Interlaboratory comparison and optimization of hepatic microsomal ethoxyresorufin O‐deethylase activity in white sucker (Catostomus commersoni) exposed to bleached kraft pulp mill effluent

Abstract Twelve samples of minced liver from white sucker (Catostomus commersoni) were delivered to 14 laboratories in Canada and the United States for the analysis of hepatic ethoxyresorufin O-deethylase (EROD). Samples were collected from a site exposed to bleached kraft pulp mill effluent (BKME) and from a reference site. All laboratories analyzing EROD activity successfully differentiated between effluent exposed fish (high activity) and reference samples (low activity). Absolute activity detected in samples from the induced site varied greatly with analysis method; assay conditions at most laboratories were suboptimal for white sucker microsomes, and absolute activity of the samples was most affected by pH, temperature, and substrate limitation. The method used to quantify protein was also thought to contribute greatly to the observed variability. However, the induction level (mean exposed divided by mean reference values) was similar. Relative induction did not appear to be affected by storage time, pH, buffer, assay time, or assay temperature. The induction level was not affected by whether postmitochondrial supernatant (PMS; 10,000 g) or microsomal fractions (100,000 g) were used in analysis, although absolute activity was 3.5 times higher with microsomal preparations. There was a statistically significant agreement in the rankings of high-activity samples using microsomal preparations; a corresponding ranking of PMS values was nonsignificant. The presence of cytochrome P4501A enzyme was detected using monoclonal antibody 1-12-3 against scup P4501A1. Immunoblotting showed 8.4-fold differences in the measured levels of the single cross-reacting band, confirming the distinction between exposed and reference fish determined by EROD assay.