1 Introduction

The rise in human population has driven agricultural intensification to meet growing food demands (van Dijk et al. 2021), causing increased strain on vital natural resources, particularly the soil (Kopittke et al. 2019). In line with the circular economy concept, which advocates for reducing, reusing, and recycling, several solutions aim to mitigate the impact of agricultural intensification on soil health (Selvan et al. 2023). One approach involves integrating organic residues into the soil to efficiently introduce organic matter and essential nutrients like nitrogen and phosphorus (Leip et al. 2019), while addressing environmental concerns tied to improper disposal (Zubair et al. 2020). On one hand, these organic amendments contribute to soil health, crop yield and environmental conservation (Rastogi et al. 2023). However, on the other hand, they may pose some challenges such as the introduction of contaminants into the soil, including endocrine disrupting compounds (EDCs), which pose risks to both the ecosystem and human health (Xu et al. 2018; Jauregi et al. 2021). Of particular concern are farm animals’ excretions, comprising faeces and urine, which contribute significantly to the presence of natural steroid hormones in the environment (Adeel et al. 2017). Bio-waste and wastewater by-products also present additional challenges in effective waste management (Qin et al. 2015), raising concerns about contaminants and the presence of EDCs. Responsible waste management practices are essential to mitigate the environmental risks associated with applying these as organic amendments. Green manure and crop residues are also alternative organic amendments and essential sources of organic matter for agricultural soils (Kruidhof et al. 2011; Turmel et al. 2015). Nevertheless, the presence of phytoestrogens in certain crops also raises concerns about soil contamination with EDCs (Lorand et al. 2010). While this contamination is known to impact the health of agricultural animals, the implications for invertebrate life is still under evaluated and needs to be considered. The discussion extends to industrial effluents, encompassing a diverse range of residual organic materials, including those from oil seeds, papermaking, sugar extraction and wood ash (Goss et al. 2013). The application of these materials as organic amendments requires careful consideration of their potential impact on soil and environmental health.

Thus, one of the concerns when applying organic residues as soil amendments should be their impact on soil invertebrate health. Earthworms, a significant portion of soil invertebrate biomass (Ganault et al. 2024), are sensitive to soil contaminants, due to chemoreceptors and sensory structures on their body surface, and usually serve as indicators of ecosystem quality, displaying responsiveness to various factors such as land use/management practices, environmental conditions, disturbances and contamination levels (Bhaduri et al. 2022). Additionally, earthworms play an active role in the decomposition of organic residues (Lubbers et al. 2017), nutrient cycling (Edwards and Arancon 2022), humus formation (Kumar et al. 2020) and improvement of soil structure, fertility, porosity and water infiltration, drainage and retention (Lemtiri et al. 2014). The earthworm Eisenia fetida, due to its short life cycle, high fecundity and easy maintenance is commonly chosen for various studies, including ecotoxicological (Guo et al. 2020), vermicomposting (Enebe and Erasmus 2023), bioaccumulation (Rich et al. 2015; Ye et al. 2016) and bioremediation (Gan et al. 2021). This earthworm species is one model organism selected by several standard protocols for soil contaminations evaluation (OECD 1984; 2016; International Standard (ISO) 2008; 2012; 2014; 2023).

Given these aspects, earthworms provide a compelling model for investigating the effects of EDCs on soil invertebrates and their sensitivity to these compounds, whether through endocrine-mediated processes or not, make them useful bioindicators of potential soil contamination by this class of compounds. This review aims to comprehensively explore evidence of the broad-spectrum impact of EDCs on earthworms, highlighting important biological changes observed in these organisms after exposure to these compounds, such as alterations in oxidative stress balance, DNA damage (genotoxicity), histopathology and the expression of reproductive-related genes. This review intends to showcase the susceptibility of non-target soil species, which have been largely overlooked in the study of this class of toxicants.

A comprehensive and systematic bibliographic search of three electronic databases (PubMed, Web of Science and Scopus) was performed using Medical Subject Headings (MeSH) and keywords related to “earthworms”, “endocrine disruptors”, “histology”, “oxidative stress”, “genotoxicity”, “gene expression”. Inclusion criteria encompassed peer-reviewed articles written in English and published up to the date of the query. The most recent search was performed on February 29th, 2024. The review process involved screening titles and abstracts for relevance, followed by a full-text assessment of potentially eligible articles.

2 Overview of endocrine disruptor compounds

Endocrine-disrupting compounds (EDCs) are substances capable of interfering with hormonal synthesis and distribution, as well as hormonal signalling in the body, mimicking hormones such as oestrogens, androgens, and thyroid hormones or blocking their receptors. These compounds belong to a heterogeneous class of exogenous chemicals (Kassotis and Trasande 2021) and can be broadly classified according to their occurrence/origin as natural EDCs (e.g., genistein, coumestrol and 17β-oestradiol), and synthesized EDCs, including industrial solvents (e.g., polychlorinated biphenyls (PCBs), polybrominated biphenyls (PBBs), dioxins), plasticizers (e.g. bisphenol derivatives, phlathates), pesticides (e.g., dichlorodiphenyltrichloroethane), fungicides (e.g., vinclozolin), and some pharmaceutical agents (e.g., diethylstilbestrol; 17α-ethinylestradiol), among others (Kabir et al. 2015). As such, establishing a direct correlation between the structural characteristics of EDCs and their effects poses a significant challenge (Karthikeyan et al. 2019). Although certain structural markers, like a phenolic ring with specific substitutions, offer clues, the intricate mechanisms of action and the potential toxicity of metabolites add complexity to identifying EDCs based solely on their structure.

Both vertebrates and invertebrates can be affected by these chemicals, although the mechanisms and outcomes can differ significantly among them (Zou 2020; Rodríguez 2024). Most studies regarding EDCs focus on oestrogen, androgen, and thyroid receptor signalling as well as steroidogenesis (also known as EATS axis), but it is known that these compounds also disrupt non-EATS pathways, which are the axis mechanisms focused on other endocrine signals required for homeostasis in hormone regulated organs (e.g., brain, heart, gastrointestinal system, liver, pancreas, and intestine) (Martyniuk et al. 2022). The effects of EDCs on vertebrates, including humans, are well-documented (Street et al. 2018). EDC exposure has been linked to various health issues in both wildlife and humans, including reproductive effects, neurobehavioral and metabolic syndrome, decreased fertility and developmental effects on the nervous system (Colborn et al. 1993; Marlatt et al. 2022). Among invertebrates, gastropods (e.g., snails) and crustaceans (e.g., crabs, shrimp) are the taxonomic groups most studied extensively regarding EDC exposure (Zou 2020). The disruptions in these species include improper growth, reproduction, and development, leading to observable anomalies such as imposex in snails (a condition where female snails develop male sexual characteristics, and linked with tin compounds exposure) (Neuparth et al. 2017) or altered sex ratios in crustaceans (Zou 2020). However, the impact of EDCs on soil invertebrates, particularly earthworms, sparks an extensive discussion.

Earthworms primarily absorb organic and inorganic compounds, such as EDCs, through ingestion of organic matter and/or direct skin exposure (Sivakumar 2015; Byambas et al. 2019). The presence of steroid receptors in these species is still debated, with only a few authors addressing the possibility of earthworms having such receptors. Regardless of whether EDCs directly influence these soil invertebrates through endocrine-mediated pathways, these organisms are susceptible to these compounds, affecting both their individual and populational health (Scott 2018). Interestingly, two aquatic annelid species (Platynereis dumerilii and Capitella capitate) have shown the ability to synthesize oestrogen and to possess oestrogen receptors that are sensitive to oestrogens and, subsequently, to estrogenic EDCs (Keay and Thornton 2009) and some invertebrates have been found to possess the capacity to metabolize these hormones to some extent (Keay and Thornton 2009; Jones et al. 2017; Scott 2018; Cuvillier-Hot and Lenoir 2020; Taubenheim et al. 2021). The functional differences observed in the nuclear receptors of invertebrates compared to vertebrates, where only a few ligand-sensitive oestrogen receptors have been described, also contribute to uncertainties in this field (Jones et al. 2017). In this context, to our knowledge, information regarding earthworm oestrogen receptors is limited to the study by Novo et al. (2019) which claimed to have identified a full ORF sequence of the oestrogen receptor in earthworms, albeit with notably low expression levels, that showed high homology to the Nucellus latipes receptor. Similarly, some researchers have identified the thyroid-stimulating hormone (TSH) and its respective receptor through immunohistological methods in E. fetida, detecting these in both neuronal and non-neuronal cells of the central nervous system and various peripheral organs (Wilhelm et al. 2006); however, it is important to acknowledge that these assays may yield false-positive results as a consequence of cross-reactivities or nonspecific binding of the antibodies to abundant proteins.

If present in earthworms, steroid receptors are most likely to be the ecdysone receptor (EcR), the membrane-associated progesterone receptor (MAPR), and the adiponectin receptor (AdipoR) (Novo et al. 2018), rather than the receptors commonly found in vertebrates. These are hypothesized to exist in earthworms, since they have been found in other invertebrate species, however their role is still unknown (Novo et al. 2018). In insects and crustaceans, EcR is the receptor for the hormone involved in moulting (Gaertner et al. 2012). MAPR has also been defined as a membrane steroid-binding protein in invertebrates (Fujii-Taira et al. 2009), although the ligand of this receptor remains unknown, while AdipoR has been described to play a role in the maintenance of germline cells in Drosophila ovaries (Laws et al. 2015).

While current knowledge in EATS-mediated mechanisms in earthworms is limited, available studies report evidences of EDCs impact through non-EATS pathways, namely how these compounds affect homeostasis essential for normal physiological processes involved in growth, reproduction, and other functions, regardless of their interaction with steroid receptors analogous to those found in vertebrates. Table 1 briefly resumes the current knowledge regarding the effects of EDCs on both EATS and non-EATS mediated pathways in vertebrates. For the purposes of our methodology, we referenced lists from EU Member States (Belgium, Denmark, France, Netherlands, Spain, Sweden) available on the website «edlists.org» to categorize EDCs in this review. Briefly, list 1 comprises “substances identified as endocrine disruptors at EU level”, list 2 includes “substances under evaluation for endocrine disruption under an EU legislation” and list 3 contains “substances considered, by the evaluating National Authority, to have endocrine disrupting properties” (The Danish Environmental Protection Agency 2020). Unlisted compounds that have also shown endocrine-disrupting capability in literature studies were also included.

Table 1 List of (identified or potential) endocrine disrupting compounds (EDCs) mentioned in this review and the mechanisms of action in vertebrates
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3 Effects of EDC contaminants in earthworms

This section explores evidence of the impact of EDCs, which can be widely found in soils, mostly through the introduction of organic amendments, on earthworms, emphasizing histopathological abnormalities, oxidative stress, genotoxicity, molecular changes and reproductive toxicity described in the relevant literature. These evidences are summarized in Tables 2, 3, 4, 5 and 6.

Table 2 Microscopic findings in earthworms exposed to different endocrine disrupting compounds (EDCs)
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3.1 EDC-induced histopathological changes in earthworms

Earthworms, as key members of soil ecosystems, face susceptibility to EDCs, impacting their physiology as evidenced by the occurrence of histopathological changes in their tissues (Fig. 1). Numerous studies have emphasized the detrimental consequences of EDC exposure on the histopathological integrity of various organs of earthworms, as summarized in Table 2. Notably, organophosphate esters such as tricresyl phosphate and tris(2-chloroethyl) phosphate, usually used as flame retardants, negatively affect earthworm histology (Yang et al. 2018). These products leak into the environment through the sewage system from households, industries and stormwater drainage systems and are discharged into the soil when wastewater is used for irrigation and sewage sludge is applied (Mihajlović et al. 2011). Exposure of E. fetida to environmentally relevant concentrations (0.1–10 mg kg–1) of these compounds resulted in visible degradation of the digestive tract, including exfoliation of the typhlosole (Yang et al. 2018). In addition to these histopathological alterations, tris(2-chloroethyl) phosphate also caused disintegration of the longitudinal muscular layer and enlargement of the coelom. Tris(1,3-dichloro-2-propyl) phosphate, another flame retardant, was also capable of inducing histopathological changes in seminal vesicles from E. fetida exposed to environmental relevant concentrations (50–5000 ng g–1), inducing focal necrosis and cytoplasmic vacuolation, damaged epicuticle, thickened cuticle layer and muscle atrophy at the highest concentration (Zhu et al. 2019).

Fig. 1
figure 1

Classes of compounds with endocrine disrupting capacity capable of inducing histopathological alterations in earthworms. These alterations represent significant physiological responses to environmental contamination by endocrine-disrupting compounds. The figure has been designed using icons made by Biorender (www.biorender.com)

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Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea), a systemic herbicide in the urea chemical family that inhibits photosynthesis, showed gonad and reproductive changes in various species (Danio rerio, Oryzias javanicus) (Velki et al. 2017; Kamarudin et al. 2020), but did not exhibit any obvious effects on earthworm epidermis and intestine after 28 days of exposure to diuron at environmentally relevant concentrations (0.05–5 mg kg−1) (Wang et al. 2023b). Although the study’s goal was to investigate how diuron absorption, be it direct or digestive, could impact E. fetida tissues, studying the gonads’ histology may have provided insight into additional potential effects of this compound.

Hormones, including 17β-oestradiol and dihydrotestosterone, are naturally produced and excreted by all vertebrates, being found in livestock manure (Liu et al. 2012a). The application of manure, and associated hormones, onto fields to meet crop nutrient requirements causes these compounds to be found in abundance in beef and dairy manure amended fields (Havens et al. 2020). Exposure of E. andrei to these hormones (0.1-1 mg L−1) resulted in decreased numbers of mature oocytes and detached follicles in the ovaries, while the seminal vesicles exhibited significant inhibition of spermatogenesis, disordered germ cell distribution and decreased mature sperm bundles (Kwak and An 2021). These hormonal influences on earthworm gonads are crucial aspects to consider in understanding the broader impact of EDCs in earthworm reproduction.

The inorganic ion perchlorate (ClO4−), generated as both a natural and anthropogenic pollutant, has been recognized as an endocrine disruptor because it affects vertebrate thyroid glands and causes hypothyroidism by outcompeting iodide at the sodium-iodide symporter (Gholamian et al. 2011). The improper disposal of ammonium perchlorate, used in propellants, fireworks, as well as thyreostatic drugs and growth promoters in cattle fattening, contributes significantly to environmental contamination (Batjoens et al. 1993; Gupta et al. 2014). Perchlorate was also found to induce histopathological changes in E. fetida, namely circular and transversal muscle degradation, damage to the muscular layer protecting the digestive system and erosion in tissues after a 14-day exposure (Acevedo-Barrios et al. 2018). While it affected several tissues, this study did not explore its effects on gonad histology (Acevedo-Barrios et al. 2018).

Bisphenol A (BPA), an industrial synthetic chemical widely used in the production of polycarbonate plastics and epoxy resins (Kapustka et al. 2020), exhibited varied effects on earthworm histology. BPA primarily contaminates soil through the agricultural application of sewage sludges and biosolids (Yu et al. 2015). In earthworms, BPA induced adverse effects on the body wall and in the ovaries, in which vacuolization of interstitial space, theca folliculi hyperplasia and hypertrophy, detachment and predomination of granulosa cells, as well as overall follicular atresia was observed (Babić et al. 2016). Lesions to the inner tissues of E. fetida have also been reported, such as circular and transversal muscle disintegration, along with hypertrophy and hyperplasia of muscle fibres with disrupted myofibril architecture, particularly in the circular muscle (Babić et al. 2016). Additionally, ovaries showed atrophy and formation of aggregate clusters of necrotized follicles in E. fetida (Babić et al. 2016). Exposure of E. andrei to BPA led to a decreased number of mature oocytes and follicles with detached granulosa cells in earthworm ovaries and significant inhibition of spermatogenesis, disordered germ cell distribution, decreased mature sperm bundles and small vacuoles in earthworm seminal vesicles (Kwak and An 2021). These results indicate strong negative effects on earthworm reproduction with potential high impact on natural populations.

Methylparaben, found in cosmetics, personal care products (PCPs) and used as a food preservative, has also been found to exhibit estrogenic activity (Sun et al. 2016). Directly entering the environment through discarded products and food, methylparaben poses a risk to soil via sewage sludge and aquatic systems. In E. andrei, it resulted in decreased mature oocytes, follicles with cellular detachment, disordered germ cell distribution and decreased mature sperm bundles (Kwak and An 2021).

At environmentally relevant concentrations, the organic UV filter benzophenone-3 was found to induce significant histopathological changes in E. fetida tissues (Gautam et al. 2022). Widely present in plastics and various PCPs (e.g., sunscreens, lotions, shampoos and cosmetics), benzophenone-3 is prevalent in surface waters, sediments and sewage sludge (Balakrishna et al. 2017; Campos et al. 2017). Agricultural soil contamination occurs through sewage sludge disposal and irrigation with water from wastewater treatment plants, where these compounds persist (Ramos et al. 2016). Benzophenone-3 exposure leads to notable degeneration of the epidermal and muscular layers, compromising body wall and intestinal tissues (Gautam et al. 2022). Furthermore, the ovaries and seminal vesicles suffer from degeneration, necrosis and disruption of the cellular lining, resulting in a decrease in sperm concentration and disturbed germ cell distribution.

In summary, EDC exposure results in histopathological changes in several earthworm tissues, including significant damage to the digestive tract, muscle disintegration and overall reproductive organs atrophy. The accumulation of EDCs in soil through the application of sewage sludge and wastewater irrigation further exacerbates the risk to earthworm populations.

3.2 EDC-induced oxidative stress in earthworms

Assessment of oxidative stress in earthworms is crucial for understanding the impact of environmental stressors, particularly EDCs (Table 3). Even though earthworms are not traditional target organisms for these compounds, they absorb various low molecular weight chemicals through their semipermeable body walls. This makes the assessment particularly crucial in comprehending the implications of EDCs on oxidative balance. The bioaccumulation of pollutants through ingestion of contaminated organic matter also significantly influences their overall health and population dynamics (Phipps et al. 1993). These compounds have been found to impact the cellular redox cycle by diffusing freely in the cellular microenvironment and undergoing degradation in molecules that can originate reactive oxygen species (ROS) (Heger et al. 2015) (Fig. 2).

Table 3 Oxidative stress changes in earthworms exposed to different endocrine disrupting compounds (EDCs)
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Fig. 2
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Classes of compounds with endocrine disrupting capacity capable of inducing alterations on oxidative stress indicators in earthworms. The figure has been designed using icons made by Freepik, Smashicons, Good Ware, monkik, Nes_Kanyanee, Pixelmeetup from www.flaticon.com. BDE-47, 2,2′,4,4′-tetrabromodiphenyl ether; BPA, Bisphenol A; BPS, bisphenol S; BBP, butyl benzyl phthalate; DEHP, di(2-ethylhexyl) phthalate; DIBP, diisobutyl phthalate; DINP, diisononyl phthalate, DMP, dimethyl phthalate, DBP, di-n-butyl phthalate; DNOP, di-n-octyl phthalate

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Chlortetracycline, a veterinary antibiotic extensively used in farms for disease treatment and growth promotion (Santás-Miguel et al. 2020), enters agricultural systems through livestock manure application to soils (Sarmah et al. 2006; Pan and Chu 2017). This antibiotic affects steroidogenic pathways and alters sex hormone balance in human adenocarcinoma cell line (H295R) and in male medaka fish (Oryzias latipes) (Ji et al. 2010). Exposure in earthworms at 3 mg kg−1 resulted in significant increased superoxide dismutase (SOD) and catalase (CAT) activities, along with elevated malondialdehyde (MDA) content at 100 and 300 mg kg−1, up to increments of 257% and 251% relative to control, respectively (Lin et al. 2012b). These results indicate a potential to induce cellular damage by lipid peroxidation of membranes and to reduce the individual fitness.

Regarding triclocarban and triclosan, both polychlorinated aromatic antimicrobials, these compounds have been widely used for decades as antimicrobial additives and preservatives in various products (Chrz et al. 2023), migrating to the soil when present in biosolids (Sales Junior et al. 2020). These compounds are considered bisphenol analogues and thus have been linked to the damage of sexual development and reproductive functions in Pimephales promelas, fathead minnow (Brian et al. 2005). In E. andrei, triclocarban exposure resulted in significant decreased CAT activity, at 50 and 100 mg kg−1, and of glutathione S-transferase (GST) activity on the first days of experiments (21 and 28 days), remaining similar to the control group on days 35 and 42; also, glutathione (GSH) levels were decreased by the highest concentrations (Sales Junior et al. 2020). This study proposed that GSH was employed by GST to remove triclocarban potentially bioaccumulated in E. andrei tissues. E. fetida exposure to triclosan in soil and filter paper experiments increased CAT, GR, SOD activities and MDA content at 100 mg kg−1 (Lin et al. 2012a; Zaltauskaite and Miskelyte 2018). In another study, exposure of E. fetida to triclosan resulted in decreased CAT levels, after 2 and 14 days of exposure, with activity being similar to control levels at 7 days of exposure (Lin et al. 2010). The same trend was observed for GST and SOD activities, while MDA content increased in a concentration-dependent manner, particularly after 7 days of exposure (Lin et al. 2010). Evidence of oxidative damage has also been found in two other studies with E. fetida after exposure to triclosan, where Hsp70 transcript levels were increased at 50 mg kg−1 (Lin et al. 2014), as well as elevated CAT, GR, MDA, SOD enzyme activity levels (Zaltauskaite and Miskelyte 2018). Elevated MDA levels indicate enhanced lipid peroxidation and the changes observed in antioxidant enzyme activities implies a compromised defence mechanism against ROS.

Flame retardants such as tricresyl phosphate and tris(2-chloroethyl) phosphate also caused increased GSH levels in E. fetida when exposed to environmentally relevant doses (i.e. 1 and 10 mg kg−1) (Yang et al. 2018). Another widely used flame retardant, the polybrominated biphenyl ether 2,2’,4,4’-tetrabromodiphenyl ether (BDE-47), has been shown to cause endocrine disruption in zebrafish (D. rerio). This disruption led to detrimental effects on ovary development, lowered sex hormone levels, oxidative damage and changes to hypothalamic pituitary-gonad axis-related genes (Shi et al. 2022). The presence of this flame retardant in many commercial and household products can lead to soil contamination during product production, use and disposal (Zhao et al. 2011). Moreover, acute exposure to this compound induced increased SOD and GST activities in earthworms (Ji et al. 2013), suggesting induction of oxidative stress responses. Additionally, exposure to BDE-47 led to decreased cellular stress response (Hsp70 gene downregulated) (Ji et al. 2013). Alterations were also observed in intermediate filament proteins (IFP) gene expression, which may compromise cell structure and function, while reduced CAT transcript levels could result in increased oxidative stress. Conversely, increased transcription levels of SOD and GST genes suggest an intensified cellular response to counteract superoxide radicals and enhance detoxification processes, respectively (Ji et al. 2013; Xu et al. 2015b; Yang et al. 2018). These combined effects may disrupt cellular homeostasis and potentially impact the overall health of earthworms.

Galaxolide (HHCB) and tonalide (AHTN) are polycyclic musk compounds used in household and PCPs. They are considered contaminants both in aquatic and terrestrial environments (Ehiguese et al. 2021). When biosolids are used as fertilizers in agricultural practices, they can introduce these compounds into soils, thus making them available for exposure to non-target organisms like earthworms (Chen et al. 2014). Exposure of earthworms to these compounds in a filter paper contact test has been found to increase lipid peroxidation through increased MDA content and SOD activity levels at low doses (0.6 µg cm−2 for AHTN and 0.3 µg cm−2 for HHCB), indicating potential damage to membrane lipids (Chen et al. 2011b). These compounds have also been observed to influence the expression levels of several genes (SOD, CAT and Hsp70) in E. fetida (Chen et al. 2011b). Both compounds increased the expression of these genes, but while the effects of tonalide were seen as soon as after 12 h, galaxolide effects were only induced after 24 h. The gene fold alterations persisted longer in E. fetida exposed to the lowest concentration (0.6 µg cm−2) (Chen et al. 2011b). These results indicate potential increased oxidative stress and compromised cellular protection, leading to an imbalance in the cellular redox state, affecting earthworm physiology (Chen et al. 2011b). In a 28-day exposure study with E. fetida, the same compounds increased CAT and SOD gene expressions in a concentration-dependent manner (Chen et al. 2011a). While transcript levels of Hsp70 were decreased by both compounds, lower concentrations of tonalide showed more pronounced effects compared with galaxolide (Chen et al. 2011a).

Azole fungicides, such as epoxiconazole and hexaconazole, widely used in agriculture, have been found to induce endocrine disruption in several fish species (Huang et al. 2022). In earthworms, exposure to epoxiconazole led to increased hydroxide ion (OH–) content and elevated CAT, SOD and GST activities after 10 days at 1 and 10 mg kg−1 (Xue et al. 2023). Likewise, exposure to hexaconazole exposure also resulted in increased SOD and CAT activities and lipid peroxidation (MDA content), as well as decreased AChE content (Liu et al. 2021a).

Herbicides are the main class of compounds studied regarding their impact on oxidative stress parameters in earthworms. These compounds, frequently applied to agricultural soils, have been found to have endocrine-disrupting properties in Xenopus laevis (Orton et al. 2009). Acetochlor exposure has shown dual effects, increasing ROS levels, lipid peroxidation, while decreasing enzyme activity (SOD, CAT, POD) at low concentrations in E. fetida (Cao et al. 2022). Conversely, increased SOD and CAT activities were observed for the same species at high concentrations exposures (Liu et al. 2021b). Atrazine exposure generally resulted increased SOD and CAT activities, as well as elevated MDA content (Song et al. 2009; Jiang et al. 2022). In a study with L. rubellus, with concentrations reaching up to 59 mg kg–1, the authors found that GST levels were decreased after exposure to atrazine (Owen et al. 2008). E. fetida exposed to diuron presented increased ROS content and SOD, CAT and GST activities (Wang et al. 2023b), while mesotrione exposure resulted in decreased activities of these same enzymes and in increased lipid peroxidation (Zhang et al. 2019).

In the case of hormones, such as 17β-oestradiol, found widely in livestock manure, the exposure of E. fetida led to increased metallothionein (MT) levels, GPx activity and changes in the GSH to oxidized glutathione (GSSG) ratio (Heger et al. 2015). MT levels were highest at 3rd and 5th weeks and decreased after 8 weeks in a dose-dependent manner (10–100 µg kg−1). Elevated concentrations of 17β-oestradiol (50 and 100 µg kg−1) prompted a notable increase in the conversion ratio of reduced GSH to GSSG, like the effects observed with MT. The same study found that gene expression of GPx and MT followed similar profiles to the respective proteins (Heger et al. 2015). Despite these data, further investigations into the impact of hormones on the oxidative stress response of earthworms are warranted, given the limited availability of studies addressing this aspect.

Insecticides can reach groundwater from agricultural soils through subsurface flow, leaching or vertical movement in the soil (Carpio et al. 2021). Furthermore, when biosolids are applied, these insecticides may re-enter soil matrices (Clarke and Smith 2011). Cyantraniliprole and thiacloprid, extensively used in agriculture, have been found to significantly alter the oxidative stress enzymes activity profiles (SOD, POD, CAT, GST), increase lipid peroxidation (MDA content) and raise ROS levels in exposed earthworms (Qiao et al. 2019; Lackmann et al. 2021).

Organic UV filters such as 4-hydroxybenzophenone have been shown to decrease SOD activity and CuZn SOD gene expression levels at the lowest concentration applied to E. fetida (0.02 mg mL−1) (Novo et al. 2019), while benzophenone-3 resulted in decreased activities of several enzymes (SOD, CAT, GST) and reduced lipid peroxidation (Gautam et al. 2022).

Plasticizers are added to plastics to increase their flexibility. However, they can easily leach into the environment, because they are not chemically bonded to plastics (Maddela et al. 2023). Common plasticizers like BPA and phthalates have been implicated in adverse health effects in vertebrates (Oehlmann et al. 2009; Mathieu-Denoncourt et al. 2015) and have also been found to exert oxidative stress in earthworms. For example, BPA exposure led to changes in TBARS levels in E. fetida and increased POD and SOD activities in H. africanus. BPA exposure in E. fetida male reproductive organs resulted in altered expression levels of genes stress response and protein homeostasis (Hsc70 4 and MT), lowered at higher doses of BPA and higher at lower doses (Novo et al. 2018). Meanwhile, BPS decreased both SOD and CAT activities and MDA content (Qian et al. 2023). Several phthalates have also been found to exert oxidative damage in earthworms, especially butyl-benzyl-phthalate (Song et al. 2019a), di(2-ethylhexyl)-phthalate (Ma et al. 2017) and diisobutyl-phthalate (Yao et al. 2023).

In summary, the exposure to substances such as antibiotics (e.g. chlortetracycline) and antimicrobial additives (e.g. triclocarban and triclosan), as well as flame retardants and emerging contaminants (e.g. galaxolide and tonalide), among many others, has been shown to endocrine disrupt several species. This exposure often results in changes of antioxidant enzyme activities, such as SOD, CAT, GST and GPx, as well as in increased lipid peroxidation. These alterations in antioxidant defence mechanisms and oxidative balance can have profound implications for earthworms, potentially leading to population size reduction, altered community dynamics and compromised ecosystem functioning.

3.3 EDC-induced genotoxicity in earthworms

The assessment of genotoxicity in earthworms has emerged as a standard and invaluable practice, offering a straightforward, rapid and highly sensitive mean of evaluating the damage caused by clastogenic agents on DNA (de Lapuente et al. 2015). Surprisingly, despite widespread knowledge that EDCs have the capacity to induce genotoxicity, mostly through non-EATS mechanisms, triggering severe pathogenic consequences in humans, genotoxicity assessments have been minimized in research examining the effects of EDCs in earthworms (Fig. 3). However, it is worth noting that some studies have consistently highlighted the profound impact of EDCs on earthworm DNA damage (Table 4).

Fig. 3
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Classes of compounds with endocrine disrupting capacity capable of inducing DNA damage in earthworms. The figure has been designed using icons made by Freepik, Smashicons, Good Ware, monkik, Nes_Kanyanee, Pixelmeetup from www.flaticon.com. BPA, Bisphenol A; BPS, bisphenol S; BBP, butyl benzyl phthalate; DEHP, di(2-ethylhexyl) phthalate; DIBP, diisobutyl phthalate; DINP, diisononyl phthalate, DMP, dimethyl phthalate, DBP, di-n-butyl phthalate, DNOP, di-n-octyl phthalate; PCB, polychlorinated biphenyls; PFOS, perfluorooctane sulfonate; PFOA, perfluorooctanoic acid

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Table 4 Genotoxic effects in earthworms exposed to different endocrine disrupting compounds (EDCs)
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Among the substances studied, antibiotics such as chlortetracycline revealed DNA damage in the alkaline comet assay in earthworms (E. fetida) coelomocytes after a 28-day exposure to concentrations ranging from 0.3 to 300 mg kg⁻1 of soil (Lin et al. 2012b). Similarly, antimicrobial additives such as triclocarban and triclosan induced significant DNA damage in coelomocytes of E. fetida exposed to various concentrations, with the highest levels of genotoxicity generally observed at the concentrations of 50–100 mg kg−1 (Lin et al. 2010, 2012a, 2014; Sales Junior et al. 2020).

Flame retardants like tricresyl phosphate and tris(2-chloroethyl) phosphate induced DNA damage in coelomocytes of E. fetida, when the concentrations exceeded 1 mg kg⁻1 (Yang et al. 2018). Additionally, tricresyl phosphate also led to an increase in 8-hydroxy-2-deoxyguanosine (8-OHdG) content, a biomarker of oxidative DNA damage, in the full body tissue of earthworms (Yang et al. 2018). Exposure to fungicides, such as hexaconazole, and herbicides, like acetochlor and mesotrione, also resulted in an increase in 8-OHdG content in E. fetida (Zhang et al. 2019; Liu et al. 2021a, b). Surprisingly, exposure to diuron, another herbicide, induced low DNA damage (Wang et al. 2023b).

A variety of substances, namely polychlorinated biphenyls (PCBs), insecticides (cyantraniliprole and thiacloprid) and perfluoroalkyl substances (perfluorooctane sulfonate and perfluorooctanoic acid), were all found to induce DNA damage evaluated through the alkaline comet assay in coelomocytes of E. fetida, as evidenced by several studies (Xu et al. 2013; Hu et al. 2014; Feng et al. 2015; Zheng et al. 2016; Duan et al. 2017; Qiao et al. 2019).

Various plasticizers, including butyl-benzyl-phthalate, di(2-ethylhexyl)-phthalate, diisobutyl-phthalate, dimethyl-phthalate and di-n-butyl-phthalate, exhibited mostly dose-dependent induction of DNA damage in coelomocytes of E. fetida (comet assay) and increased 8-OHdG content levels in full body tissue studies, with soil exposure times ranging from 7 to 28 days (Ma et al. 2016, 2017; Wang et al. 2018; Song et al. 2019a; Yao et al. 2023).

The evidence of genotoxicity across such a broad spectrum of chemical classes and exposure conditions highlights the vulnerability of earthworms to EDCs. Furthermore, various substances, including antibiotics, antimicrobial additives, flame retardants, fungicides, herbicides, insecticides, perfluoroalkyl substances, pesticides and plasticizers, have been found to induce DNA damage in earthworm coelomocytes, the phagocytic leukocytes found within the coelom (Riedl et al. 2022). This widespread evidence of genotoxicity emphasises the vulnerability of earthworm coelomocytes to EDCs, which have been shown to impact the genetic integrity of these organisms, potentially compromising their immune system and defence mechanisms and therefore decreasing their ability to cope with environmental changes and impacts.

3.4 EDC-induced molecular changes in earthworms

Exposure to EDCs is of major concern, given the harmful effects observed in a multitude of organisms, crossing different taxa, and therefore multidisciplinary analysis are needed to fully understand their impact on ecosystems. Over the past few decades, a growing body of literature has shed light on the intricate mechanisms through which EDCs influence the expression of several genes (genes related to oxidative stress were already presented and discussed in Sect. 3.2) and molecular pathways related with cellular processes (Table 5), resulting in substantial physiological and developmental alterations (Fig. 4).

Table 5 Molecular changes in earthworms exposed to different endocrine disrupting compounds (EDCs)
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Fig. 4
figure 4

Classes of compounds with endocrine disrupting capacity capable of inducing molecular alterations in earthworms. The figure has been designed using icons made by Freepik from www.flaticon.com

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EDCs, including hormones like 17β-oestradiol (Heger et al. 2015), organic UV filters such as 4-hydroxybenzophenone (Novo et al. 2019) or phthalates and plasticizers, including BPA (Novo et al. 2018) and BPS (Qian et al. 2023) and diisononyl-phthalate (Zhang et al. 2022b) have been found to induce notable changes in key gene expression levels. BPA exposure in E. fetida male reproductive organs resulted in decreased expression levels of genes involved in epigenetic regulation (DNMT1, DNMT3b), DNA repair and genomic stability (PARP1), as well as hormonal regulation (ECR, MAPR, AdipoR), which may lead to impaired fertility and disrupted reproductive function (Novo et al. 2018).

Among other compounds classified as EDCs, those commonly used as pesticides and agrochemicals, such as atrazine (Jiang et al. 2022), imidacloprid (Wang et al. 2019b), isoprocarb (Gu et al. 2021) and triclosan (Lin et al. 2014) have been observed to disrupt the expression of critical genes. Exposure to these compounds induced a decrease in the expressions of annetocin (ANN) and calreticulin (CRT), potentially disrupting cellular processes and physiological functions. Annetocin is an oxytocin-related peptide that plays a key role in triggering stereotyped egg-laying behaviours in earthworms (Kawada 2016), while calreticulin in earthworms plays a crucial role in various cellular functions, including maintaining calcium homeostasis, acting as a chaperone, modulating gene transcription, facilitating integrin-mediated cell signalling, and promoting cell adhesion (Šilerová et al. 2007). Other physiological processes such as cellular ion transport and energy metabolism were also found to be compromised in earthworms after exposure to atrazine as observed by the decreased Na+/K+-ATPase (Jiang et al. 2022).

In the case of flame retardants such as BDE-47 (Ji et al. 2013) and tris(2-chloroethyl) phosphate (Yang et al. 2018), the impact of these compounds on the expression of critical genes has also been shown. Exposure to BDE-47 led to compromised energy production, as observed by ATP synthase gene downregulation (Ji et al. 2013). Alterations were also observed in intermediate filament proteins (IFP) gene expression, which may compromise cell structure and function, and nucleoside diphosphate kinase (NDK), which suggests an intensified cellular response to maintain nucleotide balance (Ji et al. 2013). Additionally, increased acetylcholinesterase (AchE) levels were found after a 14-day exposure to both tricresyl phosphate and tris(2-chloroethyl) phosphate (Yang et al. 2018). AchE is sensitive to neurotoxic compounds and plays an important role in nerve signal transduction. It is primarily responsible for the deactivation of acetylcholine, thereby ending the nerve transmitter stimulation at the postsynaptic membrane, but also promotes the development and regeneration of neurons (Calisi et al. 2013).

Furthermore, fragrance and PCPs, exemplified by galaxolide (HHCB) and tonalide (AHTN), have been observed to influence the expression levels of several genes in a 28-day exposure study with E. fetida. These compounds increased CRT gene expression in a concentration-dependent manner (Chen et al. 2011a). While transcript levels of ANN were decreased by both compounds, lower concentrations of tonalide showed more pronounced effects compared with galaxolide (Chen et al. 2011a).

In a recent study exploring the effects of tebuconazole (fungicide), RNA-seq has been used to explore total gene expression in E. fetida (Li et al. 2022a). This agricultural chemical was found to induce complex responses in various systems, such as nervous and immune systems. Notably, the study highlighted the induction of cytochrome P450-dependent detoxification and oxidative stress pathways, shedding light on the potential mechanisms underlying the observed toxicity. Transcriptomic analysis identified the MAPKKK gene as a key biomarker in these compounds toxicity and the involvement of the MAPK signalling pathway in the adverse effects of these pesticides (Li et al. 2022a).

The exposure to endocrine-disrupting compounds (EDCs), ranging from hormones like 17β-oestradiol to UV filters, phthalates and pesticides, presents a significant concern due to their detrimental effects on various organisms. A wealth of research, summarized in Table 5, has elucidated that EDCs impact cellular and metabolic changes observed in earthworms. Overall, the findings suggest that exposure to EDCs can lead to cellular damage, reductions in immune system function and disruptions in reproductive processes, ultimately impacting the overall fitness of these organisms.

3.5 EDC-induced reproductive toxicity in earthworms

In recent years, concerns have intensified regarding the impact of EDCs on environmental health, particularly their effects on soil ecosystems and organisms such as earthworms, potentially affecting both soil health and quality. In vertebrates, these compounds have been found to disrupt normal physiological processes, including growth and reproduction. In ecotoxicology, reproductive outcomes in many species are used as endpoints for evaluating the impact of EDCs on populations (Celino-Brady et al. 2021; Marlatt et al. 2022). However, comprehensive studies on the reproductive toxicity of EDCs in earthworms are still limited (Table 6) and with most studies describing decrease reproductive outputs in earthworms exposed to EDCs.

Table 6 Reproductive changes in earthworms exposed to different endocrine disrupting compounds (EDCs)
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Exposure of E. fetida to varying concentrations of 17β-oestradiol over a 56-day period revealed dose-dependent effects on reproductive outcomes. While lower concentrations (10, 30, and 50 μg kg−1) stimulated reproduction, higher concentrations (80 and 100 μg kg−1) significantly inhibited it, highlighting the existence of complex dose–response relationships of this hormone in earthworms (Heger et al. 2015), and potentially indicating the existence of different feedback regulatory mechanisms. Therefore, this natural hormone, commonly found in animal manures (Liu et al. 2012a) and on E. fetida natural environment, seems to support earthworm growth and reproduction to some extend at low doses, but having a negative impact when higher doses are present and a turn-over value is overpassed.

Chlortetracycline, an antibiotic, was studied at concentrations ranging from 0.3 to 300 mg kg−1. Results indicated a significant decrease in cocoon and juvenile number at higher concentrations (100 and 300 mg kg−1) (Lin et al. 2012b). Triclosan, an antimicrobial additive, was assessed at various concentrations (ranging from 0.5 to 750 mg kg−1). Higher concentrations of triclosan significantly reduced both cocoon production and juvenile hatching rates, with notable adverse effects observed at concentrations as low as 6.25 mg kg−1, indicative of its potent impact on earthworm reproduction (Lin et al. 2014; Zaltauskaite and Miskelyte 2018).

Herbicides like atrazine (10 mg kg−1) and mesotrione (10 mg kg−1) exhibited varied effects on E. fetida. While atrazine significantly reduced cocoon production, mesotrione showed no significant difference in reproductive outcomes, showing the differential toxicity profiles among herbicidal compounds (Zhang et al. 2019; Jiang et al. 2022). The insecticide imidacloprid, tested at concentrations ranging from 0.011 to 0.282 mg kg−1, in E. fetida showed a dose-dependent decrease in cocoon and juvenile numbers, indicating its potential reproductive toxicity in earthworms (Wang et al. 2019b). Exposure to benzophenone-3, a UV filter, at concentrations ranging from 3.64 to 36.4 mg kg−1 also resulted in a significant reduction in cocoon production in E. fetida, emphasizing the adverse effects of UV filters on earthworm reproductive health (Gautam et al. 2022).

Overall, these studies highlight the significant reproductive toxicity of various EDCs on earthworm populations. The effects observed, which ranged from decreased cocoon and juvenile number to inhibition of hatching rates, show the potentially high threat that EDCs pose to earthworm populations. Given the crucial role of earthworms in soil fertility and nutrient cycling, the widespread presence of EDCs in the environment raises serious concerns regarding soil health and quality and ecosystem function.

4 Concluding remarks and future perspectives

The collective findings regarding the impact of these compounds on histopathological, biochemical and reproductive endpoints in earthworms underscore the urgent need to continue pressing for stricter regulations and comprehensive strategies to mitigate the release of pollutants, including EDCs, into the environment. Triclosan, for instance, shows an increase in Hsp70 gene expression alongside elevated levels of CAT, GR, MDA and SOD activities, suggesting a multifaceted impact on cellular stress response and antioxidant activity (Lin et al. 2012a, 2014; Zaltauskaite and Miskelyte 2018). Similarly, exposure to BDE-47 results in complex changes, including decreased ATP synthase and Hsp70 expression but increased NDK, GST and SOD transcript levels, indicating alterations in energy metabolism and oxidative stress defence mechanisms. Tris(2-chloroethyl) phosphate exhibits alterations in gene expression alongside visible tissue degradation and histopathological changes in the digestive tract, emphasizing its profound physiological effects. Moreover, substances like BPA induce significant changes in gene expression related to masculine and reproductive organs, accompanied by biochemical alterations and histopathological abnormalities, highlighting potential endocrine-disrupting effects, complex regulatory mechanisms or indirect influences on cellular processes. The majority of the studied EDCs also cause decreased reproduction and/or reproductive success, indicative of potential negative impact on soil health, quality and biodiversity, but also on ecosystems function and services.

While current research has focused predominantly on the consequences of endocrine disruptor exposure in aquatic species such as Daphnia magna (Cho et al. 2022), Gammarus fossarum (Gauthier et al. 2023), and Danio rerio (Barros et al. 2022) among others, it is imperative to recognise the widespread presence of EDCs in soil matrices, exerting similar impacts on soil biota. Protecting soil invertebrates, particularly earthworm populations, is crucial for maintaining soil quality and health and ecosystems stability (Al-Maliki et al. 2021). Considering how agricultural practices can impact earthworm populations in the soil is crucial for promoting biodiversity conditions (Vršič et al. 2021). This review highlights the need for careful consideration, even in relatively sustainable practices such as the application of organic amendments, namely livestock manure, biosolids, and crop residues, rich in nutrients and organic matter. Despite these benefits, these amendments can have lasting impacts on soil biota health, leading to decreased biodiversity over time, due to the presence of compounds such as phytoestrogens, hormones and industrial pollutants.

This work highlights the importance of ongoing research in invertebrate endocrinology to understand the direct impact of such compounds on these organisms’ endocrine systems. Earthworms are clearly susceptible to a diverse range of EDCs at biochemical and cellular levels, despite not necessarily interacting with endocrine pathways found in vertebrates. The efforts by Novo et al. (2019) should be complemented by new studies to identify molecular mechanisms responsive to these compounds in earthworms. Intriguingly, several studies have already shown the ability of these compounds to affect gonad development and population dynamics, indicative of putative steroid nuclear receptors.

Ultimately, the adverse effects of EDCs on earthworms, through EATS and non-EATS pathways, have the potential to disrupt earthworm population dynamics, consequently affecting all the earthworm associated microbiome and soil ecosystems function and services. Considering the current necessity of fertile and healthy soils to answer increasing population nutritional needs and to help plants to cope with a climate change environment, it urges to increase studies and awareness to these compounds impact in soil organisms. Therefore, and in light of these considerations, effective legislation should institute rigorous control measures, tailored to the origin and nature of contamination, for responsible soil management. Even seemingly innocuous sources, such as livestock manure, may harbour hormones or antibiotics capable of exerting unanticipated effects on soil organisms. A well-considered regulatory framework is indispensable to strike a balance between agricultural practices and the preservation of soil ecosystems.