Making sense of nickel accumulation and sub-lethal toxic effects in saline waters: Fate and effects of nickel in the green crab, Carcinus maenas

Highlights

• Ni toxicity and accumulation in green crabs were both salinity- and gill-dependent.

• Ni accumulation was greatest in 20% SW exposures.

• Ni at an environmentally relevant concentration of 8.2 μg/L caused decreases in haemolymph [Ca].

Abstract

In freshwater, invertebrates nickel (Ni) is considered an ionoregulatory toxicant, but its mechanism of toxicity in marine settings, and how this varies with salinity, is poorly understood. This study investigated Ni accumulation and physiological mechanisms of sub-lethal Ni toxicity in the euryhaline green crab Carcinus maenas. Male crabs were exposed to 8.2 μg/L (the US EPA chronic criterion concentration for salt waters) of waterborne Ni (radiolabelled with ⁶³Ni) at three different salinities, 20%, 60% and 100% SW for 24 h. Whole body Ni accumulation in 20% SW was 3–5 fold greater than in 60% or 100% SW, and >80% of accumulated Ni was in the carapace at all salinities. Ni also accumulated in posterior gill 8, which showed a higher accumulation in 20% SW than in other salinities, a pattern also seen at higher exposure concentrations of Ni (500 and 3000 μg/L). Gill perfusion experiments revealed that Ni was taken up by both anterior and posterior gills, but in 20% SW the posterior gill 8, which performs ionoregulatory functions, accumulated more Ni than the anterior gill 5, which primarily has a respiratory function. The sub-lethal consequences of Ni exposure were investigated by placing crabs in Ni concentrations of 8.2, 500, and 3000 μg/L at 20, 60 or 100% SW for 24 h. In 20% SW, haemolymph Ca levels were significantly decreased by exposure to Ni concentrations of 8.2 μg/L or higher, whereas Na concentrations were depressed only at 3000 μg/L. Na⁺/K⁺-ATPase activity was inhibited at both 500 and 3000 μg/L in gill 8, but only in 20% SW. Haemolymph K, Mg, and osmolality were unaffected throughout, though all varied with salinity in the expected fashion. These data suggest that Ni impacts ionoregulatory function in the green crab, in a gill- and salinity-dependent manner.

Introduction

For aquatic biota, such as decapod crustaceans, the gills perform a number of vital physiological functions. The gills are exposed directly to the external milieu, represent only a thin barrier between the animal and the environment, and have high blood perfusion rates (for review, see Henry et al., 2012). These characteristics are ideal for important physiological processes such as gas and ionic exchange, but are also likely to promote metal uptake and toxicity (Wood, 2001). As the major regulatory organ involved in homeostasis, the properties of decapod crustacean gills change with fluctuations in their environment, for example, high ammonia (Martin et al., 2011), elevated pCO₂ (Fehsenfeld et al., 2011, Fehsenfeld and Weihrauch, 2013). Many marine crustaceans, such as the green crab (Carcinus maenas) are osmoregulators. Even when inhibiting the benthic regions of estuaries, they maintain their internal osmolality close to that of seawater (1050 mOsm; Henry et al., 2012). When faced with environmental dilution, a hyper-ionoregulating crab will attempt to maintain ionic balance by utilizing the gills to absorb ions lost by diffusion. In C. maenas, the anterior gills (2–5) are primarily engaged in respiration and excretion, while the posterior gills (6–9) are the primary site of NaCl uptake and osmoregulation (Freire et al., 2008, Mantel and Farmer, 1983, McNamara and Lima, 1997, Onken and Riestenpatt, 1998, Péqueux, 1995). Consequently, metal uptake takes place mainly in the posterior gills which are thus the most probable targets of metal toxicity in this species. Furthermore, with higher rates of active ion uptake by the posterior gills in response to decreased salinities, metal uptake and toxicity might be exacerbated in dilute seawater. This has important implications for animals that are exposed to both metals and an estuarine setting.

One metal contaminant that has been shown to accumulate in estuaries is nickel (Ni). Ni occurs naturally in the environment, but levels in aquatic settings are enriched anthropogenically due to emissions of fossil fuels, mining, and other industrial practices (Eisler, 1998, NAS, 1975, WHO, 1991). Ni concentrations in the estuarine and marine environments range from 10 to 100 μg/L in highly polluted areas to around 2 μg/L in unpolluted waters (Boyden, 1975). Mechanisms of Ni toxicity to freshwater invertebrates are relatively well described. For example, it has been shown that, in the freshwater Cladoceran Daphnia magna, Ni inhibited unidirectional Mg influx causing a large decrease in whole body Mg (Pane et al., 2003). This ion mimicry mode of toxicity is common for metals. Ionic species of Cd, Zn and Pb can cross the gill epithelium of crabs via non-specific Ca channels, while in freshwater and brackish water crustaceans, Cu and Ag compete with Na for uptake at the gill via the Na⁺/H⁺ exchanger (Bianchini and Wood, 2003, Brooks and Mills, 2003, Glover and Wood, 2005, Martins et al., 2011a, Martins et al., 2011b). However, less is known about Ni toxicity in the marine and estuarine environment, and specifically about the role, salinity may have in modifying toxic effects. One study to date indicates that an ionoregulatory mode of toxicity for Ni may persist in waters of higher salinity, with a disruption in both Na and Mg homeostasis being observed in the shrimp Litopenaeus vannamei (Leonard et al., 2011).

Owing to differences in water chemistry and the distinct physiology of marine organisms, the mechanisms of Ni uptake and toxicity may differ significantly from those in freshwater animals. In marine environments, the major factor governing Ni toxicity, apart from dissolved organic carbon (DOC) (Paquin et al., 2002), is likely to be the increase in cations associated with increases in salinity (Pyle and Couture, 2012). In estuarine settings, where salinity will fluctuate, the composition of ions will therefore vary significantly with factors such as tidal cycles and changes in freshwater inputs. These changes in water chemistry are likely to have a substantial influence on the bioavailability and toxicity of Ni. In both freshwater and seawater systems, it is thought that the dominant Ni species is the free ion form (Ni²⁺; Pyle and Couture, 2012). However, NiCO₃ and NiSO₄ cannot be discounted as significant chemical species of Ni at higher pH’s (>8) (Pyle and Couture, 2012). As Ni²⁺ is a divalent cation, it is likely that its uptake will be most greatly impacted by the presence of other divalent cations (e.g., Mg²⁺, Ca²⁺). These will offer possible protective effects as they may compete with Ni²⁺ for the binding sites on the gill (Paquin et al., 2002).

The first objective of the present study was to characterize the tissue-specific pattern of accumulation of the waterborne Ni in the green crab C. maenas, a euryhaline hyper-osmoregulating decapod crustacean, with a particular emphasis on Ni handling by different gills (anterior vs. posterior). An exposure level of 8.2 μg/L was chosen as this is the USEPA (1995) criterion continuous concentration for Ni in saltwater (the chronic guideline value), and is a value that is encompassed in the range of marine environment concentrations where site-specific toxicity testing is recommended (3.9–20.9 μg/L; DeForest and Schlekat, 2013). A second goal was to determine if the physiological mechanism of sub-lethal Ni toxicity is related to ion transport. Thirdly, we wished to determine the impacts of salinity on Ni uptake. In these latter two studies, a range of Ni concentrations was chosen (an environmentally relevant level (8.2 μg/L), a mid-range level (500 μg/L), and a high level (3000 μg/L)) to investigate the concentration-dependence of Ni effects.