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936 D.W. BEYERS AND P.I. SIKOSKI Chemical exposure Chemical concentrations were based on results of 32-d ELS toxicity tests with Colorado squawfish [7]. LOECs es- timated by ELS tests were the highest test concentrations in 24-h AChE-inhibition studies. Exposures were conducted fol- lowing prescribed methods for flow-through acute toxicity tests [8]. A continuous-flow minidiluter [9] supplied a dimin- ishing series of exposure concentrations to replicate aquaria. The diluter maintained a 0.5 dilution factor and provided a volume of 0.055 L/min to replicate aquaria. Aquaria were 10 x 20 x 15 cm high, and depth oftest solutions was 12 cm. Experimental treatments were assigned to two replicate aquaria using a randomized block design. Test animals were randomized to one of seven treatment groups: five toxicant concentrations, a solvent control, and a dilution-water control. Cool-white fluorescent lamps were the only source of illumi- nation, and a 16:8-h light:dark photoperiod was maintained. Mean wet weight and total length of Colorado squaw fish were 8.0 g and 74mm, respectively. Because of their relatively large size, only two fish were placed in each aquarium. Fish were acclimated to conditions within aquaria for 48 hand were not fed within 24 h of, or during, the 24-h exposure pe- riod. At conclusion of the exposure period, fish were sacri- ficed in ice water, weighed, measured, and prepared for brain AChE assay. Physical and chemical conditions Dilution water for all toxicity tests was supplied by a well on the Colorado State University campus (Fort Collins, CO) and was vigorously aerated for approximately 24 h while be- ing heated to a test temperature of 22 j;: lOC. Alkalinity, hardness, pH, and specific conductance were measured at the beginning of the exposure period. DO was measured at the beginning and end of the exposure period; water tempera- ture was measured continuously. Dilution water character- istics for both tests had the following ranges: DO, 8.0 to 8.2 mg/L (constant aeration); pH, 7,9 to 8.0; temperature, 21.2 to 22.0oC; alkalinity, 247 to 259 mg/L as CaC03; hardness, 361 to 379 mg/L as CaC03; and specific conduc- tance, 720 to 740 p'slcm. Toxicant solutions and analytical procedures Technical carbaryl (I-naphthyl methylcarbamate, 990/0) was obtained from Rhone-Poulenc (Research Triangle Park, NC), Technical malathion (diethyl mercaptosuccinate, S-ester with O,O-dimethyl phosphorodithioate, 93%) was obtained from American Cyanamid Company (Princeton, NJ). Stock solutions were prepared by dissolving each toxicant in pesticide-grade acetone or acetone-dilution-water mixtures. Stock solutions were delivered to the diluter via peristaltic pump. The amount of acetone in any exposure concentra- tion never exceeded 0,5 mIlL. Toxicant concentrations were measured twice during the exposure period, Samples for analysis were taken from al- ternate replicate aquaria. Toxicants were extracted with solid- phase extraction and analyzed with GC [10]. Extracted samples were stored at -40C until analyzed. Brain A ChE assay Brain AChE activity was measured immediately after con- clusion of the toxicant exposure period. Three fish were se- lected from each exposure concentration, Whole brains were excised by removing the top of the cranium, severing the spi- nal cord at the base of the medulla, and cutting all cranial nerves at their origin on the brain. Individual brains were weighed (j;: 1 mg), homogenized, diluted with distilled water, and held on ice until assayed (<30 min). Assays were conducted using a Sargent-Welch recording pH stat (Sargent- Welch Scientific Co, Skokie, IL) and the pH-stat method [11,12], Enzyme assay conditions for Colorado squawfish were those determined for the closely related Colorado roundtail chub (Gila robusta robusta) [13]: pH, 7.5; tempera- ture, 30oC; 10 mg brain tissue perreaction vessel (2.0 mg/ml); and substrate concentration, 0.011 M (acetylcholine iodide, Sigma Chemical Co., St. Louis, MO). Enzyme activity units (AU) were defined as the activity of AChE that hydrolyzed 1j.tmol of substrate/milligram brain tissue/minute, Human blood-serum reference standards (Fisher Scientific, Orange- burg, NY) were assayed periodically to provide quality as- surance. Estimates of activity of reference standards were within 5% of the reported value. .. '" Statisticalanalys~ Two methods, ANOYA and regression analysis, were used to analyze the response of AChE activity. For ANOYA, AChE activities of fish in solvent controls and dilution-water controls were compared by calculating a t statistic and com- paring it to a two-tailed Student's critical value. If effects of the solvent and dilution-water controls were not significantly different (ex = 0.05 for all statistical comparisons), data from the two treatments were pooled for subsequent analyses. After pooling control treatments, data were subjected to Shapiro-Wilk's test for normality and Bartlett's test for ho- mogeneity of variance [14]. No transformations were required to meet assumptions of normality or homogeneity of vari- ance. Subsequently, data were subjected to one-way ANOYA. Treatments that had significantly different effects compared to controls were distinguished by calculating a t statistic for comparison to a one-tailed Dunnett's critical value, and NOECs and LOECs were identified. For regression analysis, a linear-plateau regression model was fit to AChE activity as a function of toxicant concen- tration using the method described by Beyers et al. [7]. Be- fore regression analysis, measured toxicant concentrations were logz transformed. Data and residual plots were exam- ined to confirm that regression models were appropriate and statistical assumptions were not violated. All statistical anal- yses were conducted using SAS@ statistical software [15]. ., RESULTS Compared to malathion, carbaryl was a more potent in- hibitor of brain AChE in Colorado squawfish (Table 1). NOECs for Colorado squawfish were 29.3 ",g/L carbaryl and 371 ",g/L malathion, Threshold concentrations and 95% C,I.s (in parentheses) estimated by linear-plateau regression