Fig. 3. Rapid or instantaneous Cu accumulation in/on intestinal cells incubated in external Cu (Cu_o) concentrations between 0 (no added Cu control) and 800 µmol l^–1. A Cu-free EDTA control is also included (no added Cu + 1 µmol l^–1 EDTA, open symbol). Cells were exposed to solutions of various Cu_o and then immediately (<1 min to prepare all tubes) washed in a 0.1 µmol l^–1 EDTA washing solution and pelleted (1 min in a bench centrifuge). These rapid measurements represent a time 0 control for the main Cu exposures, which lasted 15 min (see Figs 4, 5, 6, 7, 8, 9, 10 for 15 min exposures), and were performed mainly as an additional check to confirm that spontaneous Cu adsorption/accumulation (Handy and Eddy, 2004) was only a small component of net Cu accumulation over a 15 min period. Fig. 3 shows time 0 rapid Cu accumulation in/on cells in the presence of normal (140 mmol l^–1, triangles) and low (11 mmol l^–1, squares) external [Na⁺] (Na⁺_o). Values are means ± S.E.M. of N=5–6 separate experiments using fresh cells from different individual fish. ^*Significantly different from the control with no added Cu within treatment by ANOVA (P <0.05). Significantly different from normal Na⁺_o at the indicated Cu_o; Student's t-test (P<0.05). Accumulation is normalised mg^–1 cell protein and h^–1 to allow comparison between plots in Figs 3 and 4.