Acute and chronic effects of diazepam on the polychaete Hediste diversicolor: Antioxidant, metabolic, pharmacologic, neurotoxic and behavioural mechanistic traits
Introduction
Over the past decades, the research community has become more aware of the gradual increase in the levels of pharmaceutical contaminants of human and veterinary origin in ecosystems. Some of these compounds are resistant to metabolic degradation, thereby persisting in the environment. In addition, pharmaceutical drugs are moderately lipophilic, and can even show some biological activity in the environment (Halling-Sørensen et al., 1998; Daughton and Ternes, 1999; Jones et al., 2005; Miao et al., 2002; Kümmerer, 2008; Kümmerer, 2010; Nunes et al., 2020), causing adverse effects on different species even at very low concentrations, which have been already reported to occur in the environment. Furthermore, simultaneous exposure to several compounds that co-exist in the same matrix can result in synergistic effects (Cleuvers, 2003). Therefore, pharmaceuticals are now regarded as pollutants of special importance.
Neuroactive agents are among the most frequently found drugs in the aquatic environment, (Trudeau et al., 2005), which, even at low levels, may condition the behaviour of aquatic organisms (Rivetti et al., 2016; Brodin et al., 2017; David et al., 2018; Fuertes et al., 2019); among these, the benzodiazepine class assumes particular significance (Brodin et al., 2017), considering the scale of their usage (Maust et al., 2019). Diazepam is a benzodiazepine currently used in human and veterinary therapeutics, with anxiolytic, sedative and muscle relaxing effects (Haefely et al., 1990; Mohler et al., 1996; Calisto and Esteves, 2009). These pharmacological activities result from the enhancement of GABAergic transmission at benzodiazepine-sensitive GABAA-receptors (Mohler et al., 1996). The toxicity of diazepam to several aquatic organisms has been already shown, namely to the cnidarian Hydra vulgaris (Pascoe et al., 2009) in which a concentration of 10 μg/L of diazepam for 72 h caused reduction of their regenerative capacity. In addition, Lorenzi et al. (2016) observed a decrease in the number of eggs produced by the temperate freshwater fish Pimephales promelas when exposed to diazepam. However, diazepam has also been shown to influence cellular redox systems (Musavi and Kakkar, 1998, 2003), and exposure to this drug caused significant adverse changes in the efficacy of the antioxidant response in the freshwater fish Gambusia holbrooki (Nunes et al., 2008). In addition, benzodiazepines in general are known for their relaxant properties, that have been shown to occur also in aquatic animals such as fish (Nunes et al., 2008; Huerta et al., 2016; Brodin et al., 2014, 2017); diazepam has also been implicated in the alteration of behavioural traits of fish, such as Gambusia holbrooki (Nunes et al., 2008), Danio rerio (Bencan et al., 2009; Oggier et al., 2010), and Pimephales promelas (Lorenzi et al., 2016), such as influencing their feeding activity (Snigirov and Sylantyev, 2017). No data are, however, available concerning the putative behavioural effects of diazepam in other taxa of aquatic animals.
Diazepam reaches aquatic systems due to its presence in effluents from wastewater treatment plants (Cunha et al., 2019), where its removal is incomplete (Sulaiman et al., 2016). Consequently, it is released into water courses, where it may attain significant levels. The presence of diazepam at concentrations up to 0.04 mg/l has been reported in effluents from many German sewage treatment plants (Ternes, 1998) and the drug attains concentrations ranging from 0.7 to 1.2 ng/l in the River Po, Italy (Zuccato et al., 2000). Also, Calisto and Esteves (2009) reported levels from 0.88 μg/l to 0.13 ng/l in various locations in Germany, including lakes, rivers and drinking water. Kosjek et al. (2012) reported levels between 20 and 111 ng/l in rivers in Slovenia.
Despite the existence of previous data in the literature concerning the effects of benzodiazepines (and most specifically, of diazepam) in fish, no data exists regarding the potential effects of this drug on marine polychaetes. The organism selected to assess the impact of diazepam was the polychaete Hediste diversicolor (Annelida: Polychaeta). This organism can be located in estuaries along the European and North American coasts, being also commonly distributed in coastal lagoons in Northern Europe and North Africa (Costa et al., 2006; Virgilio and Abbiati, 2004). Individuals of H. diversicolor live in the sediment and are resistant to significant climate shifts, such as salinity and temperature (Scaps, 2002); this species is also ecologically significant, since H. diversicolor is classified in its natural habitat as an omnivorous organism, which uses a variety of feeding methods, but typically acts as a filter and deposit feeder. In these processes, these organisms consume organic matter and debris on the surface layer of sediment and are deemed a key species in soft-bottom populations (Blaise et al., 2013; Garcia-Arberas and Rallo, 2002).
The aim of this work was to evaluate the effects of diazepam on H. diversicolor, following acute and chronic exposures to a range of concentrations (selected on criteria of ecological relevance) of this drug, in order to evaluate specific parameters related to the toxic response, namely behavioural effects and alterations in biochemical markers.
Section snippets
Chemicals
Diazepam (anxiolytic drug, CAS 439-14-5, purity ≥98 %) was obtained from Sigma Aldrich®. Bradford reagent was purchased from Biorad®, UK. All other chemicals (for media and buffers preparation, and for enzymatic assays) were obtained either from Sigma-Aldrich or Merck-Millipore.
Sampling of test organisms
For this work, the specimens of H. diversicolor were collected in a natural reserve, with very low anthropogenic impact (Ghribi et al., 2019), the Local Nature Reserve of the Douro Estuary in the bay of São Paio,
Weight of the chronically exposed animals
The weights of the chronically exposed worms are presented in Table 1. No differences were observed among the exposed animals (F[5, 48] = 0.640; p = 0.67).
Oxidative stress biomarkers
Significant differences in terms of CAT activity were observed among experimental groups in both exposures, with significant increases in animals acutely exposed to the highest diazepam concentrations; chronically exposed animals, namely those exposed to the highest levels of diazepam, also had significantly enhanced activity of this enzyme
Oxidative stress
Our data suggest that the metabolism of diazepam may result in oxidative stress, a condition in which the production of reactive oxygen species (ROS) surpasses the defensive capacity of the antioxidant defence system of living organisms. Oxidative stress in cells may result from high levels of reactive oxygen species, which are thought to be the main cause of oxidative damage, including protein denaturation, mutagenesis and lipid peroxidation in aerobic cells (Gutteridge and Halliwell, 1989).
Conclusions
The data presented in this report has evidenced the potential of diazepam to elicit oxidative stress-related alterations, and also behavioural effects, on the polychaete species H. diversicolor. The results showed that exposure to diazepam caused effects on behavioural traits, such as a decrease in the burrowing time after 96 h, and an increase of the same parameter after 28 days, which may pose additional challenges to environmentally exposed worms by conditioning their escape from predators.
CRediT authorship contribution statement
Ana Filipa Nogueira: Methodology, Validation, Formal analysis, Investigation, Data curation, Writing - original draft. Bruno Nunes: Conceptualization, Methodology, Validation, Resources, Writing - review & editing, Supervision, Project administration, Funding acquisition.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgements
Bruno Nunes is hired by “ECO-R-pharmplast - Ecotoxicity of realistic combinations of pharmaceutical drugs and microplastics in marine ecosystems”, Fundação para a Ciência e a Tecnologia, FCT (reference POCI-01-0145-FEDER-029203). This research was financially supported by CESAM (UIDB/50017/2020 + UIDP/50017/2020), by FCT/MCTES through national funds (PIDDAC), and by the co-funding by the FEDER, within the PT2020 Partnership Agreement and Compete 2020.
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