The influence of ration size on copper homeostasis during sublethal dietary copper exposure in juvenile rainbow trout, Oncorhynchus mykiss

Abstract

The influence of ration size on homeostasis and sublethal toxicity of copper (Cu) was assessed in rainbow trout (Oncorhynchus mykiss) during dietary Cu exposure in synthetic soft water. A constant dietary dose of 0.24 μmol Cu per g fish per day as CuSO₄·5H₂O was delivered via diets containing 15.75, 7.87, and 5.24 μmol Cu g⁻¹ fed at 1.5, 3.0, and 4.5% wet body weight daily ration, respectively. Juvenile rainbow trout showed clear effects of ration but not Cu on growth suggesting that growth is hardly a sensitive endpoint for detection of sublethal dietary Cu exposure. All Cu-exposed fish accumulated the same total metal load when expressed on a per fish basis. This suggests that differences in tissue and whole-body Cu concentrations among the treatments reflected the differences in the fish size rather than total Cu accumulation, and demonstrate that absorption and accumulation of Cu from the gut during dietary exposure are independent of the food quantity in which the Cu is delivered. Fish fed a high ration exhibited greater mass-specific unidirectional uptake of waterborne Cu than fish fed a low ration indicating an increased need for Cu for growth processes in rapidly growing fish. Stimulated excretion of Cu was indicated by greater Cu accumulation in the bile of the Cu-exposed fish. Branchial Na⁺, K⁺-ATPase was not affected by dietary Cu exposure or ration but gut Na⁺, K⁺-ATPase activities showed stimulatory effects of increasing ration but not of Cu exposure. The 96-h LC50 for waterborne Cu (range 0.17–0.21 μmol l⁻¹ (10.52–13.20 μg l⁻¹) was the same in all treatment groups indicating that ration size was unimportant and that dietary Cu did not induce an increase in tolerance to waterborne Cu. Taken together these results suggest that the nutritional status, fish size, and growth rates should be considered when comparing whole-body and tissue Cu concentration data for biomonitoring and risk assessment. Moreover, expressing the exposure as total metal dose rather than metal concentration in the diet is more appropriate.

Introduction

Although dietary factors have marked effects on fish physiology and metabolism (Cowey and Sargent, 1979), the possible modifying effect of food-related variables on the toxicity and homeostatic regulation of metals has been largely neglected in aquatic toxicology (Lanno et al., 1989). Most dietary studies carried out on fish have concentrated on growth–ration relationships geared toward establishing nutritional adequacy of diets for fish in aquaculture (Brett and Groves, 1979, Cho et al., 1982, Cowey, 1992). These studies have established that growth of fish is strongly regulated by the quantity of food consumed. However, metal contaminants in the diet may influence fish health negatively by inducing toxicosis or by affecting food utilization. For example some previous studies have reported that environmental pollutants affect appetite of the fish resulting in changes in the dynamics of metal/chemical uptake, metabolism, and depuration (Jiminez et al., 1987, Lanno et al., 1989, Wilson et al., 1994, D'Cruz et al., 1998). Several studies that have specifically assessed the effect of feeding/starvation (Buckley et al., 1982, Collvin, 1985, Segner, 1987) on metal toxicity employed waterborne exposures and reported variable results. However, there are indirect indications that nutritional factors such as high ration may mitigate waterborne (Taylor et al., 2000) and dietary (Kamunde et al., 2001) Cu toxicity in fish. These data highlight the need for a detailed examination of the role of nutritional status on the responses of fish to metals exposure.

There is apparently no agreement on the level of dietary Cu toxic to fish. Although earlier work by Lanno et al. (1989) determined the toxic threshold to be approximately 730 μg Cu g⁻¹ diet, recent data have not concurred with this finding (Berntssen et al., 1999, Kamunde et al., 2001). Possible causes of these discrepancies include differences in exposure periods, feeding regimes, fish size, and species. Several studies have investigated the effects of varying dietary metal concentrations in fish at constant ration (see review by Handy, 1996) but none, to our knowledge, have investigated the influence of constant metal load presented in different ration levels.

Impairment of branchial Na⁺,K⁺-ATPase during acute waterborne Cu exposure has been unambiguously characterized in rainbow trout (Laurén and McDonald, 1987, Li et al., 1998). However, it remains to be determined whether dietary Cu imparts toxicity by affecting Na⁺,K⁺-ATPase activities in the gastrointestinal tract. Furthermore, since dietary Cu has been shown to accumulate in the gills in some studies (Miller et al., 1993, Kamunde et al., 2001, Kamunde et al., 2002), it would be interesting to assess if Cu accumulated at the gill from the diet would have a similar inhibitory effect as waterborne Cu.

The aim of the present study was therefore to test the hypothesis that fish maintained on high ration have superior ability to regulate and arrest the deleterious effects of dietary Cu exposure. This was done by offering juvenile rainbow trout the same Cu load in three rations ranging from 1.5 to 4.5% wet body weight per day. We anticipated that sublethal endpoints of chronic metal toxicity such as growth would be influenced by the nutritional status of the animal and subsequently impact metal uptake, distribution, excretion, and accumulation. In addition, exposing fish to metal under different feeding regimes is environmentally realistic because food abundance and feeding indices vary with season in aquatic ecosystems (Segner, 1987, Smith and Griffith, 1994). A second objective was to establish possible connections between tissue Cu accumulation, metal dose, and toxicity for purposes of risk assessment in aquatic toxicology. Previous studies (Miller et al., 1992, Farag et al., 1995, Marr et al., 1996) have associated tissue metal residues with adverse effects and recently Bergman and Dorward-King (1997), proposed the use of tissue metal burdens for biomonitoring, risk assessment, and derivation of water quality criteria. Third, a waterborne Cu toxicity test was performed to determine if dietary Cu induced acclimation to waterborne Cu (McDonald and Wood, 1993) and whether ration size had any role in this process. Finally, we examined the possible effect of chronic sublethal dietary Cu exposure and ration size on branchial and gastrointestinal tract Na⁺,K⁺-ATPase activities.