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Article

A Bioremediation and Soil Fertility Study: Effects of Vermirediation on Soil Contaminated by Chlorpyrifos

by
Francesca Tagliabue
1,†,
Enrica Marini
1,†,
Arianna De Bernardi
1,*,
Costantino Vischetti
1,*,
Gianluca Brunetti
1,2 and
Cristiano Casucci
1
1
Department of Agricultural, Food and Environmental Sciences, Polytechnic University of Marche, 60131 Ancona, Italy
2
Future Industries Institute, University of South Australia, Mawson Lakes Boulevard, Mawson Lakes, SA 5095, Australia
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Environments 2025, 12(5), 136; https://doi.org/10.3390/environments12050136
Submission received: 27 February 2025 / Revised: 28 March 2025 / Accepted: 20 April 2025 / Published: 24 April 2025
(This article belongs to the Special Issue Editorial Board Members’ Collection Series: Soil Contamination and Remediation)
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Abstract

:
Although the broad-spectrum pesticide chlorpyrifos (CP) was banned in many developed countries, it is still widely used in developing countries. Its residues persist in the environment for unpredictable times. CP is toxic to various non-target organisms and humans and inhibits soil enzyme activity and bacterial and fungal abundance. This paper aimed to evaluate the effect of vermiremediation on soil chlorpyrifos content and soil fertility. The application of Eisenia fetida or vermicompost was studied in terms of soil chlorpyrifos concentration, microbial biomass content, and enzymatic activities in a 120-day trial. Pesticide application rates were 0, 25, and 50 ppm. The CP did not affect the earthworm survival rate at the tested doses. The earthworms markedly increased microbial biomass carbon and the activity of β-glucosamminidase, while the vermicompost had a noticeably positive effect mainly on alkaline phosphatase activity. Finally, although the vermiremediation techniques studied did not perform a bioremediation activity, they proved effective in improving the biological fertility of the soil in the presence of high concentrations of chlorpyrifos.

1. Introduction

Soil pollution is a worldwide pressing concern [1,2] that causes adverse effects on the physical, chemical, and biological properties of this important ecological compartment [3,4], affecting plants and terrestrial organisms’ health [5,6]. The global spread of contaminants leads to a growing concern for environmental and human health [7]. The latest FAO report [8] states that approximately 4.2 million tons of pesticides are used worldwide to improve agricultural production [9]. Pesticides are incredibly damaging to ecosystems due to their continuous, uncontrolled use and persistent nature [10,11], yet agriculture increasingly depends on their use [12].
Chlorpyrifos (O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphorothioate) (CP) is an organophosphorus broad-spectrum insecticide [13,14,15]. Due to its high efficiency [14], CP is the most widely used organophosphorus pesticide to control a wide range of pests [16,17]. According to several reports, the CP half-life in the soil can range from 10 to 120 days [18,19,20,21]; however, its residues may persist in the environment for unpredictable periods of time [16]. The high variability in CP half-life has been attributed to several factors, such as application rate [22,23], pesticide formulation [24], and soil characteristics like organic carbon content, temperature [22,25], pH [26,27], and microorganism content [23]. Consequently, the CP half-life in different soil environments varies widely [15] and is unpredictable. Racke et al. [22] evaluated the CP half-life in different soils, obtaining results ranging from 6 to 51 months; Jaiswal et al. [28] also reported high values from 2 weeks to over a year. Moreover, the degradation of chlorpyrifos in soils leads to the accumulation of 3,5,6-trichloropyridinol (TCP), a primary metabolite with antibacterial properties and a long persistence evaluated until 360 days [29,30].
It is well known that only a part of applied pesticide actually reaches the intended target, with estimated values ranging from 0.1% to 20% [14,31]; the rest of the product is dispersed into soils and various ecosystem components [32,33] and these amounts can potentially contaminate sites from meters to kilometers away from the application point both due to wind drift and/or surface and underground water transport [34,35]. Furthermore, CP is a non-selective insecticide that has toxic effects on several non-target organisms [36]. CP works by blocking acetylcholinesterase, an enzyme essential for the functionality of the nervous system [37,38]. CP can enter living tissues [39] and shows a tendency to bioaccumulate through food chains [40], leading to major human health concerns. CP poisoning can affect the central nervous, cardiovascular, and respiratory systems [28]. Furthermore, this pesticide has been shown to adversely affect soil microflora [41] and other organisms. De Bernardi et al. [42] demonstrated that high doses of CP cause DNA damage and reproductive decline in the earthworm Eisenia fetida. The negative effect of CP on E. fetida coelomocytes was also reported by the study of Curieses et al. [43], and other works measured a depletion in earthworms’ survival rate [44] and growth [45].
As a result, CP has faced several restrictions that culminated in bans in the European Union and the United States [46]; however, it is still widely used in developing countries such as India and China [20]. Developing countries are responsible for a quarter of the global pesticide use [47] and are the most significant producers as well [48,49].
In this scenario, bioremediation techniques represent an excellent solution to reduce the content of these pollutants in the soil ecosystem and improve its health and fertility, as they are effective, inexpensive, and environmentally friendly.
In particular, earthworm-based bioremediation technology is experiencing a significant expansion. The first studies regarding this bioremediation technique date back to the 80s [50]; however, only in the last 10 years an increasing interest in this field of research has been observed [2,51]. Vermiremediation utilises earthworms’ biotic and abiotic interaction, life cycle, burrowing, and feeding activities to transform, degrade, or remove contaminants from the environment [52] and was first defined by Rodriguez Campos et al. [53].
Earthworms are considered ecosystem engineers because they play a fundamental role in the terrestrial phase [54]; they positively influence the soil’s physical, chemical, and biological properties [55], contributing to pedogenesis and the cycle of organic matter [56,57]. Earthworms are directly in contact with soil particles and contaminants [58] thanks to their permeable cuticles and to the massive ingestion of surface soil [59]. Earthworms can remove contaminants directly, through absorption and digestion, and indirectly by promoting the growth of microorganisms and microbial and enzymatic activities [52,60]. E. fetida appears to be the species most used in vermiremediation [61] because it is easy to purchase and breed in the laboratory, reproduces very quickly, lives for a long time, and has demonstrated remarkable resistance to several types of xenobiotics [7,62,63].
This earthworm species has been successfully used to remediate soil contaminated by diesel [64], petroleum hydrocarbons [65,66], selenium [67,68], lead [69,70], and cadmium [71,72]. Regarding pesticides, several studies have applied E. fetida to bioremediate substrates contaminated by acetochlor [73], pentachlorophenol [74], metolachlor [75], diuron and oxyfluorfern [76], and atrazine [77].
The vermiremediation technique can exploit not only earthworms but also their products. Vermicompost is derived from a biodegradation process of organic residues through the synergistic action of earthworms and microorganisms [52,78]; the resulting product is rich in macro and micronutrients, enzymes, and immobilised microflora [79].
The use of vermicompost as a bioremediation agent is currently not widespread, but positive results have been obtained by Luo et al. [80] and Mohammadi-Moghadam et al. [81] for the removal of atrazine and phenanthrene, and pyrene, respectively.
This work aimed to evaluate the effectiveness of vermiremediation in reducing chlorpyrifos concentration in soil. This technique was applied through the earthworm Eisenia fetida and a vermicompost derived from the same earthworm species. Furthermore, the effects of vermiremediation on soil fertility were evaluated in terms of growth and activity of soil microbial biomass through measures of microbial biomass carbon and enzymatic activities with or without contamination.

2. Materials and Methods

2.1. Experimental Material

2.1.1. Soil Description

The starting matrix of this experiment was topsoil (0–20 cm depth) collected from a cultivated field managed with organic practices in the Marche region (Italy). The soil samples were air-dried, homogenised, passed through a 2 mm sieve, and stored at 4 °C until use. The soil was classified as a silty clay loam soil with the following characteristics: 19.6% sand, 48.7% silt, and 31.7% clay content, alkaline pH (pH(H2O) = 8.79), and an organic matter content of 1.4% (Supplementary Table S1).

2.1.2. Earthworms

E. fetida earthworms were reared under controlled laboratory conditions (20 ± 1 °C) in commercial potting soil and fed with oats and organic vegetables. Adult earthworms with a well-developed clitellum and wet body weights of at least 300 mg were selected for the experiment. Acclimatisation was carried out in the soil described in the previous paragraph during the 14 days preceding the trial start [82,83].

2.1.3. Vermicompost

The vermicompost used is a commercial product called Humuzom, which was purchased from a local company (Zomagarden, Brescia, Italy). It is derived from the degradation of cow manure by earthworms belonging to the species E. fetida and is certified for use in organic agriculture. The characteristics of the vermicompost used are reported in Supplementary Table S2.

2.1.4. Chlorpyrifos

Commercial formulation Dursban 75 WG (chlorpyrifos 750 g kg−1) was supplied by Dow AgroSciences (Milano, Italy), while the chlorpyrifos analytical standard (CAS 2921-88-2, purity ≥ 98.0%) was obtained from Sigma-Aldrich (Milano, Italy); their properties are summarised in Supplementary Table S3.

2.2. Research Methods and Experimental Scheme

2.2.1. Experimental Design

A greenhouse pot experiment was performed to explore the effect of vermiremediation on soil contaminated by chlorpyrifos. First, a portion of the starting matrix was contaminated with two different CP concentrations, 25 and 50 mg/kg. Pesticide colloidal suspensions were freshly prepared in deionised water to adjust the soil’s moisture content (20%) in chemically inert glass. The colloidal suspension was then uniformly distributed drop by drop on 2 kg soil plateaus with a thickness of 3–5 cm; this procedure allowed us to ensure uniform contamination. Then, 2 kg of polluted soil was placed per plastic pot (20 cm diameter × 16 cm height). A sample for each replicate was taken to determine the initial concentration: 24.1 ± 0.3 and 48.6 ± 0.7 mg/kg.
Therefore, three different treatments were established in the present study: a soil control without earthworms and vermicompost (S), a soil with earthworms (E), and a soil with vermicompost (V). All the described treatments were applied on uncontaminated soil (1), on soil contaminated with CP at 25 ppm (2), and at 50 ppm (3). A scheme of the experimental design is reported in Table 1 for major clarity. Each treatment was conducted in triplicate, with a total of 27 pots.
Twenty adult specimens of E. fetida were introduced into each pot with earthworm treatments and fed every 14 days with organic oats [84,85]. At the end of the test, all replicates with earthworms were sieved to evaluate their survival rate. The vermicompost was mixed in a percentage equal to 5% of the soil weight per pot, taking references from different works [80,81,86]. The vermiremediation experiment was maintained in a greenhouse (23 ± 3 °C) for 120 days, keeping the humidity of the pots stable at 20%. The soil was checked, and the moisture was corrected every 2–3 days while monitoring the weight of the pots themselves.
For CP analysis, soil samples were taken at 7, 14, 21, 28, 42, 63, 84, 105, and 120 days. For soil health analyses, sampling was performed at 0, 63, and 120 days instead. All the samples were taken from a depth of 5 cm to avoid alterations due to the presence of oats on the surface in the earthworm treatments.

2.2.2. Chlorpyrifos Extraction and Analysis

The extraction and analysis of chlorpyrifos followed the protocol described by Vischetti et al. [87] and De Bernardi et al. [42]. A total of 25 g of dry soil was collected from each pot. Analyses were performed by HPLC using a Spectra SYSTEM P 4000 (Thermo Scientific, Waltham, MA, USA), equipped with a Supelcosil C18 column (5 µm particle size, 25 cm [L] × 4.6 mm [i.d.], SUPELCO) and a UV-detector following the method recommended by Akbar et al. [88]. The flow rate was 1 mL min−1, and the eluent was acetonitrile: water 70:30. Under these conditions, the retention time was 5 min for chlorpyrifos, and the limit of detection (LOD) was 0.67 mg L−1.

2.2.3. Microbial Biomass Carbon

Microorganisms play a key role in nutrient and organic matter cycling [89], and they are sensitive to contamination and respond very quickly to changes in soil conditions [90,91]. For these reasons, they can be considered critical ecological indicators for soil quality [92]. Microbial biomass carbon (MBC) was estimated using the method described by Vance et al. [93], which involves fumigation and extraction. MBC was calculated as the difference between the amount of C in the chloroform-fumigated sample aliquots and the amount of C in the corresponding unfumigated samples [93].

2.2.4. Enzymatic Activity Analysis

The fluorescein diacetate hydrolytic activity (FDA), the alkaline phosphatase (ALKP), the β-glucosidase (BGLU), and the β-N-acetyl-glucosaminidase (NAG) were performed with the colourimetric method through a spectrophotometer.
The FDA summarises the hydrolytic activity of numerous enzymes, such as proteases, lipases, and esterases, and represents a measure of the overall potential for fungi and bacteria hydrolytic breakdown activity [94]. The amount of fluorescein diacetate hydrolysed in one hour was estimated using Schnurer and Rosswall’s method [95,96].
ALKP indicates the potential for organic P mineralisation and P availability in soil [96]. This enzymatic assay includes a large group of enzymes that catalyse the hydrolysis of phosphate esters [97]. The analysis was performed using the Eivazi and Tabatabai [98] method, quantifying the p-nitrophenol released in one hour.
The BGLU are extracellular enzymes belonging to the hydrolases class that catalyse the hydrolysis of oligosaccharides. They are involved in the C cycle and are positively associated with soil organic matter [99,100]. The method of Eivazi and Tabatabai [101] was adopted, evaluating the amounts of p-nitrophenol released in one hour.
NAG belongs to the class of hydrolytic enzymes and is involved in the N and C cycles, hydrolysing oligosaccharides, including chitin and peptidoglycan [102]. The method of Parham and Deng [103] quantifies the p-nitrophenol released in one hour.

2.2.5. Statistical Analysis

The data on CP concentrations, Microbial Biomass Carbon, and enzymatic activity were each subjected to the nonparametric method Kruskal–Wallis to assess the possible presence of statistical differences between treatments or the various sampling times. If so, Dunn’s post-hoc test was performed to understand which means differed significantly from the others. Statistical analysis was conducted in R software (R version 4.2.2) [104].

3. Results

3.1. Chlorpyrifos Trend and Reduction Percentages

At the end of the test, the earthworms’ survival rates at 25 and 50 ppm were 98.3% and 95%, respectively.
In Figure 1 about the CP concentrations during the test, the statistical analysis highlights any significant differences between the measurements at different sampling times within the same treatment (S, E or V). As is clearly visible, a decrease in soil CP concentration was observed at both spiked soils (Figure 1a,b) compared to the initial concentration. However, significant differences appeared at the end of the test (day 120). The only exception was represented by the soil treated with vermicompost at 25 ppm (V2) (Figure 1a), where the difference at a statistical level is already visible at day 105 with respect to the concentration measured in the same soil at day 7.
The trend of CP during the test did not change with the applied concentrations: the CP content in the soil as is (S) underwent a progressive decrease comparable at both 25 ppm and when the starting contamination was double (50 ppm).
The pesticide trend in treatments with earthworms is also similar, regardless of the applied concentration (E2–E3); the same is visible in treatments with vermicompost (V2–V3). It also emerged that, 7 days after the start of the test, the concentration of CP extracted from the soil in the presence of earthworms and, to a lesser extent, in the presence of vermicompost was higher than that measured in the soil as is (S2 and S3). This trend is observable in both tested contaminations.
The reduction percentages were calculated at each sampling time (Table S4, Supplementary Material), and no significant differences were found between the treatments. In all cases, percentages over 90% were reached at the end of the test. Evaluating the results obtained within this study, the CP half-life was between 28 and 42 days of experimentation.

3.2. Microbial Biomass Carbon

The pattern of the microbial biomass carbon assessed during the trial is shown in detail in Table 2 and Figure 2, which report a different statistical analysis as reported in the respective captions. In the uncontaminated control (S1), the highest mg kg−1 of microbial biomass carbon was found in the samples treated with earthworms and vermicompost. This trend is visible especially at the end of the test and, to a lesser extent, at 63 days (first column, Table 2).
At half-test (day 63), in the soil as is (S2 and S3), the MBC values decreased in the presence of both CP contamination levels (second and third columns, Table 2). Comparing the values of S2 and S3 at day 63 with respect to day 0, a contained growth emerged at 25 ppm; this increase was absent at the highest dose tested, where the MBC remains around 147 mg kg−1.
Figure 2a shows a dose-dependent trend with significant differences (A, AB, B), but only at half of the test. On day 120, MBC values returned to the initial values and did not show significant differences between the concentrations tested.
From Table 2, it emerged that both the vermiremediation agents (E2–E3 and V2–V3) led to higher MBC values than the respective soil S2 and S3, even in the presence of CP. In the case of contamination at the lowest dose (25 ppm), E. fetida had already increased the MBC significantly from day 63. Similarly, at 50 ppm, the MBC values in the presence of earthworms increased significantly from halfway through the test, as evident in Figure 2b, and remained statistically high over time (Table 2). The microbial biomass carbon values in the treatments with earthworms (E2 and E3) were higher than the soil as is (S2 and S3). Furthermore, E2 and E3 assumed higher values than the uncontaminated soil treated with E. fetida (E1) and the control soil (S1).
The treatment with vermicompost still determined higher MBC contents than the soil S2 and S3, but only at the test’s end and with values positioned between S and E.

3.3. FDA

FDA hydrolysis is a non-specific test measuring lipases, proteases and esterases activities [105,106]. As can be observed in the first column of Table 3, FDA hydrolysis remained unchanged regardless of the treatment applied (S-E-V) without contamination; in the presence of earthworms (E1) and vermicompost (V1), no significant differences were found when compared with the soil as is (S1). The same trend can be observed in the presence of CP (25 and 50 ppm), even if the values obtained are statistically lower (from 4.74 to 7.36 µg g−1 h−1) than those recorded in the starting substrate (22.23 µg g−1 h−1). The decrease in this activity was not influenced by the amount of CP applied.
Adding CP to the control soil (0 S) led to a significant reduction in total hydrolytic activity, as evident in Figure 3a. The same negative trend was also observed in soils treated with E. fetida (Figure 3b) and vermicompost at both CP concentrations (Figure 3c) throughout the trial. At the end of the test (day 120), when the soil CP content was less than 10% (as reported previously in Section 3.1), the FDA values remained statistically lower than in the uncontaminated soil for all the treatments studied.

3.4. Alkaline Phosphatase

Without the pesticide (first column Table 4), the ALKP increased at day 63 in all treatments; however, this increase was significant at the end of the test only in the soil with vermicompost addition (120 V1) compared to the initial. Even in the presence of CP, vermicompost led to higher values of this enzymatic activity than in the other treatments; the increase remained significant, with respect to the initial values, throughout the test at both insecticide-applied concentrations.
Observing in Figure 4a, the trend of ALKP in soil without the addition of vermiremediation agents, it can be deduced that CP remained in an almost static situation with regard to this enzyme. At the end of the experiment, values similar to the starting soil (0 S) were recorded; this trend was dose-dependent only at day 63. Even with E. fetida, the recorded values of this activity decreased in contaminated soils during the test (Figure 4b). On the contrary, in soils where vermicompost was added (Figure 4c), a stable, increasing trend was observed over time, independent of pesticide presence.

3.5. β-Glucosidase

BGLU activity was measured at the test’s beginning, middle, and end, as reported in Table 5. Without contamination (first column, Table 5), the activity of this enzyme in the control soil (S1) increased progressively during the test, from 77 to approximately 140 µg g−1 h−1. The same trend was recorded in the presence of both E. fetida and vermicompost. The results showed similar behaviour between the three treatments, indicating that the presence of earthworms and vermicompost did not influence this enzymatic activity where CP was not added. On the contrary, at 25 and 50 ppm CP contamination, significantly higher values were measured at day 63 in the soils treated with earthworms and vermicompost, with values that ranged from 130 to 143 µg g−1 h−1. After 120 days, the BGLU activity decreased compared to the intermediate time; in fact, no significant differences were found between the studied treatments or with respect to the starting soil (0 S).
In all treatments (Figure 5), at the last sampling time, the BGLU activity was lower in the contaminated samples (regardless of the applied dose), even if, at 120 days, the quantity of CP in the soil was residual (see Section 3.1). This decrease also occurs in the presence of earthworms and vermicompost (Figure 5b,c).

3.6. NAG

Regarding the nitrogen cycle, the activity of NAG was measured, and the results obtained are reported in Table 6. It is evident that the presence of earthworms led to significantly higher values than the starting soil (0 S) throughout the test. This trend is visible both in the presence and absence of contamination. At the highest CP concentration, the result obtained in the presence of earthworms at the intermediate time (63 E3) is significantly different from all the other treatments studied, reaching values of 88 µg g−1 h−1.
On the contrary, no differences were found between the values measured in the absence and presence of contamination in the soil (S1–S2–S3) throughout the test (Figure 6a). The same outcome could also be observed in samples with vermicompost (Figure 6c). Figure 6b shows that the rise due to E. fetida in the soil was more remarkable as the CP dose increases at 63 days; at the end of the test, there were no differences between the tested CP concentrations.

4. Discussion

4.1. Chlorpyrifos Trend and Reduction Percentages

The CP significantly decreased at the end of the test, regardless of the doses and treatments applied. The half-life times found in our soil were in line with those indicated by the Pesticides Properties DataBase (PPDB) (DT50 field—27.6 and DT90 field—113) and in other works [13,107]. However, several works have demonstrated a high variability of CP degradation, indicating half-life times ranging from two weeks to one year and more [22,28].
Taking into account the results obtained for MBC and FDA, the pesticide was probably degraded through abiotic degradation, such as hydrolysis and photolysis [23,108], and, to a lesser extent, through volatilisation [109].
The strong alkalinity of the soil used as a starting substrate could have favoured CP degradation, as Racke et al. [108] and Singh et al. [110] demonstrated. Furthermore, CP has a Koc of 5509 mL g−1 (PPDB), a value that makes it remarkably similar to organic matter [23,111]. In our work, the relatively low adsorptive capacity of the soil, related to its low organic matter content, could have led to a greater mobility of chlorpyrifos, making it more easily degradable.
The high survival rate of E. fetida has demonstrated that neither of the applied doses causes mortality in this earthworm species.
The greater quantity of CP recovered from the soils treated with vermiremediation (E and V) at the first sampling time (7 days) suggests that E. fetida and their products can temporarily mobilise this contaminant, making it promptly subject to the degradative phase. In fact, a sharp decrease in soil CP concentration was measured in the subsequent sampling times (14 and 21 days), a trend not found in the soil as is (S), where degradation accelerates after 28 days.
None of the applied bioremediation agents proved effective in reducing the soil CP content faster; no statistically significant differences were found between the vermiremediated soil and the soil as is. These results are corroborated by the experiment conducted by Sanchez-Hernandez et al. [112], where L. terrestris did not accelerate the degradation of the pesticide but had a beneficial impact on soil quality.

4.2. Microbial Biomass Carbon

The influence of earthworms on the soil microbial community and its activity is variable and depends on several factors, such as feeding behaviour and resource competition [113,114]. The presence of earthworms in the soil leads to a variation in the microbial community structure, mainly due to gut-associated processes [115]: several microorganisms are increased, others remain unchanged, and others decrease as they are directly digested during transit in the gut [116]. Therefore, the earthworm cast has a significantly higher microbial biomass content than the surrounding soil and a more relevant microbiological activity [117]. It seems that the presence of epigeic earthworms, such as the species E. fetida used in this experiment, can positively influence the MBC since they can indirectly stimulate microbial populations by incorporating organic material into the soil, and consequently increasing the surface available for microorganisms [113,115]. On the contrary, endogeic earthworms reduce the soil microbial biomass, competing with it and using it as a source of sustenance [113,118,119]. Finally, anecic species, such as Lumbricus terrestris, increase the microbial biomass at the surface and reduce it at depth [120].
In the present work, E. fetida determined an increase in MBC in uncontaminated soil, stimulating the growth of microorganisms to a greater extent and thus contributing positively to its fertility.
Vermicompost, derived from the same earthworm species, favoured the development of microbial biomass, although to a lesser extent than E. fetida. This result is not surprising, as it is known that the biodegradation of organic materials achieved by earthworms is prosperous in terms of the microfauna population and nutrients [121,122]; in fact, several authors have found an increase in MBC after the application of vermicompost to soil [123]. Furthermore, in the absence of CP contamination, it was possible to observe a stimulating effect of vermiremediation on the soil microbial biomass maintained throughout the test.
Soil microorganisms play a fundamental role in ensuring the correct functioning of the ecosystem [124,125]; therefore, environmental changes can have adverse effects on the microbial community and directly impact environmental quality [126]. In this experiment, CP had a dose-dependent repressive effect on MBC growth. The microfauna population was not affected by the application of CP at the recommended field dose [21,127]. However, repeated pesticide application for several years and exceeding the recommended dosage have an increasing impact on the microbial biomass content [23,46,127]. CP negatively affects MBC, inhibiting bacterial and fungal growth and abundance and modifying the structure of the microbial community, as demonstrated by several studies [128,129,130]. Even within this experiment, a worsening of soil quality, in terms of health and fertility, occurred at both CP concentrations tested.
The presence of E. fetida buffered the harmful effects of CP on MBC in the present experiment. This buffering effect appeared to be dose-dependent in a positive sense; in fact, this trend is evident at 50 ppm, both at 63 days and at the end of the test, where MBC was increased by 315 and 166%, respectively, compared to the initial value (0 S). Higher values were recorded with increasing doses, probably because earthworms responded more to the environmental variation, leading to a significant increase in MBC in the contaminated soil. Even if E. fetida, in this experiment, did not directly affect the CP degradation kinetics, it nevertheless ensured a strong stimulation of the microbial population in the presence of the contaminant itself.
Vermicompost applied to soil led to an increase in MBC, although to a lesser extent than treatments with E. fetida, both in the presence and absence of CP. These results are in line with the characteristics found by several authors in vermicompost, which is not only rich in microorganisms [131] but also stimulates the growth and activity of those already present in the soil where it is applied [132].

4.3. FDA

FDA hydrolysis activity is an important biological indicator of soil quality since it reflects the activity of soil microorganisms themselves [94]. The CP concentrations tested in this work had a significant detrimental effect on global hydrolytic activity. Similar results were obtained by Dutta et al. [127] by applying 50 ppm of CP, while, with 0.5 ppm, equal to the recommended field dose, no variations in FDA activity were found. Aceves-Diez et al. [133] and Wang et al. [134] also found a statistically significant inhibitory effect on FDA hydrolysis at high CP concentrations (50–100 and 70 ppm, respectively). Furthermore, it is essential to note that, in this study, the repression of total hydrolytic activity remained significant even at the end of the test, when CP was almost totally degraded. Indeed, at the end of the experiment, an average percentage enzyme reduction of 66% was recorded compared to uncontaminated soil. Probably the FDA activity maintains significantly low values and does not increase due to the permanence of 3,5,6-trichloropyridinol (TCP) in the soil [134]; TCP is one of the main degradation products of chlorpyrifos and is known for its antimicrobial properties on the active microfauna populations [135,136,137]. Aceves-Diez et al. [133] measured the soil TCP content, observing that, from day 40, its concentration remained unchanged until the end of the experiment (day 80), leading to the inhibition of the FDA hydrolysis activity. Several authors have also observed an adverse effect of CP on dehydrogenase activity [106,130,138,139,140,141], which is considered a direct indicator of microbial activity [106,142].
In this work, it also emerged that the applied vermiremediation agents did not significantly influence the global hydrolytic activity since the enzyme remained stable over time in the absence of CP. E. fetida led to slightly lower FDA hydrolysis values than those recorded in the soil as is, but not in a statistically significant way. Similar results were reported by Jia et al. [143], while Lescano et al. [144] identified a correlation between the reduction in this enzyme activity and the decline in earthworm biomass. In contaminated soils at both concentrations, E. fetida and vermicompost did not contribute to shielding the adverse effect of CP on FDA hydrolysis, the decline of which remained significant in all treatments applied until the end of the test.
By correlating the MBC data discussed above with the values obtained in the global soil hydrolytic activity, it seems that CP at the doses studied had a negative effect not on the quantity of microbial biomass as much as on its metabolic activity.

4.4. Alkaline Phosphatase

Alkaline phosphatase is key in regulating organic phosphorus mineralisation in soil [145,146]. Regarding phosphatase activity, the data obtained suggested an inhibitory effect of CP at both concentrations tested on the soil as is; in fact, the applied insecticide blocked the natural increase in the level of this enzyme due to the incubation period of the soil microorganisms. Similar behaviour was identified by Sanchez-Hernandez et al. [112] by applying 20 ppm of CP. This insecticide can suppress phosphate-solubilising bacterial growth with different degrees of inhibition [139,147]. Jastrzębska’s [138] experiment found a negative dose-dependent trend in phosphatase, dehydrogenase, and urease activities. Tejada et al. [130], testing a very high CP dose (700 ppm), obtained an inhibition of phosphatase activity that was maximal 10 days after application of the contaminant and decreased as the CP in the soil was degraded.
In this study, the presence of vermicompost in the soil contributed to enhancing the alkaline phosphatase activity, even with CP at high concentrations; the action of the applied vermicompost buffered the adverse effect of the pesticide. It is well known that the vermicomposting process releases readily usable phosphorus; therefore, this fertiliser increases the soil phosphorus content and consequently stimulates phosphatase activity [148]. This is due to the action of phosphatase enzymes inside the gut of earthworms, which determine phosphorus deposition in the vermicast [149,150]. In contrast, in the present study, it seems that E. fetida could not counteract the inhibitory impact of CP on this enzymatic activity.

4.5. β-Glucosidase

Glucosidase in soil is mainly involved in cellulose decomposition, and its activity is positively correlated with soil organic matter cycling [151,152]. In this work, the significant increase in β-glucosidase activity observed in soil without contamination depended on the microorganism’s incubation process. Vermiremediation agents did not affect this natural trend, leading to results similar to the control soil (S1) at all sampling times. On the contrary, at both CP concentrations tested, E. fetida and vermicompost had a positive buffer effect at half-test, which, however, did not persist over time. Earthworms can influence enzymatic activities [153], leading to an increase in β-glucosidase both in the soil, particularly within the cast [154], and during the vermicomposting process [155]. At the end of the experiment, an adverse effect of this contaminant on β-glucosidase activity was evident. Cheng et al. [15] found that the stress response of soil microorganisms to CP depends on the insecticide dose itself. In fact, at high concentrations (700 ppm), CP inhibits the activity of β-glucosidase [130], while, when applying lower doses (20 ppm), the recorded values were similar to those measured in free-contaminated soils [112]. Several authors [139,156] have found a decrease in β-glucosidase activity already at the recommended field application rate of 0.5 L ha−1 in the first 30 days of experimentation, with even lower results by doubling the CP concentration (10 L ha−1) [156]. It is likely that, within this study, the inhibition of β-glucosidase activity at the end of the test, when over 90% of the CP has been degraded, was caused by the permanence in the soil of CP secondary metabolites [157]; in particular, TCP is known for its antimicrobial properties [137,158,159]. This work also observed a similar behaviour in the FDA activity, as discussed above.

4.6. NAG

NAG is the enzyme responsible for the catalytic degradation of chitin, which leads to the release of nitrogen-rich amino sugars [160]. Chitin is one of the dominant forms of organic N in soil and is mainly derived from fungal cell walls and arthropod exoskeletons [161].
At both concentrations tested, CP did not inhibit the activity of NAG, unlike other works in which a detrimental effect of CP on this enzymatic activity was observed, although the decrease did not follow a dose-dependent trend [162]. Despite this, other researchers have instead measured a CP stimulatory effect on the abundance of denitrifying bacteria [163,164]. Even the treatment with vermicompost did not determine significant variations for the enzyme under discussion, perhaps because it is an already stabilised fertiliser.
On the contrary, earthworms had a stimulating effect on NAG activity in the absence of contamination, as observed for the earthworms Metaphire guillelmi and L. terrestris in the studies of Hoang et al. [165] and Liao et al. [166], respectively. This stimulus could be because nitrogen is more available in the soil after passing through the earthworm’s gut, which also releases protein-rich mucus [167,168,169]. Another explanation for this greater activity is that the passage of any fungal mycelia through their intestine increases the chitin content in soil, stimulating the activity of β-N-acetyl-glucosaminidase [170].
This positive interaction between E. fetida and NAG is stimulated by the presence of CP in a dose-dependent manner after 63 days.

5. Conclusions

From this study, it emerged that the vermiremediation agents positively affected the biological fertility of the contaminated soil. Specifically, microbial biomass increased, especially where earthworms were added to the substrate. The presence of E. fetida led to a rise in NAG activity with a similar trend to MBC; in both cases, a more significant positive effect was recorded as the dose of chlorpyriphos increased.
The addition of vermicompost at the dose applied resulted in significant stimulation of phosphatase activity both in the presence and absence of the insecticide.
The application of E. fetida or vermicompost in the present experiment did not accelerate the degradation of chlorpyrifos in soil. Despite this, at the end of the test, less than 10% of the pesticide under study was extracted from all the samples.
In conclusion, although the vermiremediation techniques studied did not carry out a significant bioremediation activity, the present work demonstrated how E. fetida and vermicompost are effective in the qualitative and quantitative improvement of the soil microbial community, contributing to increasing the resilience and health of the soil ecosystem.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/environments12050136/s1, Table S1: Summary of main physicochemical characteristics of the starting soil, analysis protocols by Italian Official Gazette n° 248; Table S2: Physical and chemical properties of vermicompost; Table S3: Physical and chemical properties of Chlorpyrifos and Dursban 75 WG. Pesticide properties obtained from the PPDB database; Table S4: CP reduction percentages, calculated for each sampling time until the end of the test. References [171,172] are cited in the supplementary materials.

Author Contributions

Conceptualisation, F.T., E.M., A.D.B. and C.C.; methodology, F.T., E.M. and A.D.B.; software, A.D.B.; validation C.C., G.B. and C.V.; formal analysis, F.T., E.M. and A.D.B.; investigation, F.T., E.M. and A.D.B.; resources, C.V. and C.C.; data curation, F.T., E.M. and A.D.B.; writing—original draft preparation, F.T., E.M. and A.D.B.; writing—review and editing, F.T., E.M., A.D.B., C.V., G.B. and C.C.; visualisation, F.T., E.M, A.D.B. and C.C.; supervision, C.C. and C.V.; project administration, C.V. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Chlorpyrifos soil concentrations in the 25 ppm (a) and 50 ppm (b) up to 120 days after spiking. Statistical analysis was performed for each contamination separately; different letters refer to significant differences in each treatment. The codes S, E, and V refer to Soil, Soil + Earthworms, and Soil + Vermicompost, respectively.
Figure 1. Chlorpyrifos soil concentrations in the 25 ppm (a) and 50 ppm (b) up to 120 days after spiking. Statistical analysis was performed for each contamination separately; different letters refer to significant differences in each treatment. The codes S, E, and V refer to Soil, Soil + Earthworms, and Soil + Vermicompost, respectively.
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Figure 2. Microbial biomass carbon trend measured for each treatment: S = Soil (a), E = Soil + Earthworms (b), and V = Soil + Vermicompost (c). Statistical analysis was performed for each time separately (0, 63 and 120 days); different letters refer to significant differences between each contamination (0 ppm code: 1, 25 ppm code: 2 and 50 ppm code: 3). In the sampling times where the letters are not present, no significant differences were found between the concentration tested.
Figure 2. Microbial biomass carbon trend measured for each treatment: S = Soil (a), E = Soil + Earthworms (b), and V = Soil + Vermicompost (c). Statistical analysis was performed for each time separately (0, 63 and 120 days); different letters refer to significant differences between each contamination (0 ppm code: 1, 25 ppm code: 2 and 50 ppm code: 3). In the sampling times where the letters are not present, no significant differences were found between the concentration tested.
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Figure 3. FDA hydrolysis trend measured for each treatment: S = Soil (a), E = Soil + Earthworms (b), and V = Soil + Vermicompost (c). Statistical analysis was performed for each time separately (0, 63, and 120 days); different letters refer to significant differences between each contamination (0 ppm code: 1, 25 ppm code: 2 and 50 ppm code: 3). In the sampling times where the letters are not present, no significant differences were found between the concentration tested.
Figure 3. FDA hydrolysis trend measured for each treatment: S = Soil (a), E = Soil + Earthworms (b), and V = Soil + Vermicompost (c). Statistical analysis was performed for each time separately (0, 63, and 120 days); different letters refer to significant differences between each contamination (0 ppm code: 1, 25 ppm code: 2 and 50 ppm code: 3). In the sampling times where the letters are not present, no significant differences were found between the concentration tested.
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Figure 4. Alkaline phosphatase trend measured for each treatment: S = Soil (a), E = Soil + Earthworms (b), and V = Soil + Vermicompost (c). Statistical analysis was performed for each time separately (0, 63, and 120 days); different letters refer to significant differences between each contamination (0 ppm code: 1, 25 ppm code: 2 and 50 ppm code: 3). In the sampling times where the letters are not present, no significant differences were found between the concentration tested.
Figure 4. Alkaline phosphatase trend measured for each treatment: S = Soil (a), E = Soil + Earthworms (b), and V = Soil + Vermicompost (c). Statistical analysis was performed for each time separately (0, 63, and 120 days); different letters refer to significant differences between each contamination (0 ppm code: 1, 25 ppm code: 2 and 50 ppm code: 3). In the sampling times where the letters are not present, no significant differences were found between the concentration tested.
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Figure 5. β-glucosidase trend measured for each treatment: S = Soil (a), E = Soil + Earthworms (b), and V = Soil + Vermicompost (c). Statistical analysis was performed for each time separately (0, 63, and 120 days); different letters refer to significant differences between each contamination (0 ppm code: 1, 25 ppm code: 2 and 50 ppm code: 3). In the sampling times where the letters are not present, no significant differences were found between the concentration tested.
Figure 5. β-glucosidase trend measured for each treatment: S = Soil (a), E = Soil + Earthworms (b), and V = Soil + Vermicompost (c). Statistical analysis was performed for each time separately (0, 63, and 120 days); different letters refer to significant differences between each contamination (0 ppm code: 1, 25 ppm code: 2 and 50 ppm code: 3). In the sampling times where the letters are not present, no significant differences were found between the concentration tested.
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Figure 6. β-N-acetyl-glucosaminidase trend measured for each treatment: S = Soil (a), E = Soil + Earthworms (b), and V = Soil + Vermicompost (c). Statistical analysis was performed for each time separately (0, 63, and 120 days); different letters refer to significant differences between each contamination (0 ppm code: 1, 25 ppm code: 2 and 50 ppm code: 3). In the sampling times where the letters are not present, no significant differences were found between the concentration tested.
Figure 6. β-N-acetyl-glucosaminidase trend measured for each treatment: S = Soil (a), E = Soil + Earthworms (b), and V = Soil + Vermicompost (c). Statistical analysis was performed for each time separately (0, 63, and 120 days); different letters refer to significant differences between each contamination (0 ppm code: 1, 25 ppm code: 2 and 50 ppm code: 3). In the sampling times where the letters are not present, no significant differences were found between the concentration tested.
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Table 1. Summary of the experimental design and code legend.
Table 1. Summary of the experimental design and code legend.
CP ConcentrationsTreatments
SoilSoil + EarthwormsSoil + Vermicompost
0 ppmS1E1V1
25 ppmS2E2V2
50 ppmS3E3V3
Table 2. Microbial biomass carbon measured at 0, 63, and 120 days. Statistical analysis was performed for each contamination separately (0 ppm code: 1, 25 ppm code: 2 and 50 ppm code: 3), and different letters refer to significant differences in each column. S, E, and V refer to Soil, Soil + Earthworms, and Soil + Vermicompost, respectively.
Table 2. Microbial biomass carbon measured at 0, 63, and 120 days. Statistical analysis was performed for each contamination separately (0 ppm code: 1, 25 ppm code: 2 and 50 ppm code: 3), and different letters refer to significant differences in each column. S, E, and V refer to Soil, Soil + Earthworms, and Soil + Vermicompost, respectively.
Sampling TimeTreatmentMBC (mg kg−1)
1 (0 ppm)2 (25 ppm)3 (50 ppm)
0 daysS147.66 ± 15.25
a		147.66 ± 15.25
ab		147.66 ± 15.25
ab
63 daysS253.67 ± 27.72
ab		213.56 ± 14.45
ab		147.29 ± 16.27
ab
E304.56 ± 36.34
ab		402.26 ± 23.87
a		612.99 ± 70.29
a
V318.91 ± 20.55
b		221.07 ± 27.06
ab		130.87 ± 15.70
b
120 daysS169.12 ± 18.27
ab		130.60 ± 1.03
b		134.21 ± 11.03
b
E264.86 ± 32.09
ab		288.43 ± 33.66
a		393.94 ± 41.93
ab
V229.00 ± 24.52
ab		183.15 ± 16.95
ab		219.96 ± 5.49
ab
Table 3. FDA hydrolysis at 0, 63, and 120 days. Statistical analysis was performed for each contamination separately (0 ppm code: 1, 25 ppm code: 2 and 50 ppm code: 3), and different letters refer to significant differences in each column. S, E, and V refer to Soil, Soil + Earthworms, and Soil + Vermicompost, respectively.
Table 3. FDA hydrolysis at 0, 63, and 120 days. Statistical analysis was performed for each contamination separately (0 ppm code: 1, 25 ppm code: 2 and 50 ppm code: 3), and different letters refer to significant differences in each column. S, E, and V refer to Soil, Soil + Earthworms, and Soil + Vermicompost, respectively.
Sampling TimeTreatmentFDA (µg g−1 h−1)
1 (0 ppm)2 (25 ppm)3 (50 ppm)
0 daysS22.23 ± 0.98
a		22.23 ± 0.98
a		22.23 ± 0.98
a
63 daysS20.94 ± 1.76
a		7.36 ± 0.90
b		5.81 ± 0.70
b
E15.14 ± 1.84
ab		5.62 ± 0.54
b		6.07 ± 0.74
b
V17.75 ± 2.10
ab		6.07 ± 0.69
b		6.71 ± 0.79
b
120 daysS18.24 ± 1.73
ab		6.84 ± 0.81
b		5.58 ± 0.67
b
E14.00 ± 1.67
ab		4.74 ± 0.57
b		6.36 ± 0.68
b
V15.08 ± 1.13
ab		6.81 ± 0.83
b		6.87 ± 0.69
b
Table 4. Alkaline phosphatase measured at 0, 63, and 120 days. Statistical analysis was performed for each contamination separately (0 ppm code: 1, 25 ppm code: 2 and 50 ppm code: 3), and different letters refer to significant differences in each column. S, E, and V refer to Soil, Soil + Earthworms, and Soil + Vermicompost, respectively.
Table 4. Alkaline phosphatase measured at 0, 63, and 120 days. Statistical analysis was performed for each contamination separately (0 ppm code: 1, 25 ppm code: 2 and 50 ppm code: 3), and different letters refer to significant differences in each column. S, E, and V refer to Soil, Soil + Earthworms, and Soil + Vermicompost, respectively.
Sampling TimeTreatmentALKP (µg g−1 h−1)
1 (0 ppm)2 (25 ppm)3 (50 ppm)
0 daysS28.71 ± 0.93
a		28.71 ± 0.93
a		28.71 ± 0.93
a
63 daysS50.10 ± 5.39
ab		34.40 ± 3.83
ab		28.50 ± 3.35
a
E50.42 ± 5.92
ab		35.57 ± 4.40
ab		42.07 ± 4.91
ab
V49.50 ± 5.99
ab		47.59 ± 5.78
b		55.59 ± 6.54
b
120 daysS39.37 ± 4.55
ab		28.41 ± 3.37
ab		27.97 ± 3.21
ab
E46.54 ± 5.60
ab		33.96 ± 2.38
ab		31.88 ± 3.41
ab
V61.18 ± 7.39
b		49.80 ± 6.15
b		51.76 ± 6.35
b
Table 5. β-glucosidase measured at 0, 63, and 120 days. Statistical analysis was performed for each contamination separately (0 ppm code: 1, 25 ppm code: 2 and 50 ppm code: 3), and different letters refer to significant differences in each column. S, E, and V refer to Soil, Soil + Earthworms, and Soil + Vermicompost, respectively.
Table 5. β-glucosidase measured at 0, 63, and 120 days. Statistical analysis was performed for each contamination separately (0 ppm code: 1, 25 ppm code: 2 and 50 ppm code: 3), and different letters refer to significant differences in each column. S, E, and V refer to Soil, Soil + Earthworms, and Soil + Vermicompost, respectively.
Sampling TimeTreatmentBGLU (µg g−1 h−1)
1 (0 ppm)2 (25 ppm)3 (50 ppm)
0 daysS77.22 ± 8.66
a		77.22 ± 8.66
a		77.22 ± 8.66
a
63 daysS118.73 ± 14.37
ab		123.00 ± 15.50
ab		116.22 ± 13.94
ab
E127.82 ± 13.61
ab		130.56 ± 13.86
b		138.60 ± 16.08
b
V131.66 ± 16.28
ab		132.74 ± 11.68
b		143.93 ± 7.01
b
120 daysS140.97 ± 9.69
b		105.44 ± 13.05
ab		109.14 ± 12.00
ab
E145.48 ± 18.54
b		114.06 ± 14.19
ab		118.63 ± 14.58
ab
V143.99 ± 17.29
b		106.67 ± 5.16
ab		117.51 ± 12.41
ab
Table 6. β-N-acetyl-glucosaminidase measured at 0, 63, and 120 days. Statistical analysis was performed for each contamination separately (0 ppm code: 1, 25 ppm code: 2 and 50 ppm code: 3), and different letters refer to significant differences in each column. S, E, and V refer to Soil, Soil + Earthworms, and Soil + Vermicompost, respectively.
Table 6. β-N-acetyl-glucosaminidase measured at 0, 63, and 120 days. Statistical analysis was performed for each contamination separately (0 ppm code: 1, 25 ppm code: 2 and 50 ppm code: 3), and different letters refer to significant differences in each column. S, E, and V refer to Soil, Soil + Earthworms, and Soil + Vermicompost, respectively.
Sampling TimeTreatmentNAG (µg g−1 h−1)
1 (0 ppm)2 (25 ppm)3 (50 ppm)
0 daysS17.78 ± 2.18
a		17.78 ± 2.18
a		17.78 ± 2.18
a
63 daysS22.99 ± 2.31
ab		20.05 ± 1.56
ab		21.36 ± 2.35
ab
E37.56 ± 3.70
b		41.71 ± 4.44
b		88.16 ± 10.61
c
V25.42 ± 2.99
ab		23.07 ± 2.49
ab		24.43 ± 2.98
ab
120 daysS23.25 ± 2.70
ab		19.26 ± 2.09
ab		23.09 ± 2.66
ab
E35.99 ± 4.08
b		37.51 ± 4.61
b		31.67 ± 3.82
b
V23.74 ± 2.84
ab		23.81 ± 2.68
ab		26.74 ± 3.30
ab
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MDPI and ACS Style

Tagliabue, F.; Marini, E.; De Bernardi, A.; Vischetti, C.; Brunetti, G.; Casucci, C. A Bioremediation and Soil Fertility Study: Effects of Vermirediation on Soil Contaminated by Chlorpyrifos. Environments 2025, 12, 136. https://doi.org/10.3390/environments12050136

AMA Style

Tagliabue F, Marini E, De Bernardi A, Vischetti C, Brunetti G, Casucci C. A Bioremediation and Soil Fertility Study: Effects of Vermirediation on Soil Contaminated by Chlorpyrifos. Environments. 2025; 12(5):136. https://doi.org/10.3390/environments12050136

Chicago/Turabian Style

Tagliabue, Francesca, Enrica Marini, Arianna De Bernardi, Costantino Vischetti, Gianluca Brunetti, and Cristiano Casucci. 2025. "A Bioremediation and Soil Fertility Study: Effects of Vermirediation on Soil Contaminated by Chlorpyrifos" Environments 12, no. 5: 136. https://doi.org/10.3390/environments12050136

APA Style

Tagliabue, F., Marini, E., De Bernardi, A., Vischetti, C., Brunetti, G., & Casucci, C. (2025). A Bioremediation and Soil Fertility Study: Effects of Vermirediation on Soil Contaminated by Chlorpyrifos. Environments, 12(5), 136. https://doi.org/10.3390/environments12050136

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