Osmoregulatory strategies and Cu toxicity in saltwater
All freshwater (FW) organisms osmoregulate to maintain the internal salt concentration above that of the external environment. They do this by an active uptake of Na+, which can be inhibited by Cu exposure 40 that may also affect the Na+ leakage pathway 41. Because this is true for both fish and invertebrates, it is one reason for the relative success of BLM in predicting acute Cu toxicity in both groups of organisms in freshwater, although the sensitivity to Cu displayed by FW organisms varies by more than three orders of magnitude among different taxa 20. However, saltwater (SW) organisms present wider intertaxa variability of osmoregulatory strategies. In particular, SW teleost fish (bony fish) are osmo- and ionoregulators, because they maintain extracellular ion concentrations well below that of the surrounding medium. For this reason, they need to intake seawater continuously and excrete the excess salts to compensate for water loss due to the osmotic gradient and the diffusion of salts into their tissues 20. Elasmobranch (cartilaginous) fish are ionoregulators and osmoconformers, because they regulate the ion concentration at approximately 50% of that in the SW, but they maintain the osmotic pressure of extracellular fluids similar to the surrounding medium by reabsorbing and retaining urea and other organic osmolytes in their tissues. This allows them to drink less seawater than teleosts, but they still face the problem of the diffusion of salts from the external SW, where the ion concentration is higher, into their body. They compensate for this by salt excretion in urine, by secretion from the rectal gland, and by salt transfer at the gill epithelium 42. Finally, most marine invertebrates are both iono- and osmoconformers, because their ionic composition and osmotic pressure are highly similar to those of the surrounding seawater 20.
Because it is known that acute Cu toxicity is mainly a consequence of its effect on ion transport 43–47, it is plausible that differences in osmoregulatory strategies may result in differences in Cu sensitivity among species and, as salinity changes, within the same species. If we assume that Cu exerts its toxic effect by disrupting the maintenance of a net Na+ gradient, we may hypothesize that osmoregulators would be more sensitive to Cu than are osmoconformers. Because the available data do not support this expectation (see Grosell et al. 20 for a review), the hypothesis that Cu acts as an osmoregulatory toxicant in seawater will be discussed.
In marine osmoconformers exposed to Cu, the principal cause of mortality appears to be either a disturbance in acid–base balance, related to impaired respiratory gas exchange, and/or an effect on ammonia excretion 48–51, although evidence also exists for Cu toxicity via oxidative stress 52, DNA damage 53, and metabolic inhibition as a secondary effect of oxygen uptake inhibition 54. In marine osmoregulators, the studies regarding the effects of Cu are equivocal. However, it is generally agreed that impaired ion regulation is the main effect of Cu exposure 44–47, 55 in both fish and invertebrates. Acute and chronic Cu exposure of the gulf toadfish (Opsanus beta) induced an increase in plasma Na+ and Cl− concentrations, due to impaired osmoregulatory capacity both in the gill and in the intestine, resulting in fluid loss from muscle tissue 44. These results are in accordance with previous studies on the seawater-adapted flounder (Platichthys flesus) and the rainbow trout (Oncorhynchus mykiss) 56, 57. However, some exceptions exist: in other studies on the killifish (Fundulus heteroclitus) and the cod (Gadus morhua), disturbances in acid–base balance and ammonia excretion were the main observed effects 22, 58. In the experiments performed on F. heteroclitus, Cu toxicity was studied across a range of salinities, from freshwater to seawater 22. The hypothesis that Cu would act via the same general mechanism, regardless of salinity, was tested, but the results did not confirm this. Indeed, in FW the expected mechanism of toxicity was observed; in particular, a decrease in plasma Na+ concentration occurred, caused by the Cu-induced inhibition of the Na+/K+ ATPase activity. However, in SW no effect was observed either on Na+ homeostasis or on Na+/K+ ATPase activity. The only parameter affected by Cu exposure was ammonia excretion, which has also been reported to be affected by Cu in other studies 57, 58.
Taken together, these observations not only demonstrate the complex pattern of physiological responses to Cu, but also suggest that the main target of Cu toxicity might be a common factor, which controls ion transport along with acid–base balance and nitrogenous waste excretion. If we assume this, we can try to give an overall explanation for the main reported effects of Cu, both in osmoconformers and osmoregulators. In osmoconformers, acid–base and ammonia excretion disturbances seem to be the main causes of Cu-induced mortality, because maintaining an osmoregulatory gradient is not an issue for this group of animals. In osmoregulators, the disturbance of ion transport is the main effect, because maintaining a Na+ gradient is critical for them; however, acid–base balance and ammonia excretion are also affected, and in some cases are more sensitive endpoints (Table 1) 22.
Table 1. Summary of the effects of copper in osmoregulators and osmoconformers
|Main effects||Osmoregulatory strategies|
|Acid-base balance disturbance caused by impaired gas exchange||•||•|
|Increase in plasma ammonia concentration caused by impaired ammonia excretion||•||•|
|Impaired intestinal and/or branchial ion transport||•|| |
|Drinking rate disturbances||•||•|
Carbonic anhydrase enzyme: A Cu target
A common denominator of the previously described mechanisms is the enzyme carbonic anhydrase (CA). In fact, this enzyme can bind Cu, which in turn can inhibit its activity, as has been shown in crustaceans exposed both in vivo and in vitro to this metal 59, 60. However, this mechanism of action in fish has been confirmed only by in vitro experiments 61, not by in vivo 22. This enzyme is present in a range of tissues, including branchial and intestinal epithelia, and is involved, directly or indirectly, in several physiological processes, including gas exchange, acid–base balance, Na+ and Cl− transport, and ammonia/ammonium excretion 62, 63, which are all reported to be affected by Cu exposure. The main function of the enzyme is to facilitate the conversion of carbon dioxide and water to bicarbonates and protons 64. In the gill of FW fish, the protons produced by the hydration of carbon dioxide are involved in Na+ transport at the apical membrane, while the bicarbonates are exchanged for chloride. The inhibition of CA, besides influencing these mechanisms, can also affect ammonia excretion by reducing the diffusive trapping mechanism 65–67. Hence, the multiple functions of CA make it a candidate as the common factor that links together ion transport, acid–base balance, and nitrogenous waste excretion, thus offering a possible explanation of the complex pattern of physiological responses to Cu exposure. This hypothesis can be supported by giving further consideration to marine osmoregulators, with some distinctions between fish and crustaceans.
Marine fish have two sites of ion transport, the gill and the intestine, which are both relevant in the maintenance of the osmotic balance. In contrast to FW fish, ion transport in SW fish is not believed to be directly associated with CA in the gill 68, but it is in the intestine, which is responsible for taking up water to compensate for the diffusive loss of water to the concentrated external environment 68, 69. Water absorption is driven by the uptake of Na+ and Cl−, which are then excreted at the gill. Ions are moved actively through the intestinal epithelium and water follows passively along the generated osmotic gradient, from the intestinal lumen into the blood. The presence of Cu (and also Ag 70–72, which has a similar toxic effect) in seawater can reduce drinking rates, depending on the exposure time, and interferes with the intestinal uptake of water 45 through the inhibition of the active ion uptake processes that drive water flux by osmosis. The driving force for the movement of ions across the intestinal epithelium is provided by the enzyme Na+/K+ ATPase; however, to date, the inhibition of its activity by metal exposure has not been demonstrated to be responsible for the impaired uptake processes in the intestine of marine fish. Therefore, it is plausible that one or several other proteins involved in ion transport are more sensitive targets. One of them is the enzyme CA, which has been shown to play a key role in osmoregulation 73. It has been demonstrated that both gene expression and enzymatic activity of CA can be modulated by salinity changes, similarly to the enzyme Na+/K+ ATPase, and that their response is tissue-specific, with significant differences between gills and intestine.
Considering the gill, the salinity dependence of CA expression and activity has been demonstrated in several studies, but this pattern still must be elucidated fully because some discrepancies exist among species. In killifish, an increase in CA gene expression was observed after transfer from intermediate salinity to freshwater 74; in the coho salmon (Oncorhynchus kisutch) and in the Mozambique tilapia (Oreochromis mossambicus), CA activity increased with increasing salinity 75, 76, while flounders kept at different salinities showed no significant differences in CA activity 77.
In the intestine, CA expression and activity increased two- to fourfold in killifish 22 after transfer to seawater. For rainbow trout, a salinity change induced a response that involved two isoforms of the enzyme CA: the cytosolic CA (CAc) and the extracellular isoform membrane-bound CA type IV (CAIV), localized at the apical region of the intestinal epithelium 78. The former usually displays the majority of the CA activity in the intestinal epithelium and provides the cellular substrate for the anion exchanger on the apical membrane 79. The latter contributes to the deposition of CaCO3 in the intestinal lumen, which reduces the osmotic concentration of the intestinal fluid and thus facilitates ion transport and water absorption through the epithelium 80. In rainbow trout, an osmotic stress, such as caused by an abrupt transfer from freshwater to 65% saltwater, induced a transient increase of mRNA expression and of enzymatic activity, both of CAc and membrane-bound CAIV 78. A recent study on CA expression and activity, both in the intestine and in the gills of the gulf toadfish following transfer to salinity of 60 ppt, demonstrated that the CA was important for tolerance to hypersalinity 73. What emerges from these studies is that the enzyme CA plays a key role in osmoregulation and the modulation of its activity and expression appears to be a response to osmotic stress. If we hypothesize that CA is the main Cu target in SW, and that in fish it displays its osmoregulatory functions mainly in the intestine, then we might assume that the Cu target in SW is not (or not only) in the gill, but (also) in the intestine, and so consider the Cu speciation and bioavailability in this medium. It has been observed that Cu becomes less bioavailable as it moves along the intestine of the gulf toadfish: this may be because the absorption/excretion mechanisms at the intestinal epithelium progressively modify the chemical composition of the intestinal fluid and thus the speciation and availability of Cu 45.
Metabolically, marine crustaceans are less complex than fish, because the only tissue in which the enzyme CA may be involved in Cu-sensitive functions is the gill. However, although marine crustaceans are invertebrates, which are generally osmoconformers, several species can shift from osmoconformity to osmoregulation below a critical salinity, typically around 25 ppt 81, 82. The enzyme CA has been characterized as one of the central components of ion uptake in the branchial epithelium of crustaceans 82, because its expression is salinity sensitive and its inhibition has been linked to the disruption of ion transport and regulation 83. The increase in CA activity in response to low salinity appears to be a central feature of the transition from osmoconformity to osmoregulation. This is believed to be a common adaptive characteristic of all euryhaline marine crustaceans capable of osmotic- and ionoregulation 62, 84. If Cu exposure affects CA activity, it may interfere with the ability of these crustaceans to respond to osmotic stress and thus result in osmoregulatory imbalance.
Conceptually, from the modeling perspective, the validation of the hypothesis presented here would mean that the biotic ligand in marine fish is not only the gill, but also the intestine (as suggested also for Ag 71–73 and the relationship between toxic effect and metal accumulation at the binding site is modulated by salinity and in particular by the osmotic gradient.
Branchial permeability and mucus secretion
Another explanation of the toxic effects of Cu is a general alteration of the epithelial function of the gill. Most processes that have been reported to be affected by Cu toxicity are associated with ion regulation at the gill; therefore, a generalized mode of action has been hypothesized, possibly caused by mucus secretion 55. Mucus is thought to act as a buffer, preventing the metal from interacting with the site of toxicity; but, if a thick layer of mucus is produced, it may increase the diffusive distance at the gill surface and thus affect branchial processes by reducing branchial permeability. However, most studies on the effects of Cu exposure reported an increase, not a decrease, in branchial permeability, due to a displacement of Ca2+ at the gill surface, which controls the passive diffusion of Na+, Cl−, and also Mg2+, all ions that demonstrated an increase in plasma concentrations after Cu exposure 44, 57.
Na+/K+ ATPase activity: Why not a target in saltwater?
An issue that may need to be elucidated is why the Na+/K+ ATPase activity is one of the targets of Cu in FW 85, whereas it is reported to be unaffected in SW 86. Because this enzyme is central to ion transport by the gills of both freshwater and marine fish, it may therefore be expected to respond similarly to Cu in the two environments. An explanation of these apparently puzzling observations can be found in a study conducted on the gill of the SW-adapted flounder 56, to which Cu was applied in both in vitro and in vivo experiments. The application of Cu to gill homogenates during the in vitro experiments caused a marked reduction in Na+/K+ ATPase activity, but the in vivo experiments showed that Cu exposure induced an increase in the number of Na+/K+ ATPase units in the gill. This increase counterbalances the reduced activity of each Na+/K+ ATPase site and thus resulted in an overall unchanged Na+/K+ ATPase activity in the gill tissue, as reported in most studies on marine fish gill physiology. Furthermore, the increase of Na+/K+ ATPase units is controlled by the production of cortisol 87, a hormone responsible for osmoregulatory balance in SW, the concentration of which is enhanced by Cu exposure. Another effect of higher levels of cortisol is the enhancement of protein catabolism 87, which increases ammonia production and thus plasma ammonia concentration, a reported effect of exposure to Cu. A further explanation of the apparent insensitivity of the Na+/K+ ATPase to Cu at high salinity is provided by the observation that rapid salinity change can induce the expression of different isoforms of the enzyme 88. It is plausible that different isoforms may display differential sensitivity to Cu 45.
Salinity changes: Relevance of the osmotic gradient
With the development of a BLM for marine and transitional waters in mind, the previous considerations imply that an estuarine/marine version of the BLM should give more relevance to the physiology, because it has been shown to be important in marine and transitional environments. At present, species differences in sensitivity are dealt with through adjustments of the stability constants characterizing the metal/gill interaction. This may be sufficient in FW, but in SW it cannot explain the variation in sensitivity to Cu shown by SW and estuarine organisms. The physiological mechanisms lying at the base of the osmotic regulation system are more various and variable in marine organisms than in freshwater ones and thus result in a large interspecies variation in Cu sensitivity and an intraspecies variation in Cu sensitivity when salinity changes 20. The second point is particularly relevant if we consider estuaries, transitional waters, or other environments characterized by fluctuating salinities.
Estuarine invertebrates display a wide range of osmoregulatory strategies. In general, at their iso-osmotic point they are osmoconformers. Above it, they are still generally osmoconformers, although a few (particularly decapod crustaceans) are able to osmoregulate 89. Below the iso-osmotic point they weakly osmoregulate up to a certain salinity level, after which they start strongly osmo- and ionoregulating 82, 90. A few estuarine invertebrates are osmo- and ionoregulators in the full tolerance range 91, 92. A good example of the transition from osmotic and ion conformity to regulation is given by the euryhaline green crab (Carcinus maenas), which is an osmoconformer in full-strength seawater, but at a critical salinity of 26 ppt, starts to actively uptake ions across the gill. The activation of the mechanisms of ion transport is correlated with an eightfold induction of the enzyme CA. The role of CA in ion transport and regulation in this species is confirmed by the observation that the ability of green crabs to regulate their hemolymph osmotic and ion concentrations is disrupted when branchial CA activity is inhibited 83. A similar mechanism was also observed in the blue crab (Callinectes sapidus) 93. If Cu exposure affects CA activity, which is a key feature of the acclimation of euryhaline organisms to salinity changes, it might be deduced that Cu exposure can theoretically affect the ability of euryhaline species to respond to osmotic stress.
Reviewing eight studies on the influence of salinity to acute Cu toxicity, Grosell et al. 20 pointed out that none of the studies showed a linear increase in tolerance with increasing salinity, as competitive interactions among cations would have suggested, and the displayed tolerance variations were not fully explained on the basis of Cu speciation. A nonlinear trend of LC50 values related to salinity gradient has also been reported in a recent study of Cu toxicity to the estuarine copepod Eurytemora affinis19. Here the lowest toxicity was observed around the iso-osmotic point of the organism, a fact also seen in the work by Grosell et al. 20. The trend of Cu toxicity related to salinity gradient displayed in this study (Fig. 2) suggests that the physiology of the organism was the driving factor influencing toxicity and thus supports the observation that euryhaline species are more tolerant to metal exposure at iso-osmotic salinities due to minimization of osmotic stress 94. By comparing measured and predicted values (calculated using the main equation of the BLM as modified by De Schampheleare et al. 95), Figure 2 also demonstrates that at present BLM predictions are inaccurate across such a wide salinity range (0–35 ppt); however, as indicated by the good agreement at high salinities, it may accurately predict toxicity in some limited situations 96. Because Cu disrupts the ability to sustain the osmotic gradient between internal fluids and the external medium, the greater the gradient, the higher the toxic effect. Including a parameter that accounts for the equilibrium potential (Ep) across the gill epithelium may thus reduce the bias between the predictions of the model and the observed toxicity, because the Ep undergoes a wide variation with salinity (Fig. 3).
Figure 2. Acute Cu toxicity to juvenile killifish (Fundulus heteroclitus) as a function of ambient salinity. The triangles represent the 96 h LC50s values obtained by acute toxicity tests (data from Grosell et al. 20). The diamonds represent the values predicted by the basic biotic ligand model (BLM) equation as modified by De Schamphelaere et al. 95. Both the values are expressed as free metal ion activity.
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Water and ions (including metals) are exchanged between the internal fluids and the external environment at different rates, in relation to the osmotic gradient and ultimately to the salinity 20, 97. This means that metal uptake and toxicity (assuming a direct relationship among metal accumulation at the biotic ligand, uptake rate, and toxic response) is modulated by the osmotic gradient. However, the mechanism is even more complex, because the mode of action of Cu (the biotic ligand in the BLM language) changes according to the osmoregulatory physiology, which varies with salinity. Thus, the relationship between Cu exposure and toxic response is influenced by salinity through a double interaction. On the one hand, Cu inhibits the ability of the organism to respond to an osmotic stress by interfering with its osmotic mechanisms (i.e., CA). Yet at the same time, a salinity variation produces an increase in Cu uptake due to a general increase in ion uptake rate, as well as, in some cases, a change in the Cu biotic ligand. At present, this complex relationship is clearly overlooked by the BLM, which assumes that the toxic response is directly related to the amount of Cu bioavailable and to its affinity for the binding sites, regardless of the water chemistry.
Conceptually, the challenge is to introduce the effect of water chemistry (salinity) on the physiology-based part of the model. Therefore, we hypothesize that at varying salinities (as in an estuary) the relationship between the [Cu]EC50 and the fraction of binding sites that need to be occupied by the metal to observe a toxic effect in 50% of the test organisms () is not linear and constant, but variable and modulated by the osmotic gradient.