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Short-term (3h) acquisition of iron (16 nmol 59FeCl3 l−1) from oxic, alkaline fresh water was assessed in rainbow trout Oncorhynchus mykiss in the presence or absence of a range of iron chelators, all of which had differing binding affinities for ferric iron [100 mol l−1 of desferrioxamine (DFO), Log10K1 32·5; citric acid Log10K1 11·9; nitrilotriacetic acid (NTA) Log10K1 15·9, CP20 and CP94 (Log10K1 > 30), as well as humic acid (HA), Log10K1 5·04, 5 mg l−1]. In the absence of chelators (control conditions) O. mykiss acquired iron from the water under laboratory lights (wavelength range of the lights 440–650 nm, peak intensity 548–626 nm) via the gill. In these conditions iron uptake onto the gill had a maximum transport capacity (Jmax) of 11·2 pmol Fe g−1 h−1 (gill organ mass) and a Km of 21·3 nmol Fe l−1 h−1. Furthermore, there were two components to iron accumulation into the carcass of these fish, a slow rate of aqueous iron uptake at low concentrations (6–24 nmol Fe l−1), followed by a faster rate of uptake at higher iron concentrations (48–96 nmol Fe l−1), suggesting that the rate-limiting step of iron uptake at low iron concentrations is the apical entry step. O. mykiss also acquired iron in the presence of HA, although the majority of the other chelators prevented iron uptake. Ultraviolet light (354 nm) treatment of Fe-DFO increased iron bioavailability. Results suggest that rainbow trout are able to access either the predicted very low concentrations (picomolar) of ferrous iron present in fresh water or the ferric oxide complexes present in oxic environments. The iron uptake rate measured (0·75 pmol g−1 h−1) would be sufficient to provide a substantial proportion (c. 85%) of the daily iron requirements of growing salmonid fry.