Integrated multi-biomarker responses of juvenile seabass to diclofenac, warming and acidification co-exposure

Highlights

• Integrated multi-biomarker responses were assessed in D. labrax co-exposed to DCF, warming and acidification.

• DCF decreased HSI, BB_ratio, erythrocyte viability and HSP70/HSC70 content in fish brain.

• DCF induced ENAs, oxidative stress, Ub synthesis in muscle, brain AChE activity and liver VTG synthesis.

• DCF deleterious effects were either enhanced or reversed/inhibited by the co-exposure to acidification and/or warming.

• IBR showed that DCF and warming co-exposure resulted in an overall higher degree of stress.

• Results highlighted the need to consider interactions between different stressors in future ecotoxicological studies.

Abstract

Pharmaceutical drugs, such as diclofenac (DCF), are frequently detected in the marine environment, and recent evidence has pointed out their toxicity to non-target marine biota. Concomitantly, altered environmental conditions associated with climate change (e.g. warming and acidification) can also affect the physiology of marine organisms. Yet, the underlying interactions between these environmental stressors (pharmaceutical exposure and climate change-related stressors) still require a deeper understanding. Comprehending the influence of abiotic variables on chemical contaminants’ toxicological attributes provides a broader view of the ecological consequences of climate change. Hence, the aim of this study was to assess the ecotoxicological responses of juvenile seabass Dicenthrachus labrax under the co-exposure to DCF (from dietary sources, 500 ± 36 ng kg⁻¹ dw), warming (ΔTºC = +5 °C) and acidification (ΔpCO₂ ∼1000 μatm, equivalent to ΔpH = -0.4 units), using an “Integrated Biomarker Response” (IBR) approach. Fish were exposed to these three stressors, acting alone or combined, for 28 days in a full cross-factorial design, and blood, brain, liver and muscle tissues were subsequently collected in order to evaluate: i) animal/organ fitness; ii) hematological parameters and iii) molecular biomarkers. Results not only confirmed the toxicological attributes of dietary exposure to DCF in marine fish species at the tissue (e.g. lower HSI), cellular (e.g. increased ENAs and lower erythrocytes viability) and molecular levels (e.g. increased oxidative stress, protein degradation, AChE activity and VTG synthesis), but also showed that such attributes are altered by warming and acidification. Hence, while acidification and/or warming enhanced some effects of DCF exposure (e.g. by further lowering erythrocyte viability, and increasing brain GST activity and Ub synthesis in muscle), the co-exposure to these abiotic stressors also resulted in a reversion/inhibition of some molecular responses (e.g. lower CAT and SOD inhibition and VTG synthesis). IBRs evidenced that an overall higher degree of stress (i.e. high IBR index) was associated with DCF and warming co-exposure, while the effects of acidification were less evident. The distinct responses observed when DCF acted alone or the animals were co-exposed to the drug together with warming and acidification not only highlighted the relevance of considering the interactions between multiple environmental stressors in ecotoxicological studies, but also suggested that the toxicity of pharmaceuticals can be aggravated by climate change-related stressors (particularly warming), thus, posing additional biological challenges to marine fish populations.

Introduction

Over the last decades, the consumption of pharmaceutically active compounds (PhACs) has increased drastically, reaching an average worldwide consumption of over 100,000 tons per year, and an average per capita consumption of 150 g per year in developed countries (Lonappan et al., 2016). As the removal of PhACs by waste water treatment plants (WWTPs) is still limited, many compounds and their metabolites are continuously introduced in the aquatic environment (concentrations ranging from ng L⁻¹ up to low mg L⁻¹), potentially representing a risk to non-target marine species (e.g. Gros et al., 2012; Gaw et al., 2014; Mezzelani et al., 2016).

Widely known as one of the most popular “pain-killers”, diclofenac (DCF; usually available in the forms of sodium or potassium salts) is a non-steroidal anti-inflammatory drug (NSAID) commonly prescribed to reduce inflammation and/or to relieve pain induced by different chronic diseases (e.g. arthritis) or injuries. Despite its intensive usage, in both humans (being in the top 50 list of most prescribed and sold pharmaceuticals; European Medicines Agency, 2013; ARSLVT, 2015) and stockbreeding (i.e. bovine and pork farming; European Medicines Agency, 2014), along with its inefficient removal at WWTPs (its parental form is removed by between 30 and 70%, leaving levels in wastewater samples in the order of μg L⁻¹; Lonappan et al., 2016), DCF’s presence in aquatic systems remains unregulated in the European Union. Still, the European Commission has recently placed DCF under the “Watch List” of emerging non-regulated aquatic pollutants, for which further monitoring and toxicological data are needed, to accurately estimate their ecological risks and decide, whether their presence in the environment (and seafood) should in the future be regulated or not (proposed maximum allowable DCF concentration of 0.1 μg/L and 0.01 μg/L in freshwater and seawater, respectively; Decision EU, 2015/495).

Although several studies have recently pointed out DCF’s toxicity to marine species (Gonzalez-Rey and Bebianno, 2014; Mezzelani et al., 2016; Gröner et al., 2017), the ecotoxicological implications of this compound are still far from being completely clarified, largely for two reasons. First, most studies carried out so far were focused on DCF exposure via water (e.g. Munari et al., 2016; Boisseaux et al., 2017; Gröner et al., 2017), disregarding other contaminant exposure pathways, such as dietary exposure (i.e. trophic transfer along the food chain), which can be particularly important in predatory fish species (Zenker et al., 2014). To the best of our knowledge, so far, only two studies have assessed the ecotoxicological implications of dietary DCF exposure (Guiloski et al., 2015; Ribas et al., 2016). Second, chemical pollution is not the sole environmental stressor that marine species are subject to, and information on the potential effects of other environmental stressors (e.g. seawater warming and acidification) that can particularly affect the bioavailability and toxicity of emerging contaminants is limited (Marques et al., 2010; Amiard-Triquet et al., 2015). So far, only two studies have accounted for the interactive effects of abiotic stressors (both following DCF exposure via water), one using the marine bivalve Ruditapes philippinarum under acidification (Munari et al., 2016), and the other using a freshwater fish species Gasterosteus aculeatus (adult individuals) under hypoxia (Lubiana et al., 2018). Yet, climate change effects can already be felt in some regions of the world and are expected to worsen in the coming 50–100 years, increasing seawater temperature as much as 5 °C (i.e. ocean warming), as well as increasing CO₂ partial pressure (pCO₂) up to 1000 μatm, which leads to a seawater pH drop (i.e. ocean acidification; IPCC, 2014; McNeil and Sasse, 2016). Thus, gathering data of environmental pollutants in multi-stressors context is urgently needed, as they will provide a better estimate of the potential ecotoxicological implications of pollutants in tomorrow’s ocean.

In the field of ecotoxicology, as well as in studies of climate change effects, the marine fish species Dicenthrachus labrax has been frequently used as a suitable model species (e.g. Hernández-Moreno et al., 2011; Maulvault et al., 2016, Maulvault et al., 2017; Barboza et al., 2018), given its ecological characteristics and economical value: i) It is a predatory fish species, inhabiting temperate estuaries and coastal areas and likely accumulating high levels of chemical contaminants (FAO, 2018). Therefore, it is a suitable bioindicator in studies following dietary exposure to chemical contaminants. ii) Since D. labrax is a commercially valuable species, the deleterious effects of environmental stressors, particularly in its early life stages (including larvae and juveniles) can potentially affect species recruitment and overall ecological success, thus,- certainly having negative impacts in fisheries and aquaculture.

Hence, given the current lack of empirical data on effects of dietary PhAC exposure and the potential interactions of these contaminants with climate change-related stressors, this study aimed to assess, for the first time, different ecotoxicological responses (i.e. animal condition, hematological parameters, genotoxicity, oxidative stress, heat shock response, protein degradation, endocrine disruption and neurotoxicity) induced by dietary DCF exposure (500 ng kg⁻¹ dw), seawater warming (ΔTºC = 5 °C) and acidification (ΔpCO₂ ∼1000 μatm, equivalent to ΔpH = -0.4 units) in different tissues (brain, liver, muscle and blood) of the European seabass Dicenthrachus labrax. Since reaching a general conclusion regarding the severity of stressors can be a challenging process, especially when multiple endpoints and interactive effects of stressors are considered, an “Integrated Biomarker Response” approach (IBR; Guerlet et al., 2010) was used in present study, as its application constitutes a practical and robust tool to be used in field and laboratory studies (e.g. Serafim et al., 2012; Ferreira et al., 2015; Madeira et al., 2016a). Such approach allowed to combine the different biomarker responses observed at the different organization levels (animal, tissue and cell), thus, providing a novel, wider and integrative understanding of the ecological impact of DCF in tomorrow’s ocean.