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Following the Water Framework Directive 2000/60/CE (WFD), each member state of the European Union must monitor compliance of its rivers with ecological quality standards through biological quality indicators. The New Italian Index of the Ecological State of Fish Communities (NISECI) was developed in 2017 for the assessment of fish communities, as directed by the WFD in Italian freshwater habitats. According to the WFD, the general reference conditions (GRCs) of NISECI must be refined on a regional scale through new calculation of its metrics and sub-metrics. In the present study we used environmental and ichthyological data from 457 fish samplings distributed among 299 sampling sites within 84 different water bodies collected from 1995 to 2012 to develop: 1) new lists of expected species for six homogeneous zones identified in the Reno basin (Italy) and in the eastern regional basins of the Emilia-Romagna region; and 2) the threshold values for their species-specific abundance. Results were set as refined reference conditions (RRCs) for two of the metrics used in the application of the NISECI index in the study area (i.e. X1, relating to indigenous species and X2,b, for the abundance of expected species). The RRCs were tested by applying NISECI to 24 monitoring sites of the regional surface water monitoring network (i.e., ARPAE) and comparing the results with the application of NISECI using the GRCs. Furthermore, the analytical power of the refined NISECI was evaluated by relating the findings to three expert-based blind assessments of fish community ecological status. The results confirmed an increase in refined NISECI values and its higher consistency with expert-based assessment, supporting the validity of the presented method for RRC development and its potential for application in other regions.

Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.