Mechanisms of Naphthenic Acid Toxicity

The Alberta Oil Sands are the third largest reserve of crude oil in the world. Most of this oil is extracted via the Clarke hot water extraction process, producing large volumes of waste water or oil sands process-affected water (OSPW). This water must be kept on oil sands sites, resulting in the creation of large tailings ponds. These ponds contain numerous toxic compounds, one of the principle ones being naphthenic acids (NAs). These are carboxylic acids that are natural components of petroleum and are thought to be responsible for much of the toxicity of tailings ponds. They have been shown to be acutely lethal and have a number of sublethal effects to a variety of aquatic organisms and other species. Despite the wide knowledge of their toxic effects, the mechanism of toxic action of naphthenic acids has remained elusive. Based on the known mechanisms of resin acids, carboxylic acids from pulp and paper mill effluent, it was hypothesized that naphthenic acids would disrupt mitochondrial energetics by uncoupling oxidative phosphorylation (OXPHOS) and inhibiting the electron transport system (ETS), which would result in oxidative stress and increased reactive oxygen species (ROS). For this thesis, NAs were extracted and purified from a 17-year-old tailings pond yielding a mixture containing 99% carboxylic acids with 90% fitting the classical NA formula (CnH2n+ZO2). Mitochondria were isolated from rainbow trout (Oncorhynchus mykiss) livers via differential centrifugation and the mitochondrial oxygen consumption, mitochondrial membrane potential, and hydrogen peroxide production were measured simultaneously using the Oroboros respirometry system following exposure to different doses of the OSPW-NA mixture. Mitochondrial membrane potential and oxidation state were also measured using real-time flow cytometry methods using fluorescent dyes, JC-1 and H2DCFDA, respectively. The effects of two model adamantane NAs, 3,5-dimethyladamantane-1-carboxylic acid and 3-hydroxy-adamantane-1-carboxylic acids, were also examined to determine if they could be used as surrogate toxicants for an NA mixture. The results showed that NAs derived from OSPW inhibited mitochondrial respiration and increased ROS. Oxidative uncoupling of the mitochondria also occurred, but to a lesser degree than the other effects. The EC50 and IC50 values calculated for the endpoints were consistent with NA levels found in tailings ponds, while the estimated EC10 and IC10 values approach environmental concentrations of NAs. The model adamantane acids that were examined showed some similar effects to the OSPW-NA mixture, suggesting that some adamantane compounds may be able to act as surrogates for oil sands-derived NAs. In conclusion, the mechanism of action of NAs appears to be complex and they likely act via multiple mechanisms, wherein mitochondria may be an important target.

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