significantly impacted Cu distribution and stability in soil.Before washing, Cu was predominantly present in the unstable carbonate-bound fraction (69.2%) of the soil, as shown in Figure 2.This is consistent with previous studies showing that Cu in the soil around smelters is mainly found in the mobile fraction [31].The HS solution washing reduced the unstable exchangeable Cu (EX-Cu) fraction from 2.9% to 0.4%, with the content of EX-Cu being 1.42 mg•kg −1 in soil.Bi et al. also found that the HS provided plenty of adsorption sites for heavy metals [32].
due to the long-term residue of chemical reagents.It can be seen from Table S1 addition of 0.2 M NH2OHHCl lowered the soil pH from 7.36 to 6.30, and there is soil acidification.
Previous studies have shown that some emerging biosurfactants and biodeg chelators can achieve comparable removal efficiency, but the costs of these washin are usually relatively high [27,28].Furthermore, using high-concentration chem gents can also cause problems such as nutrient loss [15,29,30].The Cu removal creases to 33.8% when the NH2OHHCl concentration is reduced to 0.008 M, w comparable to the first washing efficiency of HS.The combination of NH2OHH works synergistically to take advantage of both compounds.Compared to usin NH2OHHCl alone, the use of NH2OHHCl + HS leaching significantly enhance moval of Cu in the soil (53.0%).The resulting total Cu content in the soil is 566 This study showed high simultaneous Cu removal efficiency in the soil in the t washing treatment, which could be attributed to the fact that NH2OHHCl dest chemical structure of the soil, followed by HS increasing the soil organic matter (Table S1) and strengthening the complexation and adsorption of Cu [9,16].
Figure 1 demonstrates the effect of washing cycles on Cu removal at an NH2O concentration of 0.008 M/0.2 M and an HS solution concentration of 10 g•L −1 .Th show that even after the second reuse of the reducing agent, 17.7% to 30.0% of Cu be removed.

Cu Distribution and Stability in the Soil after Washing
Soil washing significantly impacted Cu distribution and stability in soil.Befor ing, Cu was predominantly present in the unstable carbonate-bound fraction (6 the soil, as shown in Figure 2.This is consistent with previous studies showing th the soil around smelters is mainly found in the mobile fraction [31].The HS solutio ing reduced the unstable exchangeable Cu (EX-Cu) fraction from 2.9% to 0.4%, content of EX-Cu being 1.42 mg•kg −1 in soil.Bi et al. also found that the HS provide of adsorption sites for heavy metals [32].On the one hand, the HS can form soluble complexes with Ex-Cu by binding with organic functional groups (i.e., carboxyl group, phenolic hydroxyl group, etc.), thereby removing it from the soil.On the other hand, a small amount of HS-Cu complexes may also be adsorbed by the soil or undergo agglomeration and precipitation, resulting in relatively stable components that remain in the soil.In contrast, elution with 0.2 M NH 2 OH•HCl significantly increased the unstable EX-Cu component from 3% to 7%, with the content of EX-Cu being 3.16 mg•kg −1 in soil.Furthermore, since NH 2 OH•HCl is an inorganic acid, its high concentration facilitates the desorption of heavy metals by decreasing the soil pH, leading to a significant reduction in the content of carbonate-bound Cu (CB-Cu).However, soil washing with a low concentration of NH 2 OH•HCl (0.008 M) effectively reduced the EX-Cu fraction to 0.6%, with a Cu content of 3.41 mg•kg −1 in soil, similar to the effect of HS washing.After undergoing two cycles of reuse, both HS and NH 2 OH•HCl solutions remained effective in reducing the unstable EX-As component to less than 1% (Figure S1).This indicates that they retained their ability to remove Cu from the soil.
Humic substances could form inner or outer sphere coordination onto clay minerals via surface carboxylic and hydroxyl functional groups, hydrogen bond, and hydrophobic forces, which can impact the retention of heavy metals and enhance the passivation effect [33].Dong et al. used 1% humic acid to reduce the exchangeable heavy metals in soil by 7% [34].The two-step soil washing process using NH 2 OH•HCl + HS primarily decreased EX-Cu from 17.3 to 5.3 mg•kg −1 (as shown in Figure 2); this finding agrees with the previous study.The decrease in EX-Cu may be attributed to the complexation of HS with the loosely bound Cu that was released during the first step.Additionally, due to the electronegativity of Cu, it can be attracted to HS through electrostatic force.
Furthermore, it has been found that Cu binding affinity is enhanced by size-fractioned humic substances rich in aromaticity [35].During the two-step soil-washing process, the content of residual Cu (RS-Cu) decreased slightly from the original 64 to 54 mg•kg −1 .This may be due to a small amount of stirred and broken soil particles suspended in the upper layer after adding the HS solution in the second step.Therefore, in future research, the physical and chemical properties and the reuse value of the eluent should be further investigated.To better understand Cu's stability in the soil after washing, we conducted an analysis using the stability ratio (I R ).The results showed that the stability of Cu in the soil increased as the RS-Cu fraction increased, and the I R values of Cu ranged from 0.32 to 0.41, indicating that Cu in the soil was moderately stable (0.3 < I R ≤ 0.5).The I R value of Cu in the unwashed soil was 0.32, and there was a slight increase in the I R value after all treatments, suggesting that both washing agents positively impacted reducing the mobility and ecological risk of Cu in the soil.However, from the perspective of the distribution in Cu fractions, CB-Cu remained the dominant component in the soil after washing.This component carries a specific risk of release and can be easily absorbed and utilized by plants [36].Therefore, it can be combined with other green stabilized materials or hyperaccumulators for synergistic restoration in the subsequent remediation process.

Changes in Cu Plant Availability and Bioaccessibility after Washing
Based on our current results, the plant availability of Cu in the soil under different treatments was found to be in the order of NH 2 OH•HCl + HS < HS < NH 2 OH•HCl, as shown by the content of DTPA-extracted heavy metals in the soil before and after washing (Figure 3a and Figure S2a).The use of 0.2 M NH 2 OH•HCl increased the content of plantavailable Cu in the soil, which is consistent with the observed increase in soil EX-Cu, as explained in Section 3.2.
Using acidified NH 2 OH•HCl harmed the original structure of the soil [37][38][39][40].Minerals such as Mg, Al, and Mn may be released after the original structure of the soil is destroyed, and it may co-dissolve with Cu to enhance the mobility of Cu [41,42], thereby leading to an increase in loosely bound Cu content [43].This is an undesirable but unavoidable effect of chemical extraction.However, HS has a strong selective complexing ability for Cu.It can form complexes with soil clay minerals through metal oxide or ion bridging [44], providing additional binding sites for Cu and reducing its reactivity and plant availability.The significant reduction in plant-available Cu content in the soil treated with NH 2 OH•HCl + HS confirms the potential applicability of HS in the remediation of heavy metal-contaminated soils.Using acidified NH2OHHCl harmed the original structure of the soil [37-4 erals such as Mg, Al, and Mn may be released after the original structure of th destroyed, and it may co-dissolve with Cu to enhance the mobility of Cu [41,42] leading to an increase in loosely bound Cu content [43].This is an undesirable voidable effect of chemical extraction.However, HS has a strong selective com ability for Cu.It can form complexes with soil clay minerals through metal oxid bridging [44], providing additional binding sites for Cu and reducing its reacti plant availability.The significant reduction in plant-available Cu content in the so with NH2OHHCl + HS confirms the potential applicability of HS in the remed heavy metal-contaminated soils.
The above analysis can also help with understanding the variations in soil C cessibility.Except for the 0.008 M NH2OHHCl treatment, all other treatments soil Cu bioavailability to varying degrees, with the effects on bioaccessibility of low to high being HS < NH2OHHCl < NH2OHHCl + HS (as shown in Figure 3b results could be attributed to Cu resorption in the soil, Cu complexation with p chemical precipitation of metals caused by the higher pH environment of the i The above analysis can also help with understanding the variations in soil Cu bioaccessibility.Except for the 0.008 M NH 2 OH•HCl treatment, all other treatments reduced soil Cu bioavailability to varying degrees, with the effects on bioaccessibility of Cu from low to high being HS < NH 2 OH•HCl < NH 2 OH•HCl + HS (as shown in Figure 3b and Figure S2b).These results could be attributed to Cu resorption in the soil, Cu complexation with pepsin, or chemical precipitation of metals caused by the higher pH environment of the intestinal compartment [31].The reducing agent's complexation with Cu makes them less soluble in the simulated gastric and intestinal environment [45].Moreover, Cu had a higher bioaccessibility in the gastric compartment than in the intestinal one, mainly because the former had a much lower pH than the latter.Most of the Cu was dissolved and released in the simulated gastric environment.As corroborated by previous research, the increased intestinal pH would cause the precipitation of dissolved Cu and its adsorption onto the intestinal compartment [46].In summary, these results demonstrate that both HS and NH 2 OH•HCl + HS washing are effective soil remediation methods for reducing the risk of Cu in soil for human health.

Effects of Soil Washing on Soil Enzyme Activities
Enzyme activities are susceptible to changes in soil environmental quality and can serve as performance indicators after soil remediation [47].Catalase and urease activities are commonly used in current research to estimate microbial-related metabolic activities, and they are also sensitive biological indicators of heavy metal pollution [48].The observed effects on enzyme activity may be due to changes in soil properties after sequential washing, such as the loss of microorganisms, soil organic matter, and soil nutrients, which can affect the activity of soil enzymes.Therefore, enzyme activity can be used as an essential indicator to evaluate the effectiveness of soil remediation.
Both high and low concentrations of NH 2 OH•HCl washing reduced the soil catalase activity compared to the original soil, while HS and NH 2 OH•HCl + HS washing had little effect on soil catalase activity (Figure 4a).Research has shown that HS can stabilize enzymes by forming complexes [49].Similarly, our findings confirm this phenomenon; there was little change in soil urease activity after treatment with HS and two-step washing.In contrast, high concentrations of NH 2 OH•HCl nearly doubled the urease activity compared to the unwashed treatment (Figure 4b).This increase in activity was likely due to the disaggregation of the loosely bound soil structure during the washing process, which provides more substrate to support increased microbial mass [50,51].These findings suggest that using HS washing has little effect on the activity of important enzymes in the soil and can effectively mitigate the adverse effects of chemical washing on enzyme activity.In the future, combined measures such as phytoremediation [52] after washing could further improve soil enzyme activity and quality.

Effects of Soil Washing on Soil Enzyme Activities
Enzyme activities are susceptible to changes in soil environmental quality and can serve as performance indicators after soil remediation [47].Catalase and urease activities are commonly used in current research to estimate microbial-related metabolic activities, and they are also sensitive biological indicators of heavy metal pollution [48].The observed effects on enzyme activity may be due to changes in soil properties after sequential washing, such as the loss of microorganisms, soil organic matter, and soil nutrients, which can affect the activity of soil enzymes.Therefore, enzyme activity can be used as an essential indicator to evaluate the effectiveness of soil remediation.
Both high and low concentrations of NH2OHHCl washing reduced the soil catalase activity compared to the original soil, while HS and NH2OHHCl + HS washing had little effect on soil catalase activity (Figure 4a).Research has shown that HS can stabilize enzymes by forming complexes [49].Similarly, our findings confirm this phenomenon; there was little change in soil urease activity after treatment with HS and two-step washing.In contrast, high concentrations of NH2OHHCl nearly doubled the urease activity compared to the unwashed treatment (Figure 4b).This increase in activity was likely due to the disaggregation of the loosely bound soil structure during the washing process, which provides more substrate to support increased microbial mass [50,51].These findings suggest that using HS washing has little effect on the activity of important enzymes in the soil and can effectively mitigate the adverse effects of chemical washing on enzyme activity.In the future, combined measures such as phytoremediation [52] after washing could further improve soil enzyme activity and quality.

Figure 1 .
Figure 1.Changes in total Cu content in the soil before and after washing/washing cycles tre with NH2OHHCl, HS, NH2OHHCl + HS.Different letters between the original soil and soil with agents indicate significant differences (p < 0.05) in the residual content.

Figure 1 .
Figure 1.Changes in total Cu content in the soil before and after washing/washing cycles treatments with NH 2 OH•HCl, HS, NH 2 OH•HCl + HS.Different letters between the original soil and soil washed with agents indicate significant differences (p < 0.05) in the residual content.

Figure 2 .
Figure 2. Changes in Cu fraction in the soil before and after washing treatments w NH2OHHCl, NH2OHHCl + HS.The reduced partition index (IR) is shown for different

Figure 2 .
Figure 2. Changes in Cu fraction in the soil before and after washing treatments with HS, NH 2 OH•HCl, NH 2 OH•HCl + HS.The reduced partition index (I R ) is shown for different types of treatment to quantify Cu stability.The classification of I R is I R ≤ 0.1, lack of stability; 0.1 < I R ≤ 0.3, low stability; 0.3 < I R ≤ 0.5, medium stability; 0.5 < I R ≤ 0.7, elevated stability; 0.7< I R ≤ 0.9, high stability; and I R > 0.9, very high stability.

Figure 3 .
Figure 3. Plant availability (a) and bioaccessibility (b) of Cu content after soil washing.Different letters present significant differences according to Tukey's test (p < 0.05).

Figure 4 .
Figure 4. Soil enzyme activities ((a) catalase activity and (b) urease activity) tests after soil washing.Different letters present significant differences according to Tukey's test (p < 0.05).

Figure 4 .
Figure 4. Soil enzyme activities ((a) catalase activity and (b) urease activity) tests after soil washing.Different letters present significant differences according to Tukey's test (p < 0.05).

Table 1 .
Physical and chemical properties of the contaminated soils.