Use of an mfo‐directed toxicity identification evaluation to isolate and characterize bioactive impurities from a lampricide formulation

Abstract Recently, a field formulation of the lampricide containing 3-trifluoromethyl-4-nitrophenol (TFM) was identified as a potent inducer of mixed-function oxygenase (MFO) detoxification enzymes in fish. It was further shown that induction was not associated with primary formulation ingredients. Using a toxicity identification evaluation (TIE) approach based on rainbow trout hepatic MFO activity, the TFM field formulation was investigated to isolate the compound(s) responsible for induction. Solid phase extraction and reverse-phase high-pressure liquid chromatography (HPLC) isolated activity in two distinct fractions, which were profiled by gas chromatography–high-resolution mass spectrometry. The major constituents in both fractions were confirmed by synthesis as nitro-, trifluoromethyl-, and/or chloro-substituted diphenyl ethers. However, fish exposures to the pure compounds failed to cause MFO induction. After further fractionations by HPLC, induction was determined in three new subfractions. Confident identification of a chloro-nitro-trifluoromethyl-substituted dibenzo-p-dioxin has been made in two of these fractions. Although the specific chemicals responsible for induction have not been confirmed, a suite of impurities including chloro-, and/or nitro-, and/or trifluoromethyl-substituted phenols, diphenyl ethers, and dibenzo-p-dioxins have been identified in the formulation. It is likely that these materials originate during industrial synthesis of TFM. These findings suggest that additional structurally related impurities are also present in this formulation.