Second‐round interlaboratory comparison of hepatic ethoxyresorufin‐O‐deethylase activity in white sucker (Catostomus commersoni) exposed to bleached‐kraft pulp mill effluent

Abstract Livers were collected from six male and six female white sucker (Catostomus commersoni) at both a bleached-kraft pulp mill effluent (BKME)-exposed site and a reference site during the 1992 spring spawning migration. A semistandardized methodology for 7-ethoxyresorufin-O-deethylase (EROD) analysis was proposed that featured similar physical and chemical conditions yet allowed for different instrumentation and techniques. Postmitochondrial supernatants (PMSs) were analyzed for EROD activity by 14 laboratories, and repeat EROD analyses were performed within one laboratory to estimate within-laboratory variability. All laboratories found significantly higher levels of EROD activity (approximately threefold) in males from the BKME-exposed site; only 5 of 14 laboratories found significantly higher levels in exposed females. Data from three labs were dropped from the analysis because of outlying values. The mean coefficient of variation (C.V.) for the remaining 11 laboratories was 34.7%, which was not significantly different from the C.V. determined for six analyses by one technician (28.4%). The mean C.V. for five analysts within one laboratory was significantly lower (16.4%) than either of the former values. There was no difference in absolute values or the variability of microplate and conventional EROD assay data. Analysis of Bradford and Lowry protein data showed that both assays have low variability (C.V.s = 16.8 and 17.4%, respectively); these two techniques produced very similar estimates of protein content. Immunochemical analysis demonstrated a significant correlation existed among microsomal P4501A concentration and EROD activity. Sources of commercially available resorufin standard were tested, and the mean molar extinction coefficient was 54.0 ± 1.1 cm−1 mM−1.