Combined effects of environmentally relevant concentrations of diclofenac and cadmium on Chironomus riparius larvae

Highlights

• Combined effects of diclofenac and cadmium were assessed in C. riparius larvae.

• The co-existence of diclofenac and cadmium reciprocally enhanced their accumulation.

• The high-level mixture induced stronger oxidative damage to larvae.

• The high-level mixture caused greater decreases in gene expressions in most cases.

Abstract

The nonsteroidal anti-inflammatory drug diclofenac (DCF) is considered a contaminant of emerging concern. DCF can co-exist with heavy metals in aquatic environments, causing unexpected risks to aquatic organisms. This study aimed to assess the combined effects of DCF and cadmium (Cd) at environmentally relevant concentrations on the bioconcentration and status of oxidative stress and detoxification in Chironomus riparius larvae. The larvae were exposed to DCF (2 and 20 μg L⁻¹) and Cd (5 and 50 μg L⁻¹) alone or in mixtures for 48 h. The combined exposure to DCF and Cd was found to reciprocally facilitate the accumulation of each compound in larvae compared with single exposures. As indicated by the antioxidant enzyme activities, reduced glutathione levels, and malondialdehyde contents, the low concentration of the mixture (2 μg L⁻¹ DCF + 5 μg L⁻¹ Cd) did not alter the oxidative stress status in larvae, while the high concentration of the mixture (20 μg L⁻¹ DCF + 50 μg L⁻¹ Cd) induced stronger oxidative damage to larvae compared with single exposures. The expression levels of eight genes (CuZnSOD, MnSOD, CAT, GSTd3, GSTe1, GSTs4, CYP4G, and CYP9AT2) significantly decreased due to the high concentration of the mixture compared with single exposures in most cases. Overall, the results suggest that the mixture of DCF and Cd might exert greater ecological risks to aquatic insects compared with their individual compounds.

Introduction

Pharmaceuticals in aquatic environments have become an emerging environmental issue in the last two decades (Ebele et al., 2017; Liu et al., 2018). Pharmaceuticals are extensively used for humans and livestock, but most of them are not completely metabolized by humans or animals (Kümmerer, 2009). Unfortunately, pharmaceuticals that enter wastewater treatment plants (WWTPs) cannot be completely removed, leading to their frequent occurrence in aquatic environments (Comber et al., 2018; Liu et al., 2020; Tang et al., 2015). Despite the relatively low concentrations in aquatic environments (ng L⁻¹ to μg L⁻¹), pharmaceuticals are considered as pseudo-persistent due to their continuous inputs (Li, 2014; Li et al., 2019).

Non-steroidal anti-inflammatory drugs (NSAIDs) are a class of commonly prescribed pharmaceuticals for both humans and domestic livestock. Diclofenac (DCF) is among the most commonly used NSAIDs. The global annual usage of DCF was estimated to be 1443 ± 58 tons during 2010–2013 (Acuña et al., 2015). DCF is among the most widespread pharmaceuticals detected in aquatic environments due to its massive consumption and limited removal by WWTPs (Vieno and Sillanpää, 2014; Sathishkumar et al., 2020). The occurrence of DCF has been reported at concentrations up to 57.16 μg L⁻¹ in surface waters (Olaitan et al., 2014). Concomitant with its wide presence in aquatic environments, DCF has been detected in many wild aquatic species, including crustaceans and vertebrates (Sathishkumar et al., 2020; Xie et al., 2015; Yang et al., 2020). Furthermore, evidence shows the adverse effects of DCF on aquatic organisms, including tissue damage in fish and mussels, immunosuppressive effects, genotoxicity, immunosuppressive effects, imbalance of redox status, and alterations in biochemical responses (He et al., 2017; Sathishkumar et al., 2020). DCF has been included in the European Water Framework Directive because of its potential ecological risk to aquatic environments (EU, 2013). More recently, DCF was identified as a priority pharmaceutical with the highest predicted environmental risk among 100 pharmaceuticals in the aquatic environment of China (Li et al., 2019).

Current toxicological studies on DCF mainly focus on the single exposure of aquatic organisms to DCF with little attention to the potential combined effects of DCF and other pollutants (Sathishkumar et al., 2020). However, in natural aquatic environments, DCF does not occur isolated but in combination with other types of pollutants, including organic compounds and heavy metals. Ecotoxicological studies have documented that pollutant mixtures exhibit toxic effects different from the effects of individual pollutants (Vasquez et al., 2014). Thus, to fully understand the true environmental risk of DCF, it is necessary to evaluate the aquatic toxicity of DCF in combination with other pollutants.

Cadmium (Cd) is a highly toxic heavy metal widely distributed in aquatic environments (Zhang and Reynolds, 2019; Kumar et al., 2019; Ding et al., 2020; Ji et al., 2020). Cd is commonly found in surface water with the global average concentration of 180.88 μg L⁻¹ (Kumar et al., 2019). Although Cd level in water is low, it accumulates in aquatic organisms and is transferred along food webs, posing threats to environmental safety and human health (DeForest and Meyer, 2015). As a nonessential element in organisms, Cd can be very toxic even at low concentrations. Exposure of aquatic organisms to Cd has been associated with oxidative damage, genotoxicity, apoptosis, immunosuppression, and reproduction limitation (Banaee et al., 2019; Martín-Folgar and Martínez-Guitarte, 2019; Pavlaki et al., 2016).

DCF and Cd can be commonly found as co-contaminants in aquatic environments, particularly in those highly impacted by human activities, such as urbanization and industries (Andreu et al., 2016). For example, DCF and Cd have both been frequently detected in some large rivers and shallow lakes in China, such as Taihu Lake, Yellow River, Pearl River and Hai River (Zhao et al., 2009; Wang et al., 2010, 2012; Cui et al., 2011; Tao et al., 2012; Xie et al., 2015; Cao et al., 2018). Although the individual effects of DCF and Cd have been extensively studied, their combined effects on aquatic organisms have been rarely explored. Recently, McRae et al. (2019) found that the co-exposure to DCF (770 μg L⁻¹) and Cd (2 or 9 μg L⁻¹) was an offset against their individual effects on the antioxidant system in inanga (Galaxias maculatus) (McRae et al., 2019). However, whether environmentally relevant concentrations of DCF can affect the toxicity of Cd to aquatic organisms remains unclear. In addition, the study by McRae et al. (2019) lacked the determination of chemical concentrations in the organism's tissues, which might provide fundamental information for the interpretation of toxic effects. Moreover, their combined effects on other aquatic organisms, such as aquatic insects, urgently need to be explored to fully reveal the interaction between DCF and Cd in aquatic environments.

Chironomus riparius larvae are widely distributed insects in the freshwater ecosystem. This species is ecologically relevant in freshwater environments because it is an important food source for fish and other macroinvertebrates (Armitage et al., 2012). The larvae of C. riparius have been widely used in ecotoxicological studies to assess the bioaccumulation and toxicity of various aquatic pollutants. The enzymes and genes related to the antioxidant system and detoxification pathways in C. riparius larvae have been characterized as sensitive biomarkers to various pollutants, including pharmaceuticals and metals (Bernabò et al., 2017; Xie et al., 2019a). Oxidative stress is defined as an imbalance when reactive oxygen species (ROS) production caused by xenobiotics overwhelms the capacity of antioxidant defense system in exposed organisms. The antioxidant defense system consists of both enzymatic and non-enzymatic components. The key enzymes responsible for the detoxification of ROS include superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase enzyme (GST). SOD is a cellular antioxidant enzyme that catalyzes the dismutation reaction of superoxide anion (O₂⁻) into hydrogen peroxide (H₂O₂). CAT further catalyzes H₂O₂ conversion into O₂ and H₂O. SOD and CAT form the first-line defense against reactive oxygen species (ROS) (Qu et al., 2014). GST acts as a catalyst in conjugation reactions between glutathione with xenobiotic compounds electrophilic centers (van der Oost et al., 2003). Reduced glutathione (GSH) is an important non-enzymatic antioxidant component. GSH can protect against oxidative stress through the quenching of oxyradicals by its sulfhydryl group (Qu et al., 2014). Lipid peroxidation (LPO) is one of the main consequences of oxidative stress (Ji et al., 2018). LPO refers to the oxidative degradation of lipids in the cell membrane, as indicated by the accumulation of malondialdehyde (MDA). The detoxification of xenobiotics is a multi-phase process, the first of which usually involves several cytochrome P450 (CYP) isoforms. Two different CYP genes belonging to families 4 and 9 have been previously identified (Martínez-Paz et al., 2012; Nair et al., 2013). It has been demonstrated that these CYP genes were responsive to many aquatic pollutants, such as Cd, nonylphenol, bisphenol A and ultraviolet filters (Martínez-Paz et al., 2012; Nair et al., 2013; Martínez-Guitarte). However, the effects of DCF alone or its mixtures with metals on these biochemical and molecular parameters in C. riparius larvae remain unknown.

Therefore, the objectives of the present study were to (1) evaluate the bioconcentrations of DCF and Cd alone or in mixtures in C. riparius larvae; (2) examine the interactive effects of DCF and Cd on the oxidative status (SOD, CAT, GST, GSH, and MDA) in C. riparius larvae; and (3) assess the responses of the genes related to oxidative stress responses (CuZnSOD, MnSOD, CAT, GSTd3, GSTe1, and GSTs4) and detoxification process (CYP4G and CYP9AT2).