How the Composition of Substrates for Seedling Production Affects Earthworm Behavior

Abstract

The constant increase in the intensity of agricultural production simultaneously increases the risk of negative effects of long-term agricultural practices. By-products of agricultural, forestry, and food production, as well as other types of organic waste, can be used as raw materials in the production of organic fertilizers and substrates for seedling cultivation through various processes of biological stabilization. In this way, the amount of waste is reduced, which contributes to the preservation of soil fertility and the sustainable use of resources. During waste processing and the stabilization of organic matter can be improved by using earthworms (vermicomposting). The aim of this study was to determine how different substrates, composed of different components and their mixtures, affect the earthworm Eisenia andrei. The effects of investigated substrates on the survival and behavior of earthworms were monitored. In addition, the effect of tested substrates on acetylcholinesterase (AChE), carboxylesterase (CES), and glutathione S-transferase (GST) activity was also assessed. The results showed that the most suitable substrates were leaves with horse manure and grape pomace alone and in combination with rock wool and sawdust. The obtained results provide important information on components and mixtures that have the greatest potential in the production of organic fertilizers and substrates for growing seedlings.

1. Introduction

The need for agricultural production is constantly growing and, at the same time, increasing the risks related to the negative effects of long-term agricultural practices, such as the usage of mineral fertilizers and pesticides. Due to the possible accumulation of these agrochemicals and residues in ecosystems and food chains, these agricultural practices may cause adverse effects on the health of animals and humans. Therefore, it is necessary to find alternatives to the usage of agrochemicals with the aim to develop a sustainable plant production system without the usage of mineral fertilizers [1,2]. The first step in this process is to determine the potential material that can be used for such a purpose. One of the options is to use by-products of agriculture, forestry, and food production, as well as various types of organic waste, as raw materials for the production of fertilizers and substrates. On one hand, these by-products and waste represent a problem since they have to be removed, but on the other hand, they are a potential reservoir of organic matter that can be usefully exploited.

Organic waste has the potential for application in the production of substrates that can be used for seedling cultivation. Different types of biowaste can be used as components in substrates and, considering their characteristics, can be mixed in different ratios in order to obtain a product of specific features. For example, animal manure, aquaculture sludge, grape pomace, rock wool, sawdust, wood chips, leaves, vegetable market waste, rice straw, etc., can be used [3,4,5,6,7]. The management of organic waste is extremely important since it provides several benefits, such as reducing the amount of waste, decreasing the production of certain toxic gases, leaching, which can cause environmental pollution, and obtaining high value products for the production of fertilizers [8,9].

Given that resources should be used sustainably and attention should be focused on organic production in these processes, vermicomposting plays an important role. Vermicomposting is the eco-biotechnological process of the decomposition of organic matter through earthworms and their symbiotic microorganisms’ activities [10,11]. As a result of this process, earthworm castings (vermicompost) are obtained. Vermicompost is a high-quality organic fertilizer that is rich in microbial activity and plant growth regulators and plant nutrients [12]. Vermicompost application can be helpful in the improvement of soil biophysical, chemical properties, and fertility, as well as in the remediation of the soil [7,13,14]. Earthworms are one of the most important animal communities in soil ecosystems. They have the capability to cultivate and aerate the soil, stimulate microbial activity, and contribute significantly to the organic matter decomposition into nutrient-rich products by improving soil properties [15]. In addition, they can act to reduce pathogens, since they possess different antimicrobial and antifungal molecules [16,17]. The digestive system of earthworms has the ability to transform various materials, such as plant, animal, industrial, and urban wastes, into beneficial vermicompost [18,19]. In the vermicomposting process, the Eisenia foetida and Lumbricus spp. species are the most preferred species due to the following characteristics. They have a short life cycle, a high reproductive rate, a low body weight, and a high resistance and tolerate variable ranges of temperatures [20,21]. Vermicomposting can also indirectly mitigate the effects of global warming and the greenhouse effect through its ability to sequester carbon in the soil [22]. The more organic carbon is retained in the soil, the more the extenuation potential of agriculture against climate change is higher. Unlike traditionally produced compost, vermicompost is characterized by excellent structure, high porosity, drainage, and water-holding capacity. It contains many useful macro and micronutrients in plant-available forms (nitrogen, phosphorus, potassium, organic carbon, calcium, sulfur, magnesium, hormones, vitamins, and enzymes) and it is an excellent alternative to mineral fertilizers [10,12,23]. Additionally, vermicompost provides many other plant-useful components such as vitamins, hormones, humic substances, and antioxidants [24]. Vermicomposting is a completely natural way of obtaining quality fertilizer which is important for soil fertility, as well as for the growth of many plants and agricultural crops [25].

Considering the potential for the usage of organic waste and the benefits resulting from the decomposition of organic matter through vermicomposting, it is important to determine which type of organic waste could be used in such a process. Even though different substrates have already been used, there are many components that could be used in different combinations and, consequently, there is a lack of information on the potentially suitable mixtures. Therefore, in the present study, different organic, as well as inorganic, components were selected for assessment and mixed in different combinations and ratios. In addition to new knowledge on the suitability of such substrates, an important aspect is also the reduction in waste that can be achieved with the usage of these substrates. This study will provide important information on the production of organic fertilizers and substrates for growing seedlings and support the principles of sustainable agriculture.

The main aim was to investigate which substrates, based on their composition, could be subjected to the vermicomposting process in order to enhance their characteristics and make them suitable for the production of organic fertilizers and substrates for growing seedlings. In order to determine that, the following aspects were examined: (1) the determination of how different substrates affect the survival of the earthworm Eisenia andrei; (2) the assessment of the behavior of the earthworm Eisenia andrei in different substrates in terms of the determination of the potential preference or avoidance of certain substrate or its components; and (3) the determination of effects of different substrates on activities of acetylcholinesterase (AChE), carboxylesterase (CES), and glutathione S-transferase (GST). Substrates suitable for vermicomposting should not cause mortality, should not be avoided by earthworms, and should not cause changes in measured biochemical biomarkers. Based on the observed responses and comparisons between results, it will be possible to determine which substrates, or specific components of a substrate, have the greatest potential for further processing in the production of organic fertilizers and substrates for growing seedlings.

2. Materials and Methods

2.1. Test Organism

Exposures were conducted using adult specimens of the earthworm Eisenia andrei [26] (Oligochaeta, Lumbricidae). Earthworms were purchased from a local supplier and placed in cultivation containers in order to acclimatize prior to experiments (at least 2 weeks at 20 °C). Prior to usage in the experimental set-up, earthworms were separated and rinsed with tap water and stored in Petri dishes on damp filter paper for 12 h, in the dark at 20 ± 2 °C, allowing to empty the gut contents [27].

2.2. Substrates

In order to investigate the effects of different substrates, i.e., specific components of the substrates, on earthworm survival, behavior, and biomarker responses, three sets of substrates were prepared. Each set is comprised of six substrates and the details are given below (

Table 1. Details of the first set of substrates.

Substrate Label	Substrate Composition	Composition Abbreviations	Ratio (v/v)	Number of Days in the Thermophilic Phase	pH
S1.1	Leaves and horse manure	a + b	1:1 (a:b)	8	9.11
S1.2	Leaves, horse manure, microorganisms, and urea	a + b + c * + d **	1:1 (a:b)	30	8.73
S1.3	Leaves, horse manure, microorganisms, wood chips, and phosphorite	a + b + c * + e + f ***	2.5:2.5:1 (a:b:e)	11	9.25
S1.4	Leaves, microorganisms, urea, and wood chips	a + c * + d ** + e	2.5:1 (a:e)	14	8.83
S1.5	Leaves and wood chips	a + e	2.5:1 (a:e)	12	8.83
S1.6	Leaves, urea, and wood chips	a + d ** + e	2.5:1 (a:e)	10+5	9.12

a—leaves; b—horse manure (straw was used as bedding); c—microorganisms; d—urea; e—wood chips; f—phosphorite. * The following microorganisms were added to S1.2: Pseudomonas, Azotobacter, Azospirillum, and Bacillus and, to S1.3 and S1.4, Pseudomonas, Azotobacter, and Azospirillum. Detailed information on the microorganisms is given in Table S2. ** Urea was added in the amount of 2.25 kg/m³. *** Phosphorite was added in the amount of 2 kg/m³.

,

Table 2. Details of the second set of substrates.

Substrate Label	Substrate Composition	Composition Abbreviations	Ratio (w/w)	Number of Days in the Thermophilic Phase	pH
S2.1	Grape pomace	g		-	7.33
S2.2	Fresh horse manure	h		-	7.71
S2.3	Grape pomace and fresh horse manure	g + h	1:1	-	7.14
S2.4	Grape pomace and rock wool	g + j	4:1	-	7.33
S2.5	Fresh horse manure and rock wool	h + j	4:1	-	8.23
S2.6	Grape pomace, fresh horse manure, and rock wool	g + h + j	2:2:1	-	7.78

g—grape pomace; h—fresh horse manure (sawdust was used as bedding); i—sawdust; j—rock wool.

and

Table 3. Details of the third set of substrates.

Substrate Label	Substrate Composition	Composition Abbreviations	Ratio (w/w)	Number of Days in the Thermophilic Phase	pH
S3.1	Fresh horse manure and sawdust	h + i	4:1	-	7.59
S3.2	Grape pomace and sawdust	g + i	4:1	-	6.42
S3.3	Grape pomace, sawdust, and rock wool	g + i + j	3:1:1	-	7.24
S3.4	Grape pomace, fresh horse manure, rock wool, and sawdust	g + h + i + j	1.5:1.5:1:1	-	7.59
S3.5	Grape pomace, fresh horse manure, and sawdust	g + h + i	2:2:1	-	6.69
S3.6	Fresh horse manure, sawdust, and rock wool	h + i + j	3:1:1	-	7.31

g—grape pomace; h—fresh horse manure (sawdust was used as bedding); i—sawdust; j—rock wool.

). Additional parameters of investigated substrates (electrical conductivity, moisture, organic content, and C/N ratio) are given in Table S1. The components of the substrates were chosen based on their potential to be used in the production of organic fertilizers and substrates.

2.3. Acute Toxicity Test

In order to determine the survival of earthworms in a particular substrate, an acute toxicity test was conducted. The test was performed following standardized OECD guidelines [26] with some modifications, as required by the research objectives. Namely, since the aim was to determine the potential toxicity of the investigated substrates, instead of in the artificial soil, earthworms were placed in the test substrates. All exposures were performed in three replicates at 20 °C. The substrates (400 g) were placed into plastic containers (18 cm length, 16 cm width, and 10 cm height), followed by the addition of ten earthworms to each container. The containers were covered with a lid with ventilation holes. Survival rates were assessed after 48 h and 14 days. In each container, surviving earthworms were sorted by hand. Earthworms were considered dead when they did not respond to gentle touching.

2.4. Avoidance Tests with Earthworms

The avoidance experiments were carried out following standardized guidelines [28]. Two separate chambers were created in a plastic container (18 cm length, 16 cm width, and 10 cm height) divided by using a metal divider that was placed in the middle of the box. Each chamber was filled with 200 g of different substrates. After the addition of substrates on both sides of the container, the metal separator was removed, and ten adult earthworms were placed on the separating line. All exposures were performed in three replicates. The containers were covered with a lid with ventilation holes and incubated at 20 °C in the dark for 48 h. At the end of the test period, substrates were again separated by inserting the metal divider and the number of earthworms on each side was determined by hand sorting.

2.5. Biomarker Assessment

2.5.1. Chemicals

All chemicals used in the study were of analytical grade. The following chemicals were used: acetonitrile (C₂H₃N, CAS 75-05-8), 1-chloro-2,4-dinitrobenzene (CDNB) (C₆H₃ClN₂O₄, CAS 97-00-7), acetylthiocholine iodide (CH₃COSCH₂CH₂N(CH₃)₃I, CAS 1866-15-5), disodium hydrogen phosphate (NaH2PO4, CAS 7558-79-4), 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) ([−SC₆H₃(NO₂)CO₂H]₂, CAS 69-78-3), 4-nitrophenyl acetate (C₈H₇NO₄, CAS 830-03-5), (2S)-2-amino-4-{[(1R)-1-[(carboxymethyl)carbamoyl]-2-sulfanylethyl]carbamoyl}butanoic acid (glutathione (GSH)) (C₁₀H₁₇N₃O₆S, CAS 70-18-8), and sodium dihydrogen phosphate dihydrate (NaH₂PO₄ × 2H₂O, CAS 13472-35-0).

2.5.2. Sample Preparation

After the exposure period ended, earthworms were removed from the soil, thoroughly cleaned with distilled water, and dried. Earthworms were then individually homogenized on ice with the addition of a cold sodium phosphate buffer (0.1 M, pH 7.2, and a ratio of 1:5 w:v) with an Ultra-Turrax T10 homogenizer (IKA, Königswinter, Germany). The homogenates were centrifuged for 30 min at 9000× g at 4 °C to yield the post-mitochondrial fraction (supernatant S9). The aliquots of the S9 samples were stored at −80 °C until biomarker measurements.

2.5.3. Enzymatic Activities Evaluation

Protein content was measured according to the method first described by Smith et al. (1985) [29] using the PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The samples were diluted in a sodium phosphate buffer (0.1 M, pH 7.2), followed by the addition of a working solution. The absorbance was measured after a 2 h incubation period at 562 nm. The amount of proteins was calculated based on the calibration curve with BSA.

Acetylcholine esterase (AChE; 3.1.7) activity was assessed according to the method of Ellman et al. (1961) [30]. The reaction mixture included sodium phosphate buffer (0.1 M, pH 7.2), DTNB (1.6 mM), acetylcholine iodide (156 mM), and the sample (S9). Changes in absorbance were recorded during 2 min at 412 nm and the specific enzyme activity was expressed as nmol of acetylthiocholine iodide hydrolyzed in one min per mg of proteins.

Carboxylesterase (CES; EC 3.1.1.1.) activity was determined according to Hosokawa and Satoh (2002) [31]. The reaction mixture was comprised of 4-nitrophenylacetate and the sample (S9). Kinetics were recorded for 1 min at 405 nm and the specific enzyme activity was expressed as nmol of 4-nitrophenol produced per min per mg of protein.

Glutathione S-transferase (GST; EC 2.5.1.18.) activity was determined according to the method of Habig et al. (1974) [32]. The assay mixture consisted of CDNB (1mM), GSH (25 mM), and the sample (S9). Kinetics was recorded for 2 min at 340 nm and the specific enzyme activity was expressed as nmol of conjugated GSH in one min per mg of protein.

All measurements were performed in technical triplicates in 96-well microplates using a Tecan Spark microplate reader (Tecan Trading AG, Männedorf, Switzerland).

2.6. Data Analysis

Data obtained from experiments performed in three independent biological replicates have been analyzed. The percentage of survival was calculated based on the number of surviving earthworms in each substrate. Data on the behavior and biomarker responses were analyzed using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). Data were first checked for homoscedasticity (the Bartlett test) and normality (the Shapiro–Wilk test). Since the normal distribution of the data was determined, parametrical tests were applied. For the avoidance behavior, significant differences in the preference of the earthworms for control or exposed soil were determined by means of Student’s t-test. Biomarker data were analyzed with one-way ANOVA followed by Tukey’s post hoc test. The probability level for statistical significance was set to p < 0.05 throughout the study.

3. Results

The first step in the experimental set-up was the investigation of the potential toxicity of tested substrates in terms of causing the mortality of earthworms. For that purpose, earthworms were placed in tested substrates for 48 h and 14 days, and the survival rate was assessed. Since the survival of earthworms is crucial for the determination of the suitability of substrate for vermicomposting, based on the mortality results following the experiments that were performed. Namely, only substrates in which earthworm survival rate was over 80% were used in the following experiments. In the case of lower survival, substrates were considered inadequate for further processing by earthworms. Further investigations included behavior assessment and measurement of biomarker responses.

3.1. Survival Rate

The survival rate results are presented in

Table 4. Survival rate (%) of the earthworms Eisenia andrei exposed to tested substrates for 48 h and 14 days.

Substrate	Survival Rate after 48 h	Survival Rate after 14 Days
S1.1	100%	100%
S1.2	0%	0%
S1.3	100%	100%
S1.4	0%	0%
S1.5	100%	100%
S1.6	100%	100%
S2.1	100%	95%
S2.2	30%	25%
S2.3	95%	85%
S2.4	90%	80%
S2.5	100%	90%
S2.6	95%	90%
S3.1	73%	50%
S3.2	100%	100%
S3.3	90%	90%
S3.4	53%	43%
S3.5	63%	53%
S3.6	57%	47%

Substrates labeled in grey were excluded from further assessments due to a low survival rate (<80%).

. The survival rate (expressed in %) was calculated from the number of earthworms that were found alive in the substrate. Missing earthworms, if any, were considered dead. Based on the survival rate results, substrates S1.2, S1.4, S2.2, S3.1, S3.4, S3.5, and S3.6 were excluded from further investigations.

In the first set of substrates, where substrates S1.2 and S1.4 were excluded from further experiments, it seems that the addition of microorganisms and/or urea caused unfavorable conditions for earthworms and, consequently, led to mortality.

In the second set of substrates, only substrate S2.2 had to be excluded, indicating that fresh horse manure is not an adequate substrate for earthworms unless other components are added.

In the third set of substrates, four substrates (S3.1, S3.4, S3.5, and S3.6) caused high mortality and were consequently excluded from further investigations. Here again, fresh horse manure was the problematic component; however, the addition of other components was not adequate to neutralize fresh horse manure’s adverse effects, indicating the importance of proper component selection.

3.2. Behavior Assessment

In order to determine whether earthworms have a preference for a particular substrate (due to different components of the substrate), the avoidance behavior of earthworms was assessed. Since three separate sets of substrates were prepared, avoidance behavior was assessed for each set separately.

3.2.1. First Set of Substrates

In the first set of substrates, after the mortality assessment, four (of six) substrates were tested for avoidance behavior. The results are presented in

Table 5. Results of the avoidance test with the earthworms Eisenia andrei exposed to a first set of tested substrates for 48 h.

Distribution		Significance	Result
S1.1	S1.3	***	preference of substrate S1.1
90%	10%
S1.1	S1.5	**	preference of substrate S1.1
80%	20%
S1.1	S1.6	**	preference of substrate S1.1
67%	33%
S1.3	S1.5	NS	-
53%	47%
S1.3	S1.6	NS	-
48%	52%
S1.5	S1.6	NS	-
43%	57%

Significant differences between substrates (t-test) are labeled with ** (p < 0.01) and *** (p < 0.001). NS—not significant.

.

The results of the behavioral assessment showed that in the first set of tested substrates, earthworms preferred substrate S1.1, i.e., a combination of horse manure and leaves. Even though horse manure and leaves were also present in some of the other substrates, in this set this combination proved to be the most adequate for earthworms.

3.2.2. Second Set of Substrates

In the second set of substrates, after the mortality assessment, five (of six) substrates were tested for avoidance behavior. The results are presented in

Table 6. Results of the avoidance test with the earthworms Eisenia andrei exposed to a second set of tested substrates for 48 h.

Distribution		Significance	Result
S2.1	S2.3	***	preference of substrate S2.1
93%	7%
S2.1	S2.4	NS	-
33%	67%
S2.1	S2.5	***	preference of substrate S2.1
100%	0%
S2.1	S2.6	**	preference of substrate S2.1
77%	23%
S2.3	S2.4	***	preference of substrate S2.4
10%	90%
S2.3	S2.5	NS	-
50%	50%
S2.3	S2.6		preference of substrate S2.6
3%	97%
S2.4	S2.5		preference of substrate S2.4
97%	3%
S2.4	S2.6		preference of substrate S2.4
97%	3%
S2.5	S2.6		preference of substrate S2.6
27%	73%

Significant differences between substrates (t-test) are labeled with ** (p < 0.01) and *** (p < 0.001). NS—not significant.

.

In the second set of substrates, the behavioral assessment showed preferences for substrates S2.1, S2.4, and S2.6. When analyzing the composition of these substrates, it is visible that all preferred substrates contained grape pomace, indicating its suitability as a substrate component.

3.2.3. Third Set of Substrates

In the third set of substrates, after the mortality assessment, two (of six) substrates were tested for avoidance behavior. The results are presented in

Table 7. Results of the avoidance test with the earthworms Eisenia andrei exposed to a third set of tested substrates for 48 h.

Distribution		Significance	Result
S3.2	S3.3	***	preference of substrate S3.3
23%	77%

Significant differences between substrates (t-test) are labeled with *** (p < 0.001).

.

In the third set, only two substrates were tested and, considering their composition it, seems that the addition of rock wool to the substrate contributed to its favorable characteristics for the earthworms.

3.3. Biomarker Responses

Responses of selected biomarkers in earthworms, exposed to all substrates that were analyzed in behavioral assessment, were evaluated. Exposure of earthworms to substrates from the second and third sets did not result in significant changes, so those results are not shown. Biomarkers measured in earthworms placed in substrates from the first set showed some significant differences, and the results are presented in Figure 1, Figure 2 and Figure 3.

Biomarker responses showed that in all three measured biomarkers, significant differences between substrates were determined. Already 48 h after exposure to tested substrates, the differences in measured activities were observed indicating that, in addition to the behavioral changes in terms of preferences for certain substrates, components of the substrates could have effects also on a biochemical level.

4. Discussion

The success of vermicomposting is significantly influenced by the type of substrate added to the process [33]. In this sense, before the vermicomposting process, it is important to carry out certain tests with selected substrates. In this way, it is possible to choose components that will be most adequate for this process. Selection of the most adequate substrate will result in the optimal processing of material and obtaining a product of high value.

In the present study, three substrate sets, each comprising six substrates with different compositions, were tested to assess their effects on the earthworm Eisenia andrei. In the first set, the substrates were first subjected to composting (days spent in the thermophilic phase are given in Table 1). After that, earthworms were placed in the obtained substrates and two substrates proved to be completely unsuitable for vermicomposting, as substantial mortality was observed already after 48 h of exposure (substrates S1.2 and S1.4). In other substrates, survival was 100%, which enabled further behavioral and biomarker testing. Regarding the mortality in mixtures of leaves, horse manure, microorganisms and urea (S1.2), leaves, microorganisms, urea, and wood chips (S1.4), it is possible that the addition of microorganisms caused unfavorable conditions and, consequently, led to the mortality of earthworms. There is also a possibility that the urea was not sufficiently balanced in the mixture, which may be the cause of mortality of earthworms in these substrate combinations. The remaining substrates (S1.1, S1.3, S1.5, and S1.6) were further tested in an avoidance test. Namely, the avoidance test is based on the fact that organisms have the ability to avoid unfavorable conditions. This test is quick, cost-effective, ecologically relevant, and has a high sensitivity. Avoidance behavior by earthworms has been recognized as a valuable endpoint in soil quality assessment [34,35]. In the avoidance test, earthworms showed a clear preference for the S1.1 substrate, indicating this substrate to be most favorable for earthworms. Obviously, the combination of horse manure and leaves (components of the S1.1 substrate) is the most adequate for the earthworms. Comparison of additional properties of substrates (electrical conductivity, moisture, organic matter, and C/N ratio) did not reveal a relationship between observed avoidance results and measured properties.

The second and third sets of substrates were not subjected to composting, yet were used for vermicomposting right after mixing the components. In the second set of substrates, only one substrate (S2.2) proved to be unsuitable for vermicomposting, as substantial mortality was observed already after 48 h of exposure. Obviously, fresh horse manure (the only component of the S2.2 substrate) is not suitable for vermicomposting, yet additional components have to be added. Again, the remaining substrates (S2.1, S2.3, S2.4, S2.5, and S2.6) were further tested in an avoidance test. The results showed that earthworms had a preference for substrates S2.1, S2.4, and S2.6. When the composition of these substrates is compared, it is clearly visible that all substrates contained grape pomace. The grape pomace (the main solid by-product of the wine industry) has high concentrations of macro-nutrients and micro-nutrients that are easily available to plants due to high solubility in water and, therefore, its use in the vermicomposting process is recommended [36]. Additionally, it is considered that the grape pomace has a favorable effect on the growth and reproductivity of earthworms [37], and has antioxidant and antimicrobial properties [38]. In the present study, grape pomace also proved to be a good choice as a substrate in vermicomposting as the only component, as well as in combination with rock wool and fresh horse manure. Many studies have shown that rock wool is a very good medium for the growth of many ornamental plants such as chrysanthemums (Chrysanthemum sp.), but also various agricultural crops, such as tomatoes (Solanaceae lycoperiscum), peppers (Capiscum annuum), lettuce (Lactuca sativa), melon (Cucumius melo), and many others [38,39,40]. Therefore, this substrate is considered very suitable for growing seedlings, fruits, and vegetables. Obviously, grape pomace and rock wool can be used separately and in a mixture as substrates for vermicomposting. The usage of earthworms in the processing of these materials can contribute to obtaining a product with better characteristics.

In the third set of substrates, substantial mortality was observed in four substrates (S3.1, S3.4, S3.5, and S3.6). All these substrates contained fresh horse manure in a large proportion. Obviously, similar to substrate S2.2, fresh horse manure is not suitable for vermicomposting. If the fresh horse manure is used in vermicomposting, the medium could be phytotoxic. Phytotoxicity occurs due to various substances that are present in fresh horse manure, such as harmful trace elements, pathogens, ammonia, organic acids, phenol, salt, and others [41,42]. It is considered that horse manure is a favorable choice because it has significant amounts of carbon; however, precomposting should last 1–2 weeks [43]. Without precomposting, horse manure can cause high earthworm mortality and disable vermicomposting. The avoidance test was performed between two remaining substrates (S3.2 and S3.3), both containing grape pomace and sawdust, but S3.3 also had rock wool. Sawdust is considered a very good additive, given that it has a very high C:N ratio [44]. Although sawdust is dry, it removes unpleasant odors, so it can be used as an addition to various fertilizers [45]. Here, a combination of grape pomace and sawdust proved to be a good substrate for vermicomposting. However, the avoidance test showed a preference for substrate S3.3, indicating that the addition of rock wool to this combination additionally favors this substrate as a vermicomposting material.

The process of vermicomposting is influenced by various physicochemical factors, among which, the toxic heavy metals are of much concern since they may adversely affect earthworm activities and the overall vermicomposting process [46]. Even though the mortality and behavior assessment does show whether the substrate could be potentially used in vermicomposting, there could be still other effects of substrates on earthworms that could affect their efficiency in vermicomposting. Therefore, a preliminary biomarker assessment was performed and responses of selected biomarkers in earthworms exposed to investigated substrates were measured. Namely, activities of acetylcholinesterase (AChE), carboxylesterase (CES), and glutathione S-transferase (GST) were selected, as these biomarkers are one of the most common ones used in investigations of the impact of environmental pollutants on earthworms [47,48]. AChE is a biomarker of neurotoxicity; CES participates in phase I metabolism and GST is a phase II enzyme, and they are also involved in oxidative stress reactions. The obtained results showed that only substrates from the first set affected the activities of AChE, CES, and GST in earthworms. Namely, for all three enzymes, significant differences in activities between earthworms being exposed to different substrates were observed. Even though it is not possible to have any firm conclusions on the adverse effects of substrates on earthworms, the obtained results show that future investigations of substrate suitability should also include biochemical endpoints in order to fully address the substrate effects. Determination of the optimal substrate mixtures, in terms of optimal conditions for earthworms, will enable vermicomposting with optimal efficiency.

5. Conclusions

In order to achieve maximum efficiency in the vermicomposting process, it is advisable to carry out preliminary tests with previously selected substrates. In this way, the losses in terms of reduced efficiency and earthworm mortality during vermicomposting would be reduced. The avoidance test and measurement of biochemical parameters in earthworms proved to be adequate for that. For a more detailed insight into the effects of the substrates on earthworms in future studies, additional endpoints can be included—from the measurement of additional biochemical biomarkers to the assessment of biomass change and the reproduction of earthworms. Investigation of substrate suitability in this study showed that the most suitable substrates were leaves with horse manure and grape pomace alone and in combination with rock wool and sawdust. Therefore, the combinations of these components have the greatest potential to be used in vermicomposting. Products obtained by vermicomposting these mixtures should be evaluated in the production of organic fertilizers and substrates for growing seedlings.