The assessment of the impact of bioaccumulable pollutants on the extracellular fraction of model organisms is crucial for understanding ecotoxicological risks. In this study, the hemolytic activity of acellular coelomic fluid from Eisenia fetida was analyzed on sheep erythrocytes as a model to monitor the functionality of extracellular proteins exposed to environmental contaminants. An integrated approach was applied, based on three distinct experimental tests: (I) a control test with unexposed coelomic fluid to establish the functional baseline, (II) a direct test involving exposure of erythrocytes to the pollutants without coelomic fluid to estimate the intrinsic toxic effect of the contaminant, and (III) a mediated test in which the coelomic fluid was pre-exposed to the pollutant before the hemolytic assay on erythrocytes, in order to evaluate changes in protein lytic activity.

The control with unexposed coelomic fluid showed significant intrinsic lytic capacity supported by pore-forming protein lysenin. The direct and mediated exposure tests made it possible to explore, respectively, the toxic potential of the pollutants on erythrocytes and the vulnerability of the coelomic protein components to functional modifications.

The hemolytic assay applied in these three complementary modes represents an innovative model for distinguishing inhibition, activation, or chemical–protein interaction effects, leaving open the possibility for further investigation of specific mechanisms of action. This approach emerges as a useful tool for ecotoxicological research and for environmental monitoring aimed at evaluating extracellular protein functionality.