Biochemical and locomotor responses of Carcinus maenas exposed to the serotonin reuptake inhibitor fluoxetine

Abstract

The aim of this study was to assess the effects of the widely used anti-depressant fluoxetine on behaviour (locomotion), moulting, neuromuscular transmission, energy production and anti-oxidant defences’ efficiency of the epibenthic crab Carcinus maenas. Crabs were individually exposed to fluoxetine concentrations for 7 d. Effects on locomotion were assessed at the end of the exposure using an open field test adapted to C. maenas in the present study. Tissue samples were later collected to evaluate fluoxetine effects on physiological functions using the activity of key enzymes and other parameters as biomarkers, namely: N-acetyl-β-glucosaminidase (NAGase) in the epidermis (moulting) and the hepatopancreas; cholinesterases (ChE) in muscle (neuromuscular cholinergic transmission); NADP⁺-dependent isocitrate dehydrogenase (IDH) and lactate dehydrogenease (LDH) in muscle (energy production); glutathione S-transferases (GST) in hepatopancreas (biotransformation and oxidative stress system); glutathione reductase (GR), and glutathione peroxidade (GPx), total glutathione levels (TG) and lipid peroxidation levels in the hepatopancreas (anti-oxidant defences and oxidative damage). Because no information on C. maenas NAGase activity was previously available, its variation during the moult cycle was also investigated. The results showed that locomotion was significantly increased at fluoxetine concentrations equal or above 120 μg L⁻¹, with animals spending more time moving, walking longer distances than controls. Levels of NAGase activity were found to vary in relation to C. maenas moult cycle, but no alterations were observed after exposure to fluoxetine. Significant increases in the activity of ChE, GST and GR enzymes, and the levels of TG were found, with a lowest observed effect concentration (LOEC) of 120 μg L⁻¹. Effects on locomotion were significantly and positively correlated to those induced on ChE activity. The results raise concern when hypothesising conditions of chronic exposure in the wild.

Introduction

Over the past years a wide range of pharmaceuticals has been detected in aquatic ecosystems, as a result of routine use or disposal of unused medicines. Several pharmaceuticals have been found in soils, and in surface and ground waters following their inappropriate disposal and/or due to inefficient degradation in sewage treatment plants (Daughton and Ternes, 1999). Although most of them are found in the ppb or ppt range, their presence in the environment raises considerable concern regarding their effects on wild organisms and potential accumulation in trophic chains since they are designed to be biologically active and several of them are persistent. Furthermore, they may be biotransformed into reactive metabolites and/or environmentally degraded generating different substances, which may be also biologically active and/or persistent in the environment.

Among pharmaceuticals, the antidepressant fluoxetine is of particular concern since it is highly consumed, often found in natural waters and municipal effluents (Metcalfe et al., 2010) and was considered as one of the most acutely toxic towards non-target species (Fent et al., 2006). Fluoxetine is primarily indicated to treat depression, but also for treating obsessive–compulsive behaviour, as well as eating and personality disorders (Simpson and Noble, 2000). In England 2.56 tonnes of fluoxetine have been consumed in the year 2000 (Sebastine and Wakeman, 2003), whereas in the United States 22,266 million prescriptions were issued in 2007 (Modern Medicine Pharmacy, 2010). Moreover, consumption is expected to increase with the growing prevalence of psychiatric disorders (Alonso et al., 2004).

Fluoxetine has been detected in the environment in surface waters and in effluents from wastewater treatment plants. For surface waters, concentrations ranging from 0.054 to 0.141 μg L⁻¹ were measured in Ontario, Canada (Metcalfe et al., 2010), whereas in the United States maximum concentrations of 0.012 μg L⁻¹ were found in several states with a frequency of detection of 1.2% (Kolpin et al., 2002). In untreated wastewaters a maximum concentration of 0.191 μg L⁻¹ was found in Ontario (Metcalfe et al., 2010). In treated wastewaters maximum concentrations of 0.122 μg L⁻¹ and 0.509 μg L⁻¹ and 0.540 μg L⁻¹ were reported, respectively, in Canada in Ontario (Metcalfe et al., 2010) and Alberta (Chen et al., 2006), and in the United States (Weston et al., 2001).

Fluoxetine is considered to be fairly stable in the environment. It is relatively resistant to hydrolysis, photolysis, and microbial degradation, with a half-life greater than 100 d at 25 °C, under UV light, in both pH 5–9 buffered solutions and natural lake waters (Kwon and Armbrust, 2006). In addition, fluoxetine dissipates from the aqueous to the sediments phase where it also appears to be persistent (Johnson et al., 2005, Kwon and Armbrust, 2006), with a potential toxic risk to aquatic organisms associated with sediments (Brooks et al., 2003, Oakes et al., 2010). As a serotonin reuptake inhibitor, fluoxetine binds to serotonin transporters inhibiting the reuptake from the synaptic cleft and potentiating its effects. In crustaceans, serotonin participates in several physiological and behavioural processes such as neurohormones release (namely crustacean hyperglycemic hormone and moult inhibiting hormone), circulatory and digestive functions, aggression, and locomotion (Weiger, 1997, Daughton and Ternes, 1999). Therefore, studying the sublethal effects of this emerging contaminant on crustaceans, particularly on species living in close association with sediments is a priority issue.

During the past decades biochemical biomarkers have been increasingly used in the early assessment of aquatic contamination, its deleterious effects on wild species, and the health status of their populations. Several relevant biomarkers are the activity of the enzymes cholinesterases (ChE), lactate dehydrogenase (LDH), NADP⁺-dependent isocitrate dehydrogenase (IDH), glutathione-S-transferases (GST), glutathione reductase (GR) and glutathione peroxidase (GPx), and the levels of total glutathiones (TG) and lipid peroxidation (LPO), as well. ChE are a family of enzymes present in vertebrate and invertebrate species (Bacq and Nachmansohn, 1937, Guilhermino et al., 1996). Among ChE, acetylcholinesterase participates in cholinergic transmission by degrading the neurotransmitter acetylcholine (ACh). Altered ChE activity has been shown to cause behavioural impairments such as abnormal locomotion and swimming (Jensen et al., 1997, Vieira et al., 2009, Almeida et al., 2010). LDH is an enzyme involved in the anaerobic pathway of energy production, that catalysis the interconversion of pyruvate to lactate in glycolysis, and may be induced by exposure to contaminants (Diamantino et al., 2001, Guimarães et al., 2009). IDH plays a role on the regeneration of NADPH, and besides contributing to cellular energy production it is directly related to the maintenance of the cellular redox balance (Lima et al., 2007). GST participates in the conjugation of electrophilic compounds with reduced glutathione (GSH), which becomes oxidised (Van der Oost et al., 2003), and is also involved in anti-oxidant defences. Besides its role in the biotransformation of electrophilic compounds, GSH is also an important antioxidant. GR merits attention because it acts on the recycling of oxidised glutathione (GSSG) into GSH (Cribb et al., 1989). GPx also has an anti-oxidant role, catalysing the conversion of hydrogen peroxide resulting from cellular metabolism into water and oxygen. The oxidation of polyunsaturated fatty acids (LPO) is a widely used biomarker of oxidative damage caused by ROS formed following toxicant exposure (Ohkawa et al., 1979, Van der Oost et al., 2003). Not so widely used but also of high relevance due to its functional role on the moult process is the activity of the enzyme N-acetyl-β-glucosaminidase (NAGase), also called chitobiase. Moulting is a hormonally-regulated process essential for crustacean development, growth, and reproduction. NAGase acts in the final step of moulting by breaking down oligomers of cuticle constituents into monomers, allowing the digestion of the old endocuticle and a successful moult (Espie and Roff, 1995). In some aquatic crustaceans, NAGase has been used as a biomarker of disruption of the moulting process by environmental contaminants, including fluoxetine (Zou and Fingerman, 1999b, Richards et al., 2008).

Behavioural traits are essential to the survival of the organism, and population and community viability. Because of this they have been considered to provide a unique toxicological perspective, linking biochemical disturbances and ecological consequences of environmental contamination (Little, 1990). Moreover, since behaviour results from the integration of genetic, biochemical, physiological and environmental cues, Peakall et al. (2002) remarked that it may be seen as a functional interface between the individual and the population, relevant either direct or indirectly for population maintenance. Hence, the assessment of behavioural traits together with biochemical and physiological biomarkers in key-species for the structuring and functioning of ecosystems has been proposed as a relevant approach to disclose risks of cascading deleterious effects at the community and ecosystems levels (Amiard-Triquet, 2009). Of the various behavioural traits of an organism, locomotion is of particular importance on its own, and because it is required for many other complex behaviours, such as borrowing, food search, reproduction and predator avoidance (Boyd et al., 2002, Amiard-Triquet, 2009). Impaired locomotion may thus also affect such behaviours leading to effects at the population, community and ecosystem levels (Roast et al., 2000, Boyd et al., 2002, Amiard-Triquet, 2009). In invertebrates, including Carcinus maenas, a number of environmental contaminants such as metals and pesticides have been shown to induce alterations of the locomotor behaviour (Hebel et al., 1997, Jensen et al., 1997). Furthermore, a recent study found impaired locomotor responses in a gammarid species following exposure to fluoxetine (De Lange et al., 2009).

C. maenas is an ecologically relevant species of European estuarine and coastal ecosystems where it occupies a key position in food webs. This omnivorous and scavenger animal has been estimated to moult approximately 18 times during its life cycle, after attaining the first crab stage (Crothers, 1967). This species has been widely used in both laboratorial (Lundebye et al., 1997, Elumalai et al., 2007, Dam et al., 2008) and field studies (Galloway et al., 2004, Martín-Díaz et al., 2004, Maria et al., 2009) to assess the toxic effects of pollutants, using a variety of physiological, biochemical and molecular parameters, such as the cardiac rate, cellular viability, cell membrane stability, and the activity of several enzymes. Furthermore, since C. maenas is an epibenthic organism it may be particularly exposed to fluoxetine, making the assessment of its potential effects on this species a relevant issue. Therefore, the aim of this study was to assess the effects of fluoxetine on C. maenas biochemical and behavioural parameters. Possible linkages between the studied parameters were also investigated. The biochemical biomarkers employed were: epidermal and hepatopancreas NAGase activity; muscle ChE, IDH and LDH activities; hepatopancreas GST, GR and GPx activities, and the levels of GT and LPO, as well. Although moulting is essential for growth and reproduction, to the best of our knowledge no baseline values of C. maenas NAGase activity are available in the literature. For this, and because the enzyme activity is expected to vary according to the moult stage (Espie and Roff, 1995), a study of its variation during the moult-cycle of wild C. maenas was first performed. Spontaneous locomotion was the behavioural trait assessed using an open field test adapted in this study for use with C. maenas. This test has been widely used with vertebrate and invertebrate species, showing a conspicuous ability to reveal subtle behavioural changes and an easy application to a great diversity of species (Boyd et al., 2002). The use of this test in the present study was based on the working hypothesis that given that shore crabs are usually able to explore new environments and eventually try to escape from overt exposure, when placed in the open field arena they would move, allowing the quantification of the locomotor behaviour.