1 Introduction

Anthropogenic activities associated with the rapid development of industry, agriculture, and urbanization have increased cadmium (Cd) contamination of soils worldwide (Mamat et al., 2020). Cd contamination is particularly serious in China, where levels range from 0.003 to 9.57 mg/kg (Sriprachote et al., 2012). In soil, the bioavailability and migration of Cd are significantly higher than for other soil contaminants (Wang et al., 2015; Wang et al., 2020). Cd exposure through the food chain is associated with a variety of toxic and carcinogenic effects, including damage to the kidneys, skeleton, and respiratory system (Fu et al., 2008; Huang et al., 2018). Furthermore, Cd has a negative impact on soil microorganisms and reduces the decomposition rate of litter, leading to reductions in soil quality and crop production (Liu et al., 2020). Therefore, remediation methods have been developed for the treatment of Cd-contaminated soil.

Physicochemical remediation and bioremediation have been employed to reduce Cd toxicity (Kumpiene et al., 2017). For physicochemical remediation, nano-zero-valent iron (nZVI) is a promising material that has undergone intensive study as a remediation agent for heavy metal pollution of water bodies and groundwater (Adeleye et al., 2016; Lei et al., 2018). Previous research has shown that its active chemical properties, large specific surface area, and surface energy confer high heavy metal reduction and adsorption capacities (Vilardi et al., 2017; Xue et al., 2018a). Previous studies have demonstrated that nZVI immobilizes Cd through adsorption and precipitation (Boparai et al., 2013; Guha et al., 2020). However, in the soil environment, the properties of nZVI are affected by the activities of animals and microorganisms, organic matter (OM), moisture content, and pH (Lei et al., 2018). These complex interactions accelerate the aging and agglomeration of nZVI particles, and thus affect remediation efficiency. Meanwhile, recent studies have addressed the biological toxicity of nZVI, which has negative effects on the soil ecosystem. For example, Liang et al. showed that 500 mg/kg nZVI had negative effects on the behavior and physiological responses of earthworms (Liang et al., 2018). Most research to date has not considered the health of soil animals and microorganisms. Therefore, the utility of nZVI as a heavy metal treatment in soil warrants further investigation.

For bioremediation, earthworms have strong potential for improving soil fertility, supporting soil nutrient cycling, modulating soil microflora, and remediating heavy metal-contaminated soil (Gong et al., 2019; Van Groenigen et al., 2019). For example, Eisenia fetida can effectively reduce soil available Cd (ACd) (Liu et al., 2020). Little information regarding the impact of nZVI on earthworm activities or cooperative effects of nZVI and earthworms on remediation of Cd-contaminated soil is available. Meanwhile, both nZVI and earthworms are good at remediating heavy metal–contaminated soils, but few studies have focused on their synergy. To fill this gap, we studied the combined effect of earthworms and nZVI on soil physicochemical properties and microecology. Our research provides new insights into the combined use of nZVI and earthworms for remediation of Cd-contaminated soil.

2 Materials and Methods

2.1 Experimental Materials

nZVI (DK-Fe-001; particle size, 30–50 nm, 99.9% (metal basis); true density, 7.87 g/cm3) (Fig. S2) was purchased from Beijing Dekedaojin Technology Co., Ltd. (Beijing, China). Cadmium chloride (CdCl2, > 99%) was obtained from Fengchuan Chemical Reagent Co., Ltd. (Tianjin, China). Red soil samples were collected in Kunming City, Yunnan Province, Southwestern China (N 24° 49′, E 102°51′; elevation, 1980 m). Humus soil was purchased from a commercial vendor. The red and humus soil were freed of plant rhizomes, seeds, and stones, dried naturally, and filtered through 2- and 0.25-mm mesh sieves. The red and humus soil were mixed at a mass ratio of 2:1. The background contents (measured in four samples) of ACd (available cadmium), Tfe (total iron), SOM (soil organic matter), total nitrogen (TN), alkali-hydrolyzable nitrogen (AN), total phosphorus (TP), and available phosphorus (AP) were 0 ± 0.00 g/kg, 54.52 ± 4.74 g/kg, 93.34 ± 3.00 g/kg, 2.74 ± 0.31 g/kg, 342.34 ± 47.36 mg/kg, 1.18 ± 0.12 g/kg, and 434.32 ± 32.22 mg/kg, respectively. Earthworms (Eisenia fetida, Daping-2) were purchased from a commercial breeder in Kunming City, and were allowed to excrete dirt and clear their intestines for 24 h (Liang et al., 2018). The earthworms were then transferred to soil, with a 2:1 mass ratio of red to humus soil and water content of 45%, for a 2-week habituation period. Healthy and segmented earthworms weighing 0.3 to 0.4g were chosen for the experiment.

2.2 Experimental Design

2.2.1 Experimental Procedures

We used indoor simulation experiments to study the efficiency and mechanism through which earthworms and nZVI act in combination to remediate Cd-contaminated soil. We used Eisenia fetida as our study species, with a density of five individuals per 500g of soil (Kostecka et al., 2020) and nZVI (at mass ratios of 0%, 0.05%, 0.25%, and 0.5%) (Yirsaw et al., 2016; Zou et al., 2016) as the research objects. An initial 500 g of soil containing Cd (30 mg/kg) was placed in a PVC tube (about 10 cm in diameter and 12 cm in height), and the moisture content was adjusted daily to 45%. After a stabilization period of 28 days, nZVI of various mass ratios was added to PVC tubes containing five Eisenia fetida or no earthworms. The temperature was maintained at 22 ± 3 °C and the day/night ratio was set to 12:12 throughout the experiment. On the 45th day of the test period, earthworms were collected from tubes in each treatment group, and counted and weighed after 24 h of intestinal clearing. The survival rate and biomass of earthworms in each sample were calculated. The soil samples collected on day 45 were analyzed for soil aggregate stability, ACd content, soil chemical properties, and soil microbial diversity.

2.2.2 Soil Agglomerate Stability and Physicochemical Properties

The aggregate size distribution (ASD) and mean weight diameter (MWD) were used as indicators to evaluate the stability of agglomerates (Kemper & Rosenau, 1986). Using the wet sieve method, water-stable soil agglomerates were obtained by immersing air-dried soil samples in distilled water for 5 min. Then, the sieve was manually shaken up and down 50 times within 2 min at an amplitude of 3 cm, and the resulting material was sorted into large agglomerates (> 250 μm), small agglomerates (53–250 μm), and clay (< 53 μm). Distilled water was used to wash soil from the sieve into a container, and the soil was then naturally air-dried and weighed. The proportions of different agglomerate sizes, ASD, and MWD were calculated. Soil chemical properties (pH, OM, TN, TP, AN, and AP) and TFe were determined according to the methods of Du and Gao (2006). Determination of ACd in soil was conducted with a flame atomic absorption spectrophotometer using DTPA (0.005 mol·L−1 diethylenetriaminepentaacetic acid, 0.1 mol·L−1 triethylamine, 0.01 mol·L−1 CaCl2) as the extractant.

2.2.3 Determination of Soil Microecology

On the 45th day of the soil microbial determination test, three devices were randomly selected from among the six devices of a given treatment, and a high-temperature-sterilized key was used to collect soil samples and place them in a high-temperature-sterilized centrifuge tube; these samples were stored at −20 °C until testing. After extraction of genomic DNA from the sample, the V3–V4 region of 16S rDNA was amplified. The primer sequences used in this study are 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The length of the amplicon was approximately 460 bp. The amplification product was cut and recovered, and then quantified with a QuantiFluor fluorometer. The purified amplification products were mixed in equal amounts, connected to sequencing adapters to construct a sequencing library, and sequenced on the Hiseq 2500 PE250 platform (Ilumina, San Diego, CA, USA). DNA/RNA/Small RNA/cDNA library sequencing was performed on the Hiseq 2500/4000 platform by Gene Denovo Biotechnology Co., Ltd. (Guangzhou, China). Bioinformatics analysis was performed using Omicsmart, a real-time interactive online platform for data analysis and storage (http://www.omicsmart.com). Redundancy analysis (RDA) was performed using the R package vegan; correlation coefficients were calculated based on species abundance tables and environmental factor tables using the R package psych and presented as heat maps; correlations between environmental factors and ACd were analyzed using the R package corrplot.

2.3 Data Processing and Analysis

One-way analysis of variance (ANOVA) was used to determine the effects of the mass ratio of nZVI on earthworms (including survival and biomass). Two-way ANOVA (followed by Duncan’s multiple range post hoc test) was used to determine the effects of nZVI concentration and earthworm activity on soil ACd content and soil physicochemical properties. All were normally distributed and met the requirements for the chi-square test. The analyses were performed using the SPSS statistical package (version 25.0; IBM Corp, Armonk, NY, USA). P < 0.05 was taken to indicate statistical significance. RDA was performed using the R package vegan. Correlations between environmental factors and species were analyzed using the R package psych based on species abundance and environmental factor tables; correlations between environmental factors and ACd were analyzed using the R package corrplot and presented as heat maps, based on Spearman’s correlation coefficients. Images were plotted and rendered using Origin 2022 software.

3 Results and Discussion

3.1 Effects of nZVI on Earthworm Survival Rate and Biomass

To determine the effects of nZVI on earthworms, various concentrations of nZVI (0, 0.05, 0.25, and 0.5%) were evaluated. Amendment with nZVI reduced the survival rate and biomass, although not significantly (Table 1). Similar to previous reports, nZVI addition to Cd-contaminated soil affected the biomass, survival rate, and physiology of earthworms (Bai et al., 2020; Liang et al., 2018). Liang et al. showed that 500 and 1000 mg/kg of nZVI significantly inhibited earthworm growth and respiration, and also enhanced the avoidance response (Liang et al., 2017). The highest earthworm biomass was detected under the 0.25% nZVI treatment, resulting in an 80% survival rate. Thus, it can be suggested that the 0.25% nZVI mass ratio has the least detrimental effect on earthworm survival.

Table 1 Survival rate and biomass of earthworms at different nZVI concentrations. Error bars are mean ± standard error (n=6). Different lowercase letters indicate significant differences within each treatment based on one-way ANOVA (P < 0.05)
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3.2 Effects of nZVI and Earthworms on Soil Physicochemical Properties

3.2.1 ACd and TFe in Soils

nZVI and earthworms effectively remediated the Cd-contaminated soil. However, the synergistic effects of ZVI and earthworms remain unclear. Thus, the effects of nZVI, alone and in combination with earthworms, on ACd in soil were evaluated in the remediation system. Amendment with nZVI, alone and in combination with earthworms, significantly reduced Cd availability (P < 0.01), as illustrated in Fig. 1a. Compared to treatment with nZVI alone, the combination of nZVI and earthworms more effectively reduced the ACd content of soil. Liu et al. (2022) reported a noteworthy decrease of 10.9% and 9.3% in available cadmium content in low-Cd and high-Cd soils, respectively, subsequent to treatment with 0.1% nZVI. Moreover, Jin et al. (2023) demonstrated a nZVI fixation efficiency of 32.05% in soil samples after 20 days of adding 5 g/kg of nZVI. Prior research has confirmed the significance of earthworms in reducing DTPA-Cd concentrations in cadmium-contaminated soils, ranging from 5 to 20 mg·kg−1 (Cheng et al., 2021).In our study, the 5–0.25% system had the lowest ACd content (7.40 mg/kg) and ACd was reduced by 75.3% in that treatment. From Fig. 1b, it is apparent that the total iron content of the soil was significantly increased with the addition of 0.05% nZVI in the absence of earthworms (P<0.05). However, no noteworthy alteration was observed in the total iron content of the soil under 0.25% and 0.5% nZVI treatments despite varying concentrations. Compared with the group without earthworms, the inclusion of earthworms and 0.05% nZVI led to a noteworthy fall in soil total iron content by 10.53%, dropping below the total iron background value of the earthworm and nZVI-free group. This suggests that earthworms possess the ability to decrease the overall iron content in soil. Consistent with these results, the 5–0.25% restoration system had little impact on earthworm survival. NZVI was oxidized to produce Fe2+ (Eqs. 1, 2) and Fe3+ (Eq. 3), and can co-precipitate with Cd2+ and anions (Eqs. 4–7) in the soil to form precipitates (Boparai et al., 2013; Li et al., 2020), and can adsorb Cd2+ via an oxide film on the outer layer of nZVI (Fajardo et al., 2020; Tasharrofi et al., 2020; Zhang et al., 2014). Thus, nZVI effectively reduces the ACd content of soil. Treatment with nZVI alone significantly reduced ACd content and alleviated its toxic effects on the earthworms. Moreover, previous studies showed that nZVI reduces the toxicity of Cd in soil (Xue et al., 2018b), thereby promoting the survival of soil organisms (Guha et al., 2020; Habish et al., 2017). Meanwhile, earthworm activity promoted the formation of aggregates and enhanced the remediation efficiency of nZVI (Al-Maliki & Scullion, 2013 ; Bottinelli et al., 2017). Additionally, a high concentration of nZVI (0.5%) negatively impacts earthworm biomass, movement, and life quality (Liang et al., 2017; Liang et al., 2018; Liu et al., 2015). Accordingly, the combination of a moderate nZVI concentration and earthworms (5–0.25%) yielded the highest remediation efficiency for Cd-contaminated soil. Overall, these results indicate that the combination of nZVI and earthworms is a promising strategy for remediation of Cd-contaminated soil. The main reaction equation is as follows:

$$2{\mathrm{Fe}}^0+{\mathrm{O}}_2+2{\mathrm{H}}_2\mathrm{O}\to 2{\mathrm{Fe}}^{2+}+4{\mathrm{O}\mathrm{H}}^{\hbox{-} }$$
(1)
$$2{\mathrm{Fe}}^0+2{\mathrm{H}}_2\mathrm{O}\to 2{\mathrm{Fe}}^{2+}+{\mathrm{H}}_2+2{\mathrm{OH}}^{\hbox{-} }$$
(2)
$$2{\mathrm{Fe}}^{2+}+2{\mathrm{H}}_2\mathrm{O}\to 2{\mathrm{Fe}}^{3+}\ {\mathrm{H}}_2+2{\mathrm{OH}}^{\hbox{-} }$$
(3)
$${\mathrm{Fe}}^{3+}+3{\mathrm{OH}}^{\hbox{-}}\to \mathrm{Fe}{\left(\mathrm{OH}\right)}_3$$
(4)
$$\mathrm{Fe}{\left(\mathrm{OH}\right)}_3\to \mathrm{Fe}\mathrm{O}\left(\mathrm{OH}\right)+{\mathrm{H}}_2\mathrm{O}$$
(5)
$${\mathrm{Cd}}^{2+}+{\mathrm{OH}}^{\hbox{-}}\to \mathrm{Cd}{\left(\mathrm{OH}\right)}^{-}$$
(6)
$$\mathrm{FeOOH}+{\left[\mathrm{Cd}\left(\mathrm{OH}\right)\right]}^{+}\to \mathrm{FeOOCd}\left(\mathrm{OH}\right)+{\mathrm{H}}^{+}$$
(7)
Fig. 1
figure 1

nZVI and its impact on total iron content in soil in the presence of earthworms (a); nZVI and its impact on soil available Cd in soil in the presence of earthworm (b). Error bars are mean ± standard error (n = 6). Difference lowercase letters indicate significant differences within each treatment based on two-way ANOVA (P < 0.05)

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3.2.2 Structure of Agglomerates in Soils

The ASD and MWD are important indicators of aggregate stability (Kemper & Rosenau, 1986). The nZVI-only and earthworm plus nZVI remediation systems in this study showed a significant difference in ASD (P < 0.05). Specifically, the proportion of large aggregates (diameter > 250 μm) was highest in the 0–0% (no earthworms and 0% nZVI) system (46.83%), while the proportion of clay (diameter < 53 μm) was lowest (12.34%) among the systems (Fig. 2a). The proportion of large aggregates in the nZVI-only remediation systems decreased with increasing nZVI concentration. After adding 0.05% nZVI, there was a significant increase in the clay content.

Fig. 2
figure 2

Effects of nZVI and earthworm activities on aggregate size distribution (a) and mean weight diameter (b). Error bars are mean ± standard error (n = 6). Difference lowercase letters indicate significant differences within each treatment based on two-way ANOVA (P < 0.05). d>250 μm represents large soil agglomerates, 53 μm<d<250 μm represents small agglomerates, d<53 μm represents clay, and MWD represents the mean equivalent diameter of the agglomerates. Difference lowercase letters indicate significant differences within each treatment based on two-way ANOVA (P < 0.05)

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As shown in Fig. 2b, the addition of various contents of nZVI to the soil significantly reduced the MWD of aggregates, but this effect leveled off after amendment with earthworms. In the system with earthworms, no significant difference was found in the MWD of soil aggregates with the addition of different low mass ratios (0%, 0.05%, 0.25%) of nZVI. Previous studies showed that reduction of toxic heavy metal content combined with increased soil animal movement was conducive to aggregate formation in soil (Aghababaei et al., 2014; Al-Maliki & Scullion, 2013; Mamat et al., 2020). Furthermore, the burrowing activity of earthworms affects soil structure and quality (Bacher et al., 2020). In summary, both nZVI and earthworm activity during the remediation of Cd-contaminated soil improve soil structure.

3.2.3 Soil Chemical Properties

The nZVI plus earthworm system altered the chemical composition of the soil, including soil pH, OM, TP, TN, AN, and AP, as shown in Fig. 3. Moreover, nZVI significantly influenced soil pH (Fig. 3a). Soil pH (6.34) was highest in the 5–0.25% remediation system. The system with low-concentration nZVI plus earthworms did not impact soil pH, while systems with moderate to high nZVI concentrations increased pH. This difference is likely attributable to alkaline substances released during the remediation process (Yang et al., 2019), and is consistent with previous studies showing that soil pH is an important factor affecting the remediation of heavy metal-contaminated soil with nZVI (Latif et al., 2020; Li et al., 2020). Increasing pH promotes the formation of Cd precipitates and thereby reduces ACd.

Fig. 3
figure 3

Effects of nZVI and earthworm activities on soil pH (a), organic matter content (b), total nitrogen (c), total phosphorus (d), alkali-hydrolyzable nitrogen (e), and available phosphorus (f). Error bars are mean ± standard error (n = 6). Difference lowercase letters indicate significant differences within each treatment based on two-way ANOVA (p < 0.05)

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The OM content was significantly higher in the combined treatment compared to the nZVI-only remediation systems (Fig. 3b). The OM content was 120.47 g/kg higher in the 5–0% (five earthworms and 0% nZVI) remediation system than other treatment groups. The addition of earthworms significantly increased the OM content of the soil (P < 0.05), while an increase in the nZVI mass ratio reduced the OM content, although not significantly (P > 0.05). Similarly, the combination of earthworms and nZVI significantly affected the TN content of soil (P < 0.01) (Fig. 3c), consistent with the observation that TN in soil is related to OM content (Zhu et al., 2017). As shown in Fig. 3d, in the treatments without earthworms, TP content was higher with 0.05% and 0.25% mass ratios of nZVI (1.47 g/kg and 1.31 g/kg, respectively) than with a 0% mass ratio. Meanwhile, the TP content was significantly lower with a 0.5% than 0% mass ratio of nZVI. As illustrated in Fig. 3e, an increase in the mass ratio of nZVI decreases the AN content of soil, but this effect was minimized with the addition of earthworms, and the combined nZVI-earthworm remediation system significantly affected the AN content of soil (P < 0.01). As shown in Fig. 3f, nZVI significantly altered the available P content of soil (P < 0.05), and the available P content of soil was positively correlated with the nZVI treatment level. The AP content of soil was highest in the 5–0.5% remediation system (519.06 mg/kg).

Figure 1b shows that earthworms combined with nZVI significantly reduced the ACd content. Yu et al. showed that a reduction in soil ACd was associated with increases in the OM, TN, and TP contents of soil (Yu et al., 2020). Thus, reducing ACd in the soil reduces its negative impact on soil quality (Boparai et al., 2013; Chen et al., 2019). Earthworms improve soil quality in both chemical and physical terms by maintaining soil structure and recycling nutrients (Bacher et al., 2020). An increase in earthworm abundance promotes the production of water-stable aggregates, which are rich in OM (Bedano et al., 2019). Based on the results of two-factor ANOVA shown in Table 2, the combined effect of earthworms and nZVI significantly affected the proportions of large aggregates, clay, MWD, TN, TP, and AN in the soil (P < 0.001), thereby significantly improving the quality of Cd-contaminated soil. Therefore, the combined effect of earthworms and nZVI remediation of Cd-contaminated soil reduces the availability and negative effects of Cd, while earthworm activities prevent the loss of C, N, and P from the soil during the restoration process.

Table 2 Effects of nZVI (N) and earthworm (E) soil physical and chemical properties, and the stability of aggregates (two-way repeated measures ANOVA, F values, and significances). ns, not significant (P > 0.05), *P < 0.05, **P < 0.01, ***P < 0.001
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3.3 Effects of nZVI and Earthworms on Soil Microorganisms

3.3.1 Analysis of Bacterial Community Structure and Diversity

Microorganisms are an important component of the soil ecosystem, and alteration of microbial abundances can affect the circulation and flow of substances in the soil ecosystem (Fierer, 2017). The bacterial community from day 45 soil samples was analyzed through high-throughput sequencing of the 16S rRNA V3–V4 region. The results showed that the Sobs index value tended to plateau with increasing depth of sequencing (Fig. S3), indicating that the data obtained from the samples were reasonable. Earthworms and nZVI significantly impacted indices of microbial diversity and species richness (P < 0.05) (Table 3). In the 0–0.05%, 5–0.25%, and 5–0.5% remediation systems, the diversity and abundance indices for soil microorganisms did not differ from the control values (0–0%). This similarity indicated that low- and moderate-concentration nZVI plus earthworms did not reduce the diversity or richness of soil microorganisms. Non-metric multidimensional scaling (NMDS) analysis revealed similarity in species composition among treatments, with a significant degree of separation associated with increasing nZVI concentrations. However, the effect of nZVl on the species distribution was dampened by the addition of earthworms (Fig. S4), indicating that amendment with earthworms reduced the impact of nZVI on the soil bacterial community.

Table 3 Effects of nZVI (mass ratios of 0%, 0.05%, 0.25%. and 0.5%) and earthworms (none or five per tube) on the diversity and abundance indices of soil microorganisms (one-way repeated measures ANOVA). Values are mean ± standard error (n = 3). Different letters indicate a significant difference among treatments at the 5% level according to Duncan’s test
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Planctomycetes, Proteobacteria, Acidobacteria, and Chloroflexi were the dominant bacterial phyla (Fig. 4a). The composition of microbes was similar among treatments, but the proportions and abundances of specific microbial taxa differed significantly. In the 5–0% remediation system, Planctomycetes was dominant (32.2%). The proportion of Planctomycetes was higher in the earthworms plus nZVI combined treatment than nZVI-only remediation system. Verrucomicrobia and Firmicutes were dominant in the 0–0.05% remediation system (5.04% and 1.1%, respectively). In the 5–0% remediation system, Patescibacteria was the most abundant taxon (8.98%). The 5–0%, 0–0.25%, and 5–0.25% remediation systems had larger proportions of Acidobacteria than other systems (18.95%, 18.93%, and 18.87%, respectively). At the genus level, as shown in Fig. 4b, the three genera with the highest relative abundances, Pirellula, Pir4_lineage, and SH-PL14, all had higher relative abundances in the 5–0.05% system, indicating that the combined effect of earthworms and low to moderate concentrations of nZVI significantly enhanced the abundance of these dominant microbial groups.

Fig. 4
figure 4

Average relative abundances of phylum (a) and genus (b) level of soil bacterial community under different treatments (at the phylum level, the relative abundance < 0.1% was classified as others; top 20 genera based on mean relative abundance rankings at the genus level)

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3.3.2 Linking the Variations of Soil Physicochemical Properties and Microbial Communities with ACd

To explore the potential mechanisms underlying the reduction of ACd associated with nZVI and earthworms, the correlations of ACd and soil physicochemical properties with species were evaluated using Spearman’s rank correlation coefficient (Fig. 5). In the earthworm-free system, ACd was negatively correlated with pH (P < 0.05), while Stenotrophobacter was negatively correlated (P < 0.05) with soil agglomerates (MWD and MA), TN, and ACd. These results indicate that nZVI reduced ACd by increasing pH and the abundance of Stenotrophobacter. Soil pH is an important factor affecting the remediation of heavy metal–contaminated soil by nZVI, and has been associated with decreased ACd due to the formation of Cd precipitates (Li et al., 2020). Stenotrophobacter belongs to the Acidobacteria, which are often enriched in heavy metal–contaminated environments and have the capacity for metal transformation (Xu et al., 2022), indicating adaptability to metal-rich environments. Therefore, we speculated that Stenotrophobacter might be involved in Cd immobilization.

Fig. 5
figure 5

Effects of various environmental factors on microbial community structure at the genus level [top 20 genera based on mean abundance rankings without earthworms (a) and with earthworms (b)]; and effects of soil physiochemical properties of ACd without earthworms (c) and with earthworms (d). Horizontal axis indicates environmental factors; vertical axis indicates species; color indicates the strength of the correlation; asterisks within the square indicate significant correlation. *P < 0.05, **P < 0.01, ***P < 0.001

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In the earthworm system, ACd was significantly positively correlated with soil organic matter (P < 0.05). Previous research has indicated that earthworms improve soil quality and stability by altering the proportion of soil macroaggregates, as well as accelerating the decomposition and transformation of OM (Jia et al., 2015). Additionally, Opitutus and Gemmatta were significantly negatively correlated (P < 0.05) with ACd. Thus, increasing AP may decrease soil ACd. These results indicate that the combination of nZVI and earthworms reduced ACd by decreasing soil OM and increasing the relative abundance of Opitutus and Gemmatta.

3.4 Mechanism Involving the Remediation of Cadmium-Contaminated Soil by the Combination nZVI and Earthworms

In our study, we found the treatment amended with nZVI or the combination of nZVI and earthworms could effectively reduce the effective Cd content in Cd-contaminated soil. Specially, the addition of nZVI decreased the effective Cd content by increasing the proportion of clay, the soil pH, abundance of Stenotrophobacter. It is well established that aggregation phenomenon of nZVI within soil matrices exerts a significant influence upon the structural configuration of the soil matrix. Upon the micron-scale aggregation of nZVI, discernible outcomes encompass the attenuation of soil hydraulic conductivity and chemical reactivity (He et al., 2007; Schrick et al., 2004). Consequently, this culminates in an observable elevation in the relative proportion of clay constituents.

The experiments conducted by Xu et al., (2019) have demonstrated that both nZVI and clay are capable of inducing the transformation of Cd within soil matrices into states of diminished biological availability. This finding corroborates with the outcomes of our own research. Similarly, the soil pH value constitutes a pivotal determinant governing the removal of positively charged metal ions by nZVI. An elevation in soil pH engenders the generation of augmented negatively charged adsorption sites on soil colloids and organic matter surfaces, consequently culminating in the diminished bioavailability of metals (Otunola & Ololade, 2020; Zhang et al., 2014). Concurrently, the experiments conducted also reveal a pronounced negative correlation between Stenotrophobacter and Cd by Cui et al., (2022).

The combination of nZVI and earthworms enhances by 30% than nZVI alone (35%) (0.25% nZVI) by decreasing soil organic matter and increasing the abundance of Gemmatta and Opitutus. Soil organic matter (SOM) serves as a carbon source that stimulates microbial proliferation, leading to increased participation of bacterial taxa in heavy metal remediation. Notably, the abundances of Gemmatta and Opitutus exhibit a significant negative correlation with the soil-available cadmium content. This observation aligns with the findings of (Li et al., 2021; Wang et al., 2021). Additionally, research has indicated the significant role of Gemmatta in cadmium-contaminated soils (Bian et al., 2023; Pei et al., 2021). The combined remediation of cadmium-contaminated soil using nZVI and earthworms demonstrates the potential for sustainable soil remediation practices. This benefits local ecosystems by improving soil quality through cadmium remediation, and the successful application of this remediation approach may have global implications.

4 Conclusions

In this study, we investigated the combined effect of earthworms and nZVI on soil physicochemical properties and microecology during the remediation of Cd-contaminated soil. The results showed that although amendment with nZVI reduced earthworm survival and biomass, the combination of nZVI and earthworms effectively reduced the ACd content of soil. Meanwhile, the combined action of earthworms and nZVI significantly improved soil properties and increased the diversity of soil microorganisms. In the earthworm-free system, nZVI reduced ACd by increasing soil pH, the proportion of clay, and the abundance of Stenotrophobacter in Cd-contaminated soil, in addition to the co-precipitation and adsorption reported in previous studies. Correlation analysis revealed that the combination of nZVI and earthworms enhanced the decrease in ACd by decreasing soil OM and increasing the relative abundance of Opitutus and Gemmatta. Overall, our study indicates that the combination of nano-zero-valent iron and earthworms is a potential system for in situ remediation of Cd-contaminated soils and provides a deep understanding of the mechanisms involved in remediation. In the future, it is worth conducting a thorough investigation into the effect of nZVI and earthworms on soil nutrient cycling and other ecosystem functions. This will facilitate a comprehensive evaluation of the remediation process’ impact on soil ecosystems. Furthermore, it is essential to apply this methodology to genuine contaminated sites to assess its practicability and sustainability.