Contents lists available at ScienceDirect Microchemical Journal journal homepage: www.elsevier.com/locate/microc An experimental approach to estimate uncertainty of diatom community analysis in the accreditation process Camilla Puccinelli ⁎ , Stefania Marcheggiani, Laura Mancini Department of Environmental and Health, Istituto Superiore di Sanità, Rome, Italy ARTICLEINFO Keywords: Diatom community Ecological methods Accreditation Quality assurance ABSTRACT Diatoms, belonging to the class Bacillariophyceae, are one of the biological elements required for ecological quality status (EQS) assessment according to the Water Frame Directive. Italy has adopted the Intercalibration Common Metric index (ICMi) to evaluate the EQS using diatoms. Developed through the InterCalibration exercise and validated at the European level, the ICMi still presents uncertainty elements related to ecological, spatial and temporal heterogeneities. Quality control assurance has become necessary for ecological tests due to their importance in data acqui- sition during monitoring activities. The aim of this work was to describe the approach used for the measurement of sampling and/or laboratory phase uncertainty associated with ICMI according to ISO/IEC 17025. Following ISO recommendations, we adopted the “top-down” approach to evaluate uncertainty from repeatability using experimental data. The study was designed to determine the variability of the method in relation to both the sampling and laboratory phases. The approach consisted of analyzing three samples, each with Bad, Moderate or High eco- logical quality status, ten times, plus ten replicates of sampling from the Moderate status site. Sampling, treatment, analysis, identifcation and counting were performed according to standard procedures. The ICMi was calculated for all samples. The experiment was performed under the following repeatability conditions: same operator, microscope and iconographic guides. We obtained a standard deviation of repeatability in each series. This study is an attempt to quantify, for the frst time, the variability of the diatom analysis process through the value of the ICMi, trying to reduce at least the main sources of errors (identifcation and counting). It represents an approach to be followed for the accreditation process of a diatom-based laboratory test method according to ISO/IEC 17025. 1. Introduction Aquatic ecosystem protection and management were radically changed in 2000 with the adoption of the European Union Water Framework Directive (WFD) [1]. The assessment of the structure and functioning of surface water ecosystems is expressed as Ecological Quality Status (EQS). This latter is based on composition and abundances of biological communities (phytobenthos, aquatic invertebrates, macrophytes, fsh fauna) analysis supported by physico-chemical and hydromorphological quality ele- ments. For the frst time, biological quality elements became the central elements and thus the basis of management decisions [2]. The ecological status classifcation for each waterbody is expressed as the Ecological Quality Ratio (EQR) that represents the deviation from the least disturbed conditions (reference conditions), divided into fve quality classes of increasing degradation, from High to Bad [1]. Diatoms, unicellular algae in the class Bacillariophyceae, were chosen as proxies for the entire phytobenthos for the ecological status assessment of freshwater in EU countries [3,4]. The use of diatoms as ecological indicators was recognized long before the WFD, thinking to the fact that the frst diatom-based indices were developed in the early 1980s [5]. They are sensitive against physico-chemical parameters and inorganic and organic compounds and consequently they are used to investigate more specifc impacts such as eutrophication and acidifcation [6–13]. Analysis procedures of each biological elements were updated by each European country to comply WFD requirements. In Italy, the sampling and sample treatment procedures was carried out by the National Working Group [14,15], furthermore a harmonized exercise was done to identifcation and diatom counting through three https://doi.org/10.1016/j.microc.2019.104078 Received 15 February 2019; Received in revised form 7 June 2019; Accepted 8 July 2019 ⁎ Corresponding author. E-mail address: camilla.puccinelli@iss.it (C. Puccinelli). Microchemical Journal 150 (2019) 104078 Available online 09 July 2019 0026-265X/ © 2019 Elsevier B.V. All rights reserved. T