Tri- and Tetra-Chloroveratrole, Metabolites Produced by Bacterial O-Methylation of Tri- and Tetra-Chloroguaiacol: An Assessment of Their Bioconcentration Potential and Their Effects on Fish Reproduction

Abstract

We have demonstrated that strains of bacteria belonging to the genera Pseudomonas and Acinetobacter possessed the ability to transform, by O-methylation, components of spent bleachery effluent such as 4,5,6-trichloroguaiacol and high molecular weight chlorinated lignin. O-Methylation could be enhanced by the presence of a cosubstrate such as succinate or 4-hydroxybenzoate. We have used zebra fish (Brachydanio rerio) to examine bioconcentration potential, toxicity to embryos and larvae, and the sensitivity of the offspring of exposed adults. Bioconcentration factors (total wet weight) on a logarithmic scale were 3.5 and 4.4 for 3,4,5-trichloroveratrole and tetrachloroveratrole, respectively. The potential for bioconcentration of the other neutral metabolites was inferred from estimated values of the octanol–water partition coefficients. 3,4,5-Trichloroveratrole, tetrachloroveratrole, pentachloroanisole, and 1,2,3-trichloro-4,5,6-trimethoxybenzene had threshold toxic concentrations to zebra fish embryos and larvae of 450, 100, 2.8, and 450 μg∙L−1, respectively, and all of them were capable of inducing, at somewhat lower concentrations, various deformations including curvature of the larvae and distortion of the notochord. Experiments on the effect of preexposure of adults to tetrachloroveratrole at a concentration of 16 μg∙L−1 showed that the offspring were adversely affected, e.g. in a decreased viability of the embryos, lowered median survival time for larvae, and greater sensitivity to tetrachloroveratrole. Both 3,4,5-trichloroveratrole (5–150 μg∙kg liver fat−1) and tetrachloroveratrole (40–400 μg∙kg liver fat−1) were detected in fish from localities subjected to discharge of bleachery effluent, and were absent in fish from uncontaminated localities. Identification of these compounds was based on gas chromatographic, and for tetrachloroveratrole, mass spectrometric, comparison with authentic compounds. We conclude that not only compounds present in bleachery effluent, but those that may be produced in the environment by bacterial O-methylation, have potentially hazardous biological activities. These results shed fresh light on a hitherto neglected aspect of environmental hazard assessment, and we suggest that data for a given compound must be supplemented with that for metabolites that could be produced in the environment.