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The ecological status classification of aquatic ecosystems using biological indices requires a number of steps, including the description and standardisation of the indices' natural variability. Here, we address this point with reference to selected Mediterranean and Black Sea lagoons, using benthic macroinvertebrates in order to: (i) explore the drivers and extent of the indices' natural variability; (ii) evaluate lagoon type-specific reference conditions and related classification boundaries; (iii) test the classification strength of the derived boundaries; and, (iv) propose recommendations for optimising ecological status classification. The considered indices showed large variation between and within the reference lagoons on both spatial and temporal scales. Among the tested descriptors of the proposed lagoon typologies, surface area, confinement and water salinity were found to be significant sources of index variability. Type-specific reference conditions and classification boundaries were then defined, improving the accuracy of ecological status assessment. At the lagoon level, classification strength increased up to 100 % in reference (least disturbed) lagoons and up to 83 % in an independent validation set of highly disturbed sites. Nevertheless, a certain degree of uncertainty was still found to affect classification at the study site level. Recommendations concerning the application of the various approaches to type-specific reference conditions and classification boundaries are given. [ABSTRACT FROM AUTHOR]