**3.1 What is a RRT?**

The identification and counting of microplastics face challenges due to the complexity and heterogeneity of the materials. These challenges are also paired with the

#### *Round Robin Test on Microplastic Counting and Identification Method DOI: http://dx.doi.org/10.5772/intechopen.109757*

lack of referenced certified methods and standards for microplastic analysis. In the absence of accredited procedures and standards, the robin round test (RRT) is one of the best approaches to identify and quantify microplastics.

The RRT is an advantageous approach since it is specific, pre-defined, and requires the involvement of several labs worldwide. It involves the presence of an organization that is able to supply samples and precise instructions and consecutively to evaluate the lab results [12]. Thus, RRT allows for the determination of the reproducibility of a process or analysis by means of multiple analyses performed independently. The statistical elaboration of the results provides a top-down evaluation of the variability by analyzing the outcomes of different labs [30]. Thus, the benefits and applicability of the RRT are diverse and can be strategically designed for various purposes.

In microplastic analysis, few studies carry out the RRT for the comparison of the results. For instance, Muller et al. presented the results of an international comparative study on the common analytical technique used in microplastic analysis [31]. Since there is a large discrepancy between the 17 labs involved in the study, the study pointed out the urgent necessity of a standardized method for microplastic analysis.

A novel approach to microplastic analysis was proposed by Mossotti et al., who found out an optimized protocol for the determination of microplastic with fibrous shape in water [29]. Three different synthetic filaments cut at predetermined lengths can be used as internal standards for microplastic identification. For the first time, an analytical method for microplastic identification was subjected to a RRT involving 18 labs around the world.

#### **3.2 Aim of STANDARD METHOD prEN ISO 4484-2 PROTOCOL**

The purpose of this RRT is to identify with adequate accuracy the number and type of standard microfilaments in suspension.

All the analyzed data of the samples were divided into polymer groups, counted, and then compared with their targets, thus obtaining the evaluation of the accuracy and reliability of the method.

#### **3.3 Round Robin test design**

#### *3.3.1 General information*

The RRT on prEN ISO 4484-2 was carried out from January to March 2022, and the trial was organized by Aquafil S.p.A (Italy) and CNR-STIIMA (Italy).

18 laboratories based in 17 European countries and a non-European one took part in the RRT. The study participants were from Italy, Germany, the U.K, Sweden, Spain, and South Korea. In **Figure 5**, the number of participants from each country is reported.

The laboratories that participated in this study represent universities, research institutions, laboratory equipment suppliers, and laboratories owned by private companies, as reported in **Figure 6**.

10 samples divided into 3 sample types called Sample 1, Sample 2, and Sample 3 (**Table 1**) were prepared for each laboratory, and in addition, 3 replicates per sample were prepared. The tenth sample corresponded to the Control Sample.

Considering the RRT membership of 18 laboratories, a total of 180 samples were prepared. The preparation of the samples, in particular the cutting of the microfilaments, is described in detail elsewhere.

**Figure 5.** *Laboratories involved from different countries.*

#### **Figure 6.**

*The participants came from various sectors, including universities, research institutions, laboratory equipment suppliers, and laboratories owned by private companies.*

After cutting, the procedure follows these steps:


*Round Robin Test on Microplastic Counting and Identification Method DOI: http://dx.doi.org/10.5772/intechopen.109757*


#### **Table 1.**

*Microfilaments used for the preparation of the standard samples.*


The described procedure is reported in **Figures 7** and **8**.

Each participating laboratory was provided with the analytical protocol (see Annex) to be followed in order to perform the determination of the microfilaments contained in the standard samples, as well as instructions for the results. For each

**Figure 7.** *Procedure for the preparation of standard sample.*

**Figure 8.** *A total of 10 samples were prepared for each lab.*

sample, the number of total microplastics present in the sample, polymer type, and physical characteristics (microfilament lengths) had to be reported. In addition, the presence of contaminants had to be described.
