<p>Freshwater salinization is increasing globally through seawater intrusion, road de-icing, and changes in anthropogenic land uses. Concurrently, freshwaters are browning with the rise in dissolved organic carbon (DOC) concentrations, while water pH is falling. Elevations in external major ion concentrations (Na⁺ or Ca²⁺) and low pH, independently disturb osmoregulatory homeostasis in freshwater organisms. Several studies have demonstrated that DOC often mitigates osmoregulatory stress responses to acidic pH. However, the interactive effects of these three water quality parameters together have been relatively understudied. Transepithelial potential (TEP), the electrical gradient across the gills between the animal and the external water, can be used as an index of osmoregulatory stress. We investigated whether DOC and exposure to elevated major ions interact with TEP responses at circumneutral and low environmental pH in the freshwater rainbow trout. Two natural DOCs, one allochthonous and the other autochthonous, were used. To aid interpretation, three model compounds of known chemical structure were also employed (tannic acid, sodium dodecyl sulfate, bovine serum albumin), based on the criteria that they structurally resemble or functionally behave like certain chemical moieties of humic or fulvic acids, major components of DOC. The Multi-Ion Toxicity Model predicts that a disturbance in absolute TEP is indicative of salt toxicity; however, recent studies have shown that ΔTEP (the change in TEP relative to the baseline) may be more predictive. Our data followed a pattern that could be described by the Michaelis–Menten equation. Therefore, considering Michaelis–Menten constants (<i>Km</i> and <i>ΔTEP</i>_<i>max</i>), absolute TEP and ΔTEP, we used a weight of evidence approach to predict how DOC and pH will influence Na⁺ or Ca²⁺ toxicity. We conclude that key chemical moieties of DOC will likely play pH-dependent roles in both Na⁺ and Ca²⁺ toxicity.</p> Graphical Abstract <p></p>