Effects of environmentally relevant concentrations of diclofenac in Mytilus trossulus
Graphical abstract
Introduction
Pharmaceuticals belong to the contaminants of the emerging concern, already present in the surface waters around the world (Borecka et al., 2015; Fisch et al., 2017). Emission of pharmaceuticals and personal care products (PPCPs) to the marine environment, mainly via rivers and surface runoffs, is becoming a serious global threat. However, the fate of pharmaceuticals in the marine ecosystems is still poorly researched. Many of these compounds are characterized by pseudo-persistent in the marine environment, thus they may constantly influence the condition of organisms, posing a serious threat to the marine fauna (Brausch et al., 2012; Fabbri and Franzellitti, 2016; Fent et al., 2006; Kümmerer, 2008; Sanderson et al., 2004). Nevertheless, the legislation controlling the release of PPCPs into the environment is still missing, which may be due to the insufficient number of studies to confirm the seriousness of the threat, as well as its potential long-term consequences (Dodder et al., 2014).
Non-steroidal anti-inflammatory drugs (NSAIDs) are predominant among pharmaceuticals detected in the aquatic environment worldwide. Recent studies have shown the presence of NSAIDs in the wild mussels collected along Spanish and Italian coasts, while many other works have confirmed their presence in different water bodies around the world (Hanif et al., 2020; Sathishkumar et al., 2020; Mezzelani et al., 2016a; Moreno-González et al., 2015). NSAIDs are characterized by anti-inflammatory, antipyretic and analgesic effects (Brune and Patrignani, 2015; He et al., 2017; Kümmerer, 2008; Nikolaou et al., 2007; Paíga et al., 2015). Over-the-counter (OTC) availability favours high consumption of these pharmaceuticals (Cleuvers, 2003; Comeau et al., 2008; Brune and Patrignani, 2015; Paíga et al., 2016; He et al., 2017; McRae et al., 2018). Among the NSAIDs, diclofenac has the highest toxicity and is the most frequently detected, thus it has been added to the European Surface Water Monitoring System according to the Water Framework Directive (Hallgren and Wallberg, 2015). Concentration of diclofenac in the marine environment is relatively low, ranging a few ng/L to several μg/L. However, continuous exposure of marine organisms to low concentrations of diclofenac may pose a risk to them (Dumas et al., 2020; Ryzhkina et al., 2020; Mezzelani et al., 2016b; Araujo et al., 2014). Many short-term laboratory studies have already shown that diclofenac has negative effect on marine organisms (Kloukinioti et al., 2020; Bonnefille et al., 2018; McRae et al., 2018; Freitas et al., 2019a; Freitas et al., 2019b; Du et al., 2016; Balbi et al., 2018; Sanderson et al., 2004). Study by Mezzelani et al. (2018) has shown that diclofenac, at environmentally relevant concentration, causes the modification of immunological parameters, modulation of lipid metabolism, genotoxic effects, as well as changes in cell turnover. On the other hand, Bonnefille et al. (2018) proved that diclofenac causes modulation on tyrosine-dopaminergic and tryptophan metabolic pathways in M. galloprovincialis, which consequently results in the disruption of osmotic regulation and reproduction processes. The fate of pharmaceuticals after entering invertebrates is a poorly researched issue. Studies on pharmacokinetics could significantly increase the knowledge concerning toxic properties of tested pharmaceuticals and more importantly, provide valuable information about the structure and activity of metabolites produced by organisms (Sathishkumar et al., 2020). Diclofenac metabolites produced by marine organisms are one of the least recognized concerns (Fu et al., 2020; Sathishkumar et al., 2020; Bonnefille et al., 2017; Świacka et al., 2019). Recently, Bonnefille et al. (2017) demonstrated that Mytilus spp. mussels metabolise diclofenac to 13 metabolites, including 4-OH and 5-OH diclofenac. Świacka et al. (2019) also confirmed the metabolism of diclofenac to 4-OH and 5-OH diclofenac by M. trossulus. However, data on diclofenac metabolites, their toxicity and potential bioaccumulation is very limited thus it is likely that these compounds may include potentially hazardous substances, and therefore it is worthwhile to scrutinize their structure and biological activity.
The Baltic Sea is characterized by a strong salinity and temperature gradient, also remaining one of the most polluted seas in the world (Voipio, 1981; Albalat et al., 2002; Bonsdorff, 2006; Korpinen et al., 2012). Baltic organisms, such as M. trossulus are forced to spend a lot of energy on osmotic regulation, therefore they may be more sensitive to any other external stressors, such as environmental pollution. M. trossulus, marine bivalve species that has been adapted to the harsh Baltic conditions, is also one of the most important links in the food chain. Furthermore, mussels are sedentary and filter-feeding organism and can remove various environmental pollutants from the water by accumulating them (James et al., 2020). Moreover, mussels are also often found near sewage outlets. Thus they are one of the most exposed groups of marine fauna to different contaminants (James et al., 2020; Caban et al., 2016; De Solla et al., 2016, Klosterhaus et al., 2013; Marigómez et al., 2013; Gillis, 2012).
The main aim of this work was to provide a more detailed information on the effect of diclofenac on marine fauna - M. trossulus, by performing exposure experiment in the laboratory controlled conditions. For the first time histological analysis was used to determine the toxicity of diclofenac in mussels. The effect of diclofenac on the general condition of mussels was also tested. A preliminary pharmacokinetic study was performed through the determination of diclofenac and its two metabolites (4-OH and 5-OH diclofenac) concentration in tissues and water during exposure to environmentally relevant concentrations of diclofenac and further depuration phases. The effect of different diclofenac concentrations has also been included. In addition, screening of selected NSAIDs (ketoprofen, ibuprofen, flurbiprofen and naproxen) in M. trossulus and water collected from the Gulf of Gdańsk was performed. Basing on previous experimental work, this study assumed that diclofenac effects may be revealed by increased frequency of tissue changes without necessarily affecting the overall condition. Although absorption of the drug in mussel tissues was expected, taking into account their ability to metabolise diclofenac, it was also anticipated that the drug could be completely excreted from the organisms during depuration. Moreover, it was assumed that the observed effects of diclofenac will be related to its concentration and seen not only at the genetic and biochemical levels but also at the tissue level, thus even more affecting the physiology of an organism.
Section snippets
Sampling
Material (1000 individuals of M. trossulus) was collected from the Gulf of Gdańsk - at Orłowo station (54°29,40 N; 18°38,60 E) in January 2019. During the sampling, temperature and salinity were measured and their values were respectively: 5.1 °C and 7.1. Mussels were sampled using the benthic drag and water samples were collected using bathometer. Additionally, 300 individuals of M. trossulus and two litres of seawater were collected for analysis of diclofenac, its metabolites (4-OH diclofenac
Concentration of diclofenac and its metabolites in the mussels and water from the environment
Diclofenac, its metabolites (4-OH and 5-OH diclofenac) and other NSAIDs (ketoprofen, ibuprofen, flurbiprofen, naproxen and nimesulide) were not detected in mussels or in water collected from Puck Bay at Orłowo station. This may indicate that there are no NSAIDs at Orłowo station or their concentrations were too low (below a few ng/L) to be detected by GC–MS.
Concentration of diclofenac measured in water and tissues of mussels
The final measured concentrations of diclofenac in the tanks with nominal concentration of 40 μg/L were as follows: 19.859, 25.314 and
Conclusion and future perspectives
Pharmaceutical was absorbed in the tissues of mussels during the exposure time, but completely eliminated after depuration phase. Although no diclofenac metabolites were detected in the study, probably due to too low (but environmentally relevant) concentration, it appears that mussels have successfully detoxified. However, it should be emphasised that the detoxification process in marine invertebrates still requires further research. The exposure to environmental concentrations of diclofenac
CRediT authorship contribution statement
Klaudia Świacka: Conceptualization, Methodology, Investigation, Data curation, Formal analysis, Writing - original draft. Katarzyna Smolarz: Conceptualization, Methodology, Supervision, Writing - review & editing. Jakub Maculewicz: Investigation, Visualization, Data curation, Writing - review & editing. Magda Caban: Methodology, Validation, Supervision, Writing - review & editing.
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.
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