Environ. Sci. Technol. 2009, 43, 7225–7229
Multimetal Interactions between Cd, Cu, Ni, Pb, and Zn Uptake from Water in the Zebrafish Danio rerio IRINA KOMJAROVA* AND RONNY BLUST Department of Biology, University of Antwerp, Groenenborgerlaan 171, Antwerp, Belgium
Received February 24, 2009. Revised manuscript received May 21, 2009. Accepted May 28, 2009.
The uptake of essential (Cu, Ni, and Zn) and nonessential (Cd and Pb) metals in the gills and whole body of zebrafish exposed to a mixture of trace elements at environmentally relevant concentrations was investigated using a stable isotope technique. Negative and positive interactions as well as nonlinear responses were observed. The Cd and Pb uptake processes were influenced the most by other metals. The uptake of Cd was inhibited by Cu, Pb, and Zn and enhanced in the presence of Ni at concentrations above 0.1 µM. Pb uptake rates were consistently increasing in the presence of Cd, Ni, and Zn in both gills and the whole body, except in one case of decreased whole body Pb uptake in the presence of Cd. The addition of Cu resulted in more complex nonlinear variations in Pb uptake rates. The addition of Pb, in turn, facilitated Cu uptake with a more pronounced effect in the gills, while Zn had a stimulating effect on the whole body level. Uptake of Ni continuously decreased with the addition of Zn, and some decline in whole body Ni accumulation was observed in the presence of Cd. In contrast, Cu increased the Ni uptake rates in both gills and the whole body. The results demonstrate the complexity of the uptake processes occurring in media containing a mixture of metals at environmentally relevant concentrations. These interactions may be of key significance in understanding and predicting metal uptake, accumulation, and toxicity in multimetal exposure scenarios.
Introduction In natural environments, aquatic organisms are often affected by trace metals due to anthropogenic activities. In contaminated environments, several metals are often present together at elevated concentrations (1). Some of these trace metals such as Cu and Zn play an important role in cellular metabolism, and their body concentrations can be regulated by the organisms. Others such as Cd and Pb are toxic even at low concentrations and tend to accumulate in the body (2, 3). Present as a mixture in ambient waters, trace metals may, however, enter the organisms via common uptake routes and interact with each other affecting uptake, bioaccumulation, and toxicity. Uptake of metals in mixtures may demonstrate competitive and noncompetitive inhibition and in some cases enhanced uptake (1). The type of interaction depends on the metals involved, their external concentration, availability and exposure scenario, length of exposure, studied species, and examined organs (4, 5). The * Corresponding author phone: +32-3-265-3347; fax: +32-3-2653497; e-mail:
[email protected]. 10.1021/es900587r CCC: $40.75
Published on Web 06/12/2009
2009 American Chemical Society
existence of metal-metal interactions occurring at low, environmentally relevant concentrations has been reported for different crustacean (6, 7) and fish species (8, 9). Thus, it is necessary to consider possible multimetal interactions, which are currently disregarded by water quality guidelines, when setting site specific water quality criteria (5). At present, the Biotic Ligand Model (BLM) is the most widespread model used for prediction of metal toxicity in aquatic systems (1). However, it was developed for single metals. Therefore, the problem of modeling metal toxicity in multimetal environments has not been solved by the BLM, and further work is required (10). The first step in the development of metal toxicity involves the initial binding and internalization of the metals, followed by the internal partitioning of the accumulated metals between metabolically active and detoxified forms (11). Thus, changes in the uptake process will be the first indicator of metal interactions occurring in the organism. The purpose of this work is to identify the presence of possible multimetal interactions in zebrafish Danio rerio, which would affect the uptake of individual metals. The experimental organisms were simultaneously exposed to a mixture of Cd, Cu, Ni, Pb, and Zn, and the accumulation of these metals was followed using a stable isotope technique. Such an approach allowed revealing any interacting effects between metals as the changes in the tissue concentrations of all five metals were determined in the same individual organism and, thus, could be directly compared. The choice of exposure concentrations was based on available information concerning environmental metal concentrations in more or less polluted waters. For example, concentrations of these metals in natural water systems in Flanders, Belgium, vary between 1-110 nM Cd, 15-470 nM Cu, 45-340 nM Ni, 0.5-5 nM Pb, and 0.7-34 µM Zn (12, 13).
Materials and Methods Test Organisms. Adult Danio rerio were obtained from Aquaria Antwerp and maintained in 20 L glass tanks containing 25-30 fish per aquarium. Fish were held at 25 ( 1 °C under a 12 h light/12 h dark regime and fed with the commercial granulated tropical fish food TetraPrima granular on a 1% of fish weight daily ration. The aquariums were equipped with trickling filters, which also served as aerators. The water was checked daily for NH4+, NO2-, and NO3-. If the concentration of any of these ions exceeded 5, 2, or 200 µM, respectively, the medium was partially renewed. The medium of the aquariums was prepared by dissolving 0.5 mM CaCl2 · 2H2O, 2 mM NaCl, 0.5 mM MgSO4 · 7H2O, 0.077 mM KCl, and 1 mM MOPS (to keep the pH at 7-7.2) in deionized water with R > 18.2 Mohms · cm and aerating for 48 h. Fish were acclimatized to the test waters for at least 20 days. Feeding was stopped 24 h prior to the start of experiments, and no food was provided during the exposure period. Experimental Procedure. All chemicals used in the experiments were reagent grade. Stable isotopes were obtained from STB Isotope GmbH, Germany. Ultra pure water (Milli-Q, R > 18.2 MΩ·cm) was used for the preparation of the medium. The experiments were conducted in 5 L of polypropylene aquaria filled with freshly prepared test media. The exposure medium was spiked with increasing amounts of 106Cd, 65Cu, 62Ni, 204Pb, and 67Zn (Table S1 of the Supporting Information) 24 h prior to conducting the experiments to allow trace metals to reach equilibrium with the test water. Acclimated organisms were randomly distributed among experimental containers with five fish per aquarium and four VOL. 43, NO. 19, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Effect of increasing Cd concentrations on metal uptake rates in gills (A) and the whole body (B) of zebrafish exposed to varying Cd concentrations and 0.025 µM Cu, 0.1 µM Ni, 0.025 µM Pb, and 0.1 µM Zn (mean ( SD, n ) 4, pH 7, 2 mM Na+, 0.078 mM K+, 0.5 mM Ca2+, 0.5 mM Mg2+, and t ) 25 °C). For each metal, the treatments resulting in statistically different uptake rates are denoted by different lower case letters.
FIGURE 2. Effect of increasing Cu concentrations on metal uptake rates in gills (A) and the whole body (B) of zebrafish exposed to varying Cu concentrations and 0.0125 µM Cd, 0.1 µM Ni, 0.025 µM Pb, 0.1 µM Zn (mean ( SD, n ) 4, pH 7, 2 mM Na+, 0.078 mM K+, 0.5 mM Ca2+, 0.5 mM Mg2+, and t ) 25 °C). For each metal, the treatments resulting in statistically different uptake rates are denoted by different lower case letters.
aquaria per condition. At 3, 21, 28, and 46 h, one fish was sampled randomly from each aquarium, allowed to swim in a fresh test water without metal additions for 10 to 15 min to wash out any metals weakly bounded to the surface, blotted dry, and killed by spinal dissection. Then, gills were carefully dissected from the fish, and the other parts were pooled. Both gills and the rest of the body were dried to a constant weight at 60 °C, weighed, and digested with a 69% nitric acid/35% hydrogen peroxide mixture (Ultrapur, Merck) in a microwave. In parallel, water samples were collected for the total dissolved metal concentrations measurements. The samples were filtered through a 0.45 µm cellulose nitrate membrane (NC45, Schleicher & Schu ¨ ll), acidified with 69% HNO3 to achieve final acid concentration of 1%, and kept in a freezer until analysis. All digested samples were analyzed for metals in batches with procedural blanks and digested certified reference material CRM 278 (mussel tissue) by inductively coupled plasma mass spectrometry (Varian Expert 700 quadrupole ICP-MS, Mulgrave, Australia) as described in the Supporting Information.
Results and Discussion During the course of the experiments the concentrations of metals (added isotopes and naturally occurring metals) were followed separately in the gills and the body of each fish, which allowed both gills and total body metal uptake rates to be quantified (Figures 1-5). The uptakes of all added metal isotopes with the exception of Zn could be described by simple linear functions at all examined conditions as demonstrated for Cd (Figure S1 of the Supporting Information). Thus, the uptake rates were equal to the slope of the linear regression analysis describing the increase in metal concentrations due to the uptake of the stable tracers (further referred to as metals) in tissues as a function of time. In the case of Zn, accumulation patterns in both gills and the body were more complicated (Figure S2 of the Supporting Information). The Zn uptake by gills was marked by a fast, 7226
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FIGURE 3. Effect of increasing Ni concentrations on metal uptake rates in gills (A) and the whole body (B) of zebrafish exposed to varying Ni concentrations and 0.0125 µM Cd, 0.025 µM Cu, 0.025 µM Pb, 0.1 mM Zn (mean ( SD, n ) 4, pH 7, 2 mM Na+, 0.078 mM K+, 0.5 mM Ca2+, 0.5 mM Mg2+, and t ) 25 °C). For each metal, the treatments resulting in statistically different uptake rates are denoted by different lower case letters. concentration dependent increase in metal concentrations within 21 to 28 h, followed by the elimination of the Zn from the tissue. The presence of a regulation mechanism was evident on a total body level. Thus, accurate quantification of Zn uptake rates could not be made because Zn uptake appeared to shut down after an initial uptake phase. However,
FIGURE 4. Effect of increasing Pb concentrations on metal uptake rates in gills (A) and the whole body (B) of zebrafish exposed to varying Pb concentrations and 0.0125 µM Cd, 0.025 µM Cu, 0.1 µM Ni, 0.1 µM Zn. (mean ( SD, n ) 4, pH ) 7, 2 mM Na+, 0.078 mM K+, 0.5 mM Ca2+ and 0.5 mM Mg2+, t)25 °C). For each metal, the treatments resulting in statistically different uptake rates are denoted by lower case letters.
FIGURE 5. Effect of increasing Zn concentrations on metal uptake rates in gills (A) and the whole body (B) of zebrafish exposed to varying Zn concentrations and 0.0125 µM Cd, 0.025 µM Cu, 0.1 µM Ni, 0.025 µM Pb (mean ( SD, n ) 4, pH 7, 2 mM Na+, 0.078 mM K+, 0.5 mM Ca2+, 0.5 mM Mg2+, and t ) 25 °C). For each metal, the treatments resulting in statistically different uptake rates are denoted by lower case letters. it was possible to determine the effect of Zn on the uptake rates of other metals. The limited accumulations of Zn in fish at higher exposure levels compared to those used in the current study were reported earlier. For example, a study performed by McGeer et al. (14) showed only a slight Zn buildup in the gills and a negligible internal accumulation
during a 10 d exposure of rainbow trout to 4 µM waterborne Zn. A decrease in Zn concentration in the gills of Tilapia sparrmanii during the first 12 h of exposure to 15.5 µM Zn was reported in another study (15). Although the amounts of Zn accumulated by Tilapia gradually increased within the next 48 h, the concentrations of Zn in gill tissue returned to a control level after 96 h. Uptake of Cd. As a general trend, the Cd uptake by gills and the whole body was significantly suppressed by all studied metals with two observed exceptions: an increased whole body Cd uptake at the highest Ni concentration and a nonlinear dependence of whole body Cd uptake as a function of Pb additions. The inhibition of Cd uptake by Pb and Zn is in agreement with well-documented competitive interactions between these metals for entry into the cell via Ca2+ channels (9, 10, 16). Reductions in Cd uptake by Zn were reported in several species including the deposit-feeding polychaete Capitella capitata, rainbow trout (18), and zebrafish (19). The observation of nonlinear Cd-Pb interactions on the body level indicates that the rates of metal detoxification and/or elimination processes are also affected. Less than additive metal-gill binding was reported in rainbow trout exposed to a mixture of Pb and Cd at environmentally relevant concentrations in soft, moderately acidic waters. At the same time, ionic disturbances caused by Cd plus Pb were reported to be greater than additive, which ultimately may lead to increased toxicity of Cd-Pb mixtures to fishes (9). The inhibitory effects of Cu and Ni on Cd accumulation were unexpected as there is no evidence that these elements share a common uptake route. Although a protective effect of Ca2+ ions against Ni toxicity has been reported in rainbow trout (20), Ni is considered to be a respiratory rather than an ion regulatory toxicant in fish (21, 22). Therefore, the occurrence of competitive Cd-Ni interactions for the binding sites at the gill surface is very unlikely. Presence of Cu-Cd interactions has been reported for different fish species (23, 24). The study by Pelgrom et al. (23) showed that Cu-Cd interactions in Tilapia were more pronounced when the organisms were exposed to metals at low concentrations (0.3 µM Cu and 0.04 µM Cd), although no explanation for the observed effects was given. It is possible that apart from Ca channels another Cd transporting system exists in a form of a transporting protein, which has lower affinity toward Cd compared to other metals. For example, Divalent Metal (Ion) Transporter 1 (DMT1), which mainly transports Fe2+, has recently been demonstrated to have affinity to Cd, Co, Cu, Mn, Ni, Zn, and Pb (25). Uptake of Pb. In contrast to Cd, the Pb uptake rates were consistently increasing in the presence of Cd, Ni, and Zn in both gills and the whole body, except in one case of decreased whole body Pb uptake in the presence of Cd. Additions of Cu resulted in more complex nonlinear variations in Pb uptake rates. The obtained results are in contradiction with an expected decrease in Pb uptake rates in the presence of Cd or Zn due to competitive Pb-Cd(Zn) interactions at the gill surface for entry via Ca2+ channels. Apparently, these interactions become important only at high Pb (∼5 µM) and low Cd (>0.01 µM) exposure concentrations as demonstrated for Cd-Pb mixtures (26). At lower exposure levels (0.01 µM Pb plus to 0.07 µM Cd), the decrease in Pb accumulation in gills of rainbow trout acclimated to soft water was insignificant (9). Wilkie et al. (27) suggested binding of Pb to nonspecific cytosolic proteins within a cell as an additional Pb uptake route. The correlation analysis of the data obtained in the present study revealed that Pb and Cd do share a common uptake system on the whole body level (Pb ) f(Cd), slope ) 2.71 ( 0.95, Pearson r ) 0.785, p ) 0.04, and N ) 7). The induction of such proteins triggered by Cd, Ni, and Zn additions may be responsible for the increased Pb uptake. However, suppressions of Pb intracellular accumulation VOL. 43, NO. 19, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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and/or increased elimination rates were indicated by the observation of a Cd-induced decrease in the whole body Pb uptake rates. A stimulating effect of Cu on Pb uptake and vice versa was reported in neon tetras during simultaneous Cu-Pb exposure (8). Studies showed that Pb causes competitive and noncompetitive inhibition of Na+ and Cl- influx in rainbow trout (28, 29). Thus, a direct competition at Na+ channels for binding sites may have contributed to a complex Pb uptake pattern in the presence of Cu. Uptakes of Cu and Ni. Compared to Cd and Pb, the uptakes of these two elements were affected considerably less by other metals. The Pb-facilitated Cu uptake, more pronounced in gills, implies the existence of a common Cu and Pb transporting system, which plays a major role in the accumulation along with a diminished contribution of competitive interactions at Na+ channels to the uptake process. Similar increases in Cu accumulation were reported in crustacean (7) and fish species (8). Moreover, the stimulating effect of Zn on the whole body Cu uptake rates observed in the present study also points to the existence of a complex interactive system. For example, Dethloff et al. (30) found that Cu concentrations in gills of rainbow trout increased in Cu-Zn mixtures compared to that of the Cu treatment. The authors proposed that the presence of Zn could have caused induction of low molecular weight protein metallothionein (MT), which had higher affinity for Cu compared to Zn and, thus, could have caused increased Cu accumulation in gills. Taking into account lower Cu elimination rates compared to Zn (30), increased production of MTs in the whole body might be responsible for the elevated Cu uptake rates observed in the present study. The uptake of Ni continuously decreased with additions of Zn, and some decline in the whole body Ni accumulation was observed with additions of Cd. Copper, in contrast, increased the Ni uptake rates in both gills and the whole body. It should be noted that Ni does not interfere with the uptake pathways of major ions such as Na+ or Ca2+ (21, 22). Therefore, observed interactions cannot be explained by competitive interactions for the binding sites and most likely involve the induction or suppression of metal transferring proteins. At this point the mechanisms underlying Ni-Zn and Ni-Cu interactions remain unclear. This study has demonstrated the existence of several stimulatory and inhibitory interactions among metals during uptake and illustrated the complexity of multimetal exposure emphasizing the need for further attention. The observation that these interactions occur at environmentally relevant concentrations implies that they have to be taken into account when performing metal risk assessments and setting environmental quality criteria.
Acknowledgments The authors are grateful for the support in trace metal analysis provided by Dr. Valentine Mubiana, EBT laboratory, University of Antwerp.
Supporting Information Available Additional information on analytical and computational procedures is available. Table S1 presents stable isotope exposure concentrations. Figure S1 illustrates as an example determination of the Cd uptake rates in gills of zebrafish exposed to a range of 106Cd total dissolved concentrations. Figure S2 presents the 67Zn accumulation profile in the gills and whole body of zebrafish. This material is available free of charge via the Internet at http://pubs.acs.org.
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