Quantifying the phenolic content of freshwaters using simple assays with different underlying reaction mechanisms

Five different assays, Gibbs, Prussian Blue, Folin-Ciocalteau, fluorescence quenching of added phenol and precipitation of phenolics with bovine serum albumin (BSA) were investigated for their suitability in measuring the phenolic content of freshwaters. Phenol and a hydrolysable tannic acid were used as standards for monophenolics and polyphenolics, respectively. The individual and simultaneous application of both standards in doubly distilled water and filtered freshwater samples showed no matrix interference for the Gibbs, the Prussian Blue and the Folin-Ciocalteau assays. The quenching of phenol fluorescence and incomplete precipitation of added tannic acid in the freshwater samples were thought to originate from complexation. The Gibbs assay was specific for monophenolics, monohydroxybenzenes, with a Criterion of Detection (CoD) of 0.027 mg l⁻¹. Evaluating the assay using twenty-two monophenolics of lignin origin showed, apart from phenol itself, the phenolic acids vanillic, isovanillic, ferulic and syringic to have a linear response between 0 and 10 µM. The other monophenolics were not responsive in the Gibbs assay. The oxidation-based assays Prussian Blue and Folin-Ciocalteau had a CoD of 0.169 and 0.025 mg l⁻¹, respectively. The ratio of response of both assays for each sample was taken as an indication of the degree of polymerisation of the phenolic content. The Folin-Ciocalteau assay was used directly on the samples, on samples spiked with tannic acid at 2 and 4 mg l⁻¹, and after precipitation of phenolics with BSA. The difference in tannic acid equivalents before and after treatment, assayed the amount of protein precipitated phenolics. The results of all assays allowed differentiation between monophenolics (Gibbs), polyphenolics (Prussian Blue), total phenolics (Folin-Ciocalteau), complexation of added phenol and protein-precipitated phenolics. The reaction mechanisms underlying the assays were matched onto those occurring during humification. The assays were applied to six filtered freshwater samples and two humic and two fulvic acids. The results showed a different pattern for each site and illustrated varying reactivity of the ‘phenolic content’ of freshwater.