Common carp exposed to binary mixtures of Cd(II) and Zn(II): A study on metal bioaccumulation and ion-homeostasis
Introduction
Trace metals are part of a wide variety of pollutants that have increased in the environment as result of anthropogenic activity (Sevcikova et al., 2011). Moreover, they have a long persistence and can accumulate in the food chain (Eisler 1993; Begum et al., 2005). Even though zinc (Zn) is an essential element, being a key component of structural components and proteins (Watanabe et al., 1997), it can cause problems if present at too high or too low concentrations in the organism. One of the main problems associated with Zn pollution is its ability to lead to disruption of physiological and biochemical mechanisms, one of which is the interference with calcium (Ca) homeostasis (Hogstrand and Wood 1996; Bury et al., 2003; Loro et al., 2014). Cadmium (Cd), in contrast to Zn, is a non-essential metal with no-known biological role (Zheng et al., 2016; Danabas et al., 2018). The toxic effects caused by this metal are related with disruption of ionoregulation (McGeer et al., 2011), oxidative stress and immunosuppression (Zhang et al., 2017).
Both Cd2+ and Zn2+ ions can compete with each other for their uptake due to their similar chemical characteristics, such as similar size, electron configuration on their outer shell and to their different affinity for the -SH (sulfhydryl) groups, which is greater for Cd2+ (Brzóska and Moniuszko-Jakoniuk 2001). It has been shown by Verbost et al. (1988) that Cd2+ can block the Ca2+ transporting ATPase. Similarly, also Zn2+ can bind the Ca2+ pump, interfering with the transport of this ion (Hogstrand et al., 1996). Moreover, once the metal species are accumulated, they can lead to the production of reactive oxygen species (ROS), causing oxidative stress including lipid peroxidation and osmoregulatory dysfunctions (Livingstone 2001; Zheng et al., 2016). In case of serious oxidative stress, apoptotic events might occur (Pellegrini and Baldari 2009). Apoptosis is induced by intracellular signaling molecules, such as caspase 9 (CASP), which mediates apoptosis through the mitochondrial pathway (Pillet et al., 2019; Wang et al., 2019).
Nonetheless, fish have a suite of defensive mechanisms to cope with increasing ROS and oxidative stress such as the enzyme superoxide dismutase (SOD), which catalyses the conversion of the superoxide radical (•O2−) into hydrogen peroxide (H2O2). The H2O2 is further converted into water (H2O) and oxygen (O2) by catalase (CAT) and glutathione peroxidase (GPx) (Livingstone 2001; Pillet et al., 2019). Furthermore, the presence of peroxiredoxin (Prdx), a family of peroxidases that reduce H2O2, organic peroxides and peroxynitrite by using cysteine residues helps in protecting the cells and tissues from the effects of oxidant molecules (Tolomeo et al., 2016; Tolomeo et al., 2019).
Moreover, glutathione (GSH) plays a crucial role as a chelating agent for metals (Freedman et al., 1989) and in ROS scavenging (Peña-Llopis et al., 2003). The levels of GSH are ensured by the presence of glutathione reductase (GR), which catalyses the reduction of glutathione disulphide (GSSG) thereby maintaining a constant ratio of GSH/GSSG, and glutathione-S-transferase, which metabolizes lipid hydroperoxides (Dautremepuits et al., 2009; Couto et al., 2016). In addition to the antioxidant system, fish utilize metal binding proteins, called metallothioneins (MTs), for protection from metal ion toxicity. The MTs are low molecular weight, cysteine-rich proteins with high affinity for metals (Cretì et al., 2010), which play a key role both in regulation of essential metal ions and sequestration (detoxification) of non-essential metal ions (Amiard et al., 2006). In vitro experiments demonstrated that these proteins exhibit a different binding strength for different metals, following the order Hg2+ > Cu+ > Cd2+ > Pb2+ > Zn2+ > Co2+ (Vašák, 1991). Furthermore, MTs can also act as a free radical scavenger (Thornalley and Vašák 1985; Sato and Bremner 1993). This is possible due to the presence of cysteine residues, which are oxidized by the scavenging of ROS, such as H202 accumulated during oxidative stress (Kumari et al., 1998; Figueira et al., 2012). Furthermore, the induction of MT in aquatic species has been considered as a biomarker for metal pollution (Amiard et al., 2006).
The main aim of this work was to investigate the effects of Cd and Zn mixtures on bioaccumulation, ionoregulation and defensive mechanisms in the common carp, Cyprinus carpio, during a short-term exposure (seven days). The nominal concentrations were 0.02, 0.05 and 0.10 µM for Cd and 3, 7.5 and 15 µM for Zn representing respectively 10%, 25% and 50% of the 96h-LC50 (the concentration lethal for the 50% of the population) for each metal, as previously determined in our lab (Delahaut et al., 2020). We hypothesized that an antagonistic-like mutual inhibition of Cd and Zn uptake would occur. Furthermore, even though both metals can compete with Ca2+ uptake, according to previous results obtained in our lab (Delahaut et al., 2020), we did not expect severe electrolyte loss in tissues. Regarding the defensive mechanisms, we anticipated that metal bioaccumulation would trigger the MT and GR response in order to protect the organism from possible deleterious effects. Lastly, based on the slope of the dose-response curves for each metal (Delahaut et al., 2020), we hypothesized that the metal mixtures would remain sub-lethal.
Finally, in Flanders, the Belgian region where this study was conducted, the water quality guideline for dissolved Cd in rivers and lakes ranges between 0.004 to 0.013 µM (or 0.45 and 1.5 µg/L) according to the water hardness, whereas the value for Zn is set to 0.30 µM (or 20 µg/L) (VLAREM II, 2010). Nevertheless, these levels are frequently exceeded. For instance according to a field study done in Flanders over 14 different locations, values for dissolved (filtered through a 0.45 µm membrane) Cd and Zn ranged respectively from 0.001 to 0.20 µM and 1.31 to 33.15 µM (Bervoets and Blust, 2003). A more recent publication reported dissolved metal concentrations in two different rivers up to 0.05 µM and 52 µM for Cd and Zn respectively (Michiels et al., 2017), making the metal concentrations range used in this study environmentally relevant.
Section snippets
Experimental animals
The experimental fish, juvenile common carp (Cyprinus carpio), obtained from Wageningen University (Netherlands), were kept for several months at a temperature of 20 °C with a photoperiod of 12 h light and 12 h dark. Fish were kept in polyethylene (PE) tanks and water quality was ensured by the presence of a biofilter. Three weeks prior to the start of each experiment 250 fish were acclimatized in 200 L of artificial EPA medium-hard water (Weber, 1991). The artificial water was prepared by
Zn bioaccumulation
In fish gills (Fig. 1.1.A and 1.2.A), Zn content showed a similar trend for both the exposure scenarios. However, a significant Zn increase can be observed in the treatment Cdfix/Zn50 and Znfix/Cd50 starting from day three. Moreover, also the group Znfix/Cd10 showed a significant increase in Zn compared with the control at day seven (Fig. 1.1.A, 1.2.A). In both the series the metal concentration at day seven was significantly higher compared to day one (Fig. 1.1.A).
As a consequence, the trend
Discussion
We hypothesized that metal bioaccumulation would take place in fish exposed to waterborne Cd-Zn mixtures and, as a consequence, that an induction of defensive mechanisms would occur. Our results showed on the one hand a delayed Zn accumulation and on the other hand a sharp Cd increase. Nonetheless common carp were able to cope with the level of stress caused by metal ions by minimizing adverse effects; in fact, no mortality was reported during the whole experiment, despite the used
Conclusions
The main goal of the present study was to assess the effects of binary waterborne metal mixtures on bioaccumulation, defensive mechanisms, ion-homeostasis and survival rate in common carp. Our main hypothesis was that metal accumulation would occur to a different extent for Zn and Cd. In addition, an antagonistic-like effect on the accumulation between the two metals was expected. As predicted, Zn accumulated quite slowly in the gills, whereas Cd accumulation was fast and occurred since day one
Funding
This project was funded by a TOP BOF project granted by the University of Antwerp Research Council (Project ID: 32252) to LB, RB and GDB which included a PhD grant to GC.
CRediT authorship contribution statement
G. Castaldo: Conceptualization, Investigation, Formal analysis, Writing – original draft, Writing – review & editing. T. Nguyễn: Investigation, Formal analysis, Writing – original draft, Writing – review & editing. R.M. Town: Conceptualization, Validation. L. Bervoets: Supervision, Funding acquisition. R. Blust: Supervision, Funding acquisition. G. De Boeck: Conceptualization, Supervision, Funding acquisition, Writing – review & editing, Formal analysis.
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.
Acknowledgments
We thank Steven Joosen and Karin Van den Bergh and for their technical assistance and the reviewers for their constructive comments and careful review,
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CG and NT contributed equally to this paper.