Increasing agricultural production and population growth have led to increased environmental pollution due to the generation of large volumes of agricultural and household waste, with considerable potential for exerting deleterious effects on non-target biota (Durigan et al., 2012). Among these chemicals, one may find emergent pollutants, which are potentially toxic compounds released into natural waters due to low removal efficiency provided by conventional systems treatment sewage, with implications for the environment still poorly understood (Durigan et al., 2012). These contaminants include a broad group of compounds including personal care products, additives, nanomaterials, and particularly drugs for human and veterinarian use, which reach the environment as metabolites or in their unchanged form (Horvat et al., 2011; Fatoki et al., 2018), in concentrations in the order of the ng/L to μg/L (Boonstra et al., 2011; Horvat et al., 2011). Among the most abundant drugs that occur in the environment, specific classes stand out according to their use. This is the case of substances used primarily in veterinary, aquaculture, and livestock, where animals are medicated with high amounts of pharmacologically active substances, which contaminate the environment on a large scale. The importance of this issue is reinforced considering specific classes of drugs, namely antiparasitics that are regularly applied to the intensive livestock production, particularly cattle, pigs, sheep and horses, and cultured fish (Fent et al., 2006; Sherer, 2006).
Antiparasitics are chemicals used to control or kill endo- or ectoparasites (namely in cattle), being mainly used against a large number of helminths (e.g. nematodes) (Sherer, 2006; Horvat et al, 2011; Wolstenholme et al, 2016) or protozoa (Chen, 2016). These drugs either kill or immobilize/expel parasites from the host's body, not causing any damage to the host (Abongwa et al., 2017). However, a potential adverse effect of these pollutants is the damage they may cause to non-target organisms environmentally exposed. This happens since these drugs have the ability to act in low concentrations (μg/L levels, or less) causing toxic chronic effects (Solomon et al., 2007; Horvat et al., 2011). Although antiparasitics are widely used, there is little data available regarding their environmental presence. However, Sherer (2006) conducted a study in the United States of America and determined their presence in concentrations of 0.12 mg/kg of doramectin, and 1.85 mg/kg of ivermectin in feces of medicated animals. At the same sites low concentrations of these compounds were found in soils, circa 0.046mg/kg Sherer, 2006). However, the information available regarding the concentration of these drugs in the environment is still limited and, since they have a wide applicability, their presence and potential impacts are expected to occur also in the aquatic environment (Horvat et al., 2011). In the particular case of the marine environment, where aquaculture activities are undertaken, estuaries are most subjected to contamination by such pollutants; animals at these locations, namely sediment-dependent organisms, are sometimes exposed throughout their life cycle (Fent et al., 2006).
Within the pharmacotherapeutic group of the antiparasitics, one may find macrocyclic lactones, constituted by avermectins (AVMs) and milbemycins, derived from natural fermentation of soil microorganisms of the genus Streptomyces (Abongwa et al., 2017). Damage caused by parasites in animals worldwide may be something significant, which makes AVMs extremely popular and necessary (Bai & Ogbourne, 2016). As a result of this massive use, large discharges of residues of these compounds have been reported, yet poorly documented. However, these pharmaceuticals have been already classified as potentially toxic to aquatic organisms, being necessary to undertake further studies on its environmental fate and effects (Maranho et al., 2014; Bai & Ogbourne, 2016), namely on aquatic organisms.
One of the most important antiparasitic drug is ivermectin (IVM), which is a legally approved drug, among those most commonly used in veterinary procedures against nematodes, namely aquaculture (e.g. sea lice, onchocerciasis; Bai & Ogbourne, 2016). The use of IVM has not always been legal, since previous evidences pointed also to massive illegal uses of IVM, prior to its approval, as described by Grant and Briggs (1998). IVM is a neurotoxin, and acts on glutamate gamma-aminobutyric acid mediated channels, present in invertebrates and vertebrates, thereby opening chloride channels (Bai & Ogbourne, 2016). This allows IVM to act as a neuromuscular inhibitor to promote hyperpolarization of the cell by anion input, hampering the transmission of nerve impulses, leading to paralysis of the muscles (Bai & Ogbourne, 2016; Chen, 2016; Crump, 2017). The inhibition of locomotion and muscle activity of parasites, is enough to interrupt the process of secretions, which is required to prevent the host immune system response (Degani-Katzav et al., 2016). The combination of such events leads to the death and expulsion of the parasite.
Because of its low metabolism, ivermectin is excreted in the feces almost unchanged (about 90% of the administered dose) and only about 2% of the dose is excreted in urine (González Canga et al., 2009). However, IVM has a high affinity for organic matter resulting in reduced bioavailability in water, with half-life DT50 of 39h in water and of 45d in sediment (Solomon et al., 2007). However, the bioavailability and half-life of IVM is seriously influenced by conditions such as low water solubility and instability when exposed to UV or visible light (Cui et al., 2018) and may be less than 2ng/L in surface waters (Solomon et al., 2007). Nevertheless, and in field conditions, namely in marine areas, IVM tends to stay for extremely long periods in the sediments, as shown by Roth et al. (1993). According to the estimates by Davies et al. (1998), the occurrence of IVM in marine sediment may exceed 100 days. A microcosm study conducted by Boonstra et al. (2011), showed that the values DT50 of IVM may vary from 1.1 to 8.3 days; predicted environmental concentrations (PEC) were of 25 to 60 ng/L, and a worst case scenario with concentrations of 1000 ng/L could also occur (Boonstra et al., 2011). However, the higher concentration of IVM reported in the literature was of 4.4 ng/L, and corresponded to runoff from farms (Nessel et al., 1989). Soil dependent bodies are exposed to IVM due to its persistence in soil, which can reach 7 days to a few months (Horvat et al., 2011). Despite its frequent and widespread use, ivermectin is not innocuous, being toxic to fish (Kennedy et al, 2014; Domingues et al, 2016; Massei et al, 2019), mammals (Trailovic & Nederljkovic 2010 ; Moreira et al., 2017; Cordeiro et al., 2018; Parisi et al., 2019) , birds (Sakin et al., 2012; Li et al., 2013; Liu et al., 2016), and insects (Strong & James, 1993; Solomon et al., 2007; Ishikawa & Iwasa, 2019). The data obtained by Black et al. (1997) showed that IVM caused a significant mortality in sediment polychaetes, suggesting that these organisms are highly susceptible to this drug, even after short exposures. In some of these studies IVM has been shown to cause oxidative stress with adverse effects mainly on mammalian behavior, reproduction and fecundity, deformation of fish embryos and larval death of some insects or even adult death, raising concerns about its fate and ecological effects. In addition, IVM has also been shown to be toxic to marine invertebrates, namely polychaetes that are responsible for bioturbation of sediments and mineralization of organic matter, as demonstrated by Black et al. (1997). Consequently, IVM presents a non-characterized risk to non-target organisms (Grant and Briggs, 1998; Lumaret et al., 2012). This scenario is even favored by a general lack of toxicity data of IVM, for aquatic species; in fact, the present day knowledge is scarce, and reliable data is still limited to a few studies, some of them with species of the genus Daphnia, which are not representative of the marine or estuarine environments; even more limited is the amount of information for marine sediment dwelling organisms (Roehr, 2011), which seem to be the major targets of this drug.
One of the already documented outcomes of IVM is oxidative stress (El-Far, 2013), a condition characterized by increased levels of reactive oxygen species (ROS), against which the antioxidant defense system is not effective to prevent damage. This imbalance causes damage to cells and macromolecules (Nunes et al., 2016). To quantify the level of oxidative stress in cells, biomarker studies that measure the levels of activation of the antioxidant defense system are used to measure biochemical sublethal changes resulting from individual exposure of organisms to xenobiotics (Hyne & Maher, 2003).
The organism Nereis diversicolor, described in 1776 by OF Muller, is a predator and filter feeder that inhabits intertidal zones in the temperate zones of the northern European and African coasts (Aberson et al., 2011; Ghribi et al., 2019), in sand or mud where it builds U-shaped or Y- channels, avoiding contact with other individuals (Patrick, 2002). Individuals of this species are key to support various predators such as crabs, prawns, fish and birds (Carvalho et al., 2013), being important in the recycling of organic matter and nutrients, and in bioturbation (Bonnard et al, 2009). It is through burial that individuals promote sediment bioturbation that will move and irrigate the sediment affecting chemical flows (nutrients, pollutants) and microbiological activity (Aberson et al., 2011). Due to its high tolerance to temperature variations and hypoxia conditions (Patrick, 2002) it is considered a key species in bottom communities in almost all European estuaries (Patrick, 2002; Moreira et al., 2006). Moreover, this species has a great commercial interest since it is used as bait for anglers (Carvalho et al, 2013), and has a great potential to be produced as fish feed in aquaculture (Patrick, 2002; Bagarrão, 2013). Due to its high abundance and ecological relevance (in trophic chains and sedimentation processes), N. diversicolor was considered the one the appropriate species to serve as a test organism to study ecosystems exposed to various pollutants (Ghribi et al., 2019). Due to its responsiveness to pollutants, N. diversicolor is suitable for biomonitoring environmental ecosystems and management programs (Ghribi et al., 2019). It has been successfully used in biomonitoring programs, such as the assessment of environmental quality and to measure contaminant concentrations in the field, and to quantify biomarkers after exposure to pollution caused to a contaminant discharge in the Bay of Cadiz (Maranho et al., 2014). Given its importance in both the ecosystem, economy, and environmental sciences, N. diversicolor is an adequate species to assess the toxic effects of anthropogenic compounds, such as drugs such as IVM.
Considering that after excretion, IVM has the potential to contaminate aquatic sediments, it is important to study the potential effects of realistic levels of this drug on sediment dwelling organisms, such as polychaetes. For this purpose the present work used the quantification of biomarkers of oxidative stress and metabolism (namely, the activities of three key enzymes catalase (CAT), glutathione S-transferase (GSTs), and lactate desidrogenase, LDH) and behavioral changes as biomarkers in individuals of the polychaete N. diversicolor. The assessment of changes in the behavior of this organism is justified since IVM operates at the neuromuscular level, compromising the mobility of affected organisms. To attain this objective, behavioral tests focused both on locomotion and burial activity of exposed organisms.