Effects of low levels of the antibiotic ciprofloxacin on the polychaete Hediste diversicolor: biochemical and behavioural effects

https://doi.org/10.1016/j.etap.2020.103505Get rights and content

Highlights

  • Behavioral and enzymatic biomarkers were responsive to CIP exposure in H. diversicolor

  • Biochemical effects were detected at higher doses of CIP, namely of antioxidant enzymes

  • CIP altered the redox state of exposed worms, but without oxidative damage

  • Increased AChE activity suggests that CIP may have triggered apoptosis cell death mechanisms

  • Behavioral traits were altered, evidencing the ecological impact of CIP on marine worms

Abstract

The release of pharmaceutical chemicals in the biosphere can have unpredictable ecological consequences, and knowledge concerning their putative toxic effects is still scarce. One example of a widely used pharmaceutical that is present in the aquatic environment is ciprofloxacin. Previous indications suggest that this drug may exert several adverse effects on exposed biota, but the characterization of a full ecotoxicological response to this drug is far from complete, especially in estuarine ecosystems. This work aimed to characterize the acute and chronic effects of ciprofloxacin in the polychaete Hediste diversicolor (Annelida: Polychaeta), exposed to environmentally relevant levels of this drug, close to the real concentrations of this pharmaceutical in surface waters. The adopted toxic endpoints were behavioral parameters, combined with a biomarker-based approach (quantification of the activities of catalase (CAT), glutathione-S-transferase (GSTs), cholinesterases (ChEs), glutathione peroxidase (GPx), and lipid peroxidation levels. Exposure to ciprofloxacin caused effects on behavioural traits, such as an increase in burrowing times and hyperactivity, alongside alterations in biomarkers, including a significant increase in CAT activity following acute exposure. In addition, and after both acute and chronic exposure, lipid peroxidation was reduced, while AChE activities were enhanced. It was possible to ascertain the occurrence of pro-oxidative alterations following exposure to low levels of ciprofloxacin, which were counteracted by the triggering of CAT activity. The meaning of the enhancement of AChE activity is not clear, but it appears to be linked with the observed behavioural changes, and may have been associated with the stimulation of the behavioural traits. These data strongly suggest that the presence of ciprofloxacin in estuarine areas is not without risks, and exposed biota, namely polychaete species, are likely to have their ecological roles affected, thereby compromising the chemical, physical and microbiological stability of sediments, which in turn alters nutrient cycles.

Introduction

Pharmaceutical products are important for the various advantages they confer in the prevention, diagnosis and treatment of many diseases (Jones et al., 2005; Huerta-Fontela et al., 2011; Koné et al., 2013). The recognition of their potential benefits has, however, led to a significant increase in their use and consumption, particularly in developing countries (Kumar & Xagoraraki, 2010; Jelic et al., 2011). Knowledge of the environmental occurrence of active pharmaceutical ingredients (APIs) has dramatically increased, especially over the past two decades, mainly due to the production and implementation of more responsive analytical techniques (Boxall et al., 2012), which enables the quantification and detection of drugs in complex mixtures, in the extremely low concentrations (in the order of μg/L to ng/L), that are commonly encountered in the environment (Gros et al., 2012). Pharmaceutical substances exhibit a variety of unique characteristics that justify their potentially deleterious environmental impact. By being primarily designed to survive metabolic breakdown, these chemicals are often not prone to environmental degradation. In addition, drugs are sufficiently lipophilic to be easily absorbed by target species (Hignite and Azarnoff, 1977; Halling-Sorensen, 2000; Daughton & Ternes, 1999; Jones et al., 2002; Kümmerer, 2010). Because they are often not susceptible to degradation, drugs are not promptly eliminated through traditional wastewater treatment procedures employed by sewage treatment plants (STPs) (Boxall, 2004; Grassi et al., 2012).

Ciprofloxacin (CIP) is an antibiotic from the class of fluoroquinolones (FQs), used for the treatment of respiratory diseases and bacterial infections; it is active against Mycobacterium tuberculosis, Mycoplasma spp. and intracellular pathogens such as Chlamydia, Chlamydophila and Legionella. (Chin & Neu, 1984; Zeiler, 1985; Zeiler and Grohe, 1986. Its action stops bacterial multiplication by disrupting the DNA replication and repairing processes. The mode of action of this drug involves the inhibition of prokaryotic DNA gyrase (Gootz et al., 1990; Hussy et al., 1986). Consequently, its influence on vertebrate DNA related enzymes is several orders of magnitude weaker (Hussy et al., 1986; Wolfson & Hooper, 1985). Nevertheless, CIP has been correlated with genotoxic effects in both prokaryotic and eukaryotic test systems (Herbold et al., 2001; Nunes et al., 2018). The genotoxicity of CIP has been demonstrated with sister chromatid exchange, unscheduled DNA synthesis (Takayama et al., 1995), and chromosomal aberrations in lymphocytes (Pino, 1995; Gorla et al., 1999). In addition, CIP can cause other signs of toxicity in vertebrates, including central nervous system (CNS) reactions (Kappel and Calissi, 2002). In fact, CIP GABA antagonistic effects are known to trigger seizures (Akahane et al., 1989; Akahane et al., 1993; Schmuck et al., 1998). CIP also leads to chondrotoxicity (cartilage damage), reproductive and developmental toxicity, genotoxicity, carcinogenicity and phototoxicity (Christ et al., 1990; Stahlmann & Lode, 1998). One reason for the detrimental effects of FQs is the activation of free radical formation (Wagai & Tawara, 1992; Hayem et al., 1994). FQs have been shown to trigger oxidative damage to cells via the development of peroxides and free radicals, contributing to disturbances such as lipid peroxidation and animal neural dysfunction (Qin & Liu, 2013). In addition, specific FQs, including CIP, are known for their detrimental environmental effects. As demonstrated by Ebert et al. (2011), CIP may pose serious environmental risks, even at already demonstrated levels in the biosphere.

According to the review by Kelly and Brooks (2018), focusing on circa 200 studies on the worldwide occurrence of CIP, this substance was shown to have a global distribution, in fresh surface water, groundwater, saltwater, treated municipal effluent, raw municipal sewage, treated hospital effluent, and raw hospital sewage. In addition to its worldwide presence and high frequency of detection, the magnitude of its levels is also noteworthy. Golet et al. (2002) reported environmental concentrations of CIP around 15 ng/L in river water from northern Switzerland. Watkinson et al. (2009) found a CIP concentration of 1.30 μg/L in water bodies of Australia, whereas Gibs et al. (2013) reported CIP levels of 0.077 μg/L in New Jersey effluents. Extremely high concentrations of this drug (above 14 mg/L) were detected in wastewater, and also in areas with little or no wastewater treatment processes (attaining levels of 6.5 mg/L in some locations; Larsson et al., 2007). In Portugal, concentrations of CIP ranged between 127 and 10962.5 ng/L in wastewaters from four different hospitals, reaching wastewater treatment plants at concentrations of 667.1 ng/L (influent) and leaving it at 309.2 ng/L (effluent) (Seifrtová et al., 2008). Besides being found in rivers and effluents, CIP has been more recently reported in estuarine waters of the River Tejo (Portugal) in concentrations ranging from 1.56 ng/L to 7.14 ng/L (Reis-Santos et al., 2018). Additionally, its presence has also been reported in estuarine waters in the United States of America, being detected in levels up to 7.3 ng/L (Meador et al., 2016).

Furthermore, FQs are strongly adsorbed to organic matter and clays (Córdova-Kreylos & Scow, 2007), which favours its persistence, namely in sediments. Golet et al. (2002) measured CIP associated with solid and aqueous samples; sludge solids of a Swiss wastewater treatment plant contained an amount more than three orders of magnitude higher than those quantified in the raw sewage-filtered effluent (Golet et al., 2002; Córdova-Kreylos & Scow, 2007). Very few studies were done on adsorption of CIP to soil or sediments, however Córdova-Kreylos & Scow (2007) showed that CIP had a high adsorption potential to salt marsh sediment.

The species chosen to study the effects of this drug was the polychaete H. diversicolor (Annelida: Polychaeta). This organism can occur in estuaries along the coast of Europe and North America, being also often widely found in coastal lagoons from Northern Europe to North Africa (Fidalgo & Costa et al. 2006; Virgilio & Abbiati, 2004). Individuals of this species reside in the sediment and are resistant to significant changes in environmental (or physical) water parameters such as salinity and temperature (Scaps, 2002). This organism is also ecologically important, in its natural environment, H. diversicolor is described as an omnivorous species, which uses diverse feeding modes but usually behaves as a filter and a deposit feeder, while foraging organic material and detritus on the top layer of sediment, and it is considered a crucial species in soft-bottom communities (Blaise et al., 2013; Garcia-Arberas and Rallo, 2002).

The aim of this work was to evaluate the effects of CIP on H. diversicolor, following acute and chronic exposures to a range of concentrations of this pharmaceutical drug, in order to evaluate specific parameters related to the toxic response: behavioral effects, alterations in biochemical markers, tissue alterations and genotoxicity.

Section snippets

Chemicals

Ciprofloxacin (hydrochloride form; ≥98%; CAS 86393-32-0) 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

The test organisms were manually collected at the Local Natural Reserve of the Douro Estuary in the bay of São Paio, Afurada, Vila Nova de Gaia, Portugal (GPS coordinates 41° 8′ 9.01″ N–8° 39′ 47.07″ W). This location

Oxidative stress and metabolic biomarkers

Significant differences in terms of CAT activity were observed among experimental groups in the acute exposure, with a significant increase in animals exposed to CIP relative to those from the control group, with no alteration in those chronically exposed (acute exposure: F[5, 52] = 4.94; p ≤ 0.001; chronic exposure: F[5, 49] = 0.33; p = 0.88) (Fig. 1). A significant increase in GPx activity in acutely exposed animals was also detected, but only with Se-dependent GPx (total GPx - acute

Oxidative stress

Our data, namely those concerning CAT activity, suggest that the metabolism of CIP may result in oxidative stress, a condition in which the production of ROS surpasses the defensive capacity of the antioxidant defence system of living organisms. Oxidative stress in cells can result from an increase in the levels of reactive oxygen species, which are considered the major cause of oxidative damage, including protein denaturation, mutagenesis and lipid peroxidation in aerobic cells (Halliwell and

Conclusions

Various studies have reported the effects of CIP when in the aquatic environment. In our study, behavioral and enzymatic biomarkers revealed several effects in H. diversicolor after exposure to CIP. Sub-lethal parameters have been successfully used, detecting biochemical effects at very low doses. Our data demonstrate the occurrence of oxidative responses, but no clear indications about the oxidative stress and damage were obtained. In addition, the increase of AChE activity, following both

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 declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

Bruno Nunes is supported 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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