Fluorescent Dyes in Hydrological Tracing: Application Methods, Ecotoxicological Effects, and Safe Application Levels.

Fluorescent dyes are commonly used as tracers in hydrological investigations to quantify transport pathways, residence times, mixing behavior, and connectivity in surface water, groundwater, and coastal systems. Despite their long history of application, the ecological implications of deliberate dye releases are not well understood. This review synthesizes current knowledge on the physico-chemical characteristics, environmental behavior, and ecotoxicological effects of major dye classes, with emphasis on rhodamines, fluorescein derivatives, and sulfonated xanthene dyes commonly used in water tracing studies. Toxicity data for algae, cyanobacteria, invertebrates, and fish show large inter-specific variability. Some dyes, particularly rhodamine B and eosin Y, show acute or sub-lethal effects at concentrations detected during poorly controlled applications. By contrast, dyes with high polarity and extensive sulfonation (e.g., rhodamine WT, sulforhodamine B, pyranine, and fluorescein) show consistently low toxicity and minimal bioaccumulation potential. Environmental fate processes, including photolysis, sorption, and transformation into potentially more reactive products, influence exposure dynamics, especially in clear, shallow, or slow-moving systems. This review also evaluates regulatory frameworks and operational guidance for safe use, identifies gaps in toxicological and fate data, and proposes recommendations for minimizing environmental impact through dye selection, mass optimization, injection design, and monitoring. The findings support the continued use of fluorescent dyes but highlight the need for more systematic assessment of transformation products, chronic and sub-lethal responses, and cumulative exposure in sensitive environments.