The screening of photodynamic toxicity of dyes by means of a bioassay using amphibian embryos

Abstract

The photodynamic action is exerted by photogenerated reactive oxygen species (ROS) produced after cellular accumulation of a photosensitizer and irradiation with visible light. AMPHITOX test was employed successfully for standardized hazard assessment studies including phototoxic effects as well as the possibility to prevent/reduce adverse effects of different environmental agents. The main purpose of this study was to evaluate the toxicity and phototoxicity of 6 dyes and to establish the relationship between adverse effects and possible photodynamic activity. For this purpose the NOEC-48 h values were evaluated by means of the AMPHITOX test conducted with Bufo arenarum embryos at the end of their development (stage 25) for the following dyes: acridine orange (AO), phloxin (Ph), phloxin B (PhB), rose bengal B (RB), toluidine blue O (TB), and pyronine Y (PY), dissolved in AMPHITOX solution (AS). In previous studies, it was determined that irradiation with white light at a dose of 43.2 J/cm2 (72 mW/cm2 for 10 min) is appropriate to explore the photodynamic action of chemicals. Thus, for the photodynamic experiments, embryos were treated with a range of concentrations bellow the NOEC values and after 48 h of exposure they were thoroughly washed and irradiated. The NOEC-48 h values (in mg/L) for chemical toxicity and NOEC-48 h values after light irradiation were: AO 1 and 0.5; PhB 1 and 0.7; RB 5 and 1; TB 10 and 5; Ph 10 and 10; PY 20 and 20, respectively. The survival of control embryos (maintained in AS and irradiated for 10 min) was not affected. By comparing the chemical/photochemical toxicity ratios of these substances it results that the order for photodynamic action could be expressed as RB > TB > AO > PhB, the remaining dyes showing no phototoxicity. These results point out the possibility to evaluate the photodynamic effect of chemicals by means of a simple toxicity test employing amphibian embryos.