Local connected fractal dimension analysis in gill of fish experimentally exposed to toxicants

An operator-neutral method was implemented to objectively assess European seabass, Dicentrarchuslabrax (Linnaeus, 1758) gill pathology after experimental exposure to cadmium (Cd) and terbuthylazine(TBA) for 24 and 48 h. An algorithm-derived local connected fractal dimension (LCFD) frequency measurewas used in this comparative analysis. Canonical variates (CVA) and linear discriminant analysis (LDA)were used to evaluate the discrimination power of the method among exposure classes (unexposed,Cd exposed, TBA exposed). Misclassification, sensitivity and specificity, both with original and cross-validated cases, were determined. LCFDs frequencies enhanced the differences among classes whichwere visually selected after their means, respective variances and the differences between Cd and TBAexposed means, with respect to unexposed mean, were analyzed by scatter plots. Selected frequencieswere then scanned by means of LDA, stepwise analysis, and Mahalanobis distance to detect the most dis-criminative frequencies out of ten originally selected. Discrimination resulted in 91.7% of cross-validatedcases correctly classified (22 out of 24 total cases), with sensitivity and specificity, respectively, of 95.5%(1 false negative with respect to 21 really positive cases) and 75% (1 false positive with respect to 3 reallynegative cases). CVA with convex hull polygons ensured prompt, visually intuitive discrimination amongexposure classes and graphically supported the false positive case. The combined use of semithin sections,which enhanced the visual evaluation of the overall lamellar structure; of LCFD analysis, which objec-tively detected local variation in complexity, without the possible bias connected to human personnel;and of CVA/LDA, could be an objective, sensitive and specific approach to study fish gill lamellar pathol-ogy. Furthermore this approach enabled discrimination with sufficient confidence between exposureclasses or pathological states and avoided misdiagnosis.