Is Rockwool Potentially Harmful to the Soil Environment as a Nursery Substrate? Taking Eisenia fetida as an Example for Toxicological Analysis

Abstract

We studied the effect of rockwool matrix on the conventional physical and chemical properties of soil and analyzed its toxicological effect on Eisenia fetida. The physical and chemical properties of rockwool were studied with characterization tests. By measuring earthworm enzymes and earthworm intestinal microorganisms, the effects of different rockwool particle sizes and additive amount on Eisenia fetida were analyzed. The results indicate that a low concentration of rockwool (<30 g/kg) had little effect on the soil physicochemical properties and the activity of Eisenia fetida, and played a positive role in improving the soil porosity. A high concentration of rockwool (>100 g/kg) reduced the exchangeable Ca and Mg content in the soil, and had a significant impact on the enzyme activity of Eisenia fetida. Mechanism studies have shown that high concentrations of rockwool (>200 g/kg) can have a significant impact on the nervous system of earthworm tissue. In addition, small particle size and low concentration of rockwool is conducive to the increase in intestinal microbial species of Eisenia fetida. This study clarifies the effects of emerging rockwool substrates on soil and soil organisms and provides theoretical support for the safe and reliable application of rockwool substrates in agricultural production.

1. Introduction

Cultivation substrates are commonly used in modern greenhouse agriculture. The physicochemical properties of the substrate determine the water and nutrient absorption performance and air content of the substrate, which not only affects the crop root system’s ability to absorb and transport water and nutrients, but also affects the growth and development of the plant root system. The substrate materials used should have the following characteristics: (1) excellent permeability and water-holding capacity; (2) low impact on irrigation water and no effect on the recycling of drainage fluid; (3) no pollution; and (4) low cost [1]. Commonly used inorganic substrates include rockwool, vermiculite, sand, and perlite, and commonly used organic substrates include peat, rice husk charcoal, and bark [2,3,4]. One of the most used and successful soilless substrates is rockwool. The main reason for its success is that it is perfectly matched to the irrigation systems of modern greenhouses. For example, 95% of tomatoes in European regions are grown in greenhouses, and the key to this relies on rockwool slabs as a substrate as well as excellent water management and nutrient management [5]. In the Nordic regions, such as the Netherlands, where greenhouse cultivation is the main agricultural production, rockwool substrate is one of the most commonly used substrates [6], and rockwool, as an inorganic substrate, prevents the growth of some pathogens [7]. Since peat is a non-renewable resource, it is no longer used as a substrate in numerous countries [8], and it has been shown that crushed rockwool can replace peat and mixtures with alternative components in peat substrates with an optimal replacement ratio of 35% [9].

Rockwool was introduced in China in the 1990s and was popular with horticultural growers at the time, but is still mostly grown for vegetables. In the early 21st century, China used rockwool as a rice seedling substrate, but due to the immaturity of the technology and residual problems, rockwool seedling entered a relatively awkward position. In recent years, due to the improvement of the rockwool production process and the maturity of the substrate seedling technology, rockwool seedling has gradually developed.

Rockwool seedlings are soilless seedlings cultivated using rockwool as a substrate. Rockwool is an inert substrate, making it possible to control the nutrient availability and the root environment. Rockwool is weakly alkaline (pH = 7.5) and almost ion-free (electric conductivity = 50–100 μS/cm) [10]. The production process is conducted at a high temperature. Rockwool is sterile and non-toxic and is made of basalt, dolomite, and other raw materials via melting, high-speed centrifugation, blowing gas into flocculent fibers, and the addition of phenolic resin and other adhesives via pendulum, wrinkling, drying, and other technical processes [11]. Rockwool is light in texture, with a porosity of 90–96%. The water absorption rate of rockwool can reach 90%, and rockwool exhibits strong permeability, uniform pore spaces, and better permeability than other substrates, such as mudstone [12]. Moreover, the application of rockwool saves labor and effort in seedling cultivation; rockwool also inhibits weed growth and can be sown, mulched, and fertilized at the same time to improve efficiency. The residual amount of rockwool was also reduced to 0.8 g/m² when mechanical planting was performed. Since the main components of rockwool are basalt, dolomite, and other soil components and are not easily degraded, rockwool can be collected and treated together with the roots of rice.

The current research on rockwool is mostly focused on its application as a substrate for seedlings and partly on its effect on the microbial community. For example, Gundersen V studied the comparative effect of rockwool as a substrate on the concentration of major elements and for elements in tomatoes [6]; G. Carmassi used rockwool as a cultivation substrate to establish a comprehensive model of water requirements for closed-loop soilless cultivation of room-temperature tomatoes [13], studying the comparison of rockwool and coir fiber on the growth of greenhouse cucumbers [14]; and Prommart K studied the quantitative characteristics of four different substrates, including rockwool, on the population density and population dynamics of indigenous microorganisms [15]. However, due to the non-degradable nature of rockwool, researchers have not specifically investigated the physicochemical properties of rockwool on soil and its toxicological effects on earthworms. Therefore, basic data on the effects of rockwool on soil physicochemical properties and on earthworm toxicology need to be obtained.

Hence, the aim of this study was to investigate the effects of rockwool on the physicochemical properties of soil and the toxicological effects on earthworms. To address the abovementioned issues, this work selected the common earthworm Eisenia fetida as an earthworm representative and used an integrated approach of determining the earthworm biomass and enzyme activity, as well as conducting 16SrRNA sequencing and macrogenetic analysis [16,17,18], to assess the risk of rockwool in regard to soil biotoxicity. The effects of rockwool addition on earthworm activity in the presence of earthworms and on soil physicochemical properties were evaluated. This study provides the necessary data to guide the rational application of rockwool as a seedling substrate, and the results obtained will help the safe and reliable application of rockwool substrate in agricultural production.

2. Materials and Methods

2.1. Test Materials

The test materials were soil, rockwool, and earthworms.

Soil: The soil was collected from the practical training base of Yangzhou University, Jiangsu Province, China. The soil used in the experiment was Anthrosols-Plaggic. The soil type was chalky loam with low nutrient content. The basic physicochemical properties were pH 7.42, an electrical conductivity of 139.5 μS/cm, 12.85 g/kg organic matter, 0.67 g/kg total nitrogen, and 12.77 mg/kg effective phosphorus. The indicators are based on soil dry weight. The soil was air-dried, sieved through a 2 mm sieve, and prepared for earthworm cultivation.

Rockwool: Rockwool was obtained from Huaian Hand Agricultural Technology Co. (Huai’an City, China).

Earthworms: Earthworms were purchased from Wang Jun Earthworm Farm in Jurong City, Jiangsu Province, China. The earthworms were placed in a plastic box lined with moist filter paper, sealed with a perforated plastic lid, and placed in a constant temperature and humidity incubator (temperature 20 °C, humidity 60–70%) for 24 h. The filter paper was changed during the test, and the earthworms were removed and washed after the test.

2.2. Experimental Design

The experimental design was divided into 11 treatments, and each treatment was added to the corresponding rockwool, as shown in

Table 1. Earthworm treatments.

No.	Treatment	Explanation
1	CK	1 kg of soil without rockwool
2	T10M1	Add 10 g of 10-mesh sieved rockwool to 1 kg of soil
3	T10M3	Add 30 g of 10-mesh sieved rockwool to 1 kg of soil
4	T10M5	Add 50 g of 10-mesh sieved rockwool to 1 kg of soil
5	T10M10	Add 100 g of 10-mesh sieved rockwool to 1 kg of soil
6	T10M20	Add 200 g of 10-mesh sieved rockwool to 1 kg of soil
7	T20M1	Add 10 g of 20-mesh sieved rockwool to 1 kg of soil
8	T20M3	Add 30 g of 20-mesh sieved rockwool to 1 kg of soil
9	T20M5	Add 50 g of 20-mesh sieved rockwool to 1 kg of soil
10	T20M10	Add 100 g of 20-mesh sieved rockwool to 1 kg of soil
11	T20M20	Add 200 g of 20-mesh sieved rockwool to 1 kg of soil

. Three parallel trials were set up for each treatment. A black opaque plastic box of 40 cm × 20 cm × 30 cm was used as the culture container for the experiment, and each pot was filled with 1 kg of air-dried soil. Deionized water was added to the soil to maintain the soil moisture content at 60% of the maximum water-holding capacity, and the soil was fully stirred. Forty washed earthworms were taken after 24 h, weighed, and placed on the soil surface. Soil without rockwool was used as the control group. During the experiment, the boxes were placed in an incubator at 20 ± 2 °C and covered with a perforated plastic cover to ensure adequate ventilation. Soil moisture was weighed every other day and water was added if needed. Because rockwool is an inert and hard-to-degrade substrate, this study evaluated the response of earthworms to rockwool exposure in the med-term (28 days sub-chronic phase) [19].

2.3. Characterization and Determination of Rockwool Morphology

The morphology of the rockwool was observed and analyzed using a field emission scanning electron microscope (Hitachi S-4800, Chiyoda City, Japan) equipped with energy-dispersive X-ray energy spectroscopy (EDS). The specific surface area and pore size distribution of the rockwool were obtained by measuring the N₂ adsorption–desorption curves using a specific surface analyzer (BET, Autosorb-iQ, Shanghai City, China). The water retention capacity of the rockwool at high temperatures was simulated with an oven.

2.4. Determination of Physical and Chemical Properties of Soils

The organic matter content of the soil was determined based on the national standard “Determination of soil organic matter” (NY/T 85-1988) [20]. The Ca²⁺ and Mg²⁺ contents of the soil were determined based on the national standard “Determination of cation exchange and exchangeable salt group in neutral soil” (NY/T 295-1995) [21]. The SiO₂ content in the soil was determined based on the standard “Determination of effective silicon in soil” (NY/T 1121.15-2006) [22].

2.5. Determination of Earthworm Avoidance Rate

The earthworm avoidance rate behavior test was performed using the two-chamber method of the International Organization for Standardization ISO 2007. A clear plastic box of 40 cm × 20 cm × 30 cm was selected, a baffle was inserted in the middle, and 500 g of test soil was placed on one side and clean uncontaminated soil on the other side. The baffle was removed and 10 earthworms were placed in the middle line and sealed with gauze to ensure earthworm movement. After 48 h of incubation, the baffles were inserted back into place and the number of earthworms in the soil on each side was counted based on the position of the earthworm’s head pointing. Each group was set up in three parallels. The formula for calculating the earthworm avoidance rate is shown below:

$$NR=\frac{C-T}{N}\times 100\%$$

(1)

where NR is the net earthworm avoidance rate; C is the number of earthworms in the control soil; T is the number of earthworms in the test group; and N is the total number of earthworms added.

Earthworms in the fasted state were removed and washed the day before the test, placed in a plastic box lined with moistened filter paper, sealed with a perforated plastic lid, placed in a constant temperature and humidity incubator (25 °C, 60–70% humidity), and purged for 24 h. The filter paper was changed during the period, and the earthworms were removed and washed after purging and placed in the test box. Earthworms in a satiated state did not need to be fasted and were cleaned and placed directly into the test box.

2.6. Determination of Enzyme Activity of Earthworms

The determination of earthworm enzymatic activity is uniformly performed by the kit-microdosing method. The test was performed by Suzhou Kemin Bio on behalf of Suzhou Kemin Biological Company (Suzhou, China). In order to reflect the effect of rockwool on earthworms, the highest and lowest concentrations of 10-mesh rockwool and 20-mesh rockwool were selected for the assay of enzyme activity, i.e., five treatments of CK, T10M1, T10M20, T20M1, and T20M20.

Earthworm soluble protein content: under alkaline conditions, cysteine, cystine, tryptophan, tyrosine, and peptide bonds in the protein can reduce Cu²⁺ to Cu⁺; two molecules of bicinchoninic acid combine with Cu⁺ to produce a purple complex with absorption peaks at 540–595 nm, with the strongest absorption peak at 562 nm.

GST: the spectrophotometer/enzymatic standard was preheated for 30 min, and the wavelength was adjusted to 340 nm and zeroed with distilled water. Reagent III was kept at 25 °C (general species) or 37 °C (mammalian) to keep it warm. GST catalyzes the binding of glutathione to 2,4-dinitrochlorobenzene (CDNB), and the light absorption peak of its binding product is at 340 nm; the GST activity can be calculated by measuring the rate of absorbance rise at 340 nm.

SOD: the superoxide anion (O²⁻) is produced by xanthine and the xanthine oxidase reaction system. O²⁻ can react with WST-8 to produce the water-soluble dye methyl wax, which shows absorption at 450 nm, and the SOD activity can be calculated by measuring the absorbance at 450 nm.

CAT: H₂O₂ has a characteristic absorption peak at 240 m. CAT is able to decompose H₂O₂ so that the absorbance of the reaction solution at 240 nm decreases with reaction time, and the CAT activity can be calculated from the rate of change in absorbance.

T-AOC: the total antioxidant capacity is reflected by the ability of antioxidant substances to reduce Fe³⁺-tripyridyltriacridine (Fe³⁺-TPTZ) to produce blue Fe²⁺-TPTZ in an acidic environment.

MDA: MDA was condensed with thiobarbituric acid (TBA) to produce a red product with a maximum absorption peak at 532 nm, which can be used to estimate the content of lipid peroxide in the sample after colorimetric analysis; in addition, the absorbance at 600 nm was measured, and the difference between the absorbance at 532 and 600 nm was used to calculate the MDA content.

CL: the anthrone colorimetric method was used to determine the content of reducing sugars produced by the CL-catalyzed degradation of sodium carboxymethyl cellulose.

AchE: AchE catalyzes the hydrolysis of Ach to produce choline, which interacts with dithiobenzoic acid (DTNB) to produce 5-mercapto-nitrobenzoic acid (TNB), which has an absorption peak at 412 nm. The AchE activity was calculated by measuring the rate of increase in absorbance at 412 nm.

2.7. Determination of Earthworm Macrogenes

Fresh tissue samples were taken, washed with phosphate-buffered saline to remove surface microorganisms, and wiped with anhydrous ethanol. The tissues were then blocked, snap-frozen using liquid nitrogen, and stored at −80 °C for subsequent sequencing. Macrogenome sequencing was conducted based on Illumina high-throughput sequencing, using the paired-end sequencing method to construct small fragment libraries for sequencing, and was performed by Beijing BioMarker Technologies Co., Ltd. (Beijing, China). After the sample genomic DNA was tested and qualified, the DNA was broken and the fragmented DNA was subjected to end repair. The 3′-end addition of A, ligation of sequencing connectors, ligation of product purification sieves, library amplification, and product purification were performed to construct a sequencing library, which was sequenced using the Illumina sequencer after qualified library quality control. The raw reads obtained from sequencing were quality-controlled and filtered to obtain clean reads for subsequent bioinformatics analysis. The clean reads were assembled by splicing, predicting coding genes, constructing non-redundant gene sets, and performing functional annotation and taxonomic analysis on the non-redundant gene sets in both the general and specialized databases, counting the sample species composition and abundance information, and completing the analysis closure report based on BMKCloud.

Treatments for the assay: in order to represent the effect of rockwool on earthworms, the highest and lowest concentrations of 10-mesh rockwool and 20-mesh rockwool were selected for the macrogenome assay, i.e., five treatments of CK, T10M1, T10M20, T20M1, and T20M20.

2.8. Statistical Analysis

The data were statistically analyzed using Excel software and SPSS 23.0 software, and using the one-way analysis of variance model test. Differences between treatments were shown to be significant using the new complex polarization method (Duncan analysis), p < 0.05. OriginPro 9.1 software was used for plotting. The macrogenetic sequencing data were analyzed on the BMKCloud platform.

3. Results and Discussion

3.1. Material Characterization

3.1.1. Morphological and Elemental Analysis of Rockwool

According to the scanning electron microscopy images, elemental mapping, and EDS spectra of rockwool shown in Figure 1 and Figure 2, it can be seen that rockwool has a fibrous structure and contains irregular spindles inside, which are mainly composed of C⁴⁺, O²⁻, Si⁴⁺, Ca²⁺, and Mg²⁺, among which the O²⁻, Si^4+, and Ca²⁺ contents are higher and the C⁴⁺ and Mg²⁺ contents are lower.

3.1.2. BET Analysis

N₂ isothermal adsorption experiments were conducted to further investigate the porosity and specific surface area of the materials. As shown in

Table 2. BET surface area and pore characteristics of rockwool.

Materials	Specific Surface Area (m2·g−1)	Pore Volume (cm3·g−1)	Average Pore Size (nm)
Rockwool	2.74	0.01	4.31

, the specific surface area of rockwool was 2.74 m²·g⁻¹, the average pore volume was 0.01 cm³·g⁻¹, and the average pore size was 4.31 nm.

3.1.3. Water Retention Capacity of Rockwool

The same thickness of material and the same mass of pure water were placed into Petri dishes, and a high-temperature evaporation experiment was conducted to simulate the water retention capacity of various materials. As shown in Figure 3, at 30 °C, the rockwool contained 19.41 g of water after 8 h of evaporation, the vermiculite substrate with a water retention agent added had 18.92 g of water remaining, and the amounts of water remaining in the Petri dishes of pure vermiculite substrate, pure soil, and pure water were 17.66, 17.86, and 17.87 g, respectively. At 40 °C, evaporation was faster in the first 2 h because of the high surface moisture of each material, but after that the decline of the residual moisture was linear up to 8 h. At 8 h, rockwool was the best water retention material with 13.01 g of water remaining, followed by vermiculite matrix with a water retention agent added with 12.13 g of water remaining, pure vermiculite matrix with 11.07 g of water remaining, pure soil with 14.50 g of water remaining, and pure water with 9.35 g of water remaining. This experiment demonstrated that rockwool had the best water retention capacity, followed by vermiculite substrate with a water retention agent, and pure soil had the worst water retention capacity. The higher the temperature, the more significant the water retention capacity of rockwool.

3.1.4. Summary

The above characterization indicates that rockwool has a fibrous structure, high porosity, and good air permeability in addition to an outstanding water retention capacity. According to the elemental analysis, its elemental composition is basically the same as that of the soil, and it contains Ca²⁺, Mg²⁺, and SiO₂, which are beneficial to plant growth and meet the requirements for use as a substrate [1]. In addition, silicon fertilizer and calcium–magnesium phosphate fertilizer are used for rice in the form of SiO₂, CaO, and MgO, which are the main components of rockwool. It has been found that the application of silica fertilizer can thicken rice stalks, improve the mechanical strength of stalks, and effectively enhance the resistance of rice to collapse [23]. Therefore, the use of rockwool as a seedling substrate may promote the growth and development of seedlings.

3.2. Analysis of Soil Physical and Chemical Properties

3.2.1. Changes in Organic Matter Content in the Soil

Soil organic matter (SOM) alters the physicochemical reactions that affect the active composition of micronutrients [24,25]. As shown in Figure 4, the SOM content tended to decrease as the rockwool content in the soil increased, but the particle size of rockwool had no significant effect on the SOM content. This suggests that the increase in rockwool content has an effect on the normal activities of earthworms, thereby decreasing the degree of soil fecalization and thus the SOM content. However, the SOM content was slightly increased at low concentrations of 10% rockwool, indicating that rockwool at that concentration could be used as an additive to change the soil interstices without affecting the activities of earthworms.

3.2.2. Changes of Ca²⁺, Mg²⁺, and SiO₂ in the Soil

As shown in Figure 5a,b, as the content of rockwool in soil increased, the calcium and magnesium content in soil exhibited a decreasing trend, but the change in calcium and magnesium content only differed significantly at the highest rockwool concentration. The effect of 20-mesh rockwool on the calcium–magnesium–silica content of soil was smaller than that of 10-mesh rockwool. This indicates that the increase in rockwool content has little effect on the calcium and magnesium content in soil. As shown in Figure 5c, the concentration content of rockwool in the soil has a large effect on the effective silicon content in the soil. After the addition of rockwool, the effective silica content in the soil was showed a decreasing trend compared to the blank control. The decrease of 10-mesh rockwool was 7.25~17.10%, and the decrease of 10-mesh rockwool was 15.22~23.42%.

From the three figures, it is clear that the high concentration of rockwool addition has the highest effect on the effective state of Ca²⁺, Mg²⁺, and SiO₂ in the soil. The reason for this is that the high concentration of rockwool reduces the activity of earthworms, thus affecting the Ca²⁺-Mg²⁺- SiO₂ content in the soil.

3.3. Effect of Rockwool on the Survival of Earthworms

3.3.1. Survival of Earthworms in Rockwool-Amended Soil

From Figure 6, it can be seen that the survival rate of earthworms decreased after the addition of 10% rockwool, but there was no significant change in the survival rate of earthworms with the increase in the amount of rockwool. After the addition of 20% rockwool, the survival rate of earthworms decreased gradually with the increase in the amount of rockwool, and the lowest survival rate was 59.17% with the addition of 20% rockwool. However, the lowest earthworm survival rate after the addition of powdered rockwool was still higher than that after the addition of most granular rockwool, indicating that the size of rockwool had a significant effect on the survival rate of earthworms, and the larger the granules, the lower the survival rate of earthworms. The possible reason for this is that the granular rockwool (10-mesh) is more irritating to earthworms.

3.3.2. Earthworm Avoidance Rate Test

Earthworms exhibit rejection behaviors for unfavorable soil, and different behavior patterns when they are satiated and starved. As can be seen from Figure 7, when the rockwool consisted of large particles and the concentration was 10 g/kg and below, i.e., under low-concentration conditions, the earthworm avoidance rate was less than 20% and the avoidance behavior was not obvious, and the pollutant probably had no obvious toxic effect on the earthworms and did not constitute pollution stress. With the addition of rockwool, the avoidance behavior of earthworms gradually became obvious, indicating that the coercive effect of rockwool on earthworms gradually increased with an increasing amount of rockwool. There was no significant difference between the avoidance rate of earthworms in the satiated and starved states.

3.3.3. Summary

Earthworm avoidance behavior is considered to be convincing proof for soil quality assessment [26]. Due to the incorporation of rockwool, the survival and avoidance results shown by earthworms in soils containing high concentrations of rockwool were consistent with a greater risk to earthworms. However, there was little variability between satiated and starved earthworms, suggesting that high concentrations of rockwool are for the most part a valid hazard for earthworms [27,28]. In contrast, low concentrations of rockwool can be beneficial to earthworms in specific situations [29].

3.4. Effect of Rockwool on the Enzymatic Activity of Earthworms

3.4.1. Changes in the SP Content of Earthworm Tissues

As shown in Figure 8, the content of earthworm tissue SP gradually increased with increasing rockwool concentrations at 10-mesh levels, and the highest tissue SP content was observed at the highest rockwool concentration. This indicated that as the concentration of rockwool increased, the earthworm tissues accelerated the breakdown of metabolic proteins to obtain energy to resist the stress caused by the addition of rockwool. Similarly, Yang [30] found that SP levels in gophers were increased when they were subjected to external stimuli. With the addition of 20-mesh rockwool, the SP content of earthworm tissues increased and then decreased, and was highest at the lowest concentration, but was still lower than the highest value under the 10-mesh treatment, indicating a strong adaptation of earthworms to the powdered rockwool. This may have occurred because the 20-mesh-treated rockwool was less stimulating to earthworms than the 10-mesh-treated rockwool.

3.4.2. Changes in the Antioxidant Capacity of Earthworm Tissues

Using earthworms as test organisms to assess the toxicity of pollutants to soil, biomarkers are often used as diagnostic and predictive early warning indicators of soil environmental pollution stress. Antioxidant activities, such as SOD and CAT activities, scavenge excess reactive oxygen species (ROS), thereby protecting earthworm cells from the adverse effects of ROS [31]. Normally, SOD and CAT activities can be kept in dynamic balance to meet the needs of the organism and eliminate ROS; however, this balance can be easily disrupted if the organism is subjected to environmental stress [32,33]. GST is a powerful detoxifying enzyme in biological systems that promotes the binding of GSH to electrophilic substrates in toxic components to produce thiol uric acid and excrete it from the body [34]. As shown in Figure 9, the level of enzyme activities of earthworm tissues differed among treatments, and the activities of T-AOC and SOD did not change significantly among the five treatments. In the 10-mesh rockwool environment, earthworm tissue GST activity gradually decreased with the increasing rockwool concentration, and under the influence of the high rockwool concentration there was a significant decrease in GST activity of 37.5% compared with the control (CK). Meanwhile, CAT activity was directly decreased and all earthworm tissues in the 10-mesh treatment exhibited significant differences in CAT activity compared with CK, with decreases of 34.3% and 35.6%, respectively, in the M1 and M20 treatments. In the 20-mesh rockwool environment, the GST activity of earthworm tissues was the lowest in the M1 treatment, but was still higher than the lowest value in the 10-mesh treatment, while the GST content of earthworm tissues increased instead under the influence of high rockwool concentration, although the GST content was not significantly different from that in CK. By contrast, the CAT activity gradually decreased with the increasing rockwool concentration, but the lowest value in the 20-mesh treatment was still higher than the CAT activity in the 10-mesh treatment [35,36].

In summary, the variation of the antioxidant capacity of earthworm tissues showed that the effect of a high concentration of 10-mesh rockwool on earthworms was higher than that of 20-mesh rockwool, but the effect of a low concentration of 10-mesh rockwool on earthworms was less than that of 20-mesh rockwool. The large particles of 10-mesh rockwool, compared with 20-mesh powdered rockwool, were more likely to affect the normal activities of earthworms in the soil and disrupt the internal balance of earthworms, while the high concentration of 20-mesh rockwool excluding CAT showed a tendency to return to CK treatment. The reason for this may be that the high concentration of 20-mesh rockwool is in powder form and when mixed with soil, it allows earthworms to adapt to the mixed soil faster than the 1% concentration treatment due to the 20% concentration share, therefore returning the earthworm’s antioxidant capacity to near-normal values.

3.4.3. Changes in Oxidative Damage Indicators in Earthworm Tissues

The MDA content reflects the degree of peroxidation of body lipids in earthworms and briefly reflects the degree of cellular damage, where the higher the content, the greater the degree of damage [31,33,37]. As shown in Figure 10 (left), the MDA activity of earthworm tissues increased significantly with the addition of rockwool, especially at the 1% concentration, from 10.73 to 12.81 nmol/g, but there was no significant difference in the MDA content of all rockwool addition treatments [36]. This finding showed that the addition of rockwool caused cell damage in the earthworms, but that the effect was not dependent on the amount of rockwool added.

AchE is a key enzyme in biological nerve conduction. Between cholinergic synapses, the enzyme degrades acetylcholine, terminating the excitatory effect of the neurotransmitter on the postsynaptic membrane and ensuring the normal transmission of nerve signals in biological tissues [38]. As shown in Figure 10 (right), only the highest concentration of 20-mesh powdered rockwool affected the nervous system of earthworms, with AchE increasing from 75.87 to 138.29 nmol/min/g, with no significant change in the remaining treatments, similar to the findings of LUO Y-R [39]. This finding showed that the addition of a high concentration of 20-mesh rockwool caused significant changes in AchE content, indicating significant effects on the nervous system of earthworms, but the addition of 10-mesh rockwool had no significant effect on the AchE content of earthworm bodies.

3.4.4. Changes in CL Activity of Earthworm Tissues

CL is an important digestive enzyme that directly affects the ability of earthworms to break down plant litter and other cellulosic material and is therefore widely used as a biomarker to detect the effects of pollutants on earthworms [39,40]. As can be seen from Figure 11, compared with CK, there was a significant increase in CL content in all treatments except for under the low concentration of 20-mesh powdered rockwool, indicating that the earthworm bodies in these groups produced higher CL content in their tissues to obtain more energy to survive to obtain sufficient nutrients. KIM S study found that earthworms had upregulated cellulase activity after attack by LPS, Gram-negative, or Gram-positive bacteria [41]. This finding showed that the 10-mesh granular rockwool and the high concentration of 20-mesh powdered rockwool had an effect on the survival of earthworms.

3.4.5. Summary

Measurement of the enzyme activity of earthworms showed that 10-mesh rockwool had a greater effect on earthworms than 20-mesh rockwool. The results are consistent with the survival and avoidance rates of earthworms described above. In the experiment, the external stimulation of earthworms by rockwool caused some damage to their nervous system [30]. In contrast, the addition of 20-mesh high concentration of rockwool reduced some of the enzyme activities of the earthworms to control levels. It was shown that earthworms were more adapted to the contaminated soil than to the soil with 10-mesh rockwool added during the 28-day test period [42]. Therefore, the optimal rockwool addition concentration is 10 g/kg of 20-mesh rockwool, taking into account the survival and evasion rates.

3.5. Determination of Macrogenes in the Earthworm Intestine

3.5.1. Analysis of Species Composition

The microbial composition of the earthworm gut varied among treatments at the species level (Figure 12), with 1016 microorganisms common to each treatment and 43, 25, 11, 52, and 15 microorganisms unique to the six treatments CK, T10M1, T10M20, T20M1, and T20M20, respectively. This indicated that the addition of rockwool changed the microbial composition of the earthworm gut. In addition, 43 unique microbial communities were observed in CK, indicating that these microorganisms were sensitive to rockwool addition. From Figure 13, it can be seen that the more rockwool added, the fewer the microbial species, and the smaller the rockwool particle size, the more microbial species. This demonstrated that a small amount of small-particle-size rockwool had an elevated effect on the earthworm gut microorganisms. This may have been due to the fact that a small amount of small-particle-size rockwool had less of an effect on earthworms, but had a greater effect on the gut microorganisms due to the lower concentration and no decline in microbial species, but an increase in species for self-protection.

In terms of beta diversity analysis (Figure 14), non-metric multidimensional scaling suggested the separation of T10M20 and the remaining four groups of treated specimens, indicating that the addition of large particle sizes and a high concentration of rockwool resulted in significant changes in the structure of the earthworm gut microbial community. This finding was consistent with the previous results of enzyme activity analysis. According to the hierarchical clustering tree, the clusters of T10M20 were significantly separated from the clusters of the remaining four groups of treatments. The clusters of CK, T10M1, T20M1, and T20M20 were grouped together.

3.5.2. Characterization of Functional Genes in Earthworms

The highest abundance of ko01100, ko01110, ko00190, and ko01130 in the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway is shown in Figure 15, where ko01100, ko01110, and ko01130 belong to the global and overview maps of the secondary metabolic pathway and ko00190 belongs to the energy metabolism secondary metabolic pathway. The abundances of most metabolism-related pathways were higher in the T20M1-treated soils than in the remaining treatments, while there was no significant difference between the remaining treatments.

In addition, as shown by the abundance of the top 20 dominant pathways listed in

Table 3. Differences in abundance of the 20 dominant pathways at the second level (“a” indicates the variability between the data).

KEGG Pathway Level 1	KEGG Pathway Level 2	Abundance
		CK	T10M1	T10M20	T20M1	T20M20
Cellular Processes	Cell growth and death	283.75 ± 23.75 a	299.25 ± 34.75 a	266.00 ± 25.50 a	324.00 ± 46.50 a	330.25 ± 32.25 a
	Transport and catabolism	1150.5 ± 60.5 a	1120 ± 69.5 a	1085.5 ± 59.5 a	1206.25 ± 113.25 a	1110.75 ± 76.25 a
Genetic Information Processing	Translation	1033 ± 49 a	873 ± 38 a	811 ± 45.5 a	962 ± 29.5 a	923.5 ± 80 a
	Transcription	2354	1612.5	1356	1491	1554.5
	Folding, sorting, and degradation	1051.25 ± 89.75 a	993.75 ± 138.75 a	916.5 ± 154 a	1109 ± 178 a	952.5 ± 205 a
Metabolism	Global and overview maps	4996.67 ± 2221.16 a	4784.83 ± 2064.17 a	4981.75 ± 2121.26 a	6747 ± 2685.31 a	4798.25 ± 2196.11 a
	Energy metabolism	4947	4095.5	4158	4937	5239
	Nucleotide metabolism	1355 ± 387.5 a	1336.25 ± 404.75 a	1408.75 ± 434.75 a	1727 ± 450.5 a	1206.75 ± 379.25 a
	Carbohydrate metabolism	959.17 ± 52.1 a	933.83 ± 32.66 a	969.33 ± 36.28 a	1173.33 ± 19.69 a	924.17 ± 40.33 a
Environmental Information Processing	Membrane transport	1369	1335.5	1801.5	3000	1312.5

, many of the highly abundant pathways fell into the metabolism category, including global and overview maps (ko01100, ko01110, ko01130, ko01200, ko01230, and ko01120), energy metabolism (ko00190), nucleotide metabolism (ko00230 and ko00240), sugar metabolism (ko00500, ko00010, and ko00620), cellular transport and catabolism (ko04144 and ko04145), translation (ko03015 and ko03013), transcription (ko03040), folding, sorting, and degradation (ko04141 and ko03018), and membrane transport (ko02010).

In the experiment with five treatments, there was no significant difference between treatments at KEGG pathway level 2 due to variations in the amount of rockwool added. However, one functional gene, ko01100, was more abundant in the treatment with 10 g of 20-mesh rockwool than in the remaining treatments. This functional gene belonged to the metabolic pathways, which indicated a significant effect on the metabolic pathways of earthworms with the addition of low concentrations of 20-mesh rockwool. However, based on the previous analysis, although low concentration of 20-mesh rockwool changed the abundance of earthworm metabolic pathways, it had a relatively small impact on earthworm survival activity. The size of rockwool particles is still the main reason for affecting earthworm activity.

4. Conclusions

As an inorganic substrate prepared from rock, rockwool has a high degree of elemental overlap with soil, and its porous nature ensures water retention when it is used as a substrate. The addition of a low concentration of rockwool (10 g/kg) to the soil had a minimal effect on the soil and Eisenia fetida and increased the soil porosity; however, the addition of a high concentration of rockwool (200 g/kg) significantly affected the SOM and calcium–magnesium–silica content of the soil and had a greater effect on the enzyme activity of Eisenia fetida, making its toxic effect significantly higher than that of the blank test group. The findings showed that the residual particle size of the rockwool was too large, even at low levels, to irritate and thus affect the activity of the earthworm Eisenia fetida, but had less of an effect on the earthworm gut microorganisms, indicating that it was the surface of the earthworm that was affected rather than the body. In summary, rockwool, as an unbiodegradable inorganic substrate, affects the physicochemical properties of soil, and the addition of a high concentration of rockwool particles has a toxicological effect on earthworms. In agricultural production, rockwool as a seedling substrate can be used safely with regular removal.