Elsevier

Aquatic Toxicology

Volume 218, January 2020, 105363
Aquatic Toxicology

Investigating the effects of a sub-lethal metal mixture of Cu, Zn and Cd on bioaccumulation and ionoregulation in common carp, Cyprinus carpio

https://doi.org/10.1016/j.aquatox.2019.105363Get rights and content

Highlights

  • Common carp exposed to environmentally relevant concentrations of copper (Cu), zinc (Zn) and cadmium (Cd).

  • Significant Cu and Cd accumulation in the gills, whereas Zn levels remained stable.

  • General Na decrease in all tissues counteracted by induction of H+-ATPase gene expression.

  • Strong induction of metallothionein gene expression.

  • Antagonistic mutual inhibition of Cd and Zn uptake.

Abstract

The aquatic environment is continuously under threat because it is the final receptor and sink of waste streams. The development of industry, mining activities and agriculture gave rise to an increase in metal pollution in the aquatic system. Thus a wide occurrence of metal mixtures exists in the aquatic environment. The assessment of mixture stress remains a challenge considering that we can not predict the toxicity of a mixture on the basis of single compounds. Therefore the analysis of the effects of environmentally relevant waterborne mixtures is needed to improve our understanding of the impact of metal pollution in aquatic ecosystems. Our aim was to assess whether 10 % of the concentration of the 96 h LC50 (the concentration that is lethal to 50 % of the population in 96 h) of individual metal exposures can be considered as a “safe” concentration when applied in a trinomial mixture. Therefore, common carp were exposed to a sublethal mixture of Cu 0.07 ± 0.001 μM (4.3 ± 0.6 μg/L), Zn 2.71 ± 0.81 μM (176.9 ± 52.8 μg/L) and Cd 0.03 ± 0.0004 μM (3.0 ± 0.4 μg/L) at 20 °C for a period of one week. Parameters assessed included survival rate, bioaccumulation and physiological biomarkers related to ionoregulation and defensive mechanisms such as MT induction. Our results showed a sharp increase in Cu and Cd concentration in gills within the first day of exposure while Zn levels remained stable. The accumulation of these metals led to a Na drop in gills, liver and muscle as well as a decreased K content in the liver. Biomarkers related to Na uptake were also affected: on the first day gene expression for H+-ATPase was transiently increased while a concomitant decreased gene expression of the Na+/H+ exchanger occurred. A fivefold induction of metallothionein gene expression was reported during the entire duration of the experiment. Despite the adverse effects on ionoregulation all fish survived, indicating that common carp are able to cope with these low metal concentrations, at least during a one week exposure.

Introduction

The main receptor of anthropogenic discharges is the aquatic ecosystem. Activities such as mining and application of pesticides lead to an increase of metals in the water. Some metals such as copper (Cu) and zinc (Zn) are considered essential because they are important components of enzymes or metalloproteins. Other metals, such as cadmium (Cd), are considered as non-essential metals because they have no role in biological systems (Kalay and Canli, 2000).

Fish living in a polluted environment can accumulate these metals via food, or via direct uptake from water through the gills (Perera et al., 2015). Generally, as the concentration of metals increases in the environment, fish accumulate higher levels in their tissues (Al-Attar, 2005). When the intake is not balanced with excretion processes and detoxification mechanisms, metals can show their toxic effects (Handy 2003). Gills, considering that they are in direct contact with the aquatic environment, are the main entrance for dissolved substances (Heath, 1995). These substances can subsequently reach different organs such as the liver, which is the main organ for metal detoxification, through the circulatory system. When the carrying capacity of the liver is exceeded, they can be stored in other tissues such as muscle. Metal accumulation in the muscle is generally low because it is not a metabolically active tissue, but it is important in transferring the metals through the food chain (Tunçsoy and Erdem, 2014).

Metals present in the aquatic environment can be taken up via common uptake routes, interact with each other, and this interaction can affect bioaccumulation and toxicity (Komjarova and Blust, 2009). For example Cd and Zn have a comparable electron configuration and a high affinity for molecules containing -SH groups, therefore a competition between Zn2+and Cd2+ ions is expected for the uptake (Brzóska and Moniuszko-Jakoniuk, 2001). Moreover Cd uptake can also be reduced by the presence of Cu (Komjarova and Blust, 2009). This reduction in Cd uptake in presence of other metals has been reported by several authors in several species. For example Stewart (1999) reported a reduction of Cd accumulation in the presence of Cu, Zn, Pb and Ni in by a freshwater mussel (Pyganodon grandis). Moreover a reduced Cd internal concentration has been found in a freshwater green alga Chlorella sp when exposed to a mixture of Cd and Cu (Franklin et al., 2002). A cadmium decrease in presence of copper has also been observed in Daphnia magna, in zebrafish and in rainbow trout (Komjarova and Blust, 2009; Kamunde and MacPhail, 2011; Komjarova and Bury, 2014).

Copper is an essential element which is required for several metabolic functions. This metal is involved in bone and tissue formation and considering that it is an enzyme cofactor, it has a role in cellular respiration as well (Pena et al., 1999; Tunçsoy and Erdem, 2014). However, when present at high concentrations, Cu can interfere with ionoregulation and increase plasma ammonia, disturbing the acid-base balance as was seen in gibel carp (Carasius auratus gibelio) and common carp (Cyprinus carpio) (De Boeck et al., 2007). Copper disturbance on ionoregulation is due to its ability to decrease the Na+/K+-adenosine triphosphate (Na+/K+-ATPase) activity (De Boeck et al., 2001; Wilson and Taylor, 1993). The uptake of Cu can be facilitated by a putative Na+-channels located on the branchial epithelial cells. Moreover an additional sodium (Na+) uptake pathways present in the gills could be a possible target for Cu-induced inhibition of Na+ uptake. These uptake pathways are the apical Na+/H+ exchanger isoforms (mainly NHE-2 and NHE-3) (Grosell, 2011). Thus, the presence of Cu can lead to a competition for the Na+ uptake sites which results in a decreased Na+ level (Grosell and Wood, 2002; Mackenzie et al., 2004; Niyogi et al., 2015).

Zinc, just as Cu, is an essential element which plays a crucial role in cellular homeostasis, immune responses and oxidative stress (Zhao et al., 2014). The ability of Zn to alter ion-homeostasis has already been demonstrated on different fish such as Nile tilapia (Atli and Canli, 2011) and galaxiid fish (McRae et al., 2016). It has been demonstrated that Zn exposure can alter ion homeostasis as a result of changes in calcium (Ca2+) influx kinetics, inhibition of Ca2+-ATPase and competition with Ca2+ for the uptake channel (Hogstrand et al., 1995; McGeer et al., 2000b).

Also Cd constitutes a threat to fish because of its presence worldwide in the aquatic environment and unlike Cu and Zn it is a non-essential metal. Gills are the uptake site for waterborne Cd and it has been demonstrated that it can alter ion-homeostasis in common carp and trout through direct competition with Ca2+ at the uptake site (Verbost et al., 1989; Reynders et al., 2006a). In addition Suresh et al. (1995) demonstrated the ability of Cd, in common carp, to reduce Na+, Ca2+ and K+ levels.

Therefore, all three metals (Cu, Cd and Zn) have in common that they are present worldwide and can interfere with ion-homeostasis in different ways.

Although there are several studies describing the adverse effects of waterborne metals, it is difficult to find information on the effects of metal mixtures. We chose a sublethal mixture of Cu, Zn and Cd at a low concentrations (Cu: 0.08 μM; Cd: 0.03 μM and Zn: 3.16 μM) which represent approximately 10 % of the 96 h LC50 (the concentration that is lethal to 50 % of the population in 96 h) earlier determined in our lab under the same exposure conditions (96 h LC50 Cu: 0.77 μM; Cd: 0.20 μM and Zn: 30 μM) (Delahaut et al., 2019a). An initial search of the 96 h LC50 values found in the EPA ecotox database (EPA, 2019) for these metals showed a high degree of variation according to the size, age and water chemistry. The 96 h LC50 values for Cu ranged from 0.6–542 μM (Deshmukh and Marathe, 1980; Ganesh et al., 2000), for Zn from 6.9–461 μM (Alam and Maughan, 1992; Radhakrishnaiah et al., 1993) and for Cd from 0.04–862 μM (Kaur and Bajwa, 1987; Witeska et al., 1995).

The 10 % of the LC50 value sometimes is considered as a relatively safe concentration for the organism, at least in a single exposure scenario. According to the ‘classic’ review by Sprague (1971) a pollutant safe concentration can be estimated by multiplying the LC50 values with an application factor of 0.1 (10 % LC50) to obtain a concentration which presumably has no sublethal or chronic effects, and these levels have been shown to allow the occurrence of fish populations in the field. Nevertheless, application factors of 0.01 (1 % LC50) have also been suggested when looking at reproduction, including for Cu and Zn. Overall, values vary between 0.1-0.4 and 0.01-0.05. According to the US-EPA (US-Environmental Protection Agency), the national recommended acute maximal metal concentrations for the protection of all freshwater aquatic life are 1.84 μM for Zn and 0.02 μM for Cd respectively (EPA, 2018) and for Cu the reported value for freshwater corresponds to 0.20 μM (EPA, 2004). The guidelines for freshwater surface waters in Flanders, the Belgian region where this study was conducted, impose maximum values of 0.004 to 0.013 μM dissolved Cd depending on water hardness (<40 to >200 mg CaCO3/L) and average dissolved values of 0.31 μM for Zn and 0.11 μM for Cu (Belgisch Staatsblad (Belgian Official Journal, 2015). According to the Flemish Environmental Agency (VMM) the highest measured concentration in 2016 were 88.69 μM for Zn, 2.05 μM for Cu and 1.06 μM for Cd (VMM, 2016), clearly exceeding the recommended maximum levels and making our exposure levels environmentally relevant.

In the present study common carp, Cyprinus carpio, has been chosen as model species for its economic importance worldwide and its use as bioindicator species in environmental pollution studies due its resistance to heavily polluted habitats (Altun et al., 2017; Rajeshkumar et al., 2017). Its availability and ease to handle makes it also a suitable species for transplantation studies for micropollutant bioaccumulation (Bervoets et al., 2009; Schoenaers et al., 2016; Delahaut et al., 2019b). The main question of the present study was: ‘Can the 10 % of the 96 h LC50 for Cu, Zn and Cd be considered as a safe concentration when applied in a mixture?’ As mentioned above, an application factor of 0.1 would result in a safe concentration for single metal exposures, however in mixed stress scenario’s the different metals could interfere and result in additive or synergistic effects resulting in detrimental effects for the fish. We will answer this question looking at fish survival and metal bioaccumulation, in combination with the assessment of additional physiological parameters determining whether there is an effect on ion-homeostasis, such as on electrolyte loss, induction of Na+/K+-ATPase, H+-ATPase and NHE gene expression, and on defensive mechanisms such as metallothionein (MT) induction, measured as gene expression responses. We hypothesize that the metal mixture remains sub-lethal, but that bioaccumulation will occur and defensive mechanisms will have to be initiated to avoid toxic effects. Even so, we expect that ion-homeostasis will be disturbed, especially for Na and Ca, as the metals use some of the same uptake routes as these ions at the gills.

Section snippets

Experimental model

Experimental animals, were obtained from the Agricultural University of Wageningen and kept in 1000 l aquaria at 20 °C with a photoperiod of 12 h light and 12 h dark for several months. Three weeks prior starting the experiment, 200 fish were divided in four 200 l polyethylene tanks (50 fish per tank) filled with EPA medium-hard water. EPA water was reconstituted using four different salts (VWR Chemicals): NaHCO3 (1.1427 mM), CaSO4.2H2O (0.35 mM), MgSO4.7H2O (0.5 mM), KCl (0.05 mM) using

Results

No mortality and no adverse behaviour were observed during the experiment. The speciation of metal ions calculated in Visual Minteq resulted in free metal ion concentration, expressed in μM, of Cu2+ 0.004, Cd2+ 0.02 and Zn2+ 2.07. The species distribution in our media showed approximately 7 % of Cu2+, 76 % Zn2+ and 85 % Cd2+. More information can be found in supplementary information table (SI) 1 and 2. Non-significant results of metal accumulation and electrolyte levels not shown in the graphs

Discussion

As previously mentioned we hypothesized that we expected bioaccumulation and induction of protective mechanisms such as MT. A parallel study showed that antioxidant mechanisms were activated under these exposure conditions in common carp, thus avoiding oxidative stress (Pillet et al., 2019). Therefore, it seems that defensive mechanisms in common carp were able to respond adequately to protect the fish from damage. Nevertheless, even with these protective mechanisms we were expecting negative

Conclusions

The used concentrations represent a sublethal concentration when considered as single compound and as a mixture, but do not qualify as a No Observed Effect Concentration (NOEC) level as a mixture. Our results showed the ability of the metal mixture to interfere with ionoregulation despite a pronounced induction in MT gene expression. Further Na losses appeared to be prevented by the ability of common carp to cope with this situation through an increased expression of the H+-ATPase gene. In a

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 and BS. MP was partially sponsored by a FWO-Flanders research project (Project ID: G053317N).

CRediT authorship contribution statement

G. Castaldo: Conceptualization, Methodology, Writing - original draft, Writing - review & editing, Formal analysis. M. Pillet: Investigation, Methodology. B. Slootmaekers: Investigation, Methodology. L. Bervoets: Supervision, Funding acquisition. R.M. Town: Conceptualization, Validation. R. Blust: Funding acquisition, Project administration. G. De Boeck: Conceptualization, Supervision, Funding acquisition, Project administration.

Declaration of Competing Interest

None.

Acknowledgments

We are grateful to Steven Joosen for his help with metal and electrolytes analysis, and to the reviewers for their constructive remarks.

References (86)

  • G. De Boeck et al.

    Differential metallothionein induction patterns in three freshwater fish during sublethal copper exposure

    Aquat. Toxicol.

    (2003)
  • G. De Boeck et al.

    Tissue-specific Cu bioaccumulation patterns and differences in sensitivity to waterborne Cu in three freshwater fish: rainbow trout (Oncorhynchus mykiss), common carp (Cyprinus carpio), and gibel carp (Carassius auratus gibelio)

    Aquat. Toxicol.

    (2004)
  • H. De Smet et al.

    Dynamics of (Cd,Zn)-metallothioneins in gills, liver and kidney of common carp Cyprinus carpio during cadmium exposure

    Aquat. Toxicol.

    (2001)
  • V. Delahaut et al.

    A multibiomarker approach for evaluating environmental contamination: Common carp (Cyprinus carpio) transplanted along a gradient of metal pollution

    Sci. Total Environ.

    (2019)
  • D.G. Dixon et al.

    Copper bioaccumulation and hepatoprotein synthesis during acclimation to copper by juvenile rainbow trout

    Aquat. Toxicol.

    (1981)
  • H.M. Espinoza et al.

    Effect of cadmium on glutathione S-transferase and metallothionein gene expression in coho salmon liver, gill and olfactory tissues

    Aquat. Toxicol.

    (2012)
  • M. Grosell

    2 - copper

  • R.D. Handy

    Chronic effects of copper exposure versus endocrine toxicity: two sides of the same toxicological process?

    Comparative Biochemistry and Physiology a-Molecular & Integrative Physiology

    (2003)
  • C. Hogstrand et al.

    Mechanisms for zinc acclimation in freshwater rainbow trout

    Mar. Environ. Res.

    (1995)
  • C. Kamunde et al.

    Metal–metal interactions of dietary cadmium, copper and zinc in rainbow trout, Oncorhynchus mykiss

    Ecotoxicol. Environ. Saf.

    (2011)
  • A. Lange et al.

    Alterations of tissue glutathione levels and metallothionein mRNA in rainbow trout during single and combined exposure to cadmium and zinc

    Comp. Biochem. Physiol. C-Toxicol. Pharmacol.

    (2002)
  • J. Li et al.

    Kinetics of Cu2+ inhibition of Na+/K+-ATPase

    Toxicol. Lett.

    (1996)
  • N.C. Mackenzie et al.

    Cloning, expression pattern and essentiality of the high-affinity copper transporter 1 (ctr1) gene in zebrafish

    Gene

    (2004)
  • A.Y. Matsuo et al.

    Effects of copper and cadmium on ion transport and gill metal binding in the Amazonian teleost tambaqui (Colossoma macropomum) in extremely soft water

    Aquat. Toxicol.

    (2005)
  • J.C. McGeer et al.

    Effects of chronic sublethal exposure to waterborne Cu, Cd or Zn in rainbow trout 2: tissue specific metal accumulation

    Aquat. Toxicol.

    (2000)
  • J.C. McGeer et al.

    Effects of chronic sublethal exposure to waterborne Cu, Cd or Zn in rainbow trout. 1: iono-regulatory disturbance and metabolic costs

    Aquat. Toxicol.

    (2000)
  • J.C. McGeer et al.

    3 - cadmium

  • N.K. McRae et al.

    Mechanisms of zinc toxicity in the galaxiid fish, Galaxias maculatus

    Comp. Biochem. Physiol.

    (2016)
  • S. Niyogi et al.

    Interactive effects of waterborne metals in binary mixtures on short-term gill–metal binding and ion uptake in rainbow trout (Oncorhynchus mykiss)

    Aquat. Toxicol.

    (2015)
  • M. Nordberg

    Metallothioneins: historical review and state of knowledge

    Talanta

    (1998)
  • M.M. Pena et al.

    A delicate balance: homeostatic control of copper uptake and distribution

    J. Nutr.

    (1999)
  • M. Pillet et al.

    Limited oxidative stress in common carp (Cyprinus carpio, L., 1758) exposed to a sublethal tertiary (Cu, Cd and Zn) metal mixture

    Comp. Biochem. Physiol.

    (2019)
  • S. Rajeshkumar et al.

    Effects of exposure to multiple heavy metals on biochemical and histopathological alterations in common carp, Cyprinus carpio L

    Fish & Shellfish Immunol.

    (2017)
  • H. Reynders et al.

    Patterns of gene expression in carp liver after exposure to a mixture of waterborne and dietary cadmium using a custom-made microarray

    Aquat. Toxicol.

    (2006)
  • G. Roesijadi

    Metallothioneins in metal regulation and toxicity in aquatic animals

    Aquat. Toxicol.

    (1992)
  • V. Sathya et al.

    Acute and sublethal effects in an Indian major carp Cirrhinus mrigala exposed to silver nitrate: Gill Na+/K+-ATPase, plasma electrolytes and biochemical alterations

    Fish Shellfish Immunol.

    (2012)
  • S. Schoenaers et al.

    Prioritization of contaminated watercourses using an integrated biomarker approach in caged carp

    Water Res.

    (2016)
  • A.K. Sinha et al.

    Expression pattern of potential biomarker genes related to growth, ion regulation and stress in response to ammonia exposure, food deprivation and exercise in common carp (Cyprinus carpio)

    Aquat. Toxicol.

    (2012)
  • A.K. Sinha et al.

    Differential modulation of ammonia excretion, Rhesus glycoproteins and ion-regulation in common carp (Cyprinus carpio) following individual and combined exposure to waterborne copper and ammonia

    Aquat. Toxicol.

    (2016)
  • J. Sprague

    Measurement of pollutant toxicity to fish—III: sublethal effects and “safe” concentrations

    Water Res.

    (1971)
  • A. Suresh et al.

    Cadmium induced changes in ion levels and ATPase activities in the muscle of the fry and fingerlings of the freshwater fish, Cyprinus carpio

    Chemosphere

    (1995)
  • M. Vašák

    Metal Removal and Substitution in Vertebrate and Invertebrate Metallothioneins

    (1991)
  • M. Witeska et al.

    The influence of cadmium on common carp embryos and larvae

    Aquaculture

    (1995)
  • Cited by (20)

    • Study of heavy metal bioaccumulation in Mytilus galloprovincialis (Lamark 1819) from heavy metal mixture using the CCF design

      2022, Environmental Technology and Innovation
      Citation Excerpt :

      Metals can become toxic when their uptake is not balanced by excretion processes and detoxifying systems (Pelletier et al., 2004; Castaldo et al., 2020). However, most of the laboratory studies about the accumulation of heavy metals were devoted to the impacts induced by each heavy metal acting alone, and less has been done to assess the toxicological interactions (antagonistic, synergistic and no interaction) caused by the mixture of heavy metals (Boudjema et al., 2014; Castaldo et al., 2020; Freitas et al., 2016; García-Navarro et al., 2017; Mersch et al., 1993; Rocha et al., 2015; Vlahogianni and Valavanidis, 2007). As well, metals are primarily accumulated in two organs in mollusks: the hepatopancreas and kidney; however some elements can be found in other tissues including gills and mantles (Azizi et al., 2018).

    • Physiological performance of common carp (Cyprinus carpio, L., 1758) exposed to a sublethal copper/zinc/cadmium mixture

      2021, Comparative Biochemistry and Physiology Part - C: Toxicology and Pharmacology
      Citation Excerpt :

      However, at both temperatures, complete acclimatisation of the carp is expected by the end of the experiment. To facilitate direct comparisons with previous observations (Castaldo et al., 2020; Pillet et al., 2019), the concentrations of metal used in the present study targeted the concentrations previously used (10% of the LC50 for single metal, as defined by Delahaut et al. (2020)), and also reflected ecologically-relevant concentrations. Common carp were obtained from the fish hatchery at the Agricultural University of Wageningen (The Netherlands) and kept at 20 °C in 200 L aquaria supplied with medium hard water (pH 8.2 ± 0.2).

    View all citing articles on Scopus
    View full text