**2. Analytical approaches for the determination of microplastics**

Microplastics are synthetic polymers of a wide variety of different shapes and colors. The choice of the analytical approach to characterize them depends on the data to be obtained in order to estimate their impact on the environment and human health. Scientific literature proposes a wide variety of techniques that provide data ranging from morphological characterization to the determination of their concentration and polymer type. The data obtained are closely related to the technique used to obtain them. Visual inspection techniques (optical and electron microscopy) are generally used for the study of morphology, color, and counting, while the study of composition is carried out by means of thermoanalytical methods, molecular spectroscopy (FT-IR and Raman), and liquid chromatography as shown in **Figure 3**.

Visual sorting is a method based on observing and counting microplastics with a stereomicroscope or optical microscope. This method allows for an error of over 70% for particles smaller than 50 μm and false positives for fibers larger than 200 μm [14, 15]. Environmental aqueous matrices are rich in cellulosic or fibrous protein material, which can be mistaken for degraded plastic material. Therefore, the lack of recognition of the chemical nature of the polymer leads to possible errors. In some cases, optical screening involves the use of dyes to increase the accuracy during visual inspection. For example, some authors identified microplastics such as PE

**Figure 2.** *Identification system for microplastics.*

**Figure 3.** *Microplastic identification methods.*

(polyethylene), PS (polystyrene), PP (polypropylene), and nylon 6 by using Nile Red fluorescent tagging [16, 17]. However, the coloring does not highlight polymers such as polyurethane (PU) and polycarbonate, and therefore, it limits its use.

In comparison with optical microscopy, scanning electron microscopy (SEM) can give images at high resolution of morphology, examine surface condition, and provide a qualitative determination of the chemical composition by energy dispersive x-ray spectroscopy (EDS) [18]. It is a technique coupled with SEM microscopy, generally used for the identification of organic material with high content of inorganic minerals and salts (Ca/Mg/Sr) and microplastics rich in chemical elements such as C/Cl/S/Ti.

At the same time, some authors proposed chromatography techniques coupled to high-resolution mass spectrometry (LCHRMS) for the quantification and chemical identification of microplastic (e.g., PS in natural waters). However, this technique does not provide data concerning the shape and size [9, 19].

Within visual sorting, the gravimetric method can be used to quantify microplastic particles or filaments with different sizes [20] in samples with high water volumes such as wastewater from laundry machines.

During a washing cycle of different synthetic standard fabrics or clothes at different operative conditions, the gravimetric method can be used for the determination of the mass of microfilaments released through a sieve at predeterminate porosity [6, 7, 21, 22].

At present, thermal techniques such as differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA), sometimes coupled with chromatographymass spectrometry (TDSGC-MS) or pyrolysis (Py-GC-MS), are used for the quantitative determination of microplastics [12, 23].

The samples are subjected to high-temperature treatments in order to produce thermal degradation, and the volatile compound products are analyzed for their polymer identification by means of a mass spectrometer. However, these techniques

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

have the disadvantage of being able to analyze only samples with a size >500 μm, and this prevents the determination of microplastics. The technique requires basic sample preparation, compared to others, but it is destructive and does not allow for multiple or parallel analyses. In this field, liquid chromatography can be used, too. However, it requires high quantities of sample volumes for the preliminary extraction step of the sample. Moreover, it only provides reliable results for a limited number of polymers such as PET and PE.

The drawback of all these analytical techniques is that they work for the identification of polymer nature and other additives such as, UV or thermal stabilizers, flame retardants, dyes antioxidants, plasticizers, and so on, but they do not give any information about the physical characteristics such as shape, number of microplastics, or color [24].

Other analytical methods available for the identification of microplastic are vibrational spectroscopic techniques, such as mid-infrared (FTIR), near-infrared (NIR), and Raman spectroscopy. FTIR and Raman spectroscopy are complementary techniques that transform the interaction of light with the sample into a spectrum that contains all the information about the chemical structure [25]. Raman spectroscopy is sensitive to the variation of the polarization of the molecule during vibrational motion, while FTIR is affected by the variation of the dipole moment in reflection, transmission, and total attenuated reflection (ATR).

Raman is a fundamental technique for the recognition of aromatic compounds and double bonds, while FTIR is used for the identification of polar functional groups of molecules such as hydroxyl, carbonyls, carboxyl, amino groups, and so on. Both are nondestructive techniques; Raman can be performed on any type of matrix, even liquid; it requires minimal sample preparation and allows for the identification of contaminants of inorganic nature, too. The Raman and FTIR techniques can be coupled with an optical microscope and, at the same time, be applied for sorting and recognizing microplastics. μ-FTIR can be used for the identification of particles larger than 10 μm and μ-Raman for particles larger than 0.2 μm [26]. Moreover, SEM or AFM (Atomic Force Microscopy) in combination with infrared spectroscopy can be used to visualize and chemically identify microplastics [27]. Although many approaches can be applied for the quantification and identification of microplastic, there are no precise guidelines to follow in relation to microfilament identification. In this chapter, a standard protocol for identification of microplastic with fibrous shape is proposed by using μ-Raman and μ-FTIR, respectively, or in a complementary approach [26, 28].

#### **2.1 Analytical approach for the determination of microplastic with fibrous shape**

The analytical approach for the determination of microplastics are summarized in **Figure 4**. Typically, it involves three main stages. The first step is related to the sampling of microplastics coming from wastewater; the second step is related to the sample preparation and the third to the choice of an appropriate detection method to identify all their characteristics.

The standard protocol proposed is used to carry out qualitative and quantitative analyses of fibrous microplastics in textile aqueous matrices by means of analytical vibrational spectroscopy techniques (μ-FTIR and μ-Raman).

Textile wastewater is a complex matrix because it is rich in organic material, microplastics with fibrous shape, dyes, salt, fiber contaminants (natural fiber), and activated charcoal from the production or finishing processes of the fiber. In order to obtain information on the nature of the polymer constituting the microplastics,

**Figure 4.** *Identification system for microplastics in the textile sector.*

particularly when spectroscopic techniques are used, it is necessary to reduce any interference from other substances. In this regard, the analytical protocol provides suitable information on the reduction of contaminants during the sample preparation.

The following steps have been identified:


In addition, a protocol for the preparation of standard microfilament suspensions was also prepared to facilitate the control of the whole laboratory tests (counting, monitoring, and identification of microplastics). This aspect is an added value of the method because standard fibrous microplastics with established dimensional and structural parameters are not available in the market. For this purpose, four standard suspensions of the most commonly used synthetic fibers such as PA6, PA 6.6, PP, and PET were prepared [29].

In conformity with ISO 5725 and ASTM E691 standard procedures, the protocol was validated by means of a round robin test (RRT). However, during the preparation, it was not possible to use samples from textile wastewater obtained from production processes or washing machines as they are not homogeneous and highly variable.

In order to reduce their variability, 3 replicates of 3 standard water suspensions of PA 6, PET, and PP at different concentrations were used.
