Major anionic interferences in the methylene blue active substances (MBAS) method

• Anionic interferences in MBAS followed the sensitivity order: nitrate > chloride >>> sulfate. • MBAS overestimated anionic surfactant concentrations in nitrate-rich and chloride-rich waters • Measurement errors reach magnitudes comparable to EC50 values for organisms across multiple trophic levels (from bacteria to fish), potentially leading to inaccurate environmental risk assessment • LC-MS/MS exhibited up to 100-fold greater sensitivity than the MBAS method. Anionic surfactants (e.g. sodium dodecyl sulfate, SDS) are widely utilised in cleaning agents, pharmaceutical formulations, and personal care products, serving as biocides, emulsifiers, or drug carriers. Following their use, they often enter aquatic environments through wastewater discharge, raising concerns about their potential ecological impacts. The methylene blue active substances (MBAS) assay is the standardised colorimetric method commonly employed to quantify the anionic surfactants for environmental monitoring purposes due to its simplicity and ease of implementation. However, this method presented significant limitations that may compromise accurate environmental risk assessment. Specifically, the presence of common inorganic ions in natural waters can interfere with MBAS measurements, potentially leading to biased surfactant quantification. This study investigated the interference of chloride (0-500 mM), nitrate (0-2 mM), or sulfate (0-30 mM) on MBAS-based quantification of anionic surfactants in the effluent of a wastewater treatment plant (WWTP), in river water, and in seawater, and its accuracy compared to targeted analysis by LC-MS/MS. The MBAS response was linearly proportional to SDS and nitrate concentrations but followed a power relationship with chloride, while sulfate produced negligible interferences. Although the method exhibited approximately 430-fold higher sensitivity to SDS than to nitrate and up to 100,000-fold higher sensitivity than to chloride, environmentally relevant concentrations of these ions caused substantial quantification errors. Specifically, nitrate (up to 1.4 mM in wastewater) and chloride (∼520 mM in seawater) generated apparent anionic surfactant overestimations of approximately 3 µM and 17 µM, respectively. This overestimation in seawater was environmentally significant, as SDS concentrations in the 7-25µM range exhibit acute toxicity across all trophic levels, including bacteria, algae, crustaceans, echinoderms, and fish. After correcting for anionic interferences, MBAS measurements fell below the detection limit (0.29 µM), consistent with LC-MS/MS results.