Nitrate-induced elevations in circulating sex steroid concentrations in female Siberian sturgeon (Acipenser baeri) in commercial aquaculture

Abstract

Although nitrate is a ubiquitous component of aquatic environments, and has become a global pollutant in a variety of aquatic systems, it has only recently begun to receive attention for its ability to alter endocrine function. Aquaculture environments with limited water exchange often contain nitrate concentrations far in excess of natural environments, yet nitrate's impact on the reproductive health and endocrine function of commercially important species residing in these environments has not been investigated. Two experiments were conducted evaluating the effects of elevated nitrate on cultured Siberian sturgeon (Acipenser baeri). The first experiment compared the effects of a 30 day exposure to 11.5 ± 0.36 and 57 ± 2.18 mg/L nitrate-N concentrations on plasma cortisol (F), glucose, 17β-estradiol (E₂), testosterone (T) and 11-ketotestosterone (11-KT) concentrations. The second experiment was similar to the first but evaluated concentrations of 1.5 ± 0.057 and 57 ± 1.52 mg/L nitrate-N. In both experiments, after 30 days of exposure to a given nitrate concentration, blood samples were obtained at time 0, and a portion of those fish were then placed under confinement stress for a period of 6 h to evaluate whether nitrate affects the associated stress response. The fish were bled at 1 h and 6 h during the confinement period. Results revealed that 57 mg/L nitrate-N exposure was associated with an increase in plasma T, 11-KT and E₂ concentrations at time 0, but did not alter the associated stress response defined by elevated plasma cortisol concentrations. An additional measure of the stress response, plasma glucose concentration, however, was altered by nitrate exposure during the 6 h period of confinement stress in Experiment 1, but was not in Experiment 2. These findings demonstrate that nitrate has the potential to disrupt endocrine function and possibly secondary stress responses in cultured Siberian sturgeon.

Introduction

Absent from most investigations assessing the endocrine disrupting effects of environmental pollutants on aquatic inhabitants, are studies examining the effects of ions, such as nitrate and nitrite, which are ubiquitous components of most aquatic ecosystems including aquaculture environments. Aquatic organisms can receive continuous exposure to environmental contaminants throughout their lives. Thus, the effects of endocrine disrupting contaminants on aquatic life have received considerable attention (Zraly et al., 1997, Guillette et al., 1999, Kime, 1999, Iguchi and Sato, 2000, Panesar and Chan, 2000, McMaster, 2001, Sumpter, 2005, Barber et al., 2007, Lerner et al., 2007). Anthropogenic activities have dramatically impacted the amount of nitrogenous compounds entering freshwater systems, and recent reports have identified agricultural non-point source pollution, often caused by nitrate laden fertilizers, as the leading cause of water quality deterioration to freshwater systems (Sampat, 2000).

Although nitrate is a ubiquitous component of aquatic environments, and has become a global pollutant in a variety of aquatic systems, it has only recently begun to receive attention for its ability to alter endocrine function (Guillette and Edwards, 2005). In contrast, the acutely toxicological effects of nitrate have long been known. As early as 1945, nitrate-induced methemoglobinemia (Blue Baby Syndrome) in humans was associated with drinking well water contaminated with nitrate (Comly, 1945). Fish are also vulnerable to methemoglobinemia (Brown Blood Disease), and in Siberian sturgeon methemoglobinemia has been associated with a significant chloride imbalance (Gisbert et al., 2004). Toxicity studies with fish have shown lethal concentrations (LC₅₀) of nitrate to vary an order of magnitude or more (Brownell, 1980, Pierce et al., 1993, Hamlin, 2006), demonstrating significant variability in response to elevated nitrate among fish species.

Sublethal effects of nitrate include endocrine alterations which have been shown to alter metabolism, reproductive function and development. Mosquitofish (Gambusia holbrooki) in north-central Florida that were exposed to nitrate concentrations of 4–5 mg/L NO₃-N experienced significant reproductive alterations, such as reduced gonopodium length and were significantly less likely to be pregnant during the reproductive season (Edwards et al., 2006b). Those that were pregnant had smaller offspring on average, compared to fish captured from springs with lower nitrate concentrations (0.2–1.7 mg/L NO₃-N). Frogs (Rana cascadae) exposed to 3.5 mg/l nitrate-N metamorphosed more slowly, and emerged from the water in a less developed state than control animals (Marco and Blaustein, 1999). Studies in Southern toad (Bufo terrestris) tadpoles showed that nitrate-induced alterations in growth and thyroxine concentrations were altered by the source of culture water used, indicating that environmental context plays a significant role in determining the effects of nitrate (Edwards et al., 2006a). Proposed mechanisms for nitrate-induced steroidogenic disturbances include mitochondrial conversion to nitric oxide (NO), altered chloride ion concentrations and altered enzymatic action by binding to the heme region of P450 enzymes associated with steroidogenesis and steroid clearance (Guillette and Edwards, 2005).

Stress effects on reproduction can be manifested at various levels of the reproductive-endocrine axis, and stress has inhibitory effects on reproduction for most aquatic species studied to date (Pickering et al., 1987, Carragher and Sumpter, 1990, Pankhurst and Van Der Kraak, 1997, Consten et al., 2002). For many species of fish, including sturgeon and other chondrosteans, cortisol (F) is the primary stress hormone (Idler and Sangalang, 1970, Barton et al., 1998) and has been implicated in mediating the inhibitory reproductive effects induced by stress (Pankhurst et al., 1995, Pankhurst and Van Der Kraak, 1997, Semenkova et al., 1999, Bayunova et al., 2002). An animal under chronic stress can demonstrate a reduced capacity to handle subsequent stress events, and studies have shown that responses of fish to multiple stressors are cumulative (Barton et al., 1986). Fish residing in laboratories or fish farms are often subjected to chronic stress (suboptimal water chemistry, crowding, confinement) followed by acute stress events (sampling, netting), which can lead to dramatic and prolonged stress responses (Rottland and Tort, 1997, Heugens et al., 2001).

Sturgeon are among the most ancient groups of Osteichthyes, and twenty-five extant species occupy the Northern Hemisphere (Birstein, 1993). The dramatic decline in sturgeon populations due to overfishing, pollution, and habitat degradation has led to the necessity of commercial aquaculture as a means to provide animals for stock enhancement, as well as food production, reducing pressures on wild populations (Beamesderfer and Farr, 1997, Waldman and Wirgin, 1997, Williot et al., 2002, Chebanov et al., 2002). The Siberian sturgeon is one of the leading species of sturgeon adapted to aquaculture (reviewed by Gisbert and Williot, 2002). It was recently discovered that Siberian sturgeon are more sensitive to nitrate toxicosis than most fish species reported to date (Hamlin, 2006). Further, Siberian sturgeon juveniles become less tolerant to nitrate as they grow, a finding of considerable importance for the commercial culture of this species, since adult populations reared in recirculation systems often experience higher nitrate concentrations than their juvenile counterparts.

The purpose of this study is to begin to determine the potential effects of elevated nitrate on endocrine function in commercial aquaculture, and investigate whether elevated nitrate alters the stress response, defined by plasma cortisol and glucose concentrations, in captive female Siberian sturgeon.