*3.2. Characterization of MPs by Size, Shape and Colour*

As explained before, the sampling procedure allowed the MP classification by size. According to Figure 3, on average, in influent samples MP ≥ 500 μm only accounted around 30% of total MPs, whereas 56% and 80% represented MPs higher than 250 μm and 100 μm, respectively. This indicates a major percentage of small MPs than usual in the influent since most WWTPs it has been reported MPs abundance with a size greater than 500 μm above 70% [18,45,46,61–63]. The variations in the percentages of MPs found in each range of size are noticeable thorough the treatment processes, i.e., the percentage of those MPs with a size greater than 500 μm decreased from 30% in the influent to 24% in the secondary effluent. At the same time, the percentage of the smallest particles (20–100 μm) increased from 20% in the influent to 23% in the secondary effluent. It should be noted that, after pre-treatment and secondary treatment, the MPs most easily eliminated were those larger than 500 μm (57%) and those with a size between 250–500 μm (52%), as can be seen in Table S2. This means that the grit and grease system and the secondary treatment removed the bigger MPs with higher efficiency than the smaller ones. Important variations in the percentages of the middle sizes were not detected and the sizes distribution in the final effluent is similar to the secondary one. In the final effluent samples, the vast majority of MPs were smaller than 500 μm, around 76%, whereas a quarter of the microplastics were smaller than 100 μm (Table S2). These results agree with other previous studies, which reported that most of the MPs in the final effluent were smaller than 500 μm. However, the percentage of MPs smaller than 100 μm in the effluents is usually over 60%, percentage higher than those found in this work [19,31,33,34,52,64–67]. Table S2 shows that, after tertiary treatment, the most easily eliminated MPs were, both, those larger than 500 μm and those with a size between 100–250 μm (approximately 30%). In addition, considering the temperature, it can be observed that in the warmest months (May–September) the MPs with a size higher of 250 μm presented abundances of 60–70%, while during those months with lower temperature (November, February–April), it is observed that the MPs with sizes less than 250 μm presented abundances of 60%. It has been reported that MP degradation are determined by the combined effect of different parameters, including temperature. Specifically, Ariza-Tarazona et al. [68] concluded that photolysis combined to low temperatures leads to plastic brittleness, which is in accordance with results commented above, since the coldest months showed a greater proportion of MPs smaller than 250 μm. Finally, it can be observed that the overall microplastic removal efficiency was higher in MPs larger than 500 μm (70%) compared to the rest of the sizes.

The morphological characteristics of MPs found in wastewater samples can be classified into five different types: fragments, fibres, microspheres or pellets, films and foams, as can be observed in Figure 4. Fragments exhibit irregular and opaque shapes, whereas fibres show a high length-width ratio. Pellets have spherical form, foams are fluffy particles and, finally, films have a relatively flat surface.

**Figure 3.** Size variation of microplastics in influent, secondary effluent, and final effluent samples during the period studied.

**Figure 4.** Examples of some microplastic particles found in this work and classified by shape and colour. (**1**): Green and yellow fibres, (**2**,**3**): Yellow and transparent fragments, (**4**): Grey film, (**5**): Black pellet, (**6**): Transparent foam.

Figure 5 shows the percentages of MP classified by shape found in the different points of sampling: influent, secondary effluent and final effluent. It can be observed that, in all samples, fibres and fragments constitute practically the totality of MPs (above 98%). According to the literature, fibres and fragments are the most predominant particle found in wastewater with a mean percentage of 56% and 34%, respectively [18,41,52,57,69–71]. Previous studies reported that fragments are the vast majority MPs [17,34,72,73], in concordance with the results obtained in this case study. Following the evolution of MPs through the wastewater treatment process (Figure 5), in influent samples the concentration of fragments and fibres ranged between 44.8–77.6% (with an average value of 64.9 ± 9.5%) and 20.0–55.2% (with an average value of 34.2 ± 10.2%), respectively. These percentages remained constant after the secondary treatment. However, in the final effluent samples, the concentration of fragments and fibres ranged between 46.1–81.4% and 18.6–61.0%, respectively, which shows a certain decrease in the abundance of fragments (with an average value of 57.3 ± 10.9%) and an increase in the percentage of fibres (with an average value of 40.3 ± 10.8%). This means that the tertiary treatment allowed a better removal of fragments (38%) than fibres (24%), as can be seen in Table S2. It has been reported that the high length-width ratio allows fibres to remain in water masses for more time than particles with other morphologies [2]. In addition, the overall removal efficiency shows a better elimination of fragments in comparison with fibres (67% vs. 56%). Finally, it is noteworthy that films, pellets and foams only account for 1–2%.

Respect to the MPs colour, white and black microparticles were the most common MPs at every sampling point, which means 81% of total MPs. The remaining percentage corresponds to red, blue, green, yellow and purple. This is agreement with previous studies that analyse MPs in WWTPs where higher abundances of white and black MPs were also detected [40,48,74].

**Figure 5.** Shape variation of microplastics in influent, secondary effluent and final effluent samples during the period studied.

#### *3.3. Chemical Composition of MPs*

The chemical composition is a relevant characteristic that determines the MP density and therefore, directly influences over the removal efficiency. Over 30 kinds of polymers have been described in wastewater samples of different WWTPs [51]. In this study PE, PP, PS, PA, PET and polyvinyl chloride (PVC) were detected in the wastewater samples (Figure 6). In the influent, on average, PP is the polymer most frequently detected with an abundance of 24.9 ± 5.5% (ranges between 15.8–37.4%), followed by PET with 23.2 ± 2.9% (27.8–18.1%), PE with 17.3 ± 4.2% (13.0–26.0%), PS with 14.5 ± 2.7% (10.4–17.3%), PA with 3.9 ± 3.4% (9.3–22.4%) and PVC with 6.2 ± 3.1% (1.5–10.7%). Different studies reported that most frequent polymers in urban wastewaters are PS (20–90%), PE (5–60%), PP (2–40%), PET (3–38%), PA (2–35%) [2,3,75] and PVC in low abundances [61]. These data are in agreement with those found here, excepting for PS that was detected in percentages lower than values described in previous works. These variations in the abundance of different type of polymers in the influent are determined by the origin of wastewater that arrives to the WWTP (urban, industrial, agricultural) [74]. As the wastewater stream progresses through the different stages of WWTP, polymers less dense than wastewater, such as PP and PE, increased their proportion, being in the final effluent in percentages around 47.4 ± 3.6% and 29.6 ± 5.0%, respectively. On the contrary, polymers denser than wastewater, such as PS, PA, PET and PVC, decreased in abundance during the treatment processes due to their facility of settling, so they represented in the final effluent around 21%, whereas in the influent their proportion was notably higher (58%). An example of the FTIR spectra for each polymer are shown in Figure S2.

**Figure 6.** Composition variation of microplastics in influent, secondary effluent and final effluent samples during the period studied.

In addition, it has been analysed the relation between the chemical composition and the shape and colour of MPs during the different treatments in WWTP. Linking chemical composition with colour (Table S3), it is noticeable that, more than 90% of PVC microparticles were purple and yellow. Linking chemical composition with shape (Table S4), in the influent samples it has been found that PVC, PP, PET and PE have percentages of fragments of 98%, 77%, 74% and 67%, respectively. Moreover, 90% of the particles that corresponded to PA were fibres. Foams, pellets and films did not represent an abundance higher than 10% for any polymer.
