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Effects of copper on olfaction of Colorado pikeminnow of a fright reaction from remaining fish was evaluated by ap- plying the criteria during the 1-min interval after introduction of the skin homogenate. Copper concentrations in Colorado pike minnow habitat Copper concentrations in the Yampa River near Maybell were described using data from a U.S. Geological Survey gage (09251000). Data were obtained from the U.S. Geological Sur- vey, Water Resources Division, Colorado District (Denver, CO, USA). All data were used for the period of record from Sep- tember 1974 to September 1991. The number of observations ranged from two to four per year with a total of 29 for total copper and 66 for dissolved copper. Detection limits for total and dissolved copper ranged from 2 to 20 and 1 to 10 fLg/L, respectively. Frequency distributions for each constituent were constructed. Scanning electron microscopy Scanning electron microscopy was used to examine the olfactory epithelium of study fish for evidence of damage from chemical exposure. Sample preparation and analysis followed Lee [20]. Three fish from the control and each exposure con- centration were fixed and preserved for SEM by immersion in 3% glutaraldehyde buffered to pH 7.2 with sodium phos- phate. Subsequently, all fish were processed as a batch to elim- inate potential bias from variation in preparation techniques. Specimens were rinsed with phosphate buffer and postfixed with 1 % osmium tetroxide (Sigma Chemical, St. Louis, MO, USA), then dehydrated in a graded acetone series and critical- point dried in a Polaron apparatus (Bio-Rad, Cambridge, MO, USA). Individual specimens were mounted on aluminum stubs with colloidal silver, sputter coated with gold (20-nm thick- ness; Hummer VII sputtercoater, Anatech, Alexandria, V A, USA), and examined using a Philips 505 SEM (Einhovn, Hol- land). Scanning electron microscopy was used only to confirm results of behavioral assays because preparation and viewing of specimens is labor and time intensive. Observations were made on fish from controls and from the lowest copper con- centrations that inhibited the fright-reaction response in all replicates. It was anticipated that olfactory receptors in ex- posed fish would be less abundant compared with control fish. To reduce potential for investigator bias, examinations of ol- factory epithelium were restricted to the same general regions of the sensory surface. Target regions were systematically sur- veyed by conducting adjacent parallel scans at x 10,000 mag- nification with a viewing field of 11.5 X 9.0 fLm for 15 min. Presence or absence of ciliated receptor cells was recorded. Statistical analysis Logistic regression was used to analyze the binomial re- sponse (present or absent) for fright reaction as a function of concentration and exposure duration. Proc Genmod (with op- tions link = logit, dist = binomial, dsca1e; [21]) was used to describe the response as a function of the independent vari- ables. The full regression model had the form logit(p) = [30 + [3jC + [32T + [33CT where p = probability of response to skin homogenate, logit(p) = naturallog[p/(l - p)], [30 = intercept, [3\, [32 = coefficients for the linear terms of main effects, C = exposure concentra- tion (fLg/L), T = exposure time (h), and [33 = coefficient of cross products. The coefficient of cross products tests for equal Environ. Toxieol. Chern. 20,2001 909 Table 1. Maximum-likelihood parameter estimates and significance probabilities for a regression model describing probability of fright- reaction response to skin homogenate by Colorado pikeminnow after exposure to copper for 24 or 96 h Standard Parameter Estimate error p 130, intercept 2.70 2.87 0.347 131> 10glO(concentration) -1.86 1.69 0.308 132> exposure time 0.144 0.0901 0.0269 133, 10glO( concentration) X exposure time -0.0792 0.0520 0.0427 a Estimates for the regression equation logit(p) = 130 + 131C + 132T + 133C T, where p = probability of response to skin homogenate, logit(p) = natural log [P/(1 - p)], C = exposure concentration (fLg/ L), and T = exposure time (h). slopes of the 24- and 96-h concentration-response relation- ships. A significant coefficient suggests that the regression lines are not parallel and further analysis is not required to demonstrate that the concentration-response relationships are different from each other. It also suggests that the effect of copper was not consistent at both durations of exposure, i.e., there was an interaction of main effects. When an interaction is detected, statistical tests of main effects cannot be simply interpreted [22]. Coefficient [31 represents the test for effects due to concentration gradient; [32 represents the test for effects due to exposure duration. Concentrations of copper estimated to inhibit olfaction in 1 and 50% of the test organisms (EC1 and EC50) were also calculated for each exposure duration using Proc Probit (with options d = logistic inversecl lackfit; [23]). In this case, the EC1 was arbitrarily selected as a conservative threshold for adverse effects on an endangered species. Transformations (lOglO) were used if they improved the fit of regression models. Graphical analyses of data and residual plots were conducted to confirm that regression models were appropriate and to evaluate compliance with statistical assumptions. RESULTS Behavioral assay Olfactory ability declined as a function of copper concen- tration after 24- or 96-h exposure, but slopes of the two con- centration-response relationships were not parallel (Table 1). There was an interaction (p = 0.0427) between the effects of copper exposure and exposure time. As a consequence of this interaction, the influence of copper on olfaction at each ex- posure time must be evaluated separately. To facilitate eval- uation, a graph of the probability of response to skin homog- enate as a function of exposure concentration was constructed using the regression model (Fig. I). At copper concentrations below 66 fLg/L (i.e., the point where the lines cross), olfaction was more sensitive to exposure at 24 h than at 96 h; at higher concentrations, the opposite relationship was observed. Behavioral assays conducted after a 14-d recovery period showed that Colorado pikeminnow exposed to 60.0 fLg/L cop- per for 96 h regained olfactory ability (Table 2). Immediately after exposure,S of 10 positive responses to skin homogenate were elicited from fish in this experimental treatment, whereas after the recovery period, positive responses were detected in eight of nine remaining replicates. The EC1s and 95% confidence limits for 24- and 96-h copper exposures were 2.61 (0.331,6.24) and 18.3 (1.58, 30.0)